CN103037893A - Treatment of coagulopathy with hyperfibrinolysis - Google Patents
Treatment of coagulopathy with hyperfibrinolysis Download PDFInfo
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Abstract
The present invention relates to the use of thrombomodulin analogues for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis, such as haemophilia disorders. These thrombomodulin analogues exhibit at therapeutically effective dosages an antifibrinolytic effect. Novel protein modifications together with methods for their identification are disclosed.
Description
The present invention relates to have the field of the coagulopathy of hyperfibrinolysis.More specifically, the present invention relates to for example treatment of haemophilia A or haemophilia B of hemophilia.The present invention requires the priority of PCT application PCT/EP2009/004218, and the disclosure of described application is incorporated this paper fully into.
Hemophilia is one group of genetic disorder of the ability of infringement health control blood clotting or blood coagulation, and described ability is used for stoping when angiorrhexis hemorrhage.Haemophilia A, modal form is because the gene mutation of Factor IX causes; Haemophilia B is also referred to as Christmas disease (Christmasdisease), because the gene mutation of factors IX causes.Haemophilia B, the same with haemophilia A, be X-linkage and account for about 12% hemophilia case.Symptom is identical with the symptom of haemophilia A: injured rear excessively hemorrhage; And spontaneous hemorrhage, spontaneous hemorrhage to weight-bearing joint, soft tissue and the mucosa especially.Extremely IA Repeated Hemorrhage causes hemarthrosis, thereby causes the serious arthrosis of making us pain that usually must carry out joint replacement.Hematoma in the soft tissue can cause comprising the false tumor (pseudo tumor) of the downright bad blood that solidifies; They can block, push or collapse in the adjacent organ and can cause infecting.In case form, hematoma just is difficult to treatment, also is like this even perform the operation.Recovery neural after the extruding is not good, thereby causes paralysis.Those bleeding episodes that involve gastrointestinal tract, central nervous system or air flue/peritoneum rear space if be not detected, can cause death.Intracranial hemorrhage is the major causes of death of hemophiliac.
According to estimates, in the U.S. 100,000 routine congenital hemophilia cases are arranged.In these congenital hemophilias, about 20,000 examples are the haemophilia B case, and this type of patient's blood lacks factors IX or famine blood plasma factor IX component fully.Therefore, there is the different orders of severity in this disease, needs weekly to treatment annual or twice.The case that lacks fully needs weekly replacement therapy; The case that part lacks only just needs treatment when bleeding episode occurs, described bleeding episode can lack to annual.In the case that congenital part lacks, bleeding episode is usually by the susceptibility of interim acquisition but not only caused by damage.The temporary transient defect correcting experimenter's of intravenous injection of sufficient a large amount of fresh plasmas or the fresh blood of equivalent defective.As if beneficial effect continued for 2 or 3 weeks usually, although by testing in vitro the blood coagulation defective of blood samples of patients measurement has only been improved 2 or 3 days.
The therapy of utilizing fresh plasma or fresh blood like this is effectively, but it has several critical defects: it needs (1) can obtain a large amount of fresh plasmas at any time; (2) need to be in hospital to use blood plasma; (3) many patients become responsive and finally cause the lethal transfusion reaction blood or the plasma infusion that repeats; (4) but blood plasma at most only partial rcsponse lack; And (5) long-term treatment or operation be impossible, because needed a large amount of blood or blood plasma will cause acute and the lethal edema.
Improved therapy comprises, utilizes the intravenous alternative medicine of Factor IX or factors IX concentrate.Yet similarly this therapy also is subject to the puzzlement of several unfavorable aspects: when the great bleeding episode for the treatment of, even carried out rapidly test-and-treat, tissue injury also still exists (1); (2) many patients become and resist thrombin and produce blocking antibody (so-called hemophilia with inhibitor) for thrombin; (3) although used improved inactivation of virus method, but still exist to increase for example the risk polluted of HIV and hepatitis C virus is (according to estimates by lethal virus, surpass 50% hemophilia colony in the U.S., surpass 10,000 people and infected HIV by the tainted blood supply); (4) thrombin, the especially recombinant blood coagulation factor that separate are very expensive, and normally unavailable in developing country.
Treating outside alternative medicine or preventing hemorrhage is a challenge, because haemophiliachemophiliac hemorrhage be a complicated pathophysiological process, this process is attributable to three defectives: (1) is in the thrombin generation of low tissue factor concentration by the minimizing of extrinsic pathways, (2) Secondary cases of the thrombin generation of the minimizing by intrinsic pathways outburst, and (3) are by the downward modulation that has defective of the fibrinolytic system of intrinsic pathways.
The thrombin generation that reduces causes the blood coagulation tendency that reduces, thereby causes the fact of the bleeding risk that increases to be widely accepted.Yet the work in past 10 years shows that Fibrinolytic downward modulation defective may also play an important role in hemophilia.Therefore, hemophilia also can be classified as the coagulopathy with hyperfibrinolysis.
Nearest publication is supported this hypothesis by the external phenomenon that shows below: when clot exhausts and is supplemented with when forming in the blood plasma (FVIII-DP) of the tPA of tissue plasminogen activator in Factor IX, fibrinolysis can not fully be reduced, clot dissolves (Broze and Higuchi too early as a result, Blood1996,88:3815-3823; Mosnier; Thromb.Haemost.2001,86:1035-1039).In addition, show, should " too early dissolving " owing to the activation of the minimizing of the fibrinolysis inhibitor (TAFI) of thrombin-can activate or do not have described activation (Broze and Higuchi, 1996), and in FVIII-DP, the mixture that comprises the TAFI of activation has increased blood-clot lysis time.Reach a conclusion: the TAFI of stabilisation can be used for treating hemophilia (WO02/099098).
TAFI plays pivotal role in Fibrinolytic downward modulation, it is that the stable clot of formation is necessary.TAFI (being also referred to as TAFI 2 or procarboxypeptidase U) is when being exposed to the soluble thrombomodulin complex, by at Arg
92On proteolysis be converted to the plasminogen that suppresses Fibrinolytic basic carboxypeptidase (bas ic carboxypeptidase) (TAFI of TAFIa or activation).It weakens fibrinolysis effectively by remove C-terminal lysine and arginine residues (its combination and activation for plasminogen is very important) from fibrin.
As discussed above, the thrombomodulin compound with thrombin (TM) is responsible for the TAFI activation.Thrombomodulin is the memebrane protein as the thrombin receptor on the endotheliocyte of liner blood vessel.Thrombin is the center enzyme in the coagulation cascade, and it changes into fibrinogen the fibrin that forms the substrate grumeleuse.At first, local damage causes producing a small amount of thrombin from its inactive precursor thrombinogen.Then, the thrombin activation platelet and, next, some thrombin comprises factor V and VIII.A rear effect produces so-called thrombin outburst (a large amount of activation of other thrombinogen molecule), and this finally causes forming stable clot.
Yet, when being incorporated into thrombomodulin, the activity of thrombin is changed indirectly: the principal character of soluble thrombomodulin complex is the ability of its activated protein c, described PROTEIN C is subsequently by the important cofactor of proteolysis deactivation, factor Va and Factor IX a reduce the coagulation cascade (people such as Esmon, Ann.N.Y.Acad.Sci. (1991) 614:30-43), thereby provide anticoagulant active.The soluble thrombomodulin complex also can activate the fibrinolysis inhibitor (TAFI) of thrombin-can activate, and it is antagonism fibrinolysis (referring to above) subsequently.
Become acquaintance TM to comprise the single polypeptide chain of 559 residues, and formed by 5 domains with following location: amino terminal " agglutinin " domain, comprise " 6EGF-sample repetitive structure territory ", O-glycosylation domain, membrane spaning domain and cytoplasmic structure territory (amino acid position is as shown in SEQ ID NO:1 or SEQ ID NO:3) that 6 epidermal growth factors (EGF) sample repeats:
| Amino acid position roughly | Domain |
| -18--1 | Signal sequence |
| 1-226 | N-end structure territory (agglutinin) |
| 227-462 | 6EGF-sample repetitive structure territory |
| 307-345 | EGF-sample repetitive structure territory 3 |
| 333-344 | The c-ring in EGF-sample repetitive structure territory 3 |
| 347-386 | EGF-sample |
| 387-422 | EGF-sample |
| 423-462 | EGF-sample repetitive structure territory 6 |
| 463-497 | The glycosylation that O-connects |
| 498-521 | Membrane spaning domain |
| 522-557 | The cytoplasmic structure territory |
Table 3: the domain structure of thrombomodulin (numbering according to SEQ ID NO:1).
The various structure function researchs that the proteolytic fragments of use rabbit TM or the deletion mutant of recombined human TM carry out have been positioned to its activity last 3 EGF samples and have repeated.The minimum mutant that can effectively promote TAFI to activate comprises the c that comprises epidermal growth factor-3 (EGF3) and encircles residue to EGF6.This mutant is than long 13 residues of the minimum mutant that activates C; The latter is by forming from encircling between the domain that connects EGF3 and EGF4 to the residue of EGF6.
As mentioned above, be used for the treatment of blood coagulation disorders for example the discontented podiatrist of haemophiliachemophiliac alternative medicine learn needs.Importantly, except being used for the thrombin of alternative medicine, fail to obtain to prevent or to treat the medicine of hemophiliac.
Therefore, although the coagulopathy that prevention or treatment are had hyperfibrinolysis, the exploitation of haemophiliachemophiliac therapy exists for a long time especially needs, and makes progress always very slowly, and still lacks safely and effectively therapy.
Therefore, the objective of the invention is, be provided for treating the novel method of the coagulopathy with hyperfibrinolysis.
This purpose is by being provided for treating mammal, and the medicament of people's the coagulopathy with hyperfibrinolysis solves especially, and described medicament is included in the thrombomodulin analog that shows the fibrinolysis effect on the treatment effective dose.Used according to the present invention particularly suitable pharmaceutically active protein matter and peptide also are provided.
Novel method is based on surprising discovery: can modify by this way thrombomodulin, described mode so that itself in addition under high plasma concentration, surpassing 15nM especially, surpass 20 especially, 30,40 or the concentration of 50nM (high to 100nM at least) under show that to surpass its fibrinolysis that causes fibrinolytic active.Therefore, this type of TM analog shows the fibrinolysis effect, thereby is suitable for according to purposes of the present invention.In most of favourable embodiments, the TM analog can in addition high to 200nM or higher, be more preferably up to that to have dominant fibrinolysis under the concentration of 300nM or 500nM active.
This fibrinolysis effect sees blood plasma from hemophiliac, and (described blood plasma has exhausted Factor IX; FVIII-DP) and suffer from the whole blood or blood plasma of Canis familiaris L. of haemophilia A.Therefore, show such thrombomodulin analog useful as therapeutics.
Up to the present, thrombomodulin is used for the treatment of haemophiliachemophiliac therapeutic use and is not taken as yet actual selection, because learn from rabbit lung thrombomodulin (rlTM), it always has (even under low concentration) fibrinolysis activity and causes fibrinolytic (referring to Mosnier and Bouma; Arterioscler.Thromb.Vasc.Biol.2006; 26:2445 – 2453; Fig. 5 especially).Be lower than under the plasma concentration of 15nM, rlTM increases blood-clot lysis time, yet is being higher than under the plasma concentration of 15nM, remarkable minimizing (causing fibrinolysis activity as final the result) (people such as Mosnier who shows dissolution time, 2001, Mosnier and Bouma, 2006).Because but the difference lethal of potential excessive administration or individual susceptibility increases the weight of, prolongs or even the event that causes bleeding, therefore this fibrinolysis activity that causes under higher concentration suppress its any therapeutic use in hemophilia.
According to the present invention, there are multiple choices, it causes showing the fibrinolysis effect, thereby is suitable for the TM analog according to treatment of the present invention.
In one embodiment, can use and have the thrombomodulin analog to the binding affinity of thrombin that reduces.Therefore, they can prolong clot dissolution in normal plasma and FVIII-DP, and are for example, high to 100nM (Fig. 4) or high to 500nM (Figure 10).
The importance of these discoveries is, even this type of thrombomodulin analog also shows the fibrinolysis effect under high concentration without harmful fibrinolysis activity that causes.This concentration is far above the treatment effective dose.Therefore, the TM analog makes it possible to treat the coagulopathy with hyperfibrinolysis.
Not bound by theory, the inventor shows that this treatment potential of TM analog can be explained by the following fact: they show the affinity to thrombin that significantly reduces.This is by the K that finds 23nM
DThe people such as the Bajzar (J.Biol.Chem1996 of value; 271:16603-16608) show, and the K of the 0.2nM that observes for the combination between thrombin and the rabbit lung thrombomodulin
DValue opposite people such as (, Ann.NY.Acad.Sci.1986,485:215-220) Esmon.
Therefore, according to one embodiment of the invention, the thrombomodulin analog can be used for treating the coagulopathy with hyperfibrinolysis, and described analog is compared with rabbit lung thrombomodulin, has the binding affinity to thrombin of minimizing.
Particularly, can use the thrombomodulin analog, described analog shows above 0.2nM, preferably surpasses the K for the thrombin combination of 1nM, 2nM, 4nM, 5nM, 7.5nM, 10nM, 12.5nM, 15nM, 17.5nM, 20nM, 22.5nM or 25nM
D, more preferably 10 to 30nM 10 to 100nM or higher scope in K
DValue.In specific embodiments, can use to have about 50,60 or the K for the thrombin combination of 70nM
DThe TM analog.
In other embodiments of the present invention, the fibrinolytic that causes of the minimizing of thrombomodulin analog can cause because of the ability (so-called " cofactor is active ") of the activated protein c that reduces.Cause Fibrinolytic rise people such as (, 2001) Mosnier because PROTEIN C activates, the cofactor activity that therefore reduces will prolong blood-clot lysis time.The strategy of the cofactor activity of the known several minimizing thrombomodulins of those skilled in the art, for example change of the change of the glycosylation of protein, secondary or tertiary structure or preferred primary structure is for example by one or more amino acid whose sudden changes.
In another embodiment, can use and thrombomodulin analog TM
EM388L compares and has the TM analog of the cofactor activity that reduces, wherein TM
EThe analog that expression only is comprised of 6 EGF domains.
According to the present invention, also can use the thrombomodulin analog of the ability (so-called " TAFI Activation Activity ") of the activation TAFI with enhancing, because activating, TAFI causes Fibrinolytic downward modulation (Mosnier and Bouma, 2006).To those skilled in the art, the strategy that has the TAFI Activation Activity of several enhancing thrombomodulins, the for example change of the change of the glycosylation of protein, secondary or tertiary structure or preferred primary structure is for example by one or more amino acid whose sudden changes.
Especially, the present invention also provides the thrombomodulin analog, described analog and thrombomodulin analog TM
EM388L TAFI Activation Activity with the remarkable increase ratio to the cofactor activity of comparing.
Notably, according to the present invention, the TM analog that is used for the treatment of coagulopathy has the one or more features in the above-mentioned feature, that is:
(i) compare with rabbit lung thrombomodulin, the binding affinity to thrombin that reduces, and/or have k above 0.2nM
DThe binding affinity to thrombin of value;
(ii) compare with the cofactor activity of TM analog TMEM388L, the cofactor that reduces is active, or
(iii) with TM analog TM
EM388L compares, and the TAFI Activation Activity of increase is to the ratio of cofactor activity.
In embodiments of the invention, thrombomodulin can be used for the treatment of the people patient who suffers from any coagulopathy, described coagulopathy is compared with the normal subjects and is occured significantly or even the slight fibrinolysis that reduces.Especially, the following disease of available thrombomodulin analogue treatment: the genetic disorder of haemophilia A, haemophilia B, C type hemophilia, von Willebrand (vWD), Of Acquired von Willebrand Disease, factor X deficiency, parahemophilia, factor I, I I, V or VII, the bleeding disorder or the acquired blood coagulation that cause because of CAC (comprise for thrombin for example the autoantibody of Factor IX) lack.
Should be understood that and to keep or the nature and extent of the disease of any particular patient is successfully depended in the treatment that realizes by therapy of the present invention.
Specific embodiments of the present invention relates to the prophylactic treatment of the hemorrhage coagulopathy of prevention or the acute treatment (" as required ") that carries out when hemorrhage the generation.The bleeding episode of stand-by thrombomodulin analogue treatment can be in each organ or tissue of organism, occurs in space or the soft tissue behind (for example intracranial hemorrhage), joint, muscle, gastrointestinal tract, respiratory tract, the peritoneum in the central nervous system most importantly.
For prophylactic treatment, can with the TM analog prolong the time interimly regularly use to the patient.Yet, also be possible at quite limited time interim multiple dosing (" inferior chronic treatment ").
In one embodiment of the invention, before for example performing the operation or have tooth pulled out, higher bleeding risk gives the thrombomodulin analog.
In other embodiments of the present invention, give and to be difficult to standard treatment for example blood or plasma infusion or use the patient of the replacement therapy of thrombin to use the thrombomodulin analog.
According to the present invention, can be preferably interim when being shorter than 1 thoughtful 4 weeks total, once a day, but also can per 2 days once, or per 3,4,5,6 or 7 days once, more preferably in the mode of chronic administration, use the TM analog with a plurality of dosage.Therefore, according to the present invention, provide the pharmaceutical composition of repeatedly using of suitable permission thrombomodulin analog.
The mode of preferably using with parenteral is for example passed through intravenous or subcutaneous administration, the non-TM analog of using orally.Intravenous or subcutaneous group annotate and use also is possible.Therefore, according to the present invention, the pharmaceutical composition that provides the parenteral that is suitable for thrombomodulin to use.
In one embodiment of the invention, the thrombomodulin analog is soluble TM analog, the TM analog that wherein the cytoplasmic structure territory is lacked and membrane spaning domain is lacked wholly or in part especially.
In a preferred embodiment of the invention, the thrombomodulin analog comprises at least one and is selected from following domain: EGF3, EGF4, EGF5 or EGF6, EGF domain EGF1 to EGF6 preferably, more preferably EGF domain EGF3 to EGF6, EGF domain EGF4 to EGF6 most preferably, the c that comprises especially epidermal growth factor-3 (EGF3) encircles the fragment to EGF6.
The various forms of soluble thrombomodulin is well known by persons skilled in the art, for example by Asahi Corporation (Tokyo, Japan) the so-called ART-123 of exploitation or at present just by PAION Deutschland GmbH, the recombinant soluble human thrombomodulin Solulin of Aachen (Germany) exploitation.The recombinant soluble thrombomodulin, that is, the soluble thrombomodulin that does not have the modification of aminoacid sequence is the theme of Asahi patent EP0312598B1.
Solulin is the solubility of human thrombomodulin and the analog of protease inhibitor and oxidation, thereby shows in vivo the long life-span.The principal character of Solulin is its widely mechanism of action, because its Trombin inhibiting not only.It also activates TAFI and native protein C/ Protein S approach.Because its thrombin combination that reduces, Solulin even under high concentration, still suppress fibrinolysis.Solulin and modified forms thereof are specific embodiments of the present invention.
Solulin is European patent EP 0641215B1 especially, the theme of EP0544826B1 and EP0527821B1.Solulin compares with the sequence (SEQ.ID NO.1) of natural human thrombomodulin and comprises modification at lower column position: the termination on removal, M388L, R456G, H457Q, S474A and the P490 of G-3V, amino acid/11-3.This numbering system is according to the natural thrombomodulin of SEQ.ID NO.1 and SEQ ID NO:3.Sequence as the Solulin of a preferred embodiment of the present invention is shown among the SEQ ID NO:2.
Yet, notably, according to the present invention, also can use only to comprise in the above-mentioned character or the thrombomodulin analog of the one or more character in the character shown in above-mentioned European patent EP 0544826B1, EP0641215B1 and the EP0527821B1.
In addition, according to the present invention, can use only to comprise in the above-mentioned character or the people such as Wnag the thrombomodulin analog of the one or more character in the character shown in the publication of 2000, J.Biol CHem.275:22942-22947.
Particularly preferred thrombomodulin analog that can be used according to the invention is the thrombomodulin analog with one or more following features:
(i) they show the oxidation resistance,
(ii) they show protease resistant,
(iii) they have N-or the C-end of homogeneous,
(iv) they have carried out post translational modification, for example glycosylation of at least some glycosylation sites by natural thrombomodulin (SEQID NO:1),
(v) they have Linear Double thrombin binding property reciprocal,
(vi) they are soluble in the aqueous solution of the detergent that contains relatively low amount, and usually lack the cross-film sequence,
(vii) they lack glycosaminoglycan chains.
The preparation of this type of analog that uses among the present invention is disclosed in the above-mentioned European patent that relates to Solulin.
In one embodiment of the invention, can only use 6 EGF domains of Solulin, the Solulin fragment that is formed by EGF4 to EGF6 domain especially.
In one embodiment, can use the thrombomodulin analog (as from WO93/25675A1, knowing) with the cofactor activity that reduces.Described a series of thrombomodulin analog herein, it has contrast human soluble thrombomodulin (TM
EM388L) about 50% or cofactor still less is active.
More particularly, described thrombomodulin analog shows and TM when bind thrombin
EThe combination of M388L is compared, and the cofactor of being less than or equal to 50% modification is active, and one or more positions of the amino acid position that described analog provides in corresponding to SEQ I D NO:1 or SEQ I D NO:3 have amino acid replacement:
ab)
355Asn；
ae)
359Gln；
af)
363Leu；
ai)
368Tyr；
aj)
371Val；
ak)
374Glu；
al)
376Phe；
am)
384His；
an)
385Arg；
ba)
387Gln；
bb)
389Phe；
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bf)
403Thr；
bg)
408Glu；
bh)
411Glu；
bi)
413Tyr；
bj)
414Ile；
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
bn)
420Ile;
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
ce)
428Glu；
cf)
429Asp；
cg)
432Phe；
ch)
434Ser；
ci)
436Val；
cj)
438His；
ck)
439Asp；
cl)
440Leu；
cm)
443Thr；
cn)
444Phe；
co)
445Glu；
cp)
456Arg；
Cq)
458Ile; Or
cr)
461Asp
The TM analog that most preferably only has one of displacement listed above.In one embodiment of the invention, the TM analog is the one or more sudden changes that have in the said mutation, the Solulin of a preferred sudden change (SEQ.ID NO:2).Therefore, the invention still further relates to and have at least one said mutation, in specific embodiment, only have the protein according to SEQ ID NO:2 of a said mutation.In one embodiment, an aminoacid on the given position is lacked but not is replaced.In other embodiments, the present invention includes the Solulin fragment with one of said mutation, the c by EGF3 to EGF6 domain or EGF3 encircles the Solulin fragment that forms to EGF6 especially.Solulin or Solulin fragment can comprise in the said mutation at least one or just what a sudden change (for example sudden change on the position 376) at amino acid position 371 to 389.If Solulin or Solulin fragment 376 comprise sudden change (for example F376A) in the position, the second sudden change that then is selected from said mutation is possible.
For convenience's sake, to the name on the left side, for example aa), be identical for each decorating site.First letter represents the EGF domain, and wherein a is EGF4; B is that EGF5 and c are EGF6.The second letter representative is with respect to the relative position of the modification of other residue in the tabulation.The nucleic acid of the above-mentioned TM analog of encoding also is provided herein.
Following analog has consisted of the preferred subgroup of above-mentioned given analog, and wherein analog has contrast TM
E25% or the less cofactor activity of M388L.This type of analog has one or more amino acid replacements, preferably only has an amino acid replacement (amino acid position shown in SEQ ID NO:1 or the SEQ IDNO:3):
ae)
359Gln；
aj)
371Val；
ak)
374Glu；
al)
376Phe；
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bg)
408Glu；
bi)
413Tyr；
bj)
414Ile;
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
bo)
423Asp；
bp)
424Ile；
bq)
425Asp；
cd)
426Glu；
ce)
429Asp；
ck)
439Asp；
Cn)
444Phe; Or
cr)
461Asp.
In one embodiment of the invention, the TM analog is the one or more sudden changes that have in the said mutation, the Solulin of a preferred sudden change (SEQ.ID NO:2).Therefore, the invention still further relates at least one sudden change that has in the said mutation, the protein according to SEQ ID NO:2 of what a sudden change just in specific embodiments.In one embodiment, an aminoacid on the given position is lacked but not is replaced.In other embodiments, the present invention includes the Solulin fragment with one of said mutation, the c by EGF3 to EGF6 domain or EGF3 encircles the Solulin fragment that forms to EGF6 especially.Solulin or Solulin fragment can in amino acid position 371 to 389, comprise in the said mutation at least one or just what a the sudden change (for example sudden change on the position 376).If Solulin or Solulin fragment 376 comprise sudden change (for example F376A) in the position, the second sudden change that then is selected from said mutation is possible.
Above also be applicable to above-mentioned analog with 50% the cofactor activity that is lower than contrast about the modification shown in proteinase activity, aliphatic series displacement, oxidation resistance and the homogeneous end.
Preferably above listed have an analog that is lower than 30% control activity.This type of analog is represented by the sudden change in the domain 4.This type of analog has one or more amino acid replacements, preferably only has an amino acid replacement (amino acid position that provides such as SEQ ID NO:1 or SEQ ID NO:3):
ae)
359Gln；
Aj)
371Val; Or
al)
376Phe.
In one embodiment of the invention, the TM analog is to have one or more in the said mutation, the Solulin of a preferred sudden change (SEQ.ID NO:2).Therefore, the invention still further relates at least one sudden change that has in the said mutation, the protein according to SEQ ID NO:2 of what a sudden change just in specific embodiment.In one embodiment, an aminoacid on the given position is lacked but not is replaced.In other embodiments, the present invention includes and draw together the Solulin fragment with one of said mutation, the c by EGF3 to EGF6 domain or EGF3 encircles the Solulin fragment that forms to EGF6 especially.Solulin or Solulin fragment can be in amino acid position 371 to 389 at least one that comprise in the said mutation, or just what a sudden change (for example sudden change on the position 376).If Solulin or Solulin fragment 376 comprise sudden change (for example F376A) in the position, the second sudden change that then is selected from said mutation is possible.
Also described herein and had and TM
EThe M388L basically unaltered K that compares
DThe analog of value.Known EGF5 and EGF6 to the high-affinity of thrombin in conjunction with in play an important role, and the EGF4 that has not too pivotal role in combination is vital for giving TM/ thrombin complex cofactor activity.Therefore, this type of analog that has modification in EGF repetition 5 and 6 can have and TM
EActive but the K that reduces of cofactor that M388L compares almost identical
D, for example (S406A).Repeat to have in 5 and 6 the analog of the modification that causes the cofactor activity that reduces lists in hereinafter at EGF.This type of analog has one or more, preferably only has an amino acid replacement (amino acid position that provides among SEQ ID NO:1 or the SEQ ID NO:3):
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bf)
403Thr；
bg)
408Glu；
bi)
413Tyr；
bj)
414Ile；
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
cf)
429Asp；
ck)
439Asp；
Cn)
444Phe; Or
cf)
461Asp
Above-mentioned analog also can according to they separately domain (being EGF4, EGF5 or EFG6) and divide into groups according to their relative activities separately.For example, the TM analog with EGF4 sequence modification that has a contrast cofactor activity of about 50% is (amino acid position shown in SEQ I D NO:1 or the SEQ I DNO:3):
bb)
355Asn；
ae)
359Gln；
af)
363Leu；
ai)
368Tyr；
aj)
371Val；
ak)
374Glu；
al)
376Phe；
Am)
384His; Or
an)
385Arg.
Most preferably only has the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.
Have those analog that in EGF4, have displacement that are lower than 25% contrast cofactor activity and be (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3):
ae)
359Gln；
Aj)
371Val; Or
al)
376Phe.
Most preferably only has the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.
In EGF5, following modification causes having the analog (amino acid position shown in SEQ I D NO:1 or the SEQ I D NO:3) that at least 50% cofactor activity reduces:
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bf)
403Thr；
bg)
408Glu；
bh)
411Glu；
bi)
413Tyr；
bj)
414Ile;
bk)
415Leu；
bl)
416Asp；
Bm)
417Asp; Or
bn)
420Ile.
Most preferably only has the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.In the middle of this type of analog, be wherein analog and TM
EM388L compares and has basically those analog of unaltered kCat/Km.
In EGF5, those modifications of the analog that can reduce according to the cofactor activity that causes having at least 75% further are divided into subgroup (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3) with analog:
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bg)
408Glu；
bi)
413Tyr；
bj)
414Ile；
bk)
415Leu；
Bl)
416Asp; Or
bm)
417Asp.
Most preferably only has the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.In the middle of this type of analog, for TM
EM388L compares and has basically those analog of unaltered kCat/Km.The nucleic acid of the above-mentioned analog of encoding also is provided.
About EGF6, provide hereinafter grouping.Have those analog that are lower than 50% contrast cofactor activity and be (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3):
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
ce)
428Glu；
cf)
429Asp；
cg)
432Phe；
ch)
434Ser；
ci)
436Val；
cj)
438His；
ck)
439Asp；
cl)
440Leu；
cm)
443Thr；
cn)
444Phe；
co)
445Glu；
cp)
456Arg；
Cq)
458Ile; Or
cr)
481Asp.
Most preferably only has the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.
Have the analog that is lower than 25% contrast cofactor activity and be (amino acid position shown in SEQ ID NO:1 or the SEQI D NO:3):
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
cf)
429Asp；
ck)
439Asp；
Cn)
444Phe; Or
cf)
461Asp.
Most preferred for only having the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.Preferred analog is those analog about the extra modification of dissolubility, protease resistant, oxidation resistance and homogeneous end of having shown in above.Encode the nucleic acid of this type of analog also for a part of the present invention.The same with other group, this type of analog comprises that wherein said analog has and TM
EM388L basically those analog of unaltered kCat/Km of comparing.
Also can analog further be divided into subgroup according to the amino acid whose analog that has modification in some site, wherein said analog is compared with the analog that has natural residue in described position, has the K for thrombin that basically equates
D, wherein said position is corresponding to (amino acid position shown in SEQID NO:1 or the SEQ ID NO:3):
Bb)
355Asn; Or
ae)
359Gln。
Most preferredly be the TM analog that only has one of institute's column permutation above for example Solulin or Solulin fragment.This type of analog can have the kCa t/Km of 30% the change that is lower than contrast.
Following site comprises comparing with the analog that has natural residue in described position to have the K of change
DOr the analog of the description of kCat/Km, wherein said position is corresponding to (amino acid position shown in SEQ IDNO:1 or the SEQ ID NO:3):
af)
363Leu；
aj)
371Val；
ak)
374Glu；
al)
376Phe；
am)
384His；
an)
385Arg；
bc)
398Asp；
Bd)
400Asp; Or
be)
402Asn.
Most preferredly be the TM analog that only has one of institute's column permutation above for example Solulin or Solulin fragment.This type of analog also comprises the K with change
DAnd kCat/Km (has been changed at least 20% K especially
DAnd kCat/Km) analog.
Following site has been described, and comparing with the analog that has natural residue in described position has the K that lower cofactor is active and basically equate
DOr the analog of kCat/Km, wherein said position is corresponding to (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3):
bg)
408Glu；
bh)
411Glu；
bi)
413Tyr；
bj)
414Ile；
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
bn)
420Ile;
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
ce)
428Glu；
cf)
429Asp；
cg)
432Phe；
ch)
434Ser；
ci)
436Val；
cj)
438His；
ck)
439Asp；
cl)
440Leu；
cm)
443Thr;
cn)
444Phe；
co)
445Glu；
cp)
456Arg；
Cq)
458Ile; Or
cr)
461Asp.
Most preferred for only having the above TM analog of one of institute's column permutation, for example Solulin or Solulin fragment.
Following location expression causes at least 75% cofactor activity to reduce but does not basically change the subgroup (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3) of those modifications of kcat/Km:
bg)
408Glu；
bi)
413Tyr；
bj)
414Ile；
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
ca)
423Asp；
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
Gf)
429Asp；
ck)
439Asp；
Cn)
444Phe; Or
cr)
461Asp.
The TM analog that most preferably has one of institute's column permutation above is Solulin or Solulin fragment for example.Can be further carry out the subgroup grouping to above-mentioned modification, wherein for the K of thrombin
DBe changed at least 30%.
The present invention also provides method.More specifically, disclose the method for the thrombomodulin analog that screens the Kd that shows the thrombin combination that changes herein, it comprises step:
A) (amino acid position shown in SEQ ID NO:1 or the SEQ I D NO:3) carries out amino acid replacement in following position:
bg)
408Glu；
bi)
413Tyr;
bj)
414Ile；
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
bn)
420Ile；
ca)
423Asp;
cb)
424Ile；
cc)
425Asp；
cd)
426Glu；
ce)
428Glu；
cf)
429Asp；
cg)
432Phe；
ch)
434Ser；
ci)
436Val；
cj)
438His；
ck)
439Asp；
cl)
440Leu;
cm)
443Thr；
cn)
444Phe；
co)
445Glu；
cp)
456Arg；
cq)
458Ile；
Cr)
461Asp; With
B) will be for the K of thrombin
DCompare with the contrast molecule.
So employed in the class methods, the TM analog that only has an amino acid replacement for example Solulin or Solulin fragment is preferred.Various embodiments of the present invention comprise such embodiment, wherein said K
DBe changed at least 33%, or the wherein said amino acid replacement that is modified to, or wherein said contrast molecule is TM
EM388L.Preferably being grouped into of modification (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3) that is used for the method:
bg)
408Glu；
bi)
413Tyr；
bj)
414Ile;
bk)
415Leu；
bl)
416Asp；
bm)
417Asp；
ca)
423Asp；
cb)
424Ile;
cc)
425Asp；
cd)
426Glu；
cf)
429Asp；
ck)
439Asp；
Cn)
444Phe; Or
cr)
461Asp.
So employed in the class methods, the TM analog that only has an amino acid replacement for example Solulin or Solulin fragment is preferred.
Described another method herein, described method is used for screening when the thrombomodulin analog of the cofactor activity that has change when thrombin is combined, and it comprises step:
A) (amino acid position shown in SEQ ID NO:1 or the SEQ ID NO:3) carries out amino acid replacement in following position:
bb)
355Asn;
Ae)
359Gln; With
B) ratio of the cofactor activity after thrombin is combined is compared with the ratio of contrast molecule.
So employed in the class methods, the TM analog that only has an amino acid replacement for example Solulin or Solulin fragment is preferred.
In a preferred embodiment of the invention, the thrombomodulin analog in the position the 376 modification (Phe376X with phenylalanine residue; SEQ ID NO:1 or SEQ ID NO:3).This residue can utilize the method for well known to a person skilled in the art to carry out chemistry or biochemical modification or disappearance.Preferably, use aliphatic amino acid, more preferably use glycine, alanine, valine, leucine or isoleucine, most preferably replace phenylalanine residue with alanine.Shown that alanine is to Phe
376Displacement (" F376A ") significantly to have reduced the cofactor of thrombomodulin analog active, keep simultaneously TAFI Activation Activity (referring to Fig. 7).Therefore, the F376A-TM analog has the TAFI Activation Activity of increase to the ratio of cofactor activity.In one embodiment of the invention, Solulin comprises Phe376X, especially F376A displacement.
In other embodiments of the present invention, the thrombomodulin analog modification that 387 (SEQ ID NO:1 or SEQ ID NO:3) have glutamine residue in the position.Glutamine residue is preferably by following aminoacid (by (referring to Fig. 8 A) arranged sequentially of the cofactor activity of successively decreasing of gained mutant Gln387X-TM analog) displacement: Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys.In one embodiment of the invention, Solulin comprises this Gln387X displacement; In other embodiments, the displacement in the Solulin comprises Gln387X displacement and above-mentioned F376X displacement.
In another embodiment of the invention, 388 (SEQ ID NO:1 or SEQ ID NO:3) have the modification of methionine residues to the thrombomodulin analog in the position.Methionine residues is preferably by following aminoacid (by (referring to Fig. 8 B) arranged sequentially of the cofactor activity of successively decreasing of gained mutant Me t388X-TM analog) displacement: Gln, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys.In one embodiment, Solulin comprises one or two in this displacement and above-mentioned Phe376X and the Gln387X displacement.
In other embodiments of the present invention, the thrombomodulin analog has the modification of phenylalanine residue at aminoacid 389 (SEQ ID NO:1 or SEQ ID NO:3).Phenylalanine residue is preferably by following aminoacid (by (referring to Fig. 8 C) arranged sequentially of the cofactor activity of successively decreasing of gained mutant Phe389X-TM analog) displacement: Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro.In one embodiment, Solulin can comprise this displacement, and what described displacement also can be with above-mentioned Phe376X, Gln387X or Met388X displacement is one or more, preferred all combinations.
In another embodiment of the invention, by 3 aminoacid Gln
387, Met
388And Phe
389The TM that forms for example encircle between the domain of Solulin and is partially or completely lacked, or has inserted one or more aminoacid (preferably alanine residue) (referring to Fig. 8 D).
For at position Phe
376, Gln
387, Met
388Or Phe
389On have the TM analog of modification, the TM analog can be total length or soluble T M analog, it comprises EGF domain EGF1 to EGF6, preferably comprises EGF domain EGF3 to EGF6.In preferred embodiments, this type of analog is included in the displacement shown in the TM analog Solulin.In a more preferred embodiment, the TM analog that this type of Solulin derives only is comprised of EGF1 to EGF6, only is comprised of EGF domain EGF3 to EGF6 especially or is encircled to EGF6 by the c of EGF3 to form (these 3 fragment called after Solulin fragments).
In one embodiment of the invention, the thrombomodulin analog uses with its oxidised form.The several technology that are used for the controlled oxidation of protein are well known by persons skilled in the art.The TM analog preferably carries out oxidation with toluene-sodium-sulfonchloramide, hydrogen peroxide or sodium metaperiodate.
The invention still further relates to the method that is used for the treatment of the TM analog of the coagulopathy with hyperfibrinolysis for screening.The method comprises first step: by preferably in EGF domain EGF1 to EGF6, more preferably in EGF domain EGF3 to EGF6, most preferably at amino acid position Asp
349With Asp
461Between insert, lack or replace the aminoacid sequence that one or more aminoacid are modified thrombomodulin.Several technology of the known modifying protein sequence of those skilled in the art (for example by direct mutagenesis or random mutagenesis, selecting subsequently).
In second step, be selected from TM analog and the control protein that following feature is relatively modified with regard to one or more: to the binding affinity (K of thrombin
DValue), the effect of the ratio of active, the TAFI Activation Activity of cofactor or TAFIa potential, TAFI Activation Activity and cofactor activity, protein oxidation, in external test in time on impact or the effect in the blood coagulation relevant animal models of clot dissolution.
Protein in contrast uses thrombomodulin or analog, preferred rabbit lung thrombomodulin or comprise the people TM analog of 6 EGF domains.The TM analog can have natural acid sequence or selectively can have for example M388L displacement of one or more modifications.
The invention still further relates to the method that treatment has the coagulopathy of hyperfibrinolysis, comprise the thrombomodulin analog of the demonstration fibrinolysis effect of administering therapeutic effective dose.
Especially, this type of Therapeutic Method comprises that comparing with control protein shows the TM analog of one or more following features: the binding affinity to thrombin that reduces, k
DValue greater than the binding affinity to thrombin of 0.2nM, the cofactor that significantly reduces is active or the TAFI Activation Activity that increases to the ratio of cofactor activity.Protein in contrast uses thrombomodulin or analog, preferred rabbit lung thrombomodulin or comprise the people TM analog of 6 EGF domains.The TM analog can have natural acid sequence or selectively can have for example M388L displacement of one or more modifications.
In other embodiments, the present invention relates to when when thrombin is combined, with TM
EM338L compares, and has the thrombomodulin analog of the cofactor activity that reduces, and it has:
A) according to the aminoacid sequence of SEQ ID NO:2, or
B) according to the aminoacid sequence of SEQ ID NO:4, or
C) with according to the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 have at least 90%, more preferably at least 95%, the aminoacid sequence of at least 98% homogeneity most preferably,
D) thrombomodulin fragment, it is comprised of following basically: SEQ ID NO:2,6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO:1) of SEQ IDNO:3 or SEQ ID NO:4, SEQ ID NO:2,3 to EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO:1) or the SEQ ID NO:2 of SEQ ID NO:3 or SEQ ID NO:4, the c in the EGF-sample repetitive structure territory 3 of SEQ ID NO:3 or SEQ IDNO:4 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO:1)
Wherein the phenylalanine on the position 376 (as according to SEQ ID NO:1 numbering) is lacked or is replaced by glycine, alanine, leucine, isoleucine or valine.
In another embodiment of the invention, thrombomodulin also comprises disappearance or the displacement of the glutamine residue on the position 387 (as numbering among the SEQ ID NO:1), and wherein displacement is preferably by Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys displacement.
In other embodiments, the present invention relates to also (number in such as SEQ ID NO:1) in position 388 comprise the disappearance of methionine residues or the thrombomodulin of displacement, wherein methionine residues is preferably by Gln, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys displacement.
In other embodiments, thrombomodulin also comprises disappearance or the displacement of the phenylalanine residue on the position 389 (as numbering among the SEQ ID NO:1), wherein, phenylalanine is preferably by Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro displacement.
In another embodiment, thrombomodulin comprises such as first and second of description in the table 4 the amino acid modified combinations.
In another embodiment, thrombomodulin comprises such as first, second, and third of description in the table 5 amino acid modified combination.
The invention still further relates to the thrombomodulin analog and be used for the treatment of the purposes of the coagulopathy with hyperfibrinolysis, described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin, and comprises one or more in the following sequence modification:
A) removal of amino acid/11-3;
b)M388L;
c)R456G;
d)H457Q;
E) S474A, and the termination on P490,
Wherein, this thrombomodulin analog also comprises the sequence modification of one or more following amino acid positions (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1):
a)
359Gln；
b)
398Asp;
c)
400Asp；
d)
402Asn；
e)
408Glu；
f)
413Tyr；
g)
414Ile;
h)
415Leu，
i)
417Asp；
j)
439Asn.
Of the present invention other preferred aspect, a) to j) listed aminoacid replaced by alanine.
The invention still further relates to the thrombomodulin analog and be used for the treatment of the purposes of the coagulopathy with hyperfibrinolysis, described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more in the following sequence modification:
A) amino acid/11-3 removes;
b)R456G;
c)H457Q;
D) S474A, and the termination on P490,
Wherein, this thrombomodulin analog also comprises the sequence modification of one or more following amino acid positions (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1):
A)
388Met has except the modification to the change of Leu
b)
416Asp;
c)
423Asp；
d)
425Asp；
e)
426Glu；
f)
429Asp；
g)
440Leu；
h)
461Asp.
In one aspect of the invention, a) to h) listed aminoacid replaced by alanine.
The invention still further relates to the thrombomodulin analog and be used for the treatment of the purposes of the coagulopathy with hyperfibrinolysis, described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin, and comprises one or more in the following sequence modification:
A) amino acid/11-3 removes;
b)R456G;
c)H457Q;
D) S474A, and the termination on P490,
Wherein, this thrombomodulin analog comprises
388The oxidation of Met residue (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog and is used for the treatment of the medical usage of the coagulopathy with hyperfibrinolysis, and described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more in the following modification:
A) amino acid/11-3 removes;
b)M388L;
c)R456G;
d)H457Q;
E) S474A, and the termination on P490,
Wherein, this analog comprises aminoacid
387The sudden change of Gln, it is selected from the amino acid replacement of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog and is used for the treatment of the medical usage of the coagulopathy with hyperfibrinolysis, and described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more in the following modification:
A) removal of amino acid/11-3;
b)R456G;
c)H457Q;
D) S474A, and the termination on P490,
Wherein, this analog comprises aminoacid
388The sudden change of Met, described aminoacid is selected from the amino acid replacement of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or described aminoacid is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog and is used for the treatment of the medical usage of the coagulopathy with hyperfibrinolysis, and described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more in the following sequence modification:
A) removal of amino acid/11-3;
b)M388L;
c)R456G;
d)H457Q;
E) S474A, and the termination on P490,
Wherein, this analog comprises aminoacid
389The sudden change of Phe, described aminoacid is selected from the amino acid replacement of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or described institute aminoacid is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog and is used for the treatment of the medical usage of the coagulopathy with hyperfibrinolysis, and described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more modifications in the following sequence modification:
A) removal of amino acid/11-3;
b)M388L;
c)R456G;
d)H457Q;
E) S474A, and the termination on P490,
Wherein this analog comprises hydrophobic amino acid, preferred Ala following aminoacid to one of between insertion:
386Cys and
387Gln,
387Gln and
388Leu,
388Leu and
389Phe,
389Phe and
390Cys (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog and is used for the treatment of the medical usage of the coagulopathy with hyperfibrinolysis, and described analog has the aminoacid sequence of the aminoacid sequence (describing among the SEQ ID NO:1) corresponding to ripe thrombomodulin and comprises one or more modifications in the following sequence modification:
A) removal of amino acid/11-3;
b)M388L;
c)R456G;
d)H457Q;
E) S474A, and the termination on P490,
Wherein, this analog comprises aminoacid
376The sudden change of Phe, described sudden change are preferably Phe376Ala sudden change (aminoacid sequence of the ripe thrombomodulin of describing among numbering and the SEQ ID No:1 is relevant).
Of the present invention more preferably aspect, relate to the Solulin that describes among the SEQID NO:2, it comprises one or more, preferably the sequence modification of what a following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQID No:1) just:
a)
359Gln；
b)
398Asp；
c)
400Asp；
d)
402Asn；
e)
408Glu；
f)
413Tyr；
g)
414Ile;
h)
415Leu，
i)
417Asp；
j)
439Asn.
A) to j) listed above-mentioned aminoacid preferably replaced by alanine.
In one embodiment, the Solulin that describes among the SEQID NO:2 comprises one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1), the modification of a preferred position:
A)
388Met has except the modification to the change of Leu,
b)
416Asp；
c)
423Asp；
d)
425Asp；
e)
426Glu；
f)
429Asp；
g)
440Leu；
h)
461Asp.
In one aspect of the invention, a) to h) listed aminoacid replaced by alanine.
The Solulin that uses among the present invention can comprise aminoacid
387The sudden change of Gln, described aminoacid is selected from the amino acid replacement of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQID No:1).
In other side of the present invention, Solulin can comprise aminoacid
388The sudden change of Met, described aminoacid is selected from the amino acid replacement of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQID No:1).
In addition, the present invention relates to comprise aminoacid
389The Solulin of the sudden change of Phe, described aminoacid can be selected from the amino acid replacement of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, in one embodiment of the invention, Solulin comprises hydrophobic amino acid, preferred Ala following aminoacid to one of between insertion:
386Cys and
387Gln,
387Gln and
388Leu,
388Leu and
389Phe,
389Phe and
390Cys (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
Can modify Solulin to comprise oxidation
388Met residue (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).In addition, it can comprise aminoacid
376The sudden change of Phe, described sudden change are preferably Phe376Ala sudden change (aminoacid sequence of the ripe thrombomodulin of describing among numbering and the SEQ ID No:1 is relevant).
Of the present invention preferred aspect, relate to the thrombomodulin analog, described analog is comprised of following basically: by 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2, its can comprise following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1) in one or more, the sequence modification of a preferred position:
a)
359Gln;
b)
398Asp;
c)
400Asp；
d)
402Asn；
e)
408Glu；
f)
413Tyr；
g)
414Ile；
h)
415Leu，
i)
417Asp；
j)
439Asn.
In one aspect of the invention, a) to j) listed aminoacid replaced by alanine.
TM analog of the present invention also can be basically be comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2, wherein, each this type of fragment can comprise one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1), the sequence modification of a preferred position:
A)
388Met has except the modification to the change of Leu;
b)
416Asp；
c)
423Asp；
d)
425Asp；
e)
426Glu；
f)
429Asp；
g)
440Leu；
h)
461Asp.
A) to h) listed aminoacid preferably replaced by alanine.
The invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
387The sudden change of Gln, described aminoacid can be selected from the amino acid replacement of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
388The sudden change of Met, described aminoacid can be selected from the amino acid replacement of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
389The sudden change of Phe, described aminoacid can be selected from the amino acid replacement of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise hydrophobic amino acid, preferred Ala following aminoacid to one of between insertion:
386Cys and
387Gln,
387Gln and
388Met,
388Met and
389Phe,
389Phe and
390Cys (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise oxidation
388Met residue (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the TM analog, it is comprised of 6 EGF domains shown in the residue 224 to 459 of the residue 227 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
376The sudden change of Phe, described sudden change are preferably Phe376Ala sudden change (aminoacid sequence of the ripe thrombomodulin of describing among numbering and the SEQ ID No:1 is relevant).
In one embodiment of the invention, the TM analog has been described, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1), the modification of a preferred position:
a)
359Gln；
b)
398Asp；
c)
400Asp；
d)
402Asn；
e)
408Glu；
f)
413Tyr；
g)
414Ile；
h)
415Leu，
i)
417Asp；
j)
439Asn.
A) to j) listed aminoacid can be replaced by alanine.
In one embodiment of the invention, the TM analog has been described, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1), the modification of a preferred position:
A)
388Met has except the modification to the change of Leu;
b)
416Asp；
c)
423Asp；
d)
425Asp；
e)
426Glu；
f)
429Asp；
g)
440Leu；
h)
461Asp.
A) to h) listed aminoacid can be replaced by alanine.
The invention still further relates to the thrombomodulin analog, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
387The sudden change of Gln, described aminoacid can be selected from the amino acid replacement of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The invention still further relates to the thrombomodulin analog, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
388The sudden change of Met, described aminoacid can be selected from the amino acid replacement of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the thrombomodulin analog, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQ IDNO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
389The sudden change of Phe, described aminoacid can be selected from the amino acid replacement of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or it is lacked (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the thrombomodulin analog, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQ IDNO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise hydrophobic amino acid, preferred Ala following aminoacid to one of between insertion:
386Cys and
387Gln,
387Gln and
388Met,
388Met and
389Phe,
389Phe and
390Cys (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In other side of the present invention, the thrombomodulin analog has been described, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise oxidation
388Met residue (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In other side of the present invention, relate to the thrombomodulin analog, it is comprised of the EGF domain 3 to 6 shown in the residue 308 to 459 of the residue 311 to 462 of SEQID NO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can comprise aminoacid
376The sudden change of Phe, described sudden change are preferably Phe376Ala sudden change (aminoacid sequence of the ripe thrombomodulin of describing among numbering and the SEQ ID No:1 is relevant).
Of the present invention preferred aspect, the thrombomodulin analog has been described, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment comprises one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of bolt adjusting albumen in the mature blood that SEQ ID No:1 describes), the modification of a preferred position:
a)
359Gln;
b)
398Asp；
c)
400Asp；
d)
402Asn；
e)
408Glu；
f)
413Tyr；
g)
414Ile；
h)
415Leu，
i)
417Asp；
j)
439Asn.
A) to j) listed aminoacid can be replaced by alanine.
In one aspect of the invention, the thrombomodulin analog has been described, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment comprises one or more in the following amino acid position (numbering is relevant with the aminoacid sequence of bolt adjusting albumen in the mature blood that SEQ ID No:1 describes), the modification of a preferred position:
A)
388Met has except the modification to the change of Leu;
b)
416Asp;
c)
423Asp；
d)
425Asp；
e)
426Glu；
f)
429Asp;
g)
440Leu；
h)
461Asp.
A) to h) listed aminoacid preferably replaced by alanine.
The invention still further relates to the thrombomodulin analog, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can be wrapped aminoacid
387The sudden change of Gln, described aminoacid can be selected from the amino acid replacement of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro, or it can be by disappearance (numbering be relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The invention still further relates to the thrombomodulin analog, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can be wrapped aminoacid
388The sudden change of Met, described aminoacid can be selected from the amino acid replacement of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or it can be by disappearance (numbering be relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
In addition, the invention still further relates to the thrombomodulin analog, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ IDNO:1 or SEQ ID:NO2 basically, and wherein, each fragment of this type of fragment can be wrapped aminoacid
389The sudden change of Phe, described aminoacid can be selected from the amino acid replacement of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or it can be by disappearance (numbering be relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQID No:1).
In addition, the invention still further relates to the thrombomodulin analog, it is comprised of c ring and the EGF structure 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ IDNO:1 or SEQ ID:NO2 basically, wherein this thrombomodulin analog comprises hydrophobic amino acid, preferred Ala following aminoacid to one of between insertion:
386Cys and
387Gln,
387Gln and
388Met,
388Met and
389Phe,
389Phe and
390Cys (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQID No:1).
Of the present invention preferred aspect, the thrombomodulin analog has been described, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise oxidation
388Met residue (numbering is relevant with the aminoacid sequence of the ripe thrombomodulin described among the SEQ ID No:1).
The present invention preferably relates to the thrombomodulin analog, it is comprised of c ring and the EGF domain 4 to 6 of the EGF domain 3 shown in the residue 330 to 459 of the residue 333 to 462 of SEQ ID NO:1 or SEQ ID:NO2 basically, wherein, each fragment of this type of fragment can comprise aminoacid
376The sudden change of Phe, described sudden change are preferably Phe376Ala sudden change (aminoacid sequence of the ripe thrombomodulin of describing among numbering and the SEQ ID No:1 is relevant).
According to the present invention, can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified ") according to the TM analog of SEQ ID NO:2, it is described in the table 4.
In another embodiment of the invention, can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified ") according to the TM analog of SEQ ID NO:4, described modification is described in the table 4.
In another embodiment of the invention, can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified ") according to the TM analog of SEQ ID NO3, described modification is described in the table 4.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO2, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO2 or the EGF-sample repetitive structure territory 3 of SEQ IDNO2 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 4.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO3, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO3 or the EGF-sample repetitive structure territory 3 of SEQ IDNO3 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO:1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 4.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO4, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO4 or the EGF-sample repetitive structure territory 3 of SEQ IDNO4 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 4.
| Numbering | First is amino acid modified | Second is amino acid modified | |
| 1 | | Gln387Lys | |
| 2 | Phe376Ala | Gln387Gly | |
| | Phe376Ala | Gln387Ile | |
| | Phe376Ala | Gln387Trp | |
| 5 | Phe376Ala | Gln387Tyr | |
| 6 | Phe376Ala | Gln387Leu | |
| | Phe376Ala | Gln387Asn | |
| 8 | Phe376Ala | Gln387Phe |
| 9 | | Gln387Asp | |
| 10 | Phe376Ala | Gln387Cys | |
| 11 | Phe376Ala | Gln387Pro | |
| 12 | Phe376Ala | Met388Ile | |
| 13 | Phe376Ala | Met388Phe | |
| 14 | | Met388His | |
| 15 | Phe376Ala | Met388Arg | |
| 16 | Phe376Ala | Met388Pro | |
| 17 | Phe376Ala | Met388Val | |
| 18 | Phe376Ala | Met388Thr | |
| 19 | | Met388Ser | |
| 20 | Phe376Ala | Met388Ala | |
| 21 | Phe376Ala | Met388Trp | |
| 22 | Phe376Ala | Met388Asn | |
| 23 | Phe376Ala | Met388Lys | |
| | Phe376Ala | Met388Gly | |
| 25 | Phe376Ala | Met388Glu | |
| 26 | Phe376Ala | Met388Asp | |
| 27 | Phe376Ala | Met388Cys | |
| 28 | Phe376Ala | Phe389Ser | |
| 29 | | Phe389Arg | |
| 30 | | Phe389Lys | |
| 31 | Phe376Ala | Phe389Met | |
| 32 | Phe376Ala | Phe389Tyr | |
| 33 | Phe376Ala | Phe389Gly | |
| 34 | | Phe389Cys | |
| 35 | Phe376Ala | Phe389Pro | |
| 36 | Phe376Ala | Disappearance Gln387 | |
| 37 | Phe376Ala | Disappearance Met388 | |
| 38 | Phe376Ala | Disappearance Phe389 | |
| 39 | Phe376Ala | Ala inserts 386/387 | |
| 40 | Phe376Ala | Ala inserts 387/388 | |
| 41 | Phe376Ala | Ala inserts 388/389 | |
| 42 | Phe376Ala | Ala inserts 389/390 | |
| 43 | Phe376Val | Gln387Lys | |
| 44 | | Gln387Gly | |
| 45 | Phe376Val | Gln387Ile | |
| 46 | Phe376Val | Gln387Trp | |
| 47 | Phe376Val | Gln387Tyr | |
| 48 | Phe376Val | Gln387Leu | |
| 49 | | Gln387Asn | |
| 50 | Phe376Val | Gln387Phe | |
| 51 | Phe376Val | Gln387Asp | |
| 52 | Phe376Val | Gln387Cys |
| 53 | Phe376Val | Gln387Pro | |
| 54 | | Met388Ile | |
| 55 | Phe376Val | Met388Phe | |
| 56 | Phe376Val | Met388His | |
| 57 | Phe376Val | Met388Arg | |
| 58 | Phe376Val | Met388Pro | |
| 59 | | Met388Val | |
| 60 | Phe376Val | Met388Thr | |
| 61 | Phe376Val | Met388Ser | |
| 62 | Phe376Val | Met388Ala | |
| 63 | Phe376Val | Met388Trp | |
| 64 | | Met388Asn | |
| 65 | Phe376Val | Met388Lys | |
| 66 | Phe376Val | Met388Gly | |
| 67 | Phe376Val | Met388Glu | |
| 68 | Phe376Val | Met388Asp | |
| 69 | | Met388Cys | |
| 70 | Phe376Val | Phe389Ser | |
| 71 | Phe376Val | Phe389Arg | |
| 72 | Phe376Val | Phe389Lys | |
| 73 | Phe376Val | Phe389Met | |
| | Phe376Val | Phe389Tyr | |
| 75 | | Phe389Gly | |
| 76 | Phe376Val | Phe389Cys | |
| 77 | Phe376Val | Phe389Pro | |
| 78 | Phe376Val | Disappearance Gln387 | |
| 79 | | Disappearance Met388 | |
| 80 | Phe376Val | Disappearance Phe389 | |
| 81 | Phe376Val | Ala inserts 386/387 | |
| 82 | Phe376Val | Ala inserts 387/388 | |
| 83 | Phe376Val | Ala inserts 388/389 | |
| 84 | Phe376Val | Ala inserts 389/390 | |
| 85 | Phe376Ile | Gln387Lys | |
| 86 | Phe376Ile | Gln387Gly | |
| 87 | Phe376Ile | Gln387Ile | |
| 88 | Phe376Ile | Gln387Trp | |
| | Phe376Ile | Gln387Tyr | |
| 90 | Phe376Ile | Gln387Leu | |
| 91 | Phe376Ile | Gln387Asn | |
| 92 | Phe376Ile | Gln387Phe | |
| 93 | Phe376Ile | Gln387Asp | |
| 94 | Phe376Ile | Gln387Cys | |
| 95 | Phe376Ile | Gln387Pro | |
| 96 | Phe376Ile | Met388Ile |
| 97 | Phe376Ile | Met388Phe | |
| 98 | Phe376Ile | Met388His | |
| 99 | | Met388Arg | |
| 100 | Phe376Ile | Met388Pro | |
| 101 | Phe376Ile | Met388Val | |
| 102 | Phe376Ile | Met388Thr | |
| 103 | Phe376Ile | Met388Ser | |
| 104 | Phe376Ile | Met388Ala | |
| 105 | Phe376Ile | Met388Trp | |
| 106 | Phe376Ile | Met388Asn | |
| 107 | Phe376Ile | Met388Lys | |
| 108 | Phe376Ile | Met388Gly | |
| 109 | | Met388Glu | |
| 110 | Phe376Ile | Met388Asp | |
| 111 | Phe376Ile | Met388Cys | |
| 112 | Phe376Ile | Phe389Ser | |
| 113 | Phe376Ile | Phe389Arg | |
| 114 | Phe376Ile | Phe389Lys | |
| 115 | Phe376Ile | Phe389Met | |
| 116 | Phe376Ile | Phe389Tyr | |
| 117 | Phe376Ile | Phe389Gly | |
| 118 | Phe376Ile | Phe389Cys | |
| | Phe376Ile | Phe389Pro | |
| 120 | Phe376Ile | Disappearance Gln387 | |
| 121 | | Disappearance Met388 | |
| 122 | Phe376Ile | Disappearance Phe389 | |
| 123 | Phe376Ile | Ala inserts 386/387 | |
| 124 | Phe376Ile | Ala inserts 387/388 | |
| 125 | Phe376Ile | Ala inserts 388/389 | |
| 126 | Phe376Ile | Ala inserts 389/390 | |
| 127 | Phe376Leu | Gln387Lys | |
| 128 | Phe376Leu | Gln387Gly | |
| 129 | Phe376Leu | Gln387Ile | |
| 130 | Phe376Leu | Gln387Trp | |
| 131 | Phe376Leu | Gln387Tyr | |
| 132 | Phe376Leu | Gln387Leu | |
| 133 | Phe376Leu | Gln387Asn | |
| 134 | Phe376Leu | Gln387Phe | |
| 135 | Phe376Leu | Gln387Asp | |
| 136 | Phe376Leu | Gln387Cys | |
| 137 | Phe376Leu | Gln387Pro | |
| 138 | Phe376Leu | Met388Ile | |
| | Phe376Leu | Met388Phe | |
| 140 | Phe376Leu | Met388His |
| 141 | Phe376Leu | Met388Arg | |
| 142 | Phe376Leu | Met388Pro | |
| 143 | Phe376Leu | Met388Val | |
| 144 | Phe376Leu | Met388Thr | |
| 145 | Phe376Leu | Met388Ser | |
| 146 | Phe376Leu | Met388Ala | |
| 147 | Phe376Leu | Met388Trp | |
| 148 | Phe376Leu | Met388Asn | |
| | Phe376Leu | Met388Lys | |
| 150 | Phe376Leu | Met388Gly | |
| 151 | Phe376Leu | Met388Glu | |
| 152 | Phe376Leu | Met388Asp | |
| 153 | Phe376Leu | Met388Cys | |
| 154 | Phe376Leu | Phe389Ser | |
| 155 | Phe376Leu | Phe389Arg | |
| 156 | Phe376Leu | Phe389Lys | |
| 157 | Phe376Leu | Phe389Met | |
| 158 | Phe376Leu | Phe389Tyr | |
| | Phe376Leu | Phe389Gly | |
| 160 | Phe376Leu | Phe389Cys | |
| 161 | Phe376Leu | Phe389Pro | |
| 162 | Phe376Leu | Disappearance Gln387 | |
| 163 | Phe376Leu | Disappearance Met388 | |
| 164 | Phe376Leu | Disappearance Phe389 | |
| 165 | Phe376Leu | Ala inserts 386/387 | |
| 166 | Phe376Leu | Ala inserts 387/388 | |
| 167 | Phe376Leu | Ala inserts 388/389 | |
| 168 | Phe376Leu | Ala inserts 389/390 | |
| 169 | Phe376Ala | Asp416Ala | |
| 170 | Phe376Ala | Asp423Ala | |
| 171 | Phe376Ala | Asp425Ala | |
| 172 | Phe376Ala | Glu426Ala | |
| 173 | Phe376Ala | Asp429Ala | |
| 174 | Phe376Ala | Leu440Ala | |
| 175 | Phe376Ala | Asp461Ala | |
| 176 | Phe376Ala | Gln359Ala | |
| 177 | Phe376Ala | Asp398Ala | |
| 178 | Phe376Ala | Asp400Ala | |
| | Phe376Ala | Asn402Ala | |
| 180 | Phe376Ala | Glu408Ala | |
| 181 | Phe376Ala | Tyr413Ala | |
| 182 | Phe376Ala | Ile414Ala | |
| 183 | Phe376Ala | Leu415Ala | |
| 184 | Phe376Ala | Asp417Ala |
| 185 | Phe376Ala | Asn439Ala |
Table 4: first and second is amino acid modified
According to the present invention, can comprise separately the modification (" first, second, and third is amino acid modified ") of description in the table 5 according to the TM analog of SEQ ID NO2.Table 5 has been described the alanine displacement (" first is amino acid modified ") of Phe376.In certain embodiments of the invention, " first is amino acid modified " is made of glycine, valine, leucine or isoleucine.This type of modification is made up with " second is amino acid modified " shown in the table 5 and " triamido acid is modified " separately.This type of further embodiment of the present invention jointly is summarized as the modification of describing in the table 5.
In another embodiment of the invention, can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified ") according to the TM analog of SEQ ID NO4, described modification is described in the table 5.
In another embodiment of the invention, can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified ") according to the TM analog of SEQ ID NO3, described modification is described in the table 5.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO2, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO2 or the EGF-sample repetitive structure territory 3 of SEQ IDNO2 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 5.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO3, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO3 or the EGF-sample repetitive structure territory 3 of SEQ IDNO3 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 5.
In another embodiment, the thrombomodulin fragment is comprised of following basically: 6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO1) of SEQID NO4, the c in 3 to the EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO1) of SEQ ID NO4 or the EGF-sample repetitive structure territory 3 of SEQ IDNO4 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQ IDNO1), wherein this type of each fragment that comprises the fragment of EGF domain can comprise two sequence modifications (" first is amino acid modified " and " second is amino acid modified "), and described modification is described in the table 5.
| Numbering | The 1st is amino acid modified | The 2nd is amino acid modified | The 3rd is amino acid modified | |
| 1 | | Asp416Ala | Gln359Ala | |
| 2 | Phe376Ala | Asp416Ala | Asp398Ala | |
| 3 | | Asp416Ala | Asp400Ala | |
| 4 | | Asp416Ala | Asn402Ala | |
| 5 | Phe376Ala | Asp416Ala | Glu408Ala | |
| 6 | Phe376Ala | Asp416Ala | Tyr413Ala | |
| 7 | | Asp416Ala | Ile414Ala | |
| 8 | Phe376Ala | Asp416Ala | Leu415Ala | |
| 9 | | Asp416Ala | Asp417Ala | |
| 10 | Phe376Ala | Asp416Ala | Asn439Ala | |
| 11 | Phe376Ala | Asp416Ala | Gln387Cys | |
| 12 | Phe376Ala | Asp416Ala | Gln387Pro | |
| 13 | Phe376Ala | Asp416Ala | The Met388 of oxidation | |
| 14 | | Asp416Ala | Met388Asp | |
| 15 | Phe376Ala | Asp416Ala | Met388Cys | |
| 16 | Phe376Ala | Asp416Ala | Phe389Cys | |
| 17 | Phe376Ala | Asp416Ala | Phe389Pro | |
| 18 | Phe376Ala | Asp416Ala | Disappearance Gln387 | |
| 19 | Phe376Ala | | Disappearance Met388 | |
| 20 | Phe376Ala | Asp416Ala | Disappearance Phe389 | |
| 21 | Phe376Ala | Asp416Ala | Ala inserts 386/387 | |
| 22 | Phe376Ala | Asp416Ala | Ala inserts 387/388 | |
| 23 | Phe376Ala | Asp416Ala | Ala inserts 388/389 | |
| 24 | Phe376Ala | Asp416Ala | Ala inserts 389/390 | |
| 25 | Phe376Ala | Asp423Ala | Gln359Ala | |
| 26 | Phe376Ala | Asp423Ala | Asp398Ala |
| 27 | Phe376Ala | Asp423Ala | Asp400Ala | |
| 28 | Phe376Ala | Asp423Ala | Asn402Ala | |
| 29 | | Asp423Ala | Glu408Ala | |
| 30 | | Asp423Ala | Tyr413Ala | |
| 31 | Phe376Ala | Asp423Ala | I?le414Ala | |
| 32 | Phe376Ala | Asp423Ala | Leu415Ala | |
| 33 | Phe376Ala | Asp423Ala | Asp417Ala | |
| 34 | | Asp423Ala | Asn439Ala | |
| 35 | Phe376Ala | Asp423Ala | Gln387Cys | |
| 36 | Phe376Ala | Asp423Ala | Gln387Pro | |
| 37 | Phe376Ala | Asp423Ala | The Met388 of oxidation | |
| 38 | Phe376Ala | Asp423Ala | Met388Asp | |
| 39 | | Asp423Ala | Met388Cys | |
| 40 | Phe376Ala | Asp423Ala | Phe389Cys | |
| 41 | Phe376Ala | Asp423Ala | Phe389Pro | |
| 42 | Phe376Ala | Asp423Ala | Disappearance Gln387 | |
| 43 | Phe376Ala | Asp423Ala | Disappearance Met388 | |
| 44 | Phe376Ala | | Disappearance Phe389 | |
| 45 | Phe376Ala | Asp423Ala | Ala inserts 386/387 | |
| 46 | Phe376Ala | Asp423Ala | Ala inserts 387/388 | |
| 47 | Phe376Ala | Asp423Ala | Ala inserts 388/389 | |
| 48 | Phe376Ala | Asp423Ala | Ala inserts 389/390 | |
| 49 | | Asp425Ala | Gln359Ala | |
| 50 | Phe376Ala | Asp425Ala | Asp398Ala | |
| 51 | Phe376Ala | Asp425Ala | Asp400Ala | |
| 52 | Phe376Ala | Asp425Ala | Asn402Ala | |
| 53 | Phe376Ala | Asp425Ala | Glu408Ala | |
| 54 | | Asp425Ala | Tyr413Ala | |
| 55 | Phe376Ala | Asp425Ala | I?le414Ala | |
| 56 | Phe376Ala | Asp425Ala | Leu415Ala | |
| 57 | Phe376Ala | Asp425Ala | Asp417Ala | |
| 58 | Phe376Ala | Asp425Ala | Asn439Ala | |
| 59 | | Asp425Ala | Gln387Cys | |
| 60 | Phe376Ala | Asp425Ala | Gln387Pro | |
| 61 | Phe376Ala | Asp425Ala | The Met388 of oxidation | |
| 62 | Phe376Ala | Asp425Ala | Met388Asp | |
| 63 | Phe376Ala | Asp425Ala | Met388Cys | |
| 64 | | Asp425Ala | Phe389Cys | |
| 65 | Phe376Ala | Asp425Ala | Phe389Pro | |
| 66 | Phe376Ala | Asp425Ala | Disappearance Gln387 | |
| 67 | Phe376Ala | Asp425Ala | Disappearance Met388 | |
| 68 | Phe376Ala | Asp425Ala | Disappearance Phe389 | |
| 69 | Phe376Ala | Asp425Ala | Ala inserts 386/387 | |
| 70 | Phe376Ala | Asp425Ala | Ala inserts 387/388 |
| 71 | Phe376Ala | Asp425Ala | Ala inserts 388/389 | |
| 72 | Phe376Ala | Asp425Ala | Ala inserts 389/390 | |
| 73 | Phe376Ala | Glu426Ala | Gln359Ala | |
| 74 | | Glu426Ala | Asp398Ala | |
| 75 | | Glu426Ala | Asp400Ala | |
| 76 | Phe376Ala | Glu426Ala | Asn402Ala | |
| 77 | Phe376Ala | Glu426Ala | Glu408Ala | |
| 78 | Phe376Ala | Glu426Ala | Tyr413Ala | |
| 79 | Phe376Ala | Glu426Ala | I? |
|
| 80 | Phe376Ala | Glu426Ala | Leu415Ala | |
| 81 | Phe376Ala | Glu426Ala | Asp417Ala | |
| 82 | Phe376Ala | Glu426Ala | Asn439Ala | |
| 83 | Phe376Ala | Glu426Ala | Gln387Cys | |
| 84 | Phe376Ala | Glu426Ala | Gln387Pro | |
| 85 | Phe376Ala | Glu426Ala | The Met388 of oxidation | |
| 86 | Phe376Ala | Glu426Ala | Met388Asp | |
| 87 | Phe376Ala | Glu426Ala | Met388Cys | |
| 88 | Phe376Ala | Glu426Ala | Phe389Cys | |
| 89 | | Glu426Ala | Phe389Pro | |
| 90 | Phe376Ala | Glu426Ala | Disappearance Gln387 | |
| 91 | Phe376Ala | Glu426Ala | Disappearance Met388 | |
| 92 | Phe376Ala | Glu426Ala | Disappearance Phe389 | |
| 93 | Phe376Ala | Glu426Ala | Ala inserts 386/387 | |
| 94 | Phe376Ala | Glu426Ala | Ala inserts 387/388 | |
| 95 | Phe376Ala | Glu426Ala | Ala inserts 388/389 | |
| 96 | Phe376Ala | Glu426Ala | Ala inserts 389/390 | |
| 97 | Phe376Ala | Asp429Ala | Gln359Ala | |
| 98 | Phe376Ala | Asp429Ala | Asp398Ala | |
| 99 | | Asp429Ala | Asp400Ala | |
| 100 | Phe376Ala | Asp429Ala | Asn402Ala | |
| 101 | Phe376Ala | Asp429Ala | Glu408Ala | |
| 102 | Phe376Ala | Asp429Ala | Tyr413Ala | |
| 103 | Phe376Ala | Asp429Ala | I?le414Ala | |
| 104 | Phe376Ala | Asp429Ala | Leu415Ala | |
| 105 | Phe376Ala | Asp429Ala | Asp417Ala | |
| 106 | Phe376Ala | Asp429Ala | Asn439Ala | |
| 107 | Phe376Ala | Asp429Ala | Gln387Cys | |
| 108 | Phe376Ala | Asp429Ala | Gln387Pro | |
| 109 | Phe376Ala | Asp429Ala | The Met388 of |
|
| 110 | Phe376Ala | Asp429Ala | Met388Asp | |
| 111 | Phe376Ala | Asp429Ala | Met388Cys | |
| 112 | Phe376Ala | Asp429Ala | Phe389Cys | |
| 113 | Phe376Ala | Asp429Ala | Phe389Pro | |
| 114 | Phe376Ala | Asp429Ala | Disappearance Gln387 |
| 115 | Phe376Ala | Asp429Ala | Disappearance Met388 | |
| 116 | Phe376Ala | Asp429Ala | Disappearance Phe389 | |
| 117 | Phe376Ala | Asp429Ala | Ala inserts 386/387 | |
| 118 | Phe376Ala | Asp429Ala | Ala inserts 387/388 | |
| 119 | Phe376Ala | Asp429Ala | Ala inserts 388/389 | |
| 120 | Phe376Ala | Asp429Ala | Ala inserts 389/390 | |
| 121 | | Leu440Ala | Gln359Ala | |
| 122 | Phe376Ala | Leu440Ala | Asp398Ala | |
| 123 | Phe376Ala | Leu440Ala | Asp400Ala | |
| 124 | | Leu440Ala | Asn402Ala | |
| 125 | Phe376Ala | Leu440Ala | Glu408Ala | |
| 126 | Phe376Ala | Leu440Ala | Tyr413Ala | |
| 127 | Phe376Ala | Leu440Ala | I?le414Ala | |
| 128 | Phe376Ala | Leu440Ala | Leu415Ala | |
| 129 | Phe376Ala | Leu440Ala | Asp417Ala | |
| 130 | Phe376Ala | Leu440Ala | Asn439Ala | |
| 131 | Phe376Ala | Leu440Ala | Gln387Cys | |
| 132 | Phe376Ala | Leu440Ala | Gln387Pro | |
| 133 | Phe376Ala | Leu440Ala | The Met388 of oxidation | |
| 134 | Phe376Ala | Leu440Ala | Met388Asp | |
| 135 | Phe376Ala | Leu440Ala | Met388Cys | |
| 136 | Phe376Ala | Leu440Ala | Phe389Cys | |
| 137 | Phe376Ala | Leu440Ala | Phe389Pro | |
| 138 | Phe376Ala | Leu440Ala | Disappearance Gln387 | |
| 139 | Phe376Ala | | Disappearance Met388 | |
| 140 | Phe376Ala | Leu440Ala | Disappearance Phe389 | |
| 141 | Phe376Ala | Leu440Ala | Ala inserts 386/387 | |
| 142 | Phe376Ala | Leu440Ala | Ala inserts 387/388 | |
| 143 | Phe376Ala | Leu440Ala | Ala inserts 388/389 | |
| 144 | Phe376Ala | Leu440Ala | Ala inserts 389/390 | |
| 145 | Phe376Ala | Asp461Ala | Gln359Ala | |
| 146 | Phe376Ala | Asp461Ala | Asp398Ala | |
| 147 | Phe376Ala | Asp461Ala | Asp400Ala | |
| 148 | Phe376Ala | Asp461Ala | Asn402Ala | |
| 149 | | Asp461Ala | Glu408Ala | |
| 150 | Phe376Ala | Asp461Ala | Tyr413Ala | |
| 151 | Phe376Ala | Asp461Ala | I?le414Ala | |
| 152 | Phe376Ala | Asp461Ala | Leu415Ala | |
| 153 | Phe376Ala | Asp461Ala | Asp417Ala | |
| 154 | Phe376Ala | Asp461Ala | Asn439Ala | |
| 155 | Phe376Ala | Asp461Ala | Gln387Cys | |
| 156 | Phe376Ala | Asp461Ala | Gln387Pro | |
| 157 | Phe376Ala | Asp461Ala | The Met388 of oxidation | |
| 158 | Phe376Ala | Asp461Ala | Met388Asp |
| 159 | | Asp461Ala | Met388Cys | |
| 160 | Phe376Ala | Asp461Ala | Phe389Cys | |
| 161 | Phe376Ala | Asp461Ala | Phe389Pro | |
| 162 | Phe376Ala | Asp461Ala | Disappearance Gln387 | |
| 163 | Phe376Ala | Asp461Ala | Disappearance Met388 | |
| 164 | Phe376Ala | Asp461Ala | Disappearance Phe389 | |
| 165 | Phe376Ala | Asp461Ala | Ala inserts 386/387 | |
| 166 | Phe376Ala | Asp461Ala | Ala inserts 387/388 | |
| 167 | Phe376Ala | Asp461Ala | Ala inserts 388/389 | |
| 168 | Phe376Ala | Asp461Ala | Ala inserts 389/390 |
Table 5: first, second, and third modifies
Following table provides about modify the general introduction of the cofactor activity of the minimizing that causes because of the specific amino acids of thrombomodulin analog.
Table 6: first and second is amino acid modified
According to therapeutic treatment of the present invention
In one aspect of the invention, thrombomodulin analog disclosed herein is used for the treatment of patient's the coagulopathy with hyperfibrinolysis, described patient has anti--Factor IX antibody.But the activity of this antibody-like inhibitive factor VIII.In typical case, they are produced as isoantibody in haemophilia A patient's replacement therapy process.They can cause the inefficacy of haemophilia A patient's FVIII alternative medicine.Therefore, according to an aspect of the present invention, thrombomodulin analog disclosed herein can be used as the not rescue medicament of the hemophiliac of response factor VIII (rescue medication).
According to another aspect of the present invention, utilize the patient of the combined therapy shortage Factor IX of Factor IX and TM analog of the present invention.Can be concomitantly or one after the other use Factor IX and TM analog.Preferably, utilize the Factor IX molecule of the disappearance B domain of recombinant factor VIII or restructuring, more preferably octocog alfa (Octocog-alfa) or moroctocog alfa (moroctocog-alfa) treatment patient.The human factor VII I that in one embodiment, can use separation for example
According to the present invention, also can treat treated with Factor IX in the past or be at present hemophiliac in the Factor IX treatment.Therefore, this type of patient has pharmaceutically effectively Factor IX level.
In other side of the present invention, described thrombomodulin analog can be used for the existence of the Factor IX antibody of examination hemophiliac, changes (referring to Figure 11 A to Figure 12 A) because the existence of Factor IX antibody causes the characteristic of thrombelastogram.
Embodiment of the present invention
In one aspect of the invention, described the purposes of thrombomodulin analog for the preparation of the medicament that is used for the treatment of the coagulopathy with hyperfibrinolysis, wherein said TM analog is characterised in that, shows the fibrinolysis effect in the treatment effective dose.
In other side of the present invention, the purposes of thrombomodulin analog for the preparation of the medicament that is used for the treatment of the coagulopathy with hyperfibrinolysis described, wherein the thrombomodulin analog shows one or more following features:
The binding affinity to thrombin of (i) comparing and reducing with rabbit lung thrombomodulin, and/or k
DValue surpasses the binding affinity to thrombin of 0.2nM; And/or
The cofactor activity that (i i) compares with the cofactor activity of TM analog TMEM388L and reduce,
(iii) with TM analog TM
EThe M388L TAFI Activation Activity that the increases ratio to the cofactor activity of comparing.
In other side of the present invention, described the purposes of above-mentioned thrombomodulin analog, the coagulopathy that wherein has hyperfibrinolysis is selected from following disease: the genetic disorder of haemophilia A, haemophilia B, C type hemophilia, von Willebrand (vWD), Of Acquired von Willebrand Disease, factor X deficiency, parahemophilia, factor I, II, V or VII, the bleeding disorder or the acquired blood coagulation that cause because of CAC lack.
In other side of the present invention, described thrombomodulin analog can be used for treating one or more following bleeding episodes that are selected from: intracranial hemorrhage or other CNS is hemorrhage, hemorrhage behind the joint, the thin blood vessel of microtriche, muscle, gastrointestinal tract, respiratory tract, peritoneum in space or the soft tissue.The intracranial hemorrhage event of utilizing thrombomodulin analogue treatment of the present invention can be that (intra-axial) is hemorrhage in the axle, axle outer (extra-axial) is hemorrhage or subarachnoid hemorrhage (SAH) or dura mater on hematoma or subdural hematoma.Preferred therapeutic has the patient of SAH, more preferably treats the aneurysmal subarachnoid hemorrhage behind the SAH.
Of the present invention preferred aspect, be used in physical trauma according to thrombomodulin analog of the present invention, treat hyperfibrinolysis after the preferred CNS wound.The physical trauma of definition refers to herein, by health wound or the strike (shock) of unexpected physical damnification (as from violence or accident) generation.Physical trauma comprises multiple wound, head injury, thoracic trauma, trauma of abdomen, limb trauma, facial wound, urogenital system wound, Pelvic trauma and soft tissue injury.
In other side of the present invention, provide the thrombomodulin analog that is used for the treatment of the coagulopathy with hyperfibrinolysis with other fibrinolysis inhibitor combination.Especially, can use for example etamsylate of the hematoblastic material that normally adheres to of correction.Preferably, can use the inhibitor of proteolytic enzyme, it is the fibrinolysin inhibitor of aprotinin for example more preferably.Of the present invention more preferably aspect, the lysine-binding site of other antifibrinolytics thing blocking-up fibrinolysin, for example EACA or tranexamic acid.
Of the present invention preferred aspect, utilize the patient of thrombomodulin analogue treatment to have anti--factor VII I antibody.
In another aspect of the present invention, utilize the patient of thrombomodulin analogue treatment also to use Factor IX, the Factor IX molecule of the disappearance B domain of preferred recombinant factor VIII or restructuring, more preferably octocog alfa or moroctocog alfa are treated.
Of the present invention preferred aspect, provide with above-mentioned dosage or dosage range according to the thrombomodulin analog of blood SEQ ID NO:5 to 11.
In other side of the present invention, provide and Factor IX, the Factor IX molecule of the disappearance B domain of preferred recombinant factor VIII, restructuring, more preferably the thrombomodulin analog that is used for the treatment of the coagulopathy with hyperfibrinolysis of octocog alfa or moroctocog alfa combination.
In one aspect of the invention, when the bleeding episode of the coagulopathy with hyperfibrinolysis occurs or before the bleeding risk of increase is for example performed the operation or had tooth pulled out, use the thrombomodulin analog.
In other side of the present invention, be difficult to utilize the patient of blood/plasma infusion or thrombin replacement therapy to use the thrombomodulin analog.
In one aspect of the invention, can be preferably interim when being shorter than 1 thoughtful 4 weeks total, once a day, per 2 days be once, or per 3,4,5,6 or 7 days once, more preferably in the mode of chronic administration, use the thrombomodulin analog with a plurality of dosage.
Of the present invention preferred aspect, use thrombomodulin analog according to SEQ ID NO:5 to 11 according to above-mentioned application program.
Of the present invention preferred aspect, in the mode that parenteral is used, preferably in the mode of intravenous or subcutaneous administration, use the thrombomodulin analog.
In one aspect of the invention, be lower than 5nM/L in experimenter to be treated, to produce, preferably be lower than 3nM/L, to provide the thrombomodulin analog more preferably less than the amount of the plasma concentration of 1.5nM/L.
In other side of the present invention, titration thrombomodulin analog is so that plasma concentration is 0.1nM/L to 5nM/L, preferred 0.1nM/L to 3nM/L.
Of the present invention preferred aspect, according to the thrombomodulin analog of SEQ ID NO:5 to 11 for generation of disclosed plasma concentration above.
In another aspect of the present invention, dosage with 0.1 μ g/kg to 140 μ g/kg, preferably with the dosage of 0.5 μ g/kg to 40 μ g/kg, more preferably with the dosage of 0.5 μ g/kg to 4 μ g/kg, use the thrombomodulin analog for experimenter to be treated with the dosage (kg refers to experimenter's to be treated kg body weight) of 0.75 to μ g/kg especially.
In other side of the present invention, use the thrombomodulin analog with the dosage of 0.75,1.5,2.5 or 4.0 μ g/kg (its equal 0.6,1,3 or the dosage adjusted of 4.0mg/ patient's body weight).
Of the present invention other preferred aspect, use thrombomodulin analog according to SEQID NO:5 to 11 with above-mentioned dosage or dosage range.
Of the present invention more preferably aspect, the thrombomodulin analog is soluble T M analog.
Of the present invention more preferably aspect, thrombomodulin analog behaviour soluble T M analog.
In one aspect of the invention, described thrombomodulin analog comprises at least one domain that is selected from EGF3, EGF4, EGF5, EGF6, preferably comprises fragment EGF3-EGF6, more preferably comprises EGF domain 1-6.
In other side of the present invention, described thrombomodulin analog more preferably is comprised of EGF domain EGF3 to EGF6 EGF domain EGF1 to EGF6.
In one aspect of the invention, the thrombomodulin analog has the aminoacid sequence of the aminoacid sequence (describing among SEQ ID NO:1 or the SEQ ID NO:3) corresponding to ripe thrombomodulin and comprises one or more modifications in the following modification:
A) removal of amino acid/11-3,
b)M388L，
c)R456G，
d)H457Q，
E) S474A, and the termination on P490.
In other side of the present invention, the thrombomodulin analog has and comprises the aminoacid sequence that has the sequence of at least 85% or at least 90% or 95% sequence homogeneity with SEQ ID NO:2.
Of the present invention preferred aspect, the thrombomodulin analog has amino acid modified at the one or more lower column position (according to SEQ ID NO:1 or SEQ ID NO:3) corresponding to native sequences:
ab)
355Asn；
ae)
359Gln；
af)
361Gln；
ag)
363Leu；
ah)
364Asn；
ai)
368Tyr；
aj)
371Val；
ak)
374Glu；
al)
376Phe；
am)
384His；
an)
385Arg；
ba)
387Gln；
bb)
389Phe；
bc)
398Asp；
bd)
400Asp；
be)
402Asn；
bf)
403Thr；
bg)
408Glu；
bh)
411Glu；
bi)
413Tyr；
bj)
414Ile;
bk)
415Leu；
bl)
415Asp；
bm)
417Asp；
bn)
420Ile；
bo)
423Asp；
bp)
424Ile；
bq)
425Asp；
br)
426Glu;
ca)
428Glu；
cb)
429Asp；
cc)
432Phe；
cd)
434Ser；
ce)
436Val；
cf)
438His；
cg)
439Asp；
ch)
440Leu；
ci)
443Thr；
cj)
444Phe；
ck)
445Glu;
cl)
456Arg；
Cm)
458Ile; Or
cn)
461Asp.
In other side of the present invention, the thrombomodulin analog has the modification of phenylalanine in the position 376 according to SEQ ID NO:1 or SEQ ID NO:3, it is preferably replaced by aliphatic amino acid, more preferably by glycine, alanine, valine, leucine or isoleucine displacement, most preferably replaced by alanine.
In other side of the present invention, the thrombomodulin analog has the one or more following amino acid whose modification according to SEQ ID NO:1 or SEQ ID NO:3:
a)
387Gln;
b)
388Met;
b)
389Phe,
Wherein aminoacid is lacked, by one or more extra aminoacid insertion or preferably replaced.
In other side of the present invention, the thrombomodulin analog uses with its oxidised form (preferably by toluene-sodium-sulfonchloramide, hydrogen peroxide or sodium periodate oxidation).
In other side of the present invention, use the thrombomodulin analog, wherein the one or more methionines in the TM analog are oxidized, and (according to SEQ ID NO:1 or SEQ ID NO:3) is oxidized for the methionine residues on the optimum position 388.
In another aspect of the present invention, the method for screening the thrombomodulin analog that is suitable for treating the coagulopathy with hyperfibrinolysis has been described, wherein said thrombomodulin shows one or more following features:
(i) binding affinity to thrombin that reduces,
(ii) cofactor that reduces is active,
(iii) the TAFI Activation Activity that strengthens,
Described method comprises step:
A) produce one or more amino acid replacements of thrombomodulin sequence (SEQ ID NO:1 or SEQ ID NO:3), preferably the one or more amino acid replacements on the listed amino acid position in claim 15;
B) in one or more following characteristic aspect, the analog of modifying is compared with contrast molecule preferred rabbit lung TM or soluble human TM analog:
Ba) to the binding affinity (KD value) of thrombin;
Bb) cofactor is active;
Bc) TAFI Activation Activity or TAFIa potential;
Bd) ratio of TAFI Activation Activity and cofactor activity;
Be) effect of protein oxidation;
Bf) in external test in time on the impact of clot dissolution; Or
Bg) effect in the blood coagulation relevant animal models.
In another aspect of the present invention, the method that treatment has the coagulopathy of hyperfibrinolysis has been described, comprise the administering therapeutic effective dose according to claim 1 to 20 each thrombomodulin analog.
In another aspect of the present invention, thrombomodulin analog according to SEQ ID NO:2 has been described, 376 (according to numberings of SEQ ID NO:1) have the modification of phenylalanine in the position for it, it is preferably replaced by aliphatic amino acid, more preferably by glycine, alanine, valine, leucine or isoleucine displacement, optimum is replaced by alanine.
Of the present invention preferred aspect, use thrombomodulin analog as shown in Figure 19, it is described by aminoacid sequence SEQ ID NO:5 to SEQ ID NO:11.
This type of preferred TM analog is:
1. extend to the TM fragment with following amino acid change of aa490 from aa4: Phe376Ala, Met388Leu, Arg456Gly, His457Gln and Ser474Ala (being equal to SEQ ID NO:5).
2. extend to aa462 (=EGF1-6) the TM fragment with following amino acid change: Phe376Ala, Met388Leu, Arg456Gly and His457Gln (being equal to SEQ ID NO:6) from aa227.
3. extend to the TM fragment with following amino acid change of aa462 from aa333: Phe376Ala, Met388Leu, Arg456Gly and His457Gln (being equal to SEQ ID NO:7).
4. extend to the TM fragment with following amino acid change of aa462 from aa227: Phe376Ala and Met388Leu (being equal to SEQ ID NO:8).
5. extend to the TM fragment with following amino acid change of aa462 from aa333: Met388Ala, Arg456Gly and His457Gln (being equal to SEQ ID NO:9).
6. extend to the TM fragment with following amino acid change of aa462 from aa333: Met388Leu, Arg456Gly, His457Gln and Glu461Ala (being equal to SEQ ID NO:10).
7. extend to the TM fragment with following amino acid change of aa462 from aa333: Phe376Ala, Met388Ala, Arg456Gly and His457Gln (being equal to SEQ ID NO:11).
Definition
As employed in the description of the present invention, term " fibrinolysis effect " should refer to, the thrombomodulin analog is compared with the same measured condition of not adding described thrombomodulin analog, prolongs the ability (described in example I) of blood-clot lysis time.The fibrinolysis effect causes fibrinolytic because the fibrinolysis of TM analog is active with it and compares to preponderate and produce.
As used herein, term " causes fibrinolysis activity " and should refer to, the thrombomodulin analog is compared with the same measured condition of not adding described thrombomodulin analog in external test, significantly reduces the ability (described in example I) of blood-clot lysis time.
As used herein, term " significantly minimizing " and " significant prolongation " refer to the prolongation of blood-clot lysis time or minimizing, they are significantly different from basic value with the p=0.1 level, and/or refer to surpass 10%, and preferred 20%, more preferably 30%, most preferably 40%, 50%, 60%, 70%, 80%100%, 150% or 200% prolongation or minimizing.
As employed in the description of the present invention, word " treatment ", " treatment " or " medical treatment " refer to, any compositions of using TM analog of the present invention or comprising it is prophylactically prevented bleeding episode, or alleviate, improve or stop bleeding episode.Unless otherwise mentioned, otherwise they comprise and curing or healing and alleviate, alleviate or prevent.Similarly, as used herein, word " patient " refers to comprise the people by mammal.
As used herein, term " coagulopathy with hyperfibrinolysis " should refer to, as the coagulopathy of the disease of the coagulability that affects blood, the fibrinolysis that wherein significantly increases causes, increases the weight of or prolong bleeding episode.
As employed in the description of the present invention, term " thrombomodulin analog " refers to have protein and the peptide of the characteristic biologic activity identical with the thrombomodulin of membrane-bound or solubility.Described biologic activity is the ability as the activation of the receptor of thrombin and increase TAFI, or the other biological relevant with natural thrombomodulin learned active.
As used herein, term " binding affinity " refers to, the affine intensity of force between thrombomodulin analog and the thrombin, and by dissociation constant K
DDescribe.The K of the binding affinity between thrombin and the thrombomodulin
DValue can for example be passed through balance method (for example enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (BIACORE for example
TMAnalyze) measure.Binding affinity is preferably analyzed with the kinetic determination of describing among the embodiment of the invention II.
" K
D" refer to the RA between TM analog and the thrombin.High K
DValue representation hangs down binding affinity.Be used for measuring K
DAccurate Measurement method and method be provided in to implement among the II.
As used herein, term " cofactor active " refers to, thrombomodulin analog and thrombin are compound and give the ability of the ability of thrombin activation PROTEIN C.The algoscopy that is used for measurement cofactor activity is provided in embodiments of the invention III.
As used herein, term " TAFI Activation Activity " refers to, thrombomodulin analog and thrombin are compound and give the ability of the ability of thrombin activation TAFI.The algoscopy that is used for mensuration TAFI activity is provided in embodiments of the invention IV.
" Km " refers to Michaelis constant, produces with standard method by measuring the catalytic rate of measuring at different concentration of substrate.It equals reaction rate and is the residing concentration of substrate of its peaked half." Km " of TM analog of the present invention is by making concentration of thrombin remain on constant level (for example 1nM) and using the TM (for example 100nM or higher) of saturated level (to depend on K
D) measure.The PROTEIN C that working concentration increases progressively (for example, 1-60 μ M) is reacted.Measure Km and kcat with Lineweaver-Burke mapping or nonlinear regression analysis subsequently.
" TM
E" refer to, repeat the TM analog that (according to the aminoacid 227 to 462 of SEQ ID NO:1 or SEQ ID NO:3) forms by 6 EFG.
" TM
EM388L " refers to that repeat the TM analog that (aa227 to 462) forms by 6 EFG, the natural methionine on its position 388 (based on SEQ ID NO:3) is replaced by leucine residue.
Term " treatment effective dose " is defined as, and can reduce for example amount of the active component of bleeding episode of the symptom relevant with the coagulopathy with hyperfibrinolysis." in the treatment effectively " also refer to, compares any improvement of disease seriousness, frequency of disease development or persistent period with treatment not.
As employed in the description of the present invention, term " sequence modification " refers to, the modification of amino acid replacement, disappearance or insertion is passed through in the modification of one-level aminoacid sequence especially.If not in addition clearly definition, this term means, aminoacid by on its polarity, hydrophilic or hydrophobicity, acidity or alkalescence, size or armaticity respectively with visibly different another amino acid replacement of described aminoacid.
In view of amino acid whose general classes, the i.e. kind of acidity, alkalescence, polarity, nonpolar, electronegative, positively charged, aromatic series and aliphatic amino acid, the concept of sequence modification of the present invention preferably needs with the aminoacid of another amino acid replacement with different aminoacids kind.Aspect preferred, selection has the aminoacid of amino acid whose " opposite feature " to be replaced as displacer (subst ituent).The following aminoacid of ad hoc proposal (aa) displacement: acid aa to alkaline aa, polarity aa to nonpolar aa, electronegative aa to positively charged aa, aromatic series aa to aliphatic aa.
The specific embodiments of sequence modification of the present invention is that aliphatic amino acid for example glycine, alanine, valine, leucine and isoleucine most preferably utilizes the displacement of alanine to replacement of amino acid.
Sequence modification of the present invention also can comprise, utilizes the displacement of alpha-non-natural amino acid." alpha-non-natural amino acid " refers to, is not the aminoacid of 20 common amino acids or one of pyrrolysine or selenocysteine.Term " alpha-non-natural amino acid " includes but not limited to, the natural generation by the aminoacid (including but not limited to 20 common amino acids or pyrrolysine or selenocysteine) of modifying natural coding, but itself does not mix aminoacid in the polypeptide chain of growing by the translation complex.Non-naturally encoded naturally occurring amino acid whose example includes but not limited to, N-acetyl glucosamine base-Serine, N-acetyl glucosamine base-L-threonine and O-phosphotyrosine, ornithine, taurine.Sequence modification comprises that also other group is to amino acid whose connection.This type of group comprises acetate/ester, phosphate/ester, various lipid and carbohydrate, and it preferably changes amino acid whose chemical property.Sequence modification also comprises each aminoacid, oxidation or the reduction of preferred Met or Cys residue.
I. embodiment
Clot dissolution in the human plasma is measured
By using the model of external clot dissolution, test soluble thrombomodulin (Solulin) reduces or increases the ability of blood-clot lysis time in the mixture of the blood plasma of normal plasma and shortage Factor IX.
1. test macro
In the blood plasma compositions, by thrombin (factor IIa), calcium chloride and phosphatidylcholine/Phosphatidylserine (PCPS) vesicle are mixed to come in external initiation blood coagulation.Utilize turbidimetric analysis turbidimetry to measure and solidify and Fibrinolytic time course, measure " TAFIa potential " with functional examination.
2. experimental technique
Material.Such as the people such as Walker (J.Biol.Chem.1999; 274:5201-5212) described preparation thrombin and fibrinogen, except an exception: for the preparation of fibrinogen, make solution contain 1.2%PEG-8000 but not 2%PEG-8000 by being added on 40% in the water (w/v) PEG-8000, carry out subsequently the Beta-alanine precipitation.This of scheme changes permission and produces more fibrinogen.Such as (people such as Kim, 2008; Anal.Biochem372:32-40; The people such as Neill, 2004; Anal.Biochem.330:332-341) described QSY-FDP (covalently being connected in the fibrin catabolite of quencher QSY9C5-maleimide) and TAFIa standard for the preparation of TAFIa mensuration, and such as the people such as Horrevoets (J.Biol.Chem1997; 272:2176-2182) described, preparation recombined human Pg (S741C) and fluorescein derivative (5IAF-Pg).Such as the people such as previous Brufatto (J.Biol.Chem.2001; 276:17663-17671) described, purification S525C-thrombinogen, and carry out fluorescent labeling with 5-iodo Aminofluorescein (5IAF).QSY9C5-maleimide and 5-iodo Aminofluorescein are available from Invitrogen Canada Inc. (Burlington, ON, Canada).Fibrinolysin is available from Haematologic Technologies Inc. (Essex Junction, VT, USA), recombinant human soluble thrombomodulin (Solulin; STM) provided by Paion Deutschland GmbH (Aachen, Germany).Normal humanplasma (NP) is available from Kings ton, Ontario, the healthy donors of the blood bank of the Kingston general hospital (KGH) of Canada, lack the blood plasma (FVIII-DP) of FVIII available from Affinity Biologicals, Inc. (Hamilton, ON, Canada).Such as people such as Schneider, (J.Biol.Chem.2002; 277:1021-1030) described, the affinity chromatograph that carries out normal plasma by the pillar in fixing anti-human TAFI monoclonal antibody prepares the blood plasma (TDP) that lacks TAFI.Plasmin inhibitor D-Val-Phe-Lys chloromethyl ketone (VFKck), thrombin inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPAck) and potato tubers carboxyl peptide enzyme inhibitor (PTCI) are available from Calbiochem (San Diego, CA, USA).Tissue plasminogen activator's (alteplase; TPA) available from the pharmacy (Kingston, ON, Canada) of KGH.All other reagent have the AG quality.
3. method
The clot resolution measurement reaches the preparation of the sample of the degree that is used for mensuration TAFI activation
FVIII-DP is mixed so that the final concentration of NP is 0,1,6,10 50 or 100% (0-100%NP) with NP.Before mixing, with the optical density of each diluted plasma to 32, in the existence of 20nM thrombin or non-existent situation, it is added into isopyknic 1.5nMtPA of containing, 40 μ M PCPS and 20mM CaCl subsequently
2Solution in (final concentration: 0.75nMtPA, 20 μ M PCPS, 10mM CaCl
2, ± 10nM thrombin), sample is divided into a plurality of Eppendorf pipes, and places 37 ℃ of water-baths.Stop blood coagulation and dissolving in these pipes at different time points by adding 10 μ M PPAck and 10 μ M VFKck (respectively selective inhibition of coagulation enzyme and fibrinolysin).With the sample powerful mixing, with 16000g (room temperature) centrifugal 30 seconds subsequently, place immediately on ice to prevent the hot deactivation of TAFIa.The blood plasma of utilization shortage TAFI carries out 5 times of serial dilutions to the supernatant of each sample, uses the people (Anal.Biochem2008 such as Kim; The functional examination of 372:32-40) describing is measured TAFIa.In 96 orifice plates of adding a cover, carry out identical experiment, use SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) pass by in time to solidify and Fibrinolytic time-histories (timing) with mensuration at 400nm place monitoring turbidity.4 tPA concentration (0.25,0.75,1.5 and 3nM) upper soluble thrombomodulin (0-100nM) exist or non-existent situation under carry out similar experiment, sTM activates TAFI and the effect of dissolution time to measure.Also in the situation that 5 μ MPTCI exist, carry out these experiments, prolong to show normal and to lack the TAFIa dependency that dissolves in the blood plasma of FVIII.
The mensuration of the time course that thrombinogen activates in the normal blood plasma with lacking FVIII
Replenish with thrombinogen derivant (5IAF-II in the situation that the 10nM thrombin exists, for blood plasma (0-100%NP) normal and shortage FVIII; Final 300nM) and 20 μ M PCPS and 10mM CaCl
2With initial blood coagulation.In opaque plastic 96 orifice plates, carry out these experiments.At 37 ℃, utilize and be respectively exciting and emission wavelength of 495nm and 535nm, utilize 530-nm emission edge filter, with SpectraMax GeminiXS (Molecular Devices, Sunnyvale, CA, USA) be used for the fluorescence intensity that monitoring is pass by in time.With reflection baseline and maximum fluorescence, it is with the thrombinogen activation is relevant fully with fluorescence standard (0-1).
The mensuration of TAFIa potential
Select the TAFIa area under curve as the parameter of the TAFIa effect in the quantitative experiment process.With people (Thromb.Haemost.1993 such as Hemker; 85:5-11) " the thrombin potential " of definition is similar, this parameter is called " TAFIa potential ".TAFIa potential, the same with thrombin potential, proportional with the amount of substrate of cutting, and followingly carry out mathematic(al) treatment:
Wherein dS/dt is that base consumption speed and S are substrate.
If S is constant (that is, the consumption of limited S), then
For certain interval 0 to t,
The integration that it should be noted that formula (4) the right is the TAFIa area under curve,
4. result
Increase blood-clot lysis time by adding normal plasma to the blood plasma that lacks FVIII.
Utilize the 10nM factor IIa, 10mM CaCl
2With the initial blood coagulation of 20 μ M PCPS vesicles to produce wherein the clot structure to the insensitive model of FVIII concentration.Because regardless of FVIII concentration, the clot structure is all similar, therefore can measure FVIII to the impact of tPA-dependency (0.75nM) clot dissolution.By using this dissolving model, dissolution time increases with the increase of normal plasma percentage ratio.Fig. 1 shows the clot dissolution feature of the FVIII-DP of the normal plasma that is added with 0-100%, and dissolution time is summarized among Fig. 1 (illustration).In FVIII-DP, dissolution time is 37 minutes, and by adding normal plasma, it can increase about 50%.
10% normal plasma is enough to recover the clot dissolution among the FVIII-DP.
On 10% normal plasma, the dissolution time of the shortening relevant with FVIII-DP has been corrected to the dissolution time (referring to Fig. 1, illustration) of observing in the normal plasma.
50% TAFIa potential is enough to recover the clot dissolution among the FVIII-DP.
Measure normal plasma, the blood plasma that lacks FVIII and the TAFI in the pooled plasma and activate, with the impact of quantitative FVIII on the time course of activation.The functional examination method for the TAFIa that measures the whole time course of solidifying and decomposing, be the results are shown among Fig. 2.When being used for thrombin, calcium ion and PCPS in the initial blood coagulation of FVIII-DP, after 5 minutes, measure about 30pM TAFIa.Along with normal plasma percentage ratio increases, the peak concentration of TAFIa also increases.Although by being the recoverable dissolution time with the additional FVIII-DP of 10% normal plasma, this is not enough to proofread and correct fully TAFI and activates.By calculating TAFIa time course area under curve (Fig. 2 A), determined in normal plasma and 50% normal plasma (16800pM minute and 14100pM minute respectively), in front 50 minutes, to obtain roughly the same TAFIa potential (Fig. 2 B), but the FVIII-DP blood plasma that mixes with 10% normal plasma to have only be 50% TAFIa potential of the TAFIa potential in the normal plasma.
Between dissolution time and TAFIa potential, there is strong correlation.
For the relation between quantitative dissolution time and TAFI activate on the 0-100%FVIII scope, draw logarithm dissolution time (loglysistime) to the curve (Fig. 2 B, illustration) of logarithm TAFIa potential (log TAFIa potential).As expected, there is the strong positive correlation of dissolution time and TAFIa potential in data show in containing the blood plasma of 0-100%FVIII.Can activate (Fig. 3) by the thrombinogen in the analysed for plasma and come the TAFI among reasonable dismissal Fig. 2 A to activate feature, because thrombin is the activator of TAFI.General trend is that along with the increase of normal plasma percentage ratio, the speed that thrombinogen activates also increases (this can determine by slope of a curve among inspection Fig. 3).For normal plasma, exceptional case occurs.In normal plasma, the speed that thrombinogen activates is lower than the activation rate among the FVIII-DP that mixes with 50% normal plasma.Although speed is slower in normal plasma, the persistent period that thrombinogen activates is about 2 times of the FVIII-DP that mixes with 50% normal plasma.In each experiment, the time-histories that thrombinogen activates and TAFI activate very identical.Also use calcium ion and PCPS (but not adding thrombin) that normal plasma is solidified.Not solidifying that calcium is induced can occur immediately; In normal plasma, clot forms and has spent about 15 minutes.On this time, thrombinogen activates and has entered the amplification phase (propagation phase), and therefore, TAFI is activated.The degree that activates at TAFI aspect the clot formation is identical with time-histories, no matter blood coagulation be the thrombin that adds exist or non-existent situation under initial, this shows that TAFI activates and is the result of the thrombin that produces of original position but not adds the result of the thrombin of inducing blood coagulation.In the situation that thrombin exists, have the TAFIa potential of 16,800pM min, compared in the non-existent situation of thrombin 14,150pM min.
Soluble thrombomodulin prolongs the clot dissolution of the blood plasma of normal and shortage FVIII.
In normal plasma, in the situation that 10nM sTM exists, peak TAFIa level and TAFIa potential are from the 600pM in the non-existent situation of sTM and increased to respectively about 6000pM and 150,000pM min in 16800pM minute.This increase that TAFI activates causes the increase of 70% dissolution time.10nMsTM is similar to normal plasma to the effect of the relative prolongation of dissolving among the FVIII-DP, because when FVIII-DP solidifies and dissolve in the situation that sTM exists, dissolves and is extended 65%.In the situation that 10nM sTM exists, compare with 30pM in the non-existent situation of sTM, 750pM TAFIa appears in TAFIa concentration at the peak.Beginning from blood coagulation to the time of clot dissolution, in the situation that 10nM sTM exists, comparing with 600pM min in the non-existent situation of sTM, TAFIa potential is measured as 12800pM min.
The concentration of tPA and sTM is depended in the increase of blood-clot lysis time in the normal blood plasma with lacking FVIII.
Activate impact on dissolution time at tPA and sTM concentration range inner analysis TAFI, whether can activate to proofread and correct by stimulating TAFI to determine the dissolving defective among the FVIII-DP.Be summarized in dissolution time among Fig. 4 with relevant from the dissolution time of the similar experiment that comprises PTCI (its for the inhibitor of TAFIa).In the situation that PTCI exists, do not have functional TAFIa, so the TAFIa-dependency of the relative dissolution time representative dissolving that shows among Fig. 4 prolongs.On the least concentration (0.25nM) of tPA, when adding 1nM sTM in normal plasma, the maximum TAFIa-dependency of observing dissolving prolongs (2-doubly).Replenish the dose dependent prolongation (Fig. 4) that FVIII-DP causes dissolution time with sTM.When being added into 100nM sTM among the FVIII-DP, dissolution time is proofreaied and correct fully to the dissolution time of seeing in normal plasma.Along with the increase of tPA concentration, the maximum TAFIa-dependency that needs the sTM of higher concentration to obtain to dissolve prolongs.For example, when having 1.5nM tPA (Fig. 4), in normal plasma, need 25nM sTM to make the TAFIa dependency of dissolving prolong maximization, in FVIII-DP, then need 100nMsTM.Similarly, along with the increase of tPA, in these clot dissolutions experiment, TAFIa shows that dissolution time is had much bigger effect (compare with on the 0.25nM tPA 2.3 times, on 1.5nM tPA up to 5.2 times).Shown the increase along with tPA concentration, the concentration that any TAFIa-dependency that obtains to dissolve prolongs required sTM also increases.On 0.25nM tPA, the prolongation that in normal plasma, does not need sTM to obtain to dissolve, yet when being added into 3nM tPA (Fig. 4) in the normal plasma, the prolongation that needs 25nM sTM to obtain to dissolve.In order to show actual dissolution time is affected by tPA and sTM how, be displayed in Table 1 the dissolution time in the repressed normal blood plasma with lacking FVIII of TAFIa.
Thrombomodulin promotes very significantly TAFI to activate and prolong dissolving in the blood plasma of normal and shortage FVIII.
In normal plasma, with the non-existent situation (zero of sTM; 600pM TAFIa; Referring to Fig. 5 A) compare, in the situation that 10nM sTM exists (●; 6000pM TAFIa is on its peak level) show that the TAFI activation is significantly strengthened.Appended clot dissolution curve display, the interpolation of 10nM sTM cause the increase of 70% dissolution time.In the FVIII-DP that is supplemented with 10nM sTM, compare with 30pM in the non-existent situation of sTM, TAFIa is measured as 750pM (referring to Fig. 5 B) at its peak value.The increase that TAFI activates cause comparing with the FVIII-DP that the lacks sTM prolongation of 60% dissolving.
II. embodiment
The analysis of the binding affinity between thrombin and the thrombomodulin
By using fluorescence kinetics to measure, the affinity of measuring the combination between thrombin and the thrombomodulin analog (is expressed as K
DValue).
1. test macro
Measure the affinity of the combination between thrombin and the thrombomodulin analog with fluorescence kinetics, it is expressed as K
DValue.
2. experimental technique
Material
Such as the people such as Bajzar (J.Biol.Chem.1995; 270:14477-14484) described, from the separating plasma human thrombin.Obtain recombinant soluble thrombomodulin (Solulin) from PAION Deutschland GmbH (Aachen, Germany).Obtain all other reagent with the AG quality from Sigma.
Method
Thrombin is to the measurement of the combination of thrombomodulin and TAFI
Measure thrombin to the combination of thrombomodulin according to balance in conjunction with measuring.Will be at 0.02M Tris-HCl with a small amount of continuous equal portions, 0.15M NaCl, 5.0mM CaCl
20.01%Tween80, contain thrombin (20nM), thrombomodulin (1.54 μ M) and DAPA (20nM among the pH7.4, red sulfuryl arginine N-3-(ethyl-1,5-penta 2 bases) solution amide, the reversible thrombin inhibitor of fluorescence) is added into the other same solution that does not contain thrombomodulin.In the sample room of Perkin-Elmer LS50B type spectrofluorometer, in being equipped with the cuvette of magnetic stirrer, add.Utilization is respectively 280 and the exciting and emission wavelength continuous record intensity level of 545nm.The 430-nm edge filter is used for the emission light beam.Following analytical data.Fluorescence intensity I is assumed to intensity sum from thrombin-DAPA (TD) and soluble thrombomodulin-DAPA (TTMD).Be I=i
1[TD]+i
2[TTMD], wherein i
1And i
2Coefficient (owing to excite at 280nm, therefore can ignore from the emission of free DAPA) for the fluorescence of TD and TTMD.Because TM can not change the K that PROTEIN C activates or TAFI activates discernablely
m(referring to people such as Bajzar, 1996; J.Biol.Chem.271:16603-16608), therefore can suppose that it does not change the interactional affinity of thrombin-DAPA.
Therefore, [TD]=([T]+[TD])/(1+K
DAPA/ [DAPA])
And, [TTMD]=([TTM]+[TTMD])/(1+K
DAPA/ [DAPA]),
Wherein, K
DAPABe the interactional dissociation constant of thrombin-DAPA.
Therefore,
I=i
1·([T]+[T·D])/(1+K
DAPA/[DAPA])+i
2([T·TM]+[T·TM·D])/(1+K
DAPA/[DAPA])。
If f and b are respectively defined as the mark of thrombin free and that be combined with thrombomodulin, and [T]
0Be the total concentration of thrombin, so f=([T]+[TD])/[T]
0, b=([TTM]+[TTMD])/[T]
0And f+b=1.Pass through subsequently I=i
1F[T]
0/ (1+K
DAPA/ [DAPA])+i
2B[T]
0/ (1+K
DAPA/ [DAPA]) provide fluorescence intensity.If I
0Initial strength when being defined as not adding thrombomodulin, then f=1 and I
0=i
1[T]
0/ (1+K
DAPA/ [DAPA]).Similarly, if I
MaxBe defined as the intensity of thrombin when saturated by thrombomodulin, then b=1 and I
Max=i
2[T]
0/ (1+K
DAPA/ [DAPA]).Therefore, I=I
0F+I
MaxB.Substituting f with 1-b then provides: I=I
0+ (I
Max-I
0) b or Δ I=Δ I
MaxB.Standardization then provides (Δ I/I for initial strength
0)=(Δ I
Max/ I
0) b.If DAPA with the affinity that equates in conjunction with T and TTM, then TM with the affinity that equates in conjunction with T and TD.
Therefore, by with K
TMBe defined as the interactional dissociation constant of soluble thrombomodulin, [T] [TM]=K
TM[TTM]; [TD] [TM]=K
TM[TTMD]; And ([T]+[TD]) [TM]=K
TM([TTM]+[TTMD]).Last expression formula and f[TM]=K
TMB is identical.Because f=1-b and [TM]=[TM]
0-b[T]
0, wherein [TM]
0Be total thrombomodulin concentration, therefore obtain following formula: (1-b) ([TM]
0-b[T]
0)=K
TMB.This is the quadratic equation of b, when hereinbefore about (Δ I/I
0) expression formula in find the solution and when alternative, it provides formula: (Δ I/I
0)=(Δ I
Max/ I
0) 0.5 (K
TM+ [T]
0+ [TM]
0-((K
TM+ [T]
0+ [TM]
0)
2-4[T]
0[TM]
0)
1/2).Should after an equation expression relation between following: fluorescence intensity level, the nominal concentration of thrombomodulin and thrombin, the interactional fluorescence intensity increment of the interactional dissociation constant of soluble thrombomodulin and demonstration thrombomodulin and thrombin-DAPA.With [TM]
0As independent variable and with K
TMWith Δ I
MaxAs best fit parameters, by nonlinear regression analysis with the above-mentioned formula of intensity data match.
3. result
Thrombin is with K
DThe affinity of=23 ± 14nM is in conjunction with soluble thrombomodulin.
Thrombin utilizes the perturbation of the fluorescence of DAPA to measure to the combination of soluble thrombomodulin.Described in Fig. 6, titration curve has shown the increase of relative fluorescence in the concentration range of 0 to 75nM soluble thrombomodulin.Data analysis shows, thrombin to the combination of soluble thrombomodulin with K
D=23 ± 14nM is feature.
III. embodiment
The analysis of the cofactor activity of the thrombomodulin analog of sudden change
By using fluorescence kinetics to measure, the affinity of measuring the combination between thrombin and the thrombomodulin analog (is expressed as K
DValue).
1. experimental technique
Materials and methods
In shockates, directly measure the TM mutant as the ability of the cofactor of the activation of the thrombin-mediated of PROTEIN C.The r-aPC is from Dr.John McPherson, Genzyme Corp., Framingham, MA., and as (BioTechnology1990; 8:655-661) the described purification that carries out.Each shockate of 25 μ l is mixed in microtitration plate with isopyknic r-aPC (final concentration is 0.3 μ M) and people α thrombin (Sigma Chemicals, St.Louis, MO) (final concentration of 1nM).Employed all reagent are containing the 20mM Tris of 5mg/ml bovine serum albumin, pH7.4/100mM NaCl/3.75mM CaCl
2/ 0.1%NaN
3(w/V) dilute in.37 ℃ of lower incubations 1 hour, 800 units by adding 25 μ l/ml hirudin (Sigma Chemicals, St.Louis, MO) came cessation reaction with mixture.Chromogenic substrate D-valyl by adding 100 μ l-L-leucyl-L-arginine-p-Nitraniline. (S-2266) (1mM) is measured the amount of the PROTEIN C of activation.Use plate reader, utilize the absorbance at 405nm place to measure in time variation.With data record be milliOD unit/minute, measured the data that absorbance (carrying out 15 minutes) is measured each sample in per 10 seconds by using Molecular Devices plate reader.All mensuration comprise in triplicate shockate sample, with each of the DH5 α cell of pSELECT-1 carrier (without TM), pTHR211 (wild type) or pMJM57 (pTHR211, the methionine of position 388 is changed to leucine) transfection as internal contrast.The cofactor activity of TM mutant is expressed as the meansigma methods of the cofactor activity that obtains for pMJM57.
Statistical analysis
Measure the activity at least 2 times (for those mutants that only separated two positive colonies, measuring 3 times) of each mutant, use student t check all data to be used for measuring the significance of difference.The coefficient of variation between the plate is 16.7% (n=18).
The Western engram analysis
According to the description (Novex Inc., San Diego, CA) of manufacturer, under reducing condition with escherichia coli (E.coli) shockate electrophoresis in 10%Tris-tricine SDS PAGE.By with shockates at the sample buffer that contains the 10mM dithiothreitol, DTT (62.5mM Tris, pH6.8,2%SDS, 10% glycerol, 0.0025% bromophenol blue) boiled in 10 minutes, and then prepared the alkylating sample of reduction with 50mM iodoacetamide incubation.
Protein is transferred to nitrocellulose filter in transfering buffering liquid (192mM glycine, 25mM Tris, pH8.3,20% methanol) under 4 ℃.With the sealing buffer (at 10mMTris, pH7.5,0.9%NaCl, 0.05%NaN
3In 1% bovine serum albumin) the sealing nitrocellulose filter, be used in subsequently the mice polyclonal antiserum of sealing in the buffer (producing for the alkylating EGF domain of the reduction of human thrombomodulin) and carry out incubation.Using lavation buffer solution (10mM Tris, pH7.5,0.9%NaCl, 0.05%NaN
3, 0.05%Tween20) after the washing, the biotinylated goat that is used in the sealing buffer that contains 0.05%Tween20 resists-mouse IgG antibody incubation filter membrane.Use Vectastain ABC solution (Vector Laboratories, Burlingame, CA) and ECL detection system (Amersham Corporation, Arl ington Heights, IL) to detect protein according to the description of manufacturer.
IV. embodiment
Activate to analyze the thrombomodulin analog for TAFI and PROTEIN C
By using fluorescence kinetics to measure, measure the K that is expressed as of combination between thrombin and the thrombomodulin analog
DThe affinity of value.
1. experimental technique
Protein and reagent
Such as the people such as Parkinson (Biochem.Biophys.Res.Commun.1992; 185:567-576) described, preparation comprises Solulin (residue 4-490), TM
E(residue 227-462), TM
EC-ring 3-6 (residue 333-462) and TM
EThe clipped form of the thrombomodulin of i4-6 (residue 345-362).With TM construct transfection Sf9 cell, by utilize anion exchange, gel is overanxious and the combination of the chromatography of thrombin affinity from the culture medium isolated protein.The purity of utilizing SDS-polyacrylamide gel electrophoresis and silver dyeing to estimate is 95% or higher.Such as the people such as Bajzar (J.Biol.Chem.1995; 270:14477-14484) described separation human plasma TAFI.Such as Bajzar and Nesheim (J.Biol.Chem.1993; 268:8608-8616) described preparation human protein C and thrombin.Such as the people such as Nesheim (Biochemistry1979; 18:996-1003) red sulfuryl arginine N-(3-ethyl-1, the 5-penta 2 bases) amide (DAPA) of described synthetic thrombin inhibitor.From TM
EThe M388L construct produces the Point mutont that is obtained by Alanine-scanning.At expression in escherichia coli protein.The operation of pericentral siphon extract and the preparation by people such as Nagashima, (J.Biol.Chem.1993; 268:8608-8616) be described.HEPES, alkaline carboxypeptidase substrate hippuroyl-arginine, cyanuric chloride and Isosorbide-5-Nitrae-dioxs are available from Sigma.All other reagent have the AG quality.
Measure the speed of PROTEIN C and TAFI activation with the Point mutont of thrombomodulin analog
In order to activate TAFI, at room temperature be used in 20mM HEPES, pH7.5,150mM NaCl, 5mM CaCl
2In the 20-μ l sample aliquot 5 minutes of thrombin (final 13nM) each pericentral siphon extract of precincubation.Subsequently with mixture with the restructuring TAFI of purification (final 18nM) and substrate hippuroyl-arginine (finally 1.0mM) incubation 60 minutes in the volume of 60 μ l altogether.By measuring hippuroyl-arginine to the hydrolysis of hippuric acid, then measure the amount of the TAFI that hippuric acid to the conversion of chromogen (using 3% cyanuric acid (w/v) in the Zai diox of 80 μ l phosphate buffers (0.2M, pH8.3) and 60 μ l) quantitatively activates.After fully mixing, measure the absorbance of the supernatant of clarification at the 382nm place.For each mutant, calculate the amount that thrombin-dependency activates of TAFI by deducting the background absorbance that in the non-existent situation of thrombin, produces.Following mensuration TM
EM388L-alanine mutation body is to the activation of PROTEIN C.
All samples and reagent are diluted in APC mensuration diluent (20mM Tris-HC l, pH7.4,100mM NaCl, 2.5mM CaCl
2, 0.5%BSA) in.Before utilizing 20 μ l hirudins (0.16U/ μ l, 570nM) quencher, with sample and TM standard (0-1nM) in 96 orifice plates with 0.5 μ M PROTEIN C and 1nM thrombin with 60 μ l cumulative volumes in 37 ℃ of lower incubations 60 minutes, to produce APC.Measure the amount of the APC of formation in the hydrolysis of the monitoring S-2266 of 405nm place (100 μ l, 1mM) with 1 minute interval by using plate reader (Molecular Devices Corp., MenloPark, CA).The activity of 1U produces PROTEIN C/minute (37 ℃) of the activation of 1pmol.
All mensuration comprise with the pSelect-1 carrier (without TM
E), wild type TM
E(M388) or TM
E(M388L) extract of the DH5 α cell of transfection is as internal contrast.TM
E(M388L) the cofactor activity of alanine mutation body is expressed as TM
EThe percentage ratio of activity (M388L).Use 3 independently the extract prepared product activate with the PROTEIN C of each TM mutant of duplicate mensuration and TAFI.
2. result
The result (Fig. 7) who utilizes the TM mutant to obtain shows have 5 to have the cofactor activation that significantly reduces in 8 mutants.In these 5 mutants, there are 4 mutants also to show the Activation Activity of following the TAFI that reduces.Only the sudden change on F376A causes the remarkable loss that PROTEIN C activates, and the medium minimizing that causes TAFI to activate.Interesting ground, Phe
376The difference of the importance that activates for TAFI and PROTEIN C shows, when PROTEIN C is the substrate of soluble thrombomodulin complex, the requirement of thrombomodulin structure is restricted more.
V. embodiment
The analysis that the PROTEIN C relevant with oxidation of Met-specificity T M mutant activates
By using the specificity methionine mutant of thrombomodulin analog, activate the effect that this type of also relevant with protein oxidation residue activates cofactor of analyzing of measuring with PROTEIN C.
1. experimental technique
Protein and reagent
Human recombination protein C is from Genzyme Corp. (Bos ton, MA).Thrombin of beef is from MilesLaboratories Inc. (Dallas, TX).Such as the people such as Glaser (Prep.Biochem.11975; 5:333-348) described preparation D-Val-Leu-L-Arg-p-nitroaniline.People's α-thrombin (4,000NIH U/mg), bovine serum albumin (fraction V) and toluene-sodium-sulfonchloramide are from Sigma Chemical Co. (St.Louis, MO).
TM
E(Sf9) expression
Under 4 ℃, carry out all methods.The DNA sequence that 6 EGF samples of coding TM are repeated (aminoacid 227-462) is connected in the signal sequence of insect protein enzyme hypodermin A, and hybrid gene is placed under the control of polyhedrosis gene promoter in baculovirus shuttle vector pTMHY101.The Application standard technology produces recombinant virus.By using paramorphogen direct mutagenesis test kit (Stratagene, Inc., La Jolla, CA) to prepare described mutant analog, utilize identical method preparation virus in rhabdovirus system, to express.
Utilize purification and the oxidation of toluene-sodium-sulfonchloramide
The growth medium of mutant that comprises the secretion of TME (Sf9) by centrifugal clarification with its lyophilizing, is dissolved in 0.2%NEM-Ac with the volume of 1:10, among pH7 and the 0.008%Tween80 again.H with 5 μ l
2The 100mM toluene-sodium-sulfonchloramide of O or 5 μ l is processed sample aliquot; At room temperature incubation is 20 minutes; Remove oxidant by dilution; At NAP-5 post (20mM Tris-HCl, 0.1M NaCl, 2.5mM CaCl
2, 5mg/ml BSA, pH7.4; Pharmacia Inc.) carries out desalination on; And the activation of following mensuration PROTEIN C.
The measurement of TM cofactor activity (APC mensuration)
All samples and reagent are diluted in APC mensuration diluent (20mM Tris-HCl, pH7.4,100mM NaCl, 2.5mM CaCl
2, 0.5%BSA) in.Utilizing before 20 μ l hirudins (0.16U/ μ l, 570nM) carry out quencher, with sample and TM standard (0-1nM) in 96 orifice plates with 0.5 μ M PROTEIN C and 1nM thrombin with 60 μ l cumulative volumes in 37 ℃ of lower incubations 60 minutes to produce APC.Measure the amount of the APC of formation in the hydrolysis of the monitoring S-2266 of 405nm place (100 μ l, 1mM) with 1 minute interval by using plate reader (Molecular Devices Corp., MenloPark, CA).The activity of 1U produces PROTEIN C/minute (37 ℃) of the activation of 1pmol.
2. result
The oxidation of Met388 causes the cofactor that reduces of TM active.
At expressed in insect cells mutant and wild type TME (Sf9), process with toluene-sodium-sulfonchloramide, it is active to measure cofactor, and comparative result (table 2).When with oxidant for example toluene-sodium-sulfonchloramide process TM
EThe time, it loses about 85% cofactor active (referring to table 2).Met
291And Met
388Mutation site-specific show that the deactivation of TME (Sf9) is owing to the oxidation of single methionine.Keep Met
388Derivant by the toluene-sodium-sulfonchloramide deactivation to similar degree (〉 80%), and the Met388Leu mutant has resistance.Met
291Replaced mutant has activity but not antioxidation deactivation.
VI. embodiment
Analysis with TM analog of the sudden change (Gln387, Met388, Phe389) that encircles between the domain between EGF4 and the EGF5
By using the specific mutations body of thrombomodulin analog, activate the effect of analyzing these residues and oxidation thereof of measuring with PROTEIN C.
1. experimental technique
Plasmid construction.The following thrombomodulin fragment (TM that is only formed by EGF spline structure territory at expression in escherichia coli
E).Use primer 5'-CCG GGA TCC TCA ACA GTC GGT GCCAAT GTG GCG-3' and 5'-CCG GGA TCC TGC AGC GTG GAG AAC GGC GGC TGC-3' by the polymerase chain reaction from human gene group DNA obtain the to encode TM of total length TM
EThe dna fragmentation of (residue 227-462).This fragment is placed in pKT279 under the control of beta-lactamase promoter and signal sequence.Subsequently the EcoRV-BglII fragment of gained plasmid and the ScaI-SacI fragment of the pGEM3zf-that comprises the f1 replication origin are inserted the pSelect-1 carrier at EcoRV-BamHI and ScaI-SacI site respectively, to make up colibacillus expression plasmid pTHR211.By using the in vitro mutagenesis test kit, utilize strand pTHR211DNA template, make up the plasmid of the TM mutant on the coding site 387,388 or 389 with the site-directed mutagenesis method of describing in the site that changes.Confirm each primer of mutation site-specific by restriction analysis.
For the cofactor of measuring mutant active, each culture of Escherichia coli of expressing mutein is centrifugal, washing, and with cell precipitation at 20% sucrose, 300mM Tris-HCl, pH8.0,1mM EDTA, 0.5mM MgCl
2In carry out incubation (10 minutes, 4 ℃).By the centrifugal 0.5mM MgCl that uses
2The cell precipitation that process (10 minutes, 4 ℃) prepares Shockate, and in APC measures it is measured.Data are the meansigma methods from the result of each of 3 independent clonings.
The measurement of TM cofactor activity (APC mensuration)
All samples and reagent are diluted in APC mensuration diluent (20mM Tris-HCl, pH7.4,100mM NaCl, 2.5mM CaCl
2, 0.5%BSA) in.Before utilizing 20 μ l hirudins (0.16U/ μ l, 570nM) quencher, with sample and TM standard (0-1nM) in 96 orifice plates with 0.5 μ M PROTEIN C and 1nM thrombin with 60 μ l cumulative volumes in 37 ℃ of lower incubations 60 minutes to produce APC.Measure the amount of the APC of formation in the hydrolysis of the monitoring S-2266 of 405nm place (100 μ l, 1mM) with 1 minute interval by using plate reader (Molecular Devices Corp., MenloPark, CA).The activity of 1U produces PROTEIN C/minute (37 ℃) of the activation of 1pmol.
2. result
The sudden change of interannular domain causes the cofactor that reduces of TM active.
By using direct mutagenesis, express in the position 387, the 388 or 389 TM mutants (Fig. 8) with aminoacid, disappearance or insertion of change.The cofactor of TM mutant is active in available from the meansigma methods of 3 independent clonings and be expressed as the percentage ratio of the activity of TME (Sf9) WT.Use for the mutant of all the new mutation bodies on the position 388 and the selection on the position 387, is carried out the gel image scanning of Western trace for the polyclonal antibody of TM.These scannings have shown the roughly TM of equivalent, and this shows that differential expression can not explain the activity difference of observing.In addition, insertion and disappearance (Fig. 8 D) on the optional position of encircling between the independent displacement on position 387 (Fig. 8 A), 388 (Fig. 8 B), 389 (Fig. 8 C) or domain cause such analog, and it is generally than wild type TM in APC measures
EWeak cofactor.Gln
387The analog that is substituted by Thr, Met or Ala keeps greater than 70% cofactor active, but utilizes the displacement of Glu that described activity is decreased to 58% of contrast, and all other aminoacid cause the forfeiture greater than 50%.Only Leu is to Met
388Displacement cause than the remarkable higher cofactor of wild type active (1.8 times).Except Gln and Tyr, Met
388All other displacements cause forfeiture greater than 50% cofactor activity.TM cofactor activity is to Phe
389Amino acid replacement not too responsive, 9 reservations are arranged greater than 70% the activity of in contrast, finding in this locational Point mutont.Pro on the optional position or Cys displacement are decreased to greater than 10% activity, except the Met388Pro that keeps 30% activity.Each changes the length of encircling between domain between EGF4 and the EGF5 and causes having and be lower than 10% wild type TM by the disappearance single amino acids or with Ala inserts 4 possible positions
EThe mutant of activity.
VII. embodiment
Fibrinolytic analysis in the dog hemophilia blood plasma
By model and the thrombleastography of using external clot dissolution, in the whole blood of the Canis familiaris L. with haemophilia A or blood plasma, tested the ability that soluble thrombomodulin (Solulin) reduces or increase blood-clot lysis time.
Materials and methods
Clot dissolution is measured
Described in embodiments of the invention 1, carry out clot dissolution and measure.
Thrombleastography
Such as people such as Prasad, 2008; Described in the Blood111:672-679, carry out thrombleastography.Dog hemophilia whole blood (± Factor IX neutralizing antibody) (320L) is added into the Haemoscope that comprises 40 μ L solution (90nM thrombin, 9nM tPA and 0-390nM sTM)
In the passage of 5000 (Haemonetics Corp.Braintree, MA).After fully mixing, press contact pin (pin), and continuous monitoring is solidified and fibrinolysis.Be connected in torque on the wire (wire) of clot, Haemoscope by measuring through contact pin
5000 allow to measure clotting time, blood coagulation kinetics, clot intensity and clot stability (fibrinolysis).Along with clot forms, the torque on the contact pin increases and is expressed as the increase of amplitude (output).Similarly, in the fibrinolysis process, the degraded of clot causes reducing of torque and reducing of amplitude.
The people such as Giles, 1984; The people such as Blood63:451-456 and Tinlin, 1993; Thromb Haemost69:21-24 before described, and some Canis familiaris L.s of Queen's University hemophilia doggery (haemophilicdogcolony) produce blocking antibody (inhibitor).
Be considered to unavailable factor VI I I replacement therapy from the inhibitor titre greater than 150 bethesda's units (Bethesda Unit) (〉 5B.U.) Canis familiaris L. extract the blood plasma of the hemophilia Canis familiaris L. with inhibitor.
The result
Solulin even prolong clot dissolution in the concentration of 500nM.
In dog hemophilia blood plasma, blood-clot lysis time is prolonged, situation about existing at 25nM Solulin is downward to be about 2 times, arrives about 9 times platform (referring to Fig. 9) lower prolongation of 200-500nM Solulin (maximum dose level of its representative test).
The significant fibrinolysis effect of conclusion: Solulin in hemophilia blood plasma even under high concentration supported forcefully, the effective and safe purposes of thrombomodulin analog in hemophilia.
Solulin prolongs clot dissolution in the whole blood from the dog with haemophilia A.
In thrombelastogram, the peak swing of torsion wire is measuring of maximum clot intensity.In this type of experiment (referring to Figure 11 A), by being transferred to, dog hemophilia whole blood induce continuous clot to form in the cuvette that comprises 90nM thrombin and 9nMrt-PA and dissolving.Solulin exists with the concentration of 0-390nM.In the non-existent situation of Solulin, observe the amplitude of pact ± 85mm.100 and the Solulin of the concentration of 250nM block clot dissolution fully: amplitude is larger and remain unchanged until 80 to 90 minutes (end of experiment).Solulin concentration is to the further increase of 390nM still relevant with slower clot dissolution ((without Solulin) compares with control experiment) with larger amplitude (Figure 11 A).
In this experiment, clot dissolution area under curve (AUCL) can be used for quantitative clot hardness, wherein 100 and the Solulin of 250nM this parameter is increased surpass 5 times (Figure 11 B).The prolongation of the blood-clot lysis time of identical experiment is plotted among Figure 11 C.Under dissolution time does not exist from Solulin 20 minutes increase to 100 and 250nM Solulin under 3 hours.
Conclusion: the fibrinolysis effect of the Solulin that shows by thrombleastography is consistent with the result of above-mentioned clot dissolution mensuration.In addition, Solulin shows increases clot hardness.
Solulin prolongs from the clot dissolution in the whole blood of the dog with haemophilia A in the situation that anti--Factor IX antibody exists
As mentioned above to known comprise high titre anti--blood of Factor IX antibody (greater than 150 bethesda's units) carries out this type of thrombleastography experiment: induce continuous clot to form in the cuvette that contains 90nM thrombin and 9nMrt-PA and dissolving by dog hemophilia whole blood is transferred to.Solulin exists with the concentration of 0-3510nM.In the situation without Solulin, observe the amplitude (referring to Figure 12 A) of pact ± 20mm.Solulin dose dependent ground increases amplitude, 100 and 250nM concentration under increase amplitude to pact ± 60mm, be issued to the amplitude of pact ± 90mm at 390nM.
In addition, Solulin dose dependent ground prolongs blood-clot lysis time, and it is with about 46 minute (referring to Figure 12 As) about 18 minute extending to 390nMSolulin under of blood-clot lysis time from contrast.Also (0nM Solulin's increase of 10 times of surpassing by area under the elastic curve compares with 390nM; Referring to Figure 12 B) show the clot hardness of increase.
Conclusion: anti--Factor IX antibody suppresses to have reduced fully to Factor IX activity actual but can not suppress the ability that Solulin prolongs clot dissolution.From different angles, this tests demonstration, and the existence of residual Factor IX activity is enough to improve the efficient that Solulin prolongs clot dissolution in the hemophilia whole blood (in the non-existent situation of antibody, referring to Figure 11 A).
This has supported the purposes of described thrombomodulin analog in the hemophiliac for the treatment of with Factor IX.In addition, the patient who suffers hemophilia and still have functional component VIII concentration also can treat with described thrombomodulin analog.At last, do not exist Factor IX or show high titre anti--patient of Factor IX antibody also is obedient to the treatment that utilizes described thrombomodulin analog.
VIII. embodiment
The TAFI of different TM analog activates the ratio that PROTEIN C is activated
Produce describe among Figure 19 with several TM analog shown in the SEQ ID NO:5-11.The PROTEIN C and the TAFI that have the solulin analog of Phe376Ala change at testing in vitro activate, and compare with solulin.
Materials and methods
The measurement of TM cofactor activity (APC mensuration)
The composite coding tool is the dna fragmentation of the Solulin of sudden change selectively, and it is cloned into pGAEx carrier (carrier that standard HEK293-derives).The pGAEx carrier comprises for standard promoter sequence, targeting sequencing and the secretion sequence of HEK293 cell and the His-6-label on the C of protein end.The HEK293 cell carries out transfection (transient transfection) with the pGAEx-Solulin carrier, and with 1 liter of scale suspension culture.After the transfection 6 days, collect protein by Ni-Hi Trap post (at PBS, the linear gradient of the 20-500mM imidazoles among the 500mM NaCl).The target that is used for purification is the His-6-label.After the dialysis (for PBS, 16h), analysing protein.Coomassie blue stain quantitative protein by SDS-PAGE.Then, by Western trace (resisting-His-6-antibody of labelling) checking protein identity.
The measurement of TM cofactor (APC mensuration) and TAF I activity
At the respectively TAFI that contains 0 to 2 μ M or PROTEIN C, 0.5nM thrombin, 5mM calcium chloride and test TM analog in the mensuration buffer of 25nM solulin or solulin Phe376Ala analog respectively.
With D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) (for the TAFI experiment) or hirudin (for the PROTEIN C experiment) quencher reaction.
Use respectively N-(4-methoxyphenyl azo formoxyl)-Arg-OH (AAFR) and pyroGlu-Pro-Arg-pNAHCl (S-2366) to measure the PROTEIN C activity of TAFIa and activation.By the speed of substrate hydrolysis is compared to measure the TAFIa of generation in 10 minutes or the amount of aPC with one group of standard substance.By the Application standard product, we have measured the kinetics that the TAFI that caused by IIa-sTM and protein C activate.
The result
Phe376 causes the TAFI that increases to the ratio of PROTEIN C activation rate to the exchange of Ala, this is increased on the lower concentration of substrate more obvious.Although can measure the michaelis-Menten constant (Figure 15, upper picture frame) that TAFI activates, can not draw this type of constant (Figure 15, lower picture frame) that PROTEIN C activates.
The ratio (TAFI/ PROTEIN C) of the TAFI that solulin and Phe376Ala cause at different concentration of substrate and the activation rate of PROTEIN C is illustrated among Figure 16.The high TAFI/PC ratio of Solulin can further improve by the aminoacid exchange of Phe376 to Ala.
Conclusion:
Being increased under related substrates (TAFI or the PROTEIN C) concentration of the TAFI/ PROTEIN C ratio of measuring that can be used as the TAFI preference occurs, and the most remarkable under minimum test concentrations (250nm), this least concentration near TAFI (～75-220nM) and PROTEIN C (～70nM) physiological concentration, and proved conclusively solulin and variant thereof and all be applicable to preferential TAFI and activate.On the other hand, especially, Phe376Ala is proved to be so slow to the activation of PROTEIN C, to such an extent as to must think its can not physiology PROTEIN C concentration (～70nM) PROTEIN C is activated to be enhanced to any perceptible degree.Therefore, can observe the dependent interaction of additional mutations among the Phe376Ala in sizable reduction of TAFI and PROTEIN C activation capability, it still allows TAFI to activate under physiological condition, but has in fact got rid of the PROTEIN C activation.
In addition, these results verifications the single amino acids sudden change, the overall importance of Phe376Ala especially is because not only for the Solulin fragment that extends to EGF6 from EGF1 but also all observe the beneficial effect of this sudden change for the total length soluble thrombomodulin that comprises the glycosylation domain that N-terminal lectin-like domain, 6 EGF domains are connected with O-.
IX. embodiment
Body inner analysis fibrinolysis in haemophiliachemophiliac dog model
The purpose of this research
The reconstruction of Factor IX (fVIII) is the main pillar that treatment suffers from the patient of haemophilia A.Although effective treatment is provided in most of case, a considerable amount of patients have produced anti--fVIII neutralizing antibody (for obtainable auxiliary treatment is selected, still fail so far the problem that overcomes).Other shortcoming of fVIII therapy comprises, complicated dose determination and need intravenous to use and periodic monitoring.
The evidence that day by day increases shows: except not giving birth to the major defect of fVIII with the volume production of abundance, the unrestricted activity of tissue plasminogen activator (tPA) has also been facilitated the patient's condition.Usually, growing and the activation by the fibrinolysis inhibitor (TAFI) of thrombin-can activate of ripe clot is able to the protected tPA of avoiding degraded.This can realize by the complex that is formed by thrombin (producing in the process of vascular lesion closure) and thrombomodulin (the constitutive character protein of endothelial cell membrane).Thrombin/thrombomodulin complex changes into its activity form (TAFIa) with plasminogen TAFI, described activity form is by removing carboxyl terminal lysine from the fibrin of clot and arginine works, with weaken tPA in conjunction with and with the ability of this conformation plasminogen activation.This tPA effect be controlled in the haemophilia A availability imperfection because of thrombin generation proenzyme TAFI not enough and that reduce possibly.Therefore, one of sign of haemophilia A is the too early clot dissolution that is caused by tPA.
External work shows, depends on the concentration of tPA, and Solulin can partially or completely proofread and correct from the too early clot dissolution in the blood plasma of hemophiliac.Whether the purpose of this research is can obtain in vivo confirming by come the outer effect of test body with the good hemophilia dog model of setting up.(Pilot experiment) is definite for preliminary experiment, and the blood plasma of hemophilia Canis familiaris L. is the same with human plasma, allows Solulin to proofread and correct too early clot dissolution, thereby proves that this animal model can be used for this purpose.
The research material
Identity (Identification): Solulin
Physical state: colourless transparent solution, pH7.0
Dissolving buffer: sodium phosphate, 13.4; Potassium chloride, 2.7; Sodium chloride, 137 (all representing with mmol/l); Mannitol, 5%
The content of bottle: contain 3mg Solulin (3mg/ml) among the 1ml
Bottle operation: with the partition (septum) of ethanol bottle, subsequently sterile needle is inserted through partition to extract the volume that needs.The bottle that will have resid vol is stored in the refrigerator.
Storage requirement and time: be stored in the refrigerator of 2-6 ° of C.Resid vol can keep in refrigerator 28 days.Within this period, they can be used for other experiment, but for the independent bottle of each Canis familiaris L. recommendation.
The preparation of drug-delivery preparation
From original Solulin bottle, take out proper volume, replenish with 0.9%NaCl to obtain the administration volume of 3ml.
For each experiment day, the drug-delivery preparation that fresh preparation is final.
Animal: species and keeping
In the Canis familiaris L. that suffers from haemophilia A (haemophilia A Canis familiaris L.) that is characterized by " healing " FVIII gene therapy Canis familiaris L., study.After dog FVIII gene therapy, these Canis familiaris L.s have can not detection level plasma F VIII (＜1%), show that its whole blood clotting time shortens, and after treatment, do not experience other hematostaxis event (normal frequency is about 5 times/years) at least in 3 years.Can think these Canis familiaris L.s display part phenotypic suppression after gene transfer.They have summarized the clinical picture of record in about 10% few hemorrhage serious haemophilia A patient (plasma F VIII＜1%) best.Such feature has also reflected the result of low dosage FVIII prevention scheme.The age of animal and body weight were respectively about 3 years and 10-15kg.Except the hemophilia character, only healthy animal is used for this research.
Experimental technique
Pilot study (pharmacokinetics and external effect)
As first step, in the hemophilia Canis familiaris L. research Solulin pharmacokinetics and to solidifying/Fibrinolytic drug action.About dog plasma and the medicine moving external efficacy data of learning model of generation, group annotates by intravenous, uses Solulin with the volume of 3mL with the dosage of 0.5mg/kg based on obtainable.
According to the result who obtains in first Canis familiaris L., process second Canis familiaris L. in an identical manner or with the Solulin that is fit to dosage.Make decision during after this, when main research beginning or in the further research of carrying out pharmacokinetics.
The preparation of blood sample
Gather two not on the same group blood samples (seeing table) from contralateral leg.
Whole blood collection on citrate buffer (final volume: 1 part 3.13% sodium citrate is mixed with 9 parts of blood), and is processed (preparation that vide infra) immediately.Unless measure with time relationship (such as what indicated for independent aliquot (individual split)) closely, no person fully labelling they and in-80 ℃ of lower storages until further use.
* before had by the 1ml of reject
Gather to be discarded first milliliter in the time of upper table appointment, add citrate, and be divided into 5 aliquots.
The whole blood that 14ml is contained citrate is divided into aliquot 1 (thrombleastography x5ml whole blood) and aliquot 2 (whole blood coagulations; The 2ml whole blood).
With 1500g with centrifugal 15 minutes of remaining volume.The supernatant of gained is divided into 3 parts (aliquot 3, Solulin concentration, 0.5ml blood plasma; Aliquot 4, the TAFI of activation, 0.5ml blood plasma; Aliquot 5, PT/aPTT, 0.5ml blood plasma), and in-80 ℃ of lower storages until use.
Sample 2:
Contralateral leg collected specimens 2 (2ml from sample 1 side; 1ml will be by reject), and add citrate.Sampling time is indicated in as above showing.
This sample is used for measuring thrombin generation potential and needing SC.Importantly avoid contact activation, this means and to use the widest as far as possible pin draw samples (preferably not from butterfly wing not damaged pin (butterfly needle)).
Required blood plasma volume is 0.5ml.The detailed description that has the blood plasma preparation that is used for thrombin generation potential:
The 1st centrifugation step:
ο inputs following parameter of noncentricity:
-braking power (Break power): 9
-spinner velocity: 3790, when r=156mm
-the persistent period: 5 minutes.
ο is with the centrifugal sample of 2500g 5 minutes (for Hettich Rotina35R, this reaches during r=156mm at 3790rpm)
ο is transferred to the blood plasma supernatant in the plastic centrifuge tube of labelling with the disposable pasteur pipet of plastics.Carefully do not draw any cell from buffycoat, stay the blood plasma of 0.5cm at the top of buffycoat.
The 2nd centrifugation step:
ο uses following parameter of noncentricity:
-temperature: 18 ° of C
-centrifugal speed: 11000RPM is when r=156mm
-the persistent period: 10 minutes
ο is with the centrifugal blood plasma of 10000g 10 minutes (for Universal30RF, this reaches during r=156mm at 11000rpm)
ο topples over the content of centrifuge tube in once mobile or is transferred to pipettor in the pipe of labelling (mixing with supernatant to avoid deposit).
ο works as where applicable, and the content of several parts of identical blood plasma is combined in the large plastic tube.
Pharmacokinetics and the evaluation of pesticide effectiveness
Thrombleastography.In containing the whole blood of citrate, carry out thrombleastography.340 μ l whole bloods are added into Haemoscope
In the passage of 5000 (Haemonetics Corp.Braintree, MA), described passage comprises 20 μ L solution (1/15000 dilution (18X) of Innovin, 270mM CaCl
2With 18nM tPA).After fully mixing, press contact pin, continuous monitoring is solidified and fibrinolysis.Be connected in torque on the wire of clot, Haemoscope by measuring through contact pin
5000 allow to measure clotting time, blood coagulation kinetics, clot intensity and clot stability (fibrinolysis).
Whole blood clotting time:By containing the whole blood of citrate in 37 ℃ of lower incubations in teat glass, and the continuous monitoring clot forms, and measures whole blood clotting time.
Pharmacokinetics.Use and confirmed for dog plasma it is that effective ELISA measures plasma concentration.Under being responsible for of sponsor, carry out this research.The result is included in the final report.
The TAFI that activates.(Anal Biochem. (2008), 372 (1): method 32-40) is measured TAFIa to use the people such as Kim.In brief, the plasminogen (F-Pg) that comprises catabolite (QSY-FDP) the combined with fluorescent labelling of covalently bound quencher fibrin partly.When F-Pg was incorporated into QSY-FDP, fluorescence was by quencher.When adding TAFIa, the F-Pg binding site is removed, F-Pg thereby discharge from QSY-FDP, thus cause the increase of fluorescence.The speed that this fluorescence increases is directly proportional with TAFIa concentration.Be doped with the blood plasma of the TAFIa of concentration known by use, produce standard curve, and use it for the TAFIa concentration of measuring plasma sample.
Solidify mensuration.The PT (dPT) and the aPTT that have carried out dilution measure.Dilution PT measures and comprises, 1/5 dilution of test platelet poor plasma.TriniCLOT PT HTF Thromboplastin reagent is added in the blood plasma, carries out subsequently 180 seconds incubations, again the calcification sample.Utilize TriniCLOT Automated APTT reagent to carry out aPTT.Again, carry out 180 seconds incubation, subsequently calcification sample again.Carry out all researchs in Coag-A-Mate MAX (bioM é rieux) automatization's coagulometer.
Interior source thrombase potential.Source thrombase potential in use Calibrated Automated Thrombogram (CAT) algoscopy is measured in 96 orifice plate exometers.The CAT algoscopy is the Patent right method that has of CoagScope B.V. (Maastricht, The Netherlands).
In brief, the concentration in time of the thrombin in the blood plasma is solidified in the measurement of CAT algoscopy.The PPP reagent that utilization contains tissue factor and phospholipid triggers the blood plasma that contains citrate.The another kind of reagent that utilization contains calcium chloride and fluorogenic substrate (FluCa) is calcification blood plasma again.This substrate is changed into fluorogen by thrombin, measures subsequently fluorescence in exometer.In the situation that thrombin aligner (thrombin activity of the amount through calibrating) exists, measure simultaneously another sample of identical blood plasma.The concentration in time that relatively allows to calculate thrombin to the result of these horizontal surveies.
Main research (epidermis Hemorrhagometry (Cuticlebleeding assay))
Number of animals, drugs and dosage level
Study 3 hemophilia Canis familiaris L.s to test Solulin to blood coagulation and Fibrinolytic effect.Use impact with the dosage that the pilot study of describing according to 8.1 times is selected by intravenous bolus injection technique.Group annotates by intravenous, uses Solulin with the dosage of selecting according to 8.1 pilot studies of describing.
Anesthesia and supportive medicine
Premedicate: that monarch 50mg of hydrocortisone 100mg and benzene, IV
Keep: the 1-2% isoflurane
Postoperative pain control: buprenorphine
Time course and method
The process that has shown experiment in the following table.In anesthesia rear 15 minutes, carry out the test of baseline epidermis, be 15 minutes observation period subsequently.After this, nominally anaesthetized rear 30 minutes, based on ABW, the intravenous group that Canis familiaris L. is accepted Solulin annotates.After being exposed to Solulin30 minute, carrying out the second epidermis at the offside pawl and measure.Be 15 minutes observation period subsequently, total anesthesia duration is about 75 minutes.
For the hemorrhage test of epidermis, Canis familiaris L. is placed with lateral position, by pruning all hairs of removing carefully around nail matrix around the base portion of the pawl that is used for the epidermis bleeding time.Silicone grease (Siliconegrease) is applied to pawl below prevention blood flowing fingernail.The superiors that manifest epidermis make to be spring-loaded sliding blade guillotine shears (guillotine clipper) and to cut off fingernail near the carapace ditch.Pawl with animal places on the operation edge of table subsequently, allows blood freely fall from the epidermis that cuts.Record the drop of blood number of each minute in subsequently 15 minutes, be translated into 15 minutes accumulation scoring.After 15 minutes observation, if epidermis is still hemorrhage, then burn damage location by the local application silver nitrate.
Carry out all mensuration by identical experienced veterinary technician.
Results and discussions
Research has the Canis familiaris L. of serious haemophilia A in the body.The solulin concentration of finding effective hundreds of nM in the experiment in vitro shown in the example VI I leads lower, inject first Canis familiaris L. to observe with the dosage of 500 μ g/kg, whether the improvement of the thrombelastogram of observing behind external interpolation solulin can be verified in vivo.Against one's expectation, this dosage is not only invalid, and itself in addition suppress seriously that clot forms and intensity (Figure 17).This acts on uses appearance in rear 30 minutes, and still continues in the time of 2 hours.
As if when finished the same day, the minimizing that clot forms was still obvious, but suppress slowly to go down.Because there is flaw technically in test in 24 hours, so this experiment can be considered to failure.Nonetheless, determine still whether extracted final sample at the 48th hour recovers to observe animal.Even have the trend of the slight improvement that is better than the baseline thrombelastogram as if situation shows really so, but make us some surprised be.Be subjected to the encouragement of this idea, after using solulin, observed other sample in 72 hours, very unexpectedly, confirmed that clot forms and the obvious improvement of intensity.This causes conclusion: than the much lower dosage from vitro study expection be effectively and or even needs, because larger dosage is set up higher plasma concentration, this can worsen blood coagulation character subsequently, and this hypothesis is tested (Figure 18) in other serious hemophilia Canis familiaris L..
Second Canis familiaris L. accepted the solulin (10 μ g/kg) of the much lower dosage of first Canis familiaris L. of ratio.Hypothesis has been proved conclusively in this experiment: higher plasma concentration is invalid improving blood coagulation in nature, yet the concentration that is low to moderate 0.2nM can be improved blood coagulation character.
Marginal data
Fig. 1: clot dissolution curve and the dissolution time of blood plasma (FVIII-DP), the normal plasma (NP) of shortage Factor IX and the FVIII-DP that mixes with NP.The clot dissolution curve that has shown 0 (-), 1 (), 6 (---), 10 (---), 50 (---) and 100% (---) NP.According to solubility curve, be degraded to the residing time of a half of its highest optical density and measure dissolution time by obtaining clot.In illustration, summarized dissolution time, general trend is that dissolution time also increases along with the rising of (with the therefore amount of FVIII) of NP percentage ratio.The NP that adds to dissolution time act on 10%NP the time reach platform.
Fig. 2: comprise the activation of the fibrinolysis inhibitor (TAFI) that the thrombin in the blood plasma of FVIII of different weight percentage can activate: (A) when the blood plasma that will lack FVIII (FVIII-DP) and normal plasma (NP) when mixing, TAFI activates and is enhanced.In FVIII-DP, compare with about 600pM TAFIa among the 100%NP (Δ) with 50%NP (), on Qi Feng (), only record 30pM TAFIa.To carry out in triplicate these experiments, data are expressed as meansigma methods ± SE.The TAFIa potential that TAFIa potential (B), this paper are defined as area under the time course of the activating curve (A) from time that blood coagulation begins to last time point increases along with the increase of NP percentage ratio, and increases to platform when 50%NP.The TAFIa potential of 50%NP and 100%NP similar (respectively 14,100pM min and 16,800pM min) is although the differences in shape of their TAFI activating curve separately is very large.With the logarithm dissolution time curve of logarithm TAFIa potential is shown relation between (Fig. 2 B, illustration) dissolution time (Fig. 1, illustration) and the TAFIa potential, because itself and FVIII Horizontal correlation.As expected, data show comprises in the blood plasma of 0-100%FVIII and has strong positive correlation between the dissolution time and TAFIa potential.
Fig. 3: the thrombinogen that comprises in the blood plasma of FVIII of different weight percentage activates.The time course that has shown the thrombinogen activation of the FVIIIDP that mixes with 0 (), 1 (■), 6 (▲), 10 (zero), 50 () and 100%NP (Δ).Generally speaking, the thrombinogen activation rate increases along with the increase of NP percentage ratio.When 50%NP, thrombinogen activates with two-forty (as by checking that each bar slope of a curve measures) generation, and is presented in 15 minutes and finishes, yet 100%NP has slower thrombinogen activation rate in longer period.
Fig. 4 A-D: (0.25nM is among Fig. 4 A at sTM (0-100nM) and tPA; 0.75nM, among Fig. 4 B; 1.5nM, among Fig. 4 C; And 3nM, among Fig. 4 D) various concentration on, sTM is at normal plasma (NP) and lack the impact of the activation of the fibrinolysis inhibitor (TAFI) that can activate thrombin in the blood plasma (FVIII-DP) of Factor IX.The TAFIa-dependency defective of the prolongation dissolving among the FVIII-DP can be proofreaied and correct by 100nM sTM is added into the blood plasma that comprises 0.25nM tPA.Along with the increase of tPA concentration, in the situation that 100nM sTM exists, in FVIII-DP, only observe the partial correction of dissolving defective.These the experiment in, with potato tubers carboxyl peptide enzyme inhibitor (PTCI) for generation of the situation that does not wherein have functional TAFIa.Therefore, any increase of dissolving as by as shown in the ratio of dissolution time/dissolution time+PTCI, is that TAFIa is dependent.
Fig. 5: the TAFI in (B) activates and the clot dissolution curve at the blood plasma (FVIII-DP) that has 10nM thrombomodulin (●) or do not have normal plasma (NP) in thrombomodulin (zero's) the situation (A) and lack FVIII.Shown the clot dissolution curve (-) followed and without the clot dissolution curve display of sTM for reference to (---).To carry out in triplicate these experiments, data are expressed as meansigma methods ± SE.
Fig. 6: thrombin is to the combination of thrombomodulin.By with 1.54 μ M thrombomodulin titration 1.5ml in the same solution by 20mM TrisHCl, 150mM NaCl, 5.0mMCa
2+, the solution that the thrombin among the 0.01%Tween80 (20nM) and DAPA (20nM) form is measured thrombin to the combination of thrombomodulin.Measure fluorescence intensity (λ
Ex=280nm, λ
Em=545nm).
Fig. 7: Point mutont is active with the relative cofactor during PROTEIN C activates at TAFI.With the point mutation of alanine scanning mutagenesis for the preparation of soluble thrombomodulin.For TAFI (solid stick) and PROTEIN C (hacures stick), shown that the activation rate of PROTEIN C and TAFI is (with respect to utilizing mutant TM
EThe activation rate that M388L obtains).
The sudden change that encircles between the domain between Fig. 8: EGF4 and the EGF5.For each mutant, in escherichia coli, make up 3 independent plasmids.Preparation Shockate, it is active to utilize the APC algoscopy to measure its cofactor, and uses Western trace (not shown) analytic sample.Activity value is the meansigma methods from 3 independent clones.Picture frame A, Gln
387On the replacement mutation body; Picture frame B, the displacement on the Met386; Figure C, the replacement mutation body on the Phe389; Picture frame D, the disappearance between domain in the ring and alanine insert.The activity that has shown the measurement of the colibacillary shockate that the control plasmid pSelect that uses by oneself (not having the TM insert) transforms.About other detailed content, referring to the people such as Clarke (J.Biol.Chem.1993; 268:6309-6315).
Fig. 9: thrombomodulin is (at Mosnier and Bouma, Arterioscler.Thomb.Vasc.Biol.2006; Modify behind the 26:2445-2453) short-and the sketch map of fibrinolysis effect.The increase of blood-clot lysis time can be given the credit to the stimulation that TAFI activates on low TM concentration, and has illustrated that the fibrinolysis of TM is active.On the rabbit lung TM of higher concentration, blood-clot lysis time reduces because of the inhibition that the activation of PROTEIN C and TAFI activate; The fibrinolytic that causes of rabbit lung TM (solid line) has been described.Notice, be higher than in the situation of 15nM that the fibrinolytic that causes of rabbit lung TM surpasses the fibrinolysis activity, thereby causes totally causing fibrinolysis activity.On the contrary, soluble T M analog only shows fibrinolysis effect (dotted line).
Figure 10: the relation between the TAFIa-dependency multiple amplification of clot dissolution and the Solulin that adds.The dose dependent increase of blood-clot lysis time can be given the credit to the stimulation that TAFI activates, and has illustrated that the fibrinolysis of TM is active.
Figure 11 A: the Solulin dependency from clot outward appearance in the whole blood of the dog with haemophilia A changes (as measuring by thrombleastography).
Figure 11 B: the Solulin dependency from clot hardness in the whole blood of the dog with haemophilia A increases.Utilize thrombleastography the clot dissolution area under curve to be measured as the function of Solulin (sTM) concentration.
Figure 11 C: the Solulin dependency that dissolves in the whole blood from the dog with haemophilia A prolongs.Utilize thrombleastography blood-clot lysis time to be measured as the function of Solulin (sTM) concentration.
Figure 12 A: from have haemophilia A and inhibition anti--the Solulin dependency that dissolves in the whole blood of the dog of Factor IX antibody prolongs (measuring as utilizing thrombleastography).
Figure 12 B: inhibition anti--situation that Factor IX antibody exists under from the whole blood of the dog with haemophilia A in the Solulin dependency of clot hardness increase.Utilize thrombleastography the clot dissolution area under curve to be measured as the function of Solulin (sTM) concentration.
Figure 13: the figure of display list 1: be used for the general introduction of the data of design of graphics 4, be included in absolute dissolution time in the situation that PTCI exists (so that can measure under each condition dissolution time).In all cases, represent dissolution time with respect to the dissolution time that obtains in the situation about existing at TAFIa inhibitor PTCI.TAFI, the fibrinolysis that thrombin can activate suppresses; PTCI, the potato tubers carboxyl peptide enzyme inhibitor.
Figure 14: the figure of display list 2: TM
EThe toluene-sodium-sulfonchloramide oxidation of mutation site-specific body analog (Sf9).Result after toluene-sodium-sulfonchloramide processed is expressed as the percentage ratio of the activity after the control treatment.* meansigma methods and the deviation from average (deviation from the mean) measured in duplicate.
Figure 15: the activation rate of the TAFI that under the concentration of substrate that increases progressively, is produced by solulin and solulin mutant Phe376Ala (both existing with 25nM) (upper picture frame) and PROTEIN C (lower picture frame).
Figure 16: the TAFI of the Solulin (black stick) that compares with the Solulin analog (light grey stick) with Phe376Ala sudden change activates the ratio that PROTEIN C is activated, such as what measure in the TM concentration of 0.25,0.5,0.75,1,1.5 and 2 μ M.Rightmost stick shows the ratio with the combination of the measured value under all concentration.
Figure 17: from the thrombleastography of the blood sample of the Canis familiaris L. with serious haemophilia A.After with the dosage intravenous injection solulin of 500 μ g/kg on different time collected specimens.
Figure 18: from the bell concentration dependent of the improvement of clot formation and stability in the blood with serious haemophiliachemophiliac Canis familiaris L..With the dosage injection Solulin of 10 μ g/kg, and in the mensuration of the time of appointment collection plasma sample for thrombelastogram.On each thrombelastogram, shown the plasma concentration of sampling time and solulin.
Figure 19: about the general introduction of TAFI-specificity thrombomodulin analog.
The explanatory note of table
Table 1: be used for the general introduction of the data of design of graphics 4, be included in absolute dissolution time in the situation that PTCI exists (so that can measure under each condition dissolution time).In all cases, represent dissolution time with respect to the dissolution time that obtains in the situation about existing at TAFIa inhibitor PTCI.TAFI, the fibrinolysis inhibitor that thrombin can activate; PTCI, the potato tubers carboxyl peptide enzyme inhibitor.
Table 2:TM
EThe toluene-sodium-sulfonchloramide oxidation of mutation site-specific body analog (Sf9).
Result after toluene-sodium-sulfonchloramide processed is expressed as the percentage ratio of the activity after the control treatment.* meansigma methods and the deviation from average measured in duplicate.
Claims (34)
1. thrombomodulin analog, it is after thrombin is combined, with TM
EM338L compares, and has the cofactor activity that reduces, and described analog has
A) according to the aminoacid sequence of SEQ ID NO2, or
B) according to the aminoacid sequence of SEQ ID NO3, or
C) according to the aminoacid sequence of SEQ ID NO4, or
D) with according to SEQ ID NO2, the aminoacid sequence of SEQ ID:3 or SEQ ID NO:4 has at least 90%, more preferably at least 95%, the aminoacid sequence of at least 98% homogeneity most preferably, or
E) thrombomodulin fragment, it is comprised of following basically: SEQ ID NO:2,6 EGF sample repetitive structure territories (such as the amino acid position 227 to 462 of numbering among the SEQ ID NO:1) of SEQ ID NO:3 or SEQ ID NO:4, SEQ ID NO:2,3 to EGF-sample repetitive structure territories 6, EGF-sample repetitive structure territory (such as the amino acid position 307 to 462 of numbering among the SEQ ID NO:1) or the SEQ ID NO:2 of SEQ ID NO:3 or SEQID NO:4, the c in the EGF-sample repetitive structure territory 3 of SEQ ID NO:3 or SEQID NO:4 encircles to EGF-sample repetitive structure territory 6 (such as the amino acid position 333 to 462 of numbering among the SEQID NO:1)
Wherein the phenylalanine on the position 376 (as according to numbering among the SEQ ID NO:1) is lacked or is replaced by glycine, alanine, leucine, isoleucine.
2. the thrombomodulin of claim 1, it also (numbers) disappearance or the displacement that comprises glutamine residue in such as SEQ ID NO:1 in position 387, wherein said displacement is preferably by Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys displacement.
3. claim 1 or 2 thrombomodulin, it also (numbers) disappearance or the displacement that comprises methionine residues in such as SEQ IDNO in position 388, wherein methionine residues is preferably by Gln, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys displacement.
4. each thrombomodulin of the claims, it also (numbers) disappearance or the displacement that comprises phenylalanine residue in such as SEQ ID NO:1 in position 389, wherein phenylalanine is preferably by Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro displacement.
5. each thrombomodulin of claim, wherein said thrombomodulin comprise one or more first and second amino acid modified such as what describe in the table 4.
6. each thrombomodulin of claim, wherein said thrombomodulin comprise one or more first, second, and third amino acid modified such as what describe in the table 5.
7. the thrombomodulin analog is for the preparation of the purposes of medicament, and described medicament is used for the treatment of the coagulopathy with hyperfibrinolysis, wherein
A) each of TM analog such as above-mentioned claim defines, or
B) described TM analog is characterised in that, shows the fibrinolysis effect in the treatment effective dose.
8. the purposes of claim 7, wherein said thrombomodulin analog shows one or more following features:
(i) compare the binding affinity to thrombin that reduces, and/or k with rabbit lung thrombomodulin
DValue surpasses the binding affinity to thrombin of 0.2nM; And/or
(ii) compare with the cofactor activity of TM analog TMEM388L, the cofactor that reduces is active,
(iii) with TM analog TM
EM388L compares, and the TAF I Activation Activity of increase is to the ratio of cofactor activity.
9. claim 7 or 8 purposes, wherein said coagulopathy with hyperfibrinolysis is selected from following disease: the genetic disorder of haemophilia A, haemophilia B, C type hemophilia, von Willebrand (vWD), Of Acquired von Willebrand Disease, factor X deficiency, parahemophilia, factor I, II, V or VII, the bleeding disorder or the acquired blood coagulation that cause because of CAC lack.
10. each purposes of the claims, wherein said thrombomodulin analog is used for the treatment of one or more following bleeding episodes that are selected from: intracranial hemorrhage or other CNS is hemorrhage, hemorrhage behind the joint, the thin blood vessel of microtriche, muscle, gastrointestinal tract, respiratory tract, peritoneum in space or the soft tissue.
11. the purposes of each of the claims, wherein said patient has anti--Factor IX antibody.
12. the purposes of each of the claims, wherein said patient uses Factor IX, the Factor IX molecule of the disappearance B domain of preferred recombinant factor VIII or restructuring, and more preferably octocog alfa or moroctocog alfa are treated.
13. the thrombomodulin analog of each of the claims is used for the treatment of the purposes of the coagulopathy with hyperfibrinolysis, it is characterized in that described thrombomodulin analog and Factor IX, the Factor IX molecule of the disappearance B domain of preferred recombinant factor VIII, restructuring, more preferably octocog alfa or moroctocog alfa combined administration.
14. the purposes of each of the claims, wherein said patient is with Factor IX, preferred recombinant factor VIII, more preferably octocog alfa is treated.
15. the purposes of each of the claims is wherein used described thrombomodulin analog when bleeding episodes.
16. the purposes of each of the claims was wherein used described thrombomodulin analog before the bleeding risk that increases is for example performed the operation or had tooth pulled out.
17. the purposes of each of the claims wherein is difficult to use described thrombomodulin analog with the patient of blood/plasma infusion or thrombin replacement therapy.
18. the purposes of each of the claims is wherein preferred in the total time that was shorter than for 1 thoughtful 4 weeks, once a day, per 2 days once, or per 3,4,5,6 or 7 days once, more preferably in the mode of chronic administration, use described thrombomodulin analog with a plurality of dosage.
19. the purposes of each of the claims, the mode of wherein using with parenteral preferably in the mode of intravenous or subcutaneous administration, is used described thrombomodulin analog.
20. the purposes of each of the claims, wherein said thrombomodulin analog are soluble T M analog.
21. the purposes of claim 20, wherein said thrombomodulin analog behaviour soluble T M analog.
22. the purposes of each of the claims, wherein said thrombomodulin analog comprises at least one domain that is selected from EGF3, EGF4, EGF5, EGF6, preferably comprises fragment EGF3-EGF6, more preferably comprises EGF domain 1-6.
23. the purposes of each of the claims, wherein said thrombomodulin analog is comprised of EGF domain EGF1 to EGF6, more preferably is comprised of EGF domain EGF3 to EGF6.
24. the purposes of each of the claims, wherein said thrombomodulin analog has the aminoacid sequence of the aminoacid sequence (describing among SEQ ID NO:1 or the SEQ IDNO:3) corresponding to ripe thrombomodulin, and comprises one or more in the following modification:
A) removal of amino acid/11-3;
b)M388L；
c)R456G；
d)H457Q；
E) S474A, and the termination on P490.
25. having, the purposes of each of the claims, wherein said thrombomodulin analog comprise the aminoacid sequence that has the sequence of at least 85% or at least 90% or 95% sequence homogeneity with SEQ ID NO:2.
26. the purposes of each of the claims, wherein said thrombomodulin analog have such as upper/lower positions corresponding to native sequences (according to SEQ ID NO:1 or SEQ ID NO:3) one or more amino acid modified:
aa)
349Asp;
bb)
355Asn;
ac)
357Glu;
ad)
358Tyr;
ae)
359Gln;
af)
361Gln;
ag)
363Leu;
ah)
364Asn;
ai)
368Tyr;
aj)
371Val;
ak)
374Glu;
al)
376Phe;
am)
384His;
an)
385Arg;
ba)
387Gln;
bb)
389Phe;
bc)
398Asp;
bd)
400Asp;
be)
402Asn;
bf)
403Thr;
bg)
408Glu;
bh)
411Glu;
bi)
413Tyr;
bj)
414Ile;
bk)
415Leu;
bl)
416Asp;
bm)
417Asp;
bn)
420Ile;
bo)
423Asp;
bp)
424Ile;
bq)
425Asp;
br)
426Glu;
ca)
428Glu;
cb)
429Asp;
cc)
432Phe;
cd)
434Ser;
ce)
436Val;
cf)
438His;
cg)
439Asp;
ch)
440Leu;
ci)
443Thr;
cj)
444Phe;
ck)
445Glu;
cl)
456Arg;
Cm)
458Ile; Or
cn)
461Asp。
27. the purposes of each of the claims, wherein said thrombomodulin analog has the modification of phenylalanine in the position 376 according to SEQ ID NO:1 or SEQ ID NO:3, it is preferably replaced by aliphatic amino acid, more preferably by glycine, alanine, valine, leucine or isoleucine displacement, most preferably replaced by alanine.
28. the purposes of each of the claims, wherein said thrombomodulin analog have according to the one or more amino acid whose modification in the following aminoacid of SEQ ID NO:1 or SEQ ID NO:3:
a)
387Gln;
b)
388Met;
b)
389Phe,
Wherein said aminoacid is lacked, by one or more extra aminoacid insertion, or preferably replaced.
29. the purposes of each of the claims, wherein said thrombomodulin analog uses with its oxidised form, preferably by toluene-sodium-sulfonchloramide, hydrogen peroxide or sodium periodate oxidation.
30. the purposes of claim 19, wherein the one or more methionine residues in the TM analog are oxidized, and the methionine residues on the optimum position 388 (according to SEQ ID NO:1 or SEQ ID NO:3) is oxidized.
31. screening is applicable to treat the method for thrombomodulin analog of the coagulopathy with hyperfibrinolysis, wherein said thrombomodulin shows one or more in the following feature:
(i) binding affinity to thrombin that reduces,
(ii) cofactor that reduces is active,
(iii) the TAFI Activation Activity that strengthens,
Described method comprises step:
A) produce one or more amino acid replacements of thrombomodulin sequence (SEQ ID NO:1 or SEQ ID NO:3), preferably the one or more amino acid replacements on the listed amino acid position in claim 15;
B) one or more in the following feature, with analog and the contrast molecule modified, preferred rabbit lung TM or soluble human TM analog are compared:
Ba) to the binding affinity (KD value) of thrombin;
Bb) cofactor is active;
Bc) TAFI Activation Activity or TAFIa potential;
Bd) ratio of TAFI Activation Activity and cofactor activity;
Be) effect of protein oxidation;
Bf) in external test in time on the impact of clot dissolution; Or
Bg) effect in the blood coagulation relevant animal models.
32. treatment has the method for coagulopathy of hyperfibrinolysis, it comprise the administering therapeutic effective dose according to claim 1 to 20 each thrombomodulin analog.
33. treatment has the method for coagulopathy of hyperfibrinolysis, it comprise the body weight of using 0.75 μ g/kg to 140 μ g/kg receptor to be treated dosage according to claim 1 to 20 each thrombomodulin analog.
34. a pharmaceutical composition, its dosage with 0.75 μ g/kg to 140 μ g/kg experimenter's to be treated body weight comprise be used for the treatment of coagulopathy with hyperfibrinolysis according to claim 1 to 20 each thrombomodulin analog.
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| EPPCT/EP2010/003541 | 2010-06-14 | ||
| PCT/EP2010/003541 WO2010142461A2 (en) | 2009-06-12 | 2010-06-14 | Treatment of coagulopathy with hyperfibrinolysis |
| PCT/EP2010/007632 WO2011157283A1 (en) | 2010-06-14 | 2010-12-15 | Treatment of coagulopathy with hyperfibrinolysis |
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| JP (1) | JP2013531651A (en) |
| CN (1) | CN103037893A (en) |
| AU (1) | AU2010355558A1 (en) |
| BR (1) | BR112012031920A2 (en) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083357A (en) * | 2017-06-16 | 2017-08-22 | 四川农业大学 | A kind of method of efficient Fiber differentiation mesenchymal stem cells MSCs |
| CN107677834A (en) * | 2017-09-25 | 2018-02-09 | 辽宁迈迪生物科技股份有限公司 | The detection method of TAFIa contents, the kit for detecting TAFIa contents and the application of the two |
| CN111961137A (en) * | 2020-08-27 | 2020-11-20 | 南开大学 | Fusion protein for simulating endothelial cell function and application thereof |
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| US9334331B2 (en) | 2010-11-17 | 2016-05-10 | Chugai Seiyaku Kabushiki Kaisha | Bispecific antibodies |
| TWI831106B (en) * | 2014-06-20 | 2024-02-01 | 日商中外製藥股份有限公司 | Pharmaceutical compositions for the prevention and/or treatment of diseases that develop and/or progress due to reduced or deficient activity of coagulation factor VIII and/or activated coagulation factor VIII |
| BR112019003989A2 (en) | 2016-09-06 | 2019-05-28 | Chugai Pharmaceutical Co Ltd | methods of using a bispecific antibody that recognizes coagulation factor ix and / or activated coagulation factor ix and coagulation factor x and / or coagulation factor x activated |
| JP2019031486A (en) * | 2017-08-04 | 2019-02-28 | 公立大学法人福島県立医科大学 | Novel polypeptide and application thereof |
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| WO1993025675A1 (en) * | 1992-06-10 | 1993-12-23 | Schering Aktiengesellschaft | Mutants of the epidermal growth factor domains of human thrombomodulin |
| WO2008073884A2 (en) * | 2006-12-12 | 2008-06-19 | Eli Lilly And Company | Treating acute renal failure with soluble thrombomodulin variants |
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| ATE180496T1 (en) | 1987-01-08 | 1999-06-15 | Asahi Chemical Ind | PEPTIDE TO ACCELERATE THE ACTION OF PROTEIN-C WITH THROMBIN |
| US5256770A (en) | 1990-04-09 | 1993-10-26 | Schering Ag | Oxidation resistant thrombomodulin analogs |
| EP0544826B1 (en) | 1990-08-15 | 2002-01-02 | Paion GmbH | Superior thrombomodulin analogs for pharmaceutical use |
| PT641215E (en) | 1992-02-05 | 2004-09-30 | Paion Gmbh | PROTEASE RESISTANT TROMBOMODULIN ANALOGS |
| EP1383885A4 (en) | 2001-04-04 | 2005-11-30 | American Diagnostica Inc | Method of preparation of stabilized thrombin-activatable fibrinolysis inhibitor (tafi) and methods of use thereof |
| WO2010142309A1 (en) * | 2009-06-12 | 2010-12-16 | Paion Deutschland Gmbh | Treatment of coagulopathy with hyperfibrinolysis |
-
2010
- 2010-12-15 BR BR112012031920A patent/BR112012031920A2/en not_active IP Right Cessation
- 2010-12-15 WO PCT/EP2010/007632 patent/WO2011157283A1/en active Application Filing
- 2010-12-15 JP JP2013514555A patent/JP2013531651A/en active Pending
- 2010-12-15 AU AU2010355558A patent/AU2010355558A1/en not_active Abandoned
- 2010-12-15 CA CA2802072A patent/CA2802072A1/en not_active Abandoned
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993025675A1 (en) * | 1992-06-10 | 1993-12-23 | Schering Aktiengesellschaft | Mutants of the epidermal growth factor domains of human thrombomodulin |
| WO2008073884A2 (en) * | 2006-12-12 | 2008-06-19 | Eli Lilly And Company | Treating acute renal failure with soluble thrombomodulin variants |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083357A (en) * | 2017-06-16 | 2017-08-22 | 四川农业大学 | A kind of method of efficient Fiber differentiation mesenchymal stem cells MSCs |
| CN107083357B (en) * | 2017-06-16 | 2021-04-02 | 四川农业大学 | Method for inducing and culturing mesenchymal stem cells |
| CN107677834A (en) * | 2017-09-25 | 2018-02-09 | 辽宁迈迪生物科技股份有限公司 | The detection method of TAFIa contents, the kit for detecting TAFIa contents and the application of the two |
| CN107677834B (en) * | 2017-09-25 | 2019-07-09 | 辽宁迈迪生物科技股份有限公司 | The application of the detection method of TAFIa content, the kit for detecting TAFIa content and the two |
| CN111961137A (en) * | 2020-08-27 | 2020-11-20 | 南开大学 | Fusion protein for simulating endothelial cell function and application thereof |
| CN111961137B (en) * | 2020-08-27 | 2023-09-05 | 南开大学 | Fusion protein with bionic endothelial cell function and application thereof |
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| JP2013531651A (en) | 2013-08-08 |
| BR112012031920A2 (en) | 2017-05-16 |
| WO2011157283A1 (en) | 2011-12-22 |
| AU2010355558A1 (en) | 2013-01-24 |
| CA2802072A1 (en) | 2011-12-22 |
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