AU2010355558A1 - Treatment of coagulopathy with hyperfibrinolysis - Google Patents

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

WO 2011/157283 PCT/EP2010/007632 "Treatment of coaqulopathy with hyperfibrinolysis" The invention relates to the field of coagulopathy with hyperfibrinolysis. More particularly, this invention relates to the treatment of haemophila diseases such as haemophilia A or haemophilia B. The present invention claims priority of the PCT application PCT/EP2009/004218 which is hereby fully incorporated in terms of disclosure. Haemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation, which is used to stop bleeding when a blood vessel is broken. Haemophilia A, the most common form, results from a mutation in the gene for Factor Vill; haemophilia B, also known as Christmas disease, results from a mutation in the gene for Factor IX. Haemophilia B, like haemophilia A, is X-linked and accounts for approximately 12% of haemophilia cases. The symptoms are identical to those of haemophilia A: excessive bleeding upon injury; and spontaneous bleeding, especially into weight-bearing joints, soft tissues, and mucous membranes. Repeated bleeding into joints results in haemarthrosis, causing painful crippling arthropathy that often necessitates joint replacement. Haematomas in soft tissues can result in pseudo tumors composed of necrotic coagulated blood; they can obstruct, compress, or rupture into adjacent organs and can lead to infection. Once formed the haematomas are difficult to treat, even with surgery. Recovery of nerves after compression is poor, resulting in palsy. Those bleeding episodes that involve the gastrointestinal tract, CONFIRMATION COPY WO 2011/157283 PCT/EP2010/007632 -2 central nervous system, or airway/retroperitoneal space can lead to death if not detected. Intracranial bleeding is a major cause of death in haemophiliacs. There are estimated to be 100,000 cases of congenital haemophilia in the United States. Of these, approximately 20,000 are cases of haemophilia B, the blood of such patients being either totally devoid of Factor IX or seriously deficient in plasma Factor IX component. The disease therefore exists in varying degrees of severity, requiring therapy anywhere from every week up to once or twice a year. The completely deficient cases require replacement therapy once every week; the partially deficient cases require therapy only when bleeding episodes occur, which may be as seldom as once a year. The bleeding episodes in congenital, partially deficient cases are generally caused by a temporarily acquired susceptibility rather than by injury alone. Intravenous injection of a sufficiently large amount of fresh plasma, or an equivalent amount of fresh blood temporarily corrects the defect of a deficient subject. The beneficial effect often lasts for two or three weeks, although the coagulation defect as measured by in vitro tests on the patient's blood appears improved for only two or three days. Such therapy with fresh plasma or fresh blood is effective but it has several serious drawbacks: (1) it requires ready availability of a large amount of fresh plasma; (2) requires hospitalization for the administration of the plasma; (3) a great many of the patients become sensitized to repeated blood or plasma infusions and ultimately encounter fatal transfusion reactions; (4) at best plasma can only partially alleviate the deficiency; and (5) prolonged treatment or surgery is not possible because the large amounts of blood or plasma which are required will cause acute and fatal edema. An improved therapy includes intravenous replacement therapy with Factor VIII or Factor IX concentrates. However, also this therapy suffers from several disadvantages: (1) when treating major bleeding episodes tissue damage remains even after prompt detection and treatment; (2) a great many of the patients become refractory to the coagulation factors and develop inhibitory antibodies against the coagulation factors (so called haemophilia with inhibitors); (3) despite the improved virus inactivation methods there is still an increased risk of contamination with fatal viruses such as HIV and hepatitis C (it is estimated that more than 50% of the haemophilia population, over 10,000 people, contracted HIV from the tainted blood supply in the USA); (4), the isolated and especially the recombinant clotting factors are very expensive and not generally available in the developing world. A treatment or prevention of bleeding beyond a replacement therapy is a challenge because bleeding in haemophilia is a complex pathophysiological process that may be attributable to triple defects: (1) a reduced thrombin generation via the extrinsic pathway at low tissue factor concentration, (2) a reduced secondary burst of thrombin WO 2011/157283 PCT/EP2010/007632 -3 generation via the intrinsic pathway, and (3) a defective downregulation of the fibrinolytic system by the intrinsic pathway. The fact that a reduced thrombin generation results in a reduced clotting propensity and therefore an increased risk of bleeding is generally accepted. However, work in the past decade indicates that also a defective downregulation of the fibrinolysis may play a role in haemophilia. As a result haemophila can be also classified as a coagulopathy with hyperfibrinolysis. A recent publication supports this assumption by showing in vitro that when a clot is formed in Factor VillI depleted plasma (FVIII-DP) and supplemented with tissue plasminogen activator tPA, fibrinolysis is not adequately downregulated and as a result the clot lyses prematurely (Broze and Higuchi, Blood 1996, 88: 3815-3823; Mosnier et al.; Thromb. Haemost. 2001, 86: 1035-1039). Furthermore, it could be shown that this "premature lysis" is due to reduced or absent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) (Broze and Higuchi, 1996) and that in FVIII-DP, an activated TAFI containing mixture increases clot lysis time. It was concluded that stabilized TAFI can be used for the treatment of haemophilia (WO02/099098). TAFI plays a crucial role in the downregulation of fibrinolysis, which is required for formation of stable clots. TAFI also known as plasma procarboxypeptidase B2 or procarboxypeptidase U is a plasma zymogen that, when exposed to the thrombin thrombomodulin complex, is converted by proteolysis at Arg 9 2 to a basic carboxypeptidase (TAFla or activated TAFI) that inhibits fibrinolysis. It potently attenuates fibrinolysis by removing the C-terminal lysine and arginine residues from fibrin which are important for the binding and activation of plasminogen. As discussed above thrombomodulin (TM) in complex with thrombin is responsible for the TAFI activation. Thrombomodulin is a membrane protein that acts as a thrombin receptor on the endothelial cells lining the blood vessels. Thrombin is a central enzyme in the coagulation cascade, which converts fibrinogen to fibrin, the matrix clots are made of. Initially, a local injury leads to the generation of small amounts of thrombin from its inactive precursor prothrombin. Thrombin, in turn, activates platelets and, second, certain coagulation factors including factors V and Vill. The latter action gives rise to the so-called thrombin burst, a massive activation of additional prothrombin molecules, which finally results in the formation of a stable clot. When bound to thrombomodulin, however, the activity of thrombin is changed in direc tion: A major feature of the thrombin-thrombomodulin complex is its ability to activate protein C, which then downregulates the coagulation cascade by proteolytically inacti vating the essential cofactors Factor Va and Factor Villa (Esmon et al., Ann. N. Y.

WO 2011/157283 PCT/EP2010/007632 -4 Acad. Sci. (1991), 614:30-43), thus affording anticoagulant activity. The thrombin thrombomodulin complex is also able to activate the thrombin-activatable fibrinolysis inhibitor (TAFI), which then antagonizes fibrinolysis (see above). Mature human TM is composed of a single polypeptide chain of 559 residues and consists of five domains: an aminoterminal "lectin-like" domain, an "6 EGF-like repeat domain" comprising six epidermal growth factor (EGF)-like repeats, an 0- glycosylation domain, the transmembrane domain and a cytoplasmic domain with following localisation (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): Approx. amino acid position Domain -18--i Signal sequence 1 - 226 N-terminal domain (lectin-like) 227-462 6 EGF-like repeat domains 307 - 345 EGF-like repeat domain 3 333 - 344 c-loop of EGF-like repeat domain 3 347 - 386 EGF-like repeat domain 4 387 - 422 EGF-like repeat domain 5 423 - 462 EGF-like repeat domain 6 463 - 497 0-linked Glycosylation 498 - 521 Transmenbrane domain 522 - 557 Cytoplasmic domain Table 3: domain structure of thrombomodulin (numbered according to SEQ ID NO:1). Various structure-function studies using proteolytic fragments of rabbit TM or deletion mutants of recombinant human TM have localized its activity to the last three EGF-like repeats. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that activates C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. As discussed above the replacement therapy for treating coagulation disorders such as haemophilia does not meet the medical needs. Importantly, no drug besides the coagulation factors used for the replacement therapy is available which can prevent or treat haemophilia patients. Thus, despite the long-standing need for the development of therapies to prevent or treat coagulopathy with hyperfibrinolysis, in particular haemophilia, progress has been slow, and therapeutics that are safe and effective are still missing.

WO 2011/157283 PCT/EP2010/007632 Thus, it is the objective of the present invention to provide novel means for the treatment of coagulopathy with hyperfibrinolysis. This objective is solved by providing a medicament for the treatment of coagulopathy with hyperfibrinolysis in a mammal, in particular in humans, comprising a thrombomodulin analogue exhibiting at therapeutically effective dosages an antifibrinolytic effect. Particularly suitable pharmaceutically active proteins and peptides are also provided which can be used according to the invention. This novel approach is based on the surprising findings that a thrombomodulin can be modified in a way that it exhibits an antifibrinolytic activity that prevail its profibrinolytic activity even at high plasma concentrations, in particular at concentrations of more than 15 nM, in particular more than 20, 30, 40 or 50 nM (at least up to 100 nM). Hence these TM analogues exhibit an antifibrinolytic effect, and are thus suitable for the use according to the invention. In most favourable embodiments the TM analogues can have a prevailing antifibrinolytic activity in concentration even up to 200 nM or more, even more preferred up to 300 nM or 500 nM. This antifibrinolytic effect was shown in plasma from haemophilia patients (which is depleted for Factor Vill; FVIII-DP) and in whole blood or plasma of dogs with haemophilia ATherewith it was demonstrated that such a thrombomodulin analogue can be used as a therapeutic. So far the therapeutic use of thrombomodulin for the treatment of haemophilia was not regarded as a real option because it was known from rabbit lung thrombomodulin (rITM) that it always has both anti- and profibrinolytic activities even at rather low concentrations (see Mosnier and Bouma; Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2445 - 2453; especially Figure 5). At plasma concentrations of less than 15 nM rITM increased clot lysis time whereas at plasma concentrations greater than 15 nM a marked decrease in lysis time was demonstrated (Mosnier et al., 2001, Mosnier and Bouma, 2006) with a profibrinolytic effect as the final result. This profibrinolytic effect at higher concentrations prohibits any therapeutical use in haemophilia since a potential overdosing or individual variabilites in susceptibility would fatally aggravate, prolong or even cause bleeding events. According to the invention various options exist which lead to TM analogues that exhibit an antifibrinolytic effect and thus are suitable for the treatment according to the invention.

WO 2011/157283 PCT/EP2010/007632 -6 In one embodiment thrombomodulin analogues can be used with reduced binding affinity to thrombin. Consequently they can prolong the clot lysis in normal plasma and FVIII-DP, e.g. up to 100 nM (Figure 4) or up to 500 nM (Figure 10) The importance of these findings is that these thrombomodulin analogues exhibit an antifibrinolytic effect without a deleterious profibrinolytic effect even at high concentrations. This concentration exceeds by far the therapeutically effective dosages. Therefore the TM analogues enable the treatment of coagulopathy with hyperfibrinolysis. Without bound to this theory the inventors have shown that this therapeutic potential of the TM analogues can be explained by the fact that they show a markedly reduced affinity towards thrombin. This was shown by Bajzar et al. (J. Biol. Chem 1996; 271: 16603-16608) who found a KD value of 23 nM in contrast to the KD value of 0.2 nM observed for the binding between thrombin and rabbit lung thrombomodulin (Esmon et al., Ann. NY. Acad. Sci. 1986, 485: 215-220). Hence, according to one embodiment of the invention thrombomodulin analogues can be used for the treatment of coagulopathy with hyperfibrinolysis which have a reduced binding affinity towards thrombin compared to the rabbit lung thrombomodulin. In particular, a thrombomodulin analogue can be used which exhibits a KD for thrombin binding of more than 0.2 nM, preferably more than 1 nM, 2 nM, 4 nM, 5 nM, 7.5 nM, 10 nM, 12.5 nM, 15nM, 17.5 nM, 20 nM, 22.5nM, or 25 nM, and more preferably a KD value in a range between 10 and 30 nM or 10 and 100 nM or more. In specific embodiments TM analogues are used with a KD for thrombin binding of about 50, 60 or 70 nM. In a further embodiment of the invention, the reduced profibrinolytic activity of a thrombomodulin analogue can be due to a reduced ability to activate protein C (so called "cofactor activity"). Since the protein C activation results in an upregulation of fibrinolysis (Mosnier et al., 2001) a reduced cofactor activity will prolong the clot lysis time. The person skilled in art knows several strategies to reduce the cofactor activity of thrombomodulin, such as e.g. changes in the glycosylation, secondary or tertiary structure of the protein or preferably changes in the primary structure e.g. by mutation of one or more amino acids. In a yet another embodiment TM analogues can be used which have a reduced cofactor activity compared to the thrombomodulin analogue TMEM388L, where TME denotes to an analogue consisting of the six EGF domains only.

WO 2011/157283 PCT/EP2010/007632 -7 According to the invention a thrombomodulin analogue can also be used which has an increased ability to activate TAFI (so called "TAFI activation activity") since TAFI activation results in a downregulation of fibrinolysis (Mosnier and Bouma, 2006). For the person skilled in art there are several strategies to increase the TAFI activation activity by thrombomodulin such as changes in the glycosylation, secondary or tertiary structure of the protein or preferably changes in the primary structure e.g. by mutation of one or more amino acids. Particularly, this invention also provides for a thrombomodulin analogue which has a significantly increased ratio of TAFI activation activity to cofactor activity compared to the thrombomodulin analogue TMEM388L. Notably, according to the invention the TM analogue used for the treatment of coagulopathy has one or more of the above described features, namely: (i) a binding affinity towards thrombin that is decreased compared to the rabbit lung thrombomodulin, and/or a binding affinity towards thrombin with a kD value of more than 0.2 nM; (ii) a reduced cofactor activity compared to cofactor activity of the TM analogue TMEM388L, or (iii) an increased ratio of TAFI activation activity to cofactor activity as compared to the TM analogue TMEM388L. In an embodiment of the invention, thrombomodulin can be used to treat human patients with any coagulopathy that occurs with a prominently or even slightly reduced fibrinolysis compared to normal subjects. In particular the following diseases can be treated with the thrombomodulin analogue: haemophilia A, haemophilia B, haemophilia C, von Willebrandt disease (vWD),. acquired von Willebrandt disease, Factor X deficiency, parahaemophilia, hereditary disorders of the clotting factors I, 11, V, or VII, haemorrhagic disorder due to circulating anticoagulants (including autoantibodies against coagulation factors such as Factor VIII) or acquired coagulation deficiency. It will be understood that the therapeutic success that can be maintained or achieved by the treatment of the invention depends on the nature and the degree of the disease in any particular patient. Specific embodiments of the invention relate to the prophylactic treatment of coagulopathy to prevent bleeding or to the acute treatment when bleeding occurs ("on demand"). The bleeding events to be treated with the thrombomodulin analogue can WO 2011/157283 PCT/EP2010/007632 -8 occur in every organ or tissue in the organism, most importantly in the central nervous system e.g. as intracranial bleeding, in the joints, the muscles, the gastrointestinal tract, the respiratory tract, the retroperitoneal space or soft tissues. For the preventive treatment the TM analogue can be given to the patient at regular intervals over an extended period. However, also multiple dosing for a rather restricted time period ("subchronic treatment") is possible. In one embodiment of the invention the thrombomodulin analogue is given in advance of a higher bleeding risk, e.g. a surgery or a tooth extraction. In a further embodiment of the invention the thrombomodulin analogue is administered to patients that are refractory to standard therapy such as the transfusion of blood or plasma or the replacement therapy using coagulation factors. According to the invention the TM analogue can be administered in multiple doses preferably once daily but also bidaily, or every third, fourth, fifth, sixth or seven days over a total time period of less than one week to four weeks, more preferably as chronic administration. Thus, according to the invention a pharmaceutical composition is provided, which is suitable for allowing a multiple administration of the thrombomodulin analogue. The TM analogue is given preferably non-orally as a parenteral application e.g. by intravenous or subcutaneous application. An intravenous or subcutaneous bolus application is possible. Thus, according to the invention a pharmaceutical composition is provided, which is suitable for a parenteral administration of thrombomodulin. In one embodiment of the invention the thrombomodulin analogue is a soluble TM analogue, in particular a TM analogue where the cytoplasmic domain is deleted and the transmembrane domain is completely or partially deleted. In a preferred embodiment of the invention the thrombomodulin analogue comprises at least one structural domain selected from the group containing EGF3, EGF4, EGF5, or EGF6, preferably the EGF domains EGF1 to EGF6, more preferably the EGF domains EGF3 to EGF6 and most preferably the EGF domains EGF4 to EGF6 and particularly the fragment including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. Various forms of soluble thrombomodulin are known to the skilled person, e.g. the so called ART-123 developed by Asahi Corporation (Tokyo, Japan) or the recombinant soluble human thrombomodulin Solulin, currently under development by PAION Deutschland GmbH, Aachen (Germany). The recombinant soluble thrombomodulin, i.e.

WO 2011/157283 PCT/EP2010/007632 -9 a soluble thrombomodulin without a modification of the amino acid sequence, is subject of the Asahi patent EPO 312 598 B1. Solulin is a soluble, as well as protease and oxidation-resistant analogue of human thrombomodulin and thus exhibits a long life in vivo. Solulin's main feature lies in its broad mechanism of action since it not exclusively inhibits thrombin. It also activates TAFI and the natural protein C / protein S pathway. As a result of its reduced thrombin binding Solulin inhibits fibrinolysis even up to high concentrations. Solulin and its modifications are particular embodiments of the invention. Solulin is inter alia subject of the European patent EP 0 641 215 B1, EP 0 544 826 B1 as well as EP 0 527 821 B1. Solulin contains modifications compared to the sequence of native human thrombomodulin (SEQ. ID NO. 1) at the following positions: G -3V, Removal of amino acids 1-3, M388L, R456G, H457Q, S474A and termination at P490. This numbering system is in accordance with the native thrombomodulin of SEQ. ID NO. 1 and SEQ ID NO:3. The sequence of Solulin as one preferred embodiment of the invention is shown in SEQ ID NO: 2. However, notably, according to the invention also thrombomodulin analogues can be used, which comprise only one or more of the above mentioned properties, or of the properties outlined in the above mentioned European patent EP 0 544 826 B1, EP0 641 215 B1 and EP0 527 821 B1. Furthermore, according to the invention thrombomodulin analogues can be used, which comprise only one or more of the above mentioned properties, or of the properties outlined in the publication by Wnag et al., 2000, J. Biol CHem. 275: 22942-22947. Particularly preferred thrombomodulin analogues applicable according to the invention are those that have one or more of the following characteristics: (i) they exhibit oxidation resistance, (ii) they exhibit protease resistance, (iii) they have homogeneous N- or C-termini, (iv) they have been post-translationally modified, e.g., by glycosylation of at least some of the glycosylation sites of native thrombomodulin (SEQ ID NO: 1), (v) they have linear double-reciprocal thrombin binding properties, (vi) they are soluble in aqueous solution in relatively low amounts of detergents and typically lack a transmembrane sequence, (vii) they are lacking a glycosaminoglycan chain.

WO 2011/157283 PCT/EP2010/007632 - 10 The manufacture of these analogues used in this invention is disclosed in the above mentioned European patents relating to Solulin. In one embodiment of the invention only the six EGF domains of Solulin can be used, in particular a Solulin fragment consisting of the EGF4 to EGF6 domain. In an embodiment a thrombomodulin analogue with reduced cofactor activity as known from the W093/25675 Al can be used. A series of thrombomodulin analogues is described herein having about 50% or less of the cofactor activity of the control human soluble thrombomodulin (TMEM388L). More particularly said thrombomodulin analogues upon binding to thrombin, exhibit a modified cofactor activity as compared to binding with TMEM388L of less than or equal to 50%, said analogue having amino acid substitutions at one or more positions corresponding to the amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3: ab) 35 5 Asn; ae) 3 9 GIn; af) x 3 Leu; ai) 8 Tyr; aj) 371 Val; ak) 37 4 Glu; al) 37 6 Phe; am) 38 4 His; an) 3"Arg; ba) 3 7 Gln; bb) 3a 9 Phe; bc) 39 8 Asp; bd) 40 0 Asp; be) 40 2 Asn; bf) 403Thr; bg) 40 8 Glu; bh) 41 "Glu; bi) 41 Tyr; bj) 414 11e; bk) 4 15 Leu; bl) 4 1 6 Asp; bm) 417 Asp; bn) 420 1e; ca) 423 Asp; cb) 424 1e; cc) 42 Asp; WO 2011/157283 PCT/EP2010/007632 -11 cd) 4 26 Glu; ce) 42 8 Glu; cf) 4 29 Asp; cg) 432 Phe; ch) 4 34Ser; ci) 4 36Val; cj) 4 38His; ck) 439 Asp; cl) 4 0 Leu; cm) " 3 Thr; cn) 4 4 Phe; co) " 5 Glu; cp) 4 5 6 Arg; cq) 458 1le; or cr) 4"Asp Most preferred are TM analogues with only one of the above listed substitutions. In one embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2) with one or more, preferably one, of the above mutations. Accordingly the invention also relates to proteins according to SEQ ID NO:2 with at least one, in a specific embodiment with exactly one, of the above mutations. . In one embodiment one amin o acid at the given position is deleted instead of substituted. In a further embodiment the invention encompasses a Solulin fragment, in particular a Solulin fragment consisting of the EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above mutations. The Solulin or the Solulin fragment can contain at least one, or exactly one (e.g. a mutation in the position 376), of the above mutations in the amino acid positions 371 to 389. If the Solulin or the Solulin fragments contains a mutation in position 376 (e.g. F376A) a second mutation selected from the above mutations is possible. For convenience the designation to the left, e.g. aa) are identical for each modified site. The first letter represents the EGF domain, where a is EGF4; b is EGF5 and c is EGF6. The second letter represents the relative position of the modification with regard to other residues in the listing. Also provided herein are nucleic acids encoding the TM analogues described above. The following analogues constitute a preferred subset of the above given analogues wherein the analogues have 25% or less of the cofactor activity of the control, TMEM388L. These analogues have one or more amino acid substitutions, preferably only one (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): WO 2011/157283 PCT/EP2010/007632 -12 ae) 35 9 Gln; aj) 371Vat; ak) 374 Glu; al) 37 6 Phe; bc) 39 Asp; bd) 40OAsp; be) 402 Asn; bg) 40 Glu; bi) 413 Tyr; bj) 414 1e; bk) 415 Leu; bl) 416 Asp; bm) 41 7 Asp; bo) 423 Asp; bp) 424 ie; bq) 425 Asp; cd) 426 Glu; ce) 42 9 Asp; ck) 43 9 Asp; cn) 4 4Phe; or cr) 4"Asp. In one embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2) with one or more, preferably one, of the above mutations. Accordingly the invention also relates to proteins according to SEQ ID NO:2 with at least one, in a specific embodiment with exactly one, of the above mutations. In one embodiment one amin o acid at the given position is deleted instead of substituted. In a further embodiment the invention encompasses a Solulin fragment, in particular a Solulin fragment consisting of the EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above mutations. The Solulin or the Solulin fragment can contain at least one, or exactly one (e.g. a mutation in the position 376), of the above mutations in the amino acid positions 371 to 389. If the Solulin or the Solulin fragments contains a mutation in position 376 (e.g. F376A) a second mutation selected from the above mutations is possible. The modifications set forth above with regard to protease activity, aliphatic substitutions, oxidation resistance and uniform termini are also applicable for the above analogues having less than 50% of the cofactor activity of the control. Preferred are those listed above having less than 30% of the activity of the control. These analogues are represented by mutations in domain 4. These analogues have WO 2011/157283 PCT/EP2010/007632 -13 one or more amino acid substitutions, preferably only one (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): ae) s 9 Gln; aj) 371 Val; or al) 376 Phe. In one embodiment of the invention the TM analogue is Solulin (SEQ. ID NO: 2) with one or more, preferably one, of the above mutations. Accordingly the invention also relates to proteins according to SEQ ID NO:2 with at least one, in a specific embodiment with exactly one, of the above mutations. . In one embodiment one amin o acid at the given position is deleted instead of substituted. In a further embodiment the invention encompasses a Solulin fragment, in particular a Solulin fragment consisting of the EGF3 to EGF6 domain or c-loop of EGF3 to EGF6, with one of the above mutations. The Solulin or the Solulin fragment can contain at least one, or exactly one (e.g. a mutation in the position 376), of the above mutations in the amino acid positions 371 to 389. If the Solulin or the Solulin fragments contains a mutation in position 376 (e.g. F376A) a second mutation selected from the above mutations is possible. There are also described herein analogues having an essentially unmodified KD value compared to TMEM388L. EGF5 and EGF6 are known to play an important role in high affinity binding to thrombin, whereas EGF4 with a less critical role in binding is critical for conferring cofactor activity to the TM/thrombin complex. For this reason those analogues having modifications in the EGF repeats 5 and 6 can have almost the same cofactor activity but a reduced KD compared to TMEM388L, e.g. (S406A). Analogues having modifications in the EGF repeats 5 and 6 which resulted in reduced cofactor activity are listed below. These analogues have one or more amino acid substitutions, preferably only one (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bc) 39 8 Asp; bd) 400Asp; be) 402Asn; bf) 4 03 Thr; bg) 40 8 Glu; bi) 413 Tyr; bj) 414 11e; bk) 415 Leu; bl) 416 Asp; bm) 41 7 Asp; ca) 4 23 Asp; cb) 424 Ile; WO 2011/157283 PCT/EP2010/007632 -14 cc) 425 Asp; cd) 42 eGlu; cf) 42 9 Asp; ck) 439 Asp; cn) " 4 Phe; or cr) 46'Asp The above analogues may also grouped by their respective domains (i.e., EGF4, EGF5 or EFG6) as well as by their respective relative activity. For example the TM analogues with a EGF4 sequence modification having approximately 50% of the control cofactor activity are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bb) ' 5 Asn; ae) " 9 Gln; af) s 3 Leu; ai) 36 8 Tyr; aj) 371 val; ak) 374 Glu; al) 37 6 Phe; am) 3 4 His; or an) 38 5 Arg. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. Those in EGF4 having less than 25% of the cofactor activity of the control are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): ae) 35 9 GIn; aj) 3 71 Val; or al) 3 76 Phe. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. In EGF5, the following modifications resulted in analogues having at least a 50% reduction in cofactor activity (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bc) 398 Asp; bd) 400Asp; be) 40 2 Asn; WO 2011/157283 PCT/EP2010/007632 -15 bf) 403Thr; bg) 40 8 Glu; bh) 4 1 1 Glu; bi) 4 13 Tyr; bj) 41 4 1le; bk) 415 Leu; bl) 4 16 Asp; bm) 4 17 Asp; or bn) 420 11e. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. Among these analogues are those where the analogues have an essentially unmodified kCat/Km compared to TMEM388L. In EGF5, the analogues can be further subgrouped according to those modifications resulted in analogues having at least a 75% reduction in cofactor activity (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bc) 39 8 Asp; bd) 400Asp; be) 40 2 Asn; bg) 40 8 Glu; bi) 413 Tyr; bj) 41 4 11e; bk) 41 5 Leu; bl) 416 Asp; or bm) 41 7 Asp. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. Among these analogues are those with essentially unmodified kCat/Km compared to TMEM388L. Nucleic acids encoding the above analogues are also provided. With regard to EGF6 the groups are provided below. Those having a cofactor activity of less than 50% of the control are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): ca) 4 23 Asp; cb) 424 1ie; cc) 42 5 Asp; WO 2011/157283 PCT/EP2010/007632 -16 cd) 42 6 Glu; ce) 428 Glu; cf) 429 Asp; cg) 432 Phe; ch) 4 "Ser; ci) 4 36Val; cj) 4 38His; ck) 439 Asp; cl) 44"Leu; cm) 44 3 Thr; cn) 44 4 Phe; co) 4" 5 GIu; cp) 5 6 Arg; cq) 4 5Ile; or cr) 46 1 Asp. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. Those having a cofactor activity of less than 25% of the control are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): ca) 423 Asp; cb) 424 1e; cc) 42 5 Asp; cd) 426 Glu; cf) 42 9 Asp; ck) 439 Asp; cn) " 4 Phe; or cr) 46 1 Asp. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. The preferred analogues are those set forth above with additional modifications for solubility, protease resistance, oxidation resistance as well as uniform terminal ends. The nucleic acids encoding these analogues are also a part of the claimed invention. As with the other groups, these analogues include those wherein said analogue has an essentially unmodified kCat/Km compared to TMEM388L. The analogues can be further subgrouped according to those possessing a modified amino acid at a certain position, wherein said analogue has essentially equivalent KD WO 2011/157283 PCT/EP2010/007632 -17 for thrombin compared to an analogue having at said position the native residue, wherein said position corresponds to (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bb) 3 5 Asn; or ae) 5 9 GIn. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. These analogues may have a modified kCat/Km of less than 30% of the control. The following sites embrace described analogues having a modified KD or kCat/Km compared to an analogue having at said position the native residue, wherein said position corresponds to (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): af) m 3 Leu; aj) 371 Val; ak) 37 4 Glu; al) 376 Phe; am) 8 4 His; an) 3sArg; bc) 398 Asp; bd) 40 0 Asp; or be) 40 2 Asn. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. These further include those analogues having both a modified KD and kCat/Km, especially those having been modified by at least 20%. The following sites describe analogues having a lower cofactor activity and a KD or kCat/Km that is essentially equivalent when compared to an analogue having at said position the native residue, wherein said position corresponds to (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bg) 40 8 Glu; bh) 4 11 Glu; bi) 413 Tyr; bj) 414 11e; bk) 415 Leu; bl) 416 Asp; bm) 417 Asp; bn) 420 1e; WO 2011/157283 PCT/EP2010/007632 -18 ca) 42 3 Asp; cb) 424 le cc) 425 Asp; cd) 426 Glu; ce) 428 GIu; cf) 42 9 Asp; cg) 432 Phe; ch) 4 34Ser; ci) 4 3eVal; cj) 4 38His; ck) 43 9 Asp; cl) 4 0 Leu; cm) "Thr; cn) 4"Phe; co) 44 5 Glu; cp) 45 6 Arg; cq) 4 -9lle; or cr) " 1 Asp. Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. The following positions describe a subgrouping of those modifications which resulted in at least a 75% reduction in cofactor activity yet essentially little change in kcat/Km (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bg) 408 Glu; bi) 4 13 Tyr; bj) 41 4 11e; bk) 41 5 Leu; bl) 41 6 Asp; bm) 41 7 Asp; ca) 4 23 Asp; cb) 4 24 1e; cc) 4 2 5 Asp; cd) 426 Glu; cf) 429 Asp; ck) 439 Asp; cn) 4 Phe; or cr) 46 1 Asp.

WO 2011/157283 PCT/EP2010/007632 - 19 Most preferred are TM analogues, e.g. Solulin or Solulin fragments, with only one of the above listed substitutions. A further subgrouping can be made of the above modifications wherein the KD for thrombin is modified by at least 30%. This invention further provides for methods. More specifically there is described herein a method useful for screening for analogues of thrombomodulin which exhibit a modified Kd for thrombin binding, comprising the steps of: a) making an amino acid substitution at a position (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bg) 408 Glu; bi) 4 13 Tyr; bj) 414 11e; bk) 41 5 Leu; bl) 41 'Asp; bm) 4 17 Asp; bn) 420 1 e; ca) 423 Asp; cb) 424 1ie; cc) 42 Asp; cd) 426 Glu; ce) 428 Glu; cf) 42 9 Asp; cg) 432 Phe; ch) 4 34Ser; ci) 4 36Val; cj) 4 38His; ck) 4 39 Asp; cl) " 0 Leu; cm) " 3 Thr; cn) 4"Phe; co) " 5 Glu; cp) 4 *Arg; cq) 4 811e; cr) 46'Asp; and b) comparing the KD for thrombin to a control molecule. As used within these methods TM analogues, e.g. Solulin or Solulin fragments, with only one amino acid substitutions are preferred. Various embodiments of this invention include those wherein said KD is modified by at least 33%, or where said modification is WO 2011/157283 PCT/EP2010/007632 - 20 an amino acid substitution, or wherein said control molecule is TMEM388L. A preferred grouping of modifications for use in the method are (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3): bg) 40 8 Glu; bi) 413 Tyr; bj) 4 1 4 1le; bk) 415 Leu; bl) 4 16 Asp; bm) 417 Asp; ca) 423 Asp; cb) 424 11e; cc) 425 Asp; cd) 42 6 Glu; cf) 429 Asp; ck) 439 Asp; cn) " 4 Phe; or cr) 4 1 Asp. As used within these methods TM analogues, e.g. Solulin or Solulin fragments, with only one amino acid substitutions are preferred. Ananother method is described herein which is useful for screening for analogues of thrombomodulin which possess a modified cofactor activity upon binding to thrombin, comprising the steps of: a) making an amino acid substitution at a position (amino acid position as given in SEQ ID NO:1 or SEQ ID NO:3.): bb) 3 5 Asn; ae) 3 9 Gln; and b) comparing the rate of cofactor activity upon binding to thrombin with the rate of a control molecule. As used within these methods TM analogues, e.g. Solulin or Solulin fragments, with only one amino acid substitutions are preferred. In a preferred embodiment of the invention the thrombomodulin analogue has a modification of the phenylalanine residue at position 376 (Phe376X; SEQ ID NO:1 or SEQ ID NO:3). This residue can be chemically or biochemically modified or deleted by methods that are well known for the person skilled in art. The phenylalanine residue is preferably substituted with an aliphatic amino acid, more preferably with glycine, alanine, valine, leucine, or isoleucine and most preferably substituted with alanine. It WO 2011/157283 PCT/EP2010/007632 -21 was demonstrated that a substitution of Phe 3 76 by alanine ("F376A") substantially decreased the cofactor activity of the thrombomodulin analogue while preserving the TAFI activation activity (see Figure 7). As a result the F376A-TM analogue has an increased ratio of TAFI activation activity versus cofactor activity. In one embodiment of the invention Solulin contains the Phe376X, in particular the F376A substitution. In a further embodiment of the invention the thrombomodulin analogue has a modification of the glutamine residue at position 387 (SEQ ID NO:1 or SEQ ID NO:3). The glutamine residue is preferably substituted with the following amino acids, ordered in decreasing cofactor activity of the resulting mutant GIn387X-TM analogue (see Figure 8A): Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, lle, Tr, Tyr, Leu, Asn, Phe, Asp, Cys. In one embodiment of the invention Solulin contains this Gln387X substitution; in a further embodiment this substitution within Solulin contains the Gln387X substitution together with the above F376X substitution. In another embodiment of the invention the thrombomodulin analogue has a modification of the methionine residue at position 388 (SEQ ID NO:1 or SEQ ID NO:3). The methionine residue is preferably substituted with the following amino acids, ordered in decreasing cofactor activity of the resulting mutant Met388X-TM analogue (see Figure 8B): GIn, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys. In one embodiment Solulin contain this substitution together with one or both of the above Phe376X and Gln387X substitutions. In a further embodiment of the invention the thrombomodulin analogue has a modification of the phenylalanine residue at position 389 (SEQ ID NO:1 or SEQ ID NO:3). The phenylalanine residue is preferably substituted with the following amino acids, ordered in decreasing cofactor activity of the resulting mutant Phe389X-TM analogue (see Figure 8C): Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, lie, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro. In one embodiment Solulin can contain this substitution, which can be further combined with one or more, preferably all, of the above Phe376X, Gin387X or Met388X substitutions. In another embodiment of the invention the interdomain loop of the TM, e.g. Solulin, consisting of the three amino acids Gln38 7 , Meta 8 s and Phe38 9 is partially or completely deleted or inserted by one or more amino acids, preferably by an alanine residue (see Figure 8D). For the TM analogues with modifications at positions Phe 37 6 , Gln 3 7 , Meta 8 a or Phes 9 , the TM analogue can be a full length or a soluble TM analogue, comprising the EGF domains EGF1 to EGF6, preferably comprising the EGF domains EGF3 to EGF6. In a preferred embodiment these analogues contain the substitutions that are given in the WO 2011/157283 PCT/EP2010/007632 - 22 TM analogue Solulin. In a more preferred embodiment these Solulin-derived TM analogues consist only of EGF1 to EGF6, in particular of the EGF domains EGF3 to EGF6 or from the c-loop of EGF3 to EGF6 (these three fragments are denominated as Solulin fragments). In an embodiment of the invention the thrombomodulin analogue is used in its oxidised form. Several techniques are known to the skilled person for a controlled oxidation of proteins. The TM analogue is preferably oxidised using chloramine T, hydrogen peroxide or sodium periodate. The invention further pertains to a method that is useful for screening TM analogues to be used for the treatment of coagulopathy with hyperfibrinolysis. This method comprises a first step of modifying the amino acid sequence of thrombomodulin by insertion, deletion or substitution of one or more amino acids, preferably in the EGF domains EGF1 to EGF6, more preferably in the EGF domains EGF3 to EGF6, and most preferably between the amino acid positions Asp4 9 and Asp" 61 . For the person skilled in the art several techniques are known to modify protein sequences e.g. by site-directed mutagenesis or random mutagenesis with subsequent selection. In a second step the modified TM analogue is compared with a control protein for one or more of the following characteristics selected from the group consisting of: binding affinity to thrombin (KD value), cofactor activity, TAFI activation activity or TAFla potential, ratio of TAFI activation activity and cofactor activity, effect of protein oxidation, effect on clot lysis in time in an in vitro assay, or the effect in a coagulation associated animal model. As a control protein, a thrombomodulin protein or analogue is used, preferably a rabbit lung thrombomodulin or a human TM analogue comprising the six EGF domains. The TM analogue can have the native amino acid sequence or alternatively can possess one or more modifications such as the M388L substitution. The invention further relates to a method of treating coagulopathy with hyperfibrinolysis, comprising the administration of a therapeutically effective amount of a thrombomodulin analogue exhibiting an antifibrinolytic effect. Particularly these methods of treatment comprise TM analogues exhibiting one or more of the following features in comparison with a control protein: a decreased binding affinity towards thrombin, a binding affinity towards thrombin with a kD value of more than 0.2 nM, a significantly reduced cofactor activity, or an increased ratio of TAFI activation activity to cofactor activity. As a control protein, a thrombomodulin protein or analogue is used, preferably a rabbit lung thrombomodulin or a human TM WO 2011/157283 PCT/EP2010/007632 -23 analogue comprising the six EGF domains. The TM analogue can have the native amino acid sequence or alternatively can possess one or more modifications such as the M388L substitution. In a further embodiment the invention relates to a thrombomodulin analogue with reduced cofactor activity upon binding to thrombin as compared to TMEM338L with a.) an amino acid sequence according to SEQ ID NO:2 or b) an amino acid sequence according to SEQ ID NO:4 or c) with an amino acid sequence which has at least 90%, more preferred at least 95%, most preferred at least 98% identity to the amino acid sequences according to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. d) a thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (amino acid position 227 to 462 as numbered in SEQ ID NO:1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (amino acid position 307 to 462 as numbered in SEQ ID NO:1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (amino acid position 333 to 462 as numbered in SEQ ID NO:1), whereas the phenylalanine in position 376 (as numbered according to SEQ ID NO:1) is deleted or substituted by glycine, alanine, leucine, isoleucine or valine. In yet another embodiment of the invention the thrombomodulin further comprises a deletion or substitution of the glutamine residue at position 387 (as numbered in SEQ ID NO:1), whereas the substitution preferably is substituted with Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, lie, Tr, Tyr, Leu, Asn, Phe, Asp, Cys. In a further embodiment the invention relates to a thrombomodulin which further comprises a deletion or substitution of the methionine residue at position 388 (as numbered in SEQ ID NO:1), whereas the methionine residue preferably is substituted with GIn, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys. In a further embodiment the thrombomodulin further comprises a deletion or substitution of the phenylalanine residue at position 389 (as numbered in SEQ ID NO:1), whereas the phenylalanine preferably is substituted with Val, Glu, Thr, Ala, His, Trp, Asp, Gin, Leu, lie, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro. In another embodiment the thrombomodulin comprises a combination of a first and a second amino acid modification as depicted in table 4.

WO 2011/157283 PCT/EP2010/007632 -24 In yet another embodiment the thrombomodulin comprises a combination of a first, a second and a third amino acid modification as depicted in table 5. The invention relates further to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) M388L; c) R456G; d) H457Q; e) S474A, and terminating at P490, whereby this thrombomodulin analogue further comprises a sequence modification of one or more of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) s 9 Gln; b) 398 Asp; c) * 0 Asp; d) 4 2 Asn; e) 40 8 GIu; f) 413 Tyr; g) 414 11e; h) 415 Leu, i) 41 7 Asp; j) 43 9 Asn. In a further preferred aspect of the invention the amino acids as listed from a) to j) are substituted with alanine. The invention relates further to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) R456G; c) H457Q; d) S474A, and terminating at P490, whereby this thrombomodulin analogue further comprises a sequence modification of one or more of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) s 8 aMet, with a modification other than a change into Leu WO 2011/157283 PCT/EP2010/007632 - 25 b) 41 6 Asp; c) 423 Asp; d) 42 Asp; e) 426 Glu; f) 429 Asp; g) 44Leu; h) 46 1 Asp. In one aspect of the invention the amino acids as listed from a) to h) are substituted with alanine. The invention relates further to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) R456G; c) H457Q; d) S474A, and terminating at P490, whereby this thrombomodulin analogue comprises an oxidation of the 3 8 Met residue (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent modifications: a) removal of amino acids 1-3; b) M388L; c) R456G; d) H457Q; e) S474A, and terminating at P490, whereby this analogue comprises a mutation of the amino acid 38 7 Gln, which is substituted by an amino acid selected from the group consisting of Lys, Gly, lie, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO: 1) and comprises one or more of the subsequent modifications: WO 2011/157283 PCT/EP2010/007632 - 26 a) removal of amino acids 1-3; b) R456G; c) H457Q; d) S474A, and terminating at P490, whereby this analogue comprises a mutation of the amino acid 3 8 Met, which is substituted by an amino acid selected from the group consisting of lie, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) M388L; c) R456G; d) H457Q; e) S474A, and terminating at P490, whereby this analogue comprises a mutation of the amino acid 8 9 Phe, which is substituted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) M388L; c) R456G; d) H457Q; e) S474A, and terminating at P490, whereby this analogue comprises an insertion of a hydrophobic amino acid, preferably Ala between one of the following pairs of amino acids: 3 6 Cys and 3 7 Gln, 38 7 Gln and a 8 Leu, 33 8 Leu and 3 9 Phe, as 9 Phe and 39 0 Cys (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue and its medical use for the treatment of coagulopathy with hyperfibrinolysis that has an amino acid sequence WO 2011/157283 PCT/EP2010/007632 - 27 corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1) and comprises one or more of the subsequent sequence modifications: a) removal of amino acids 1-3; b) M388L; c) R456G; d) H457Q; e) S474A, and terminating at P490, whereby this analogue comprises a mutation of the amino acid 376 Phe, which preferably is a Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a more preferred aspect of the invention relates to Solulin as depicted in SEQ ID NO:2, which comprises sequence modifications of one or more, preferably exactly one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) ,s 9 GIn; b) m 8 Asp; c) 400 Asp; d) 4 2 Asn; e) 8 GIu; f) 413 Tyr; g) 41 4 11e; h) 41 5 Leu, i) 41 7 Asp; j) 439 Asn. The above amino acids as listed from a) to j) are preferably substituted by alanine. In one embodiment Solulin as depicted in SEQ ID NO:2 comprises modifications in one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 3esMet, with a modification other than a change into Leu b) 41 Asp; c) 423 Asp; d) 425 Asp; e) 426 GIu; f) 42 9 Asp; g) **Leu; h) 6 1 Asp.

WO 2011/157283 PCT/EP2010/007632 -28 On one aspect of the invention the amino acids as listed from a) to h) are substituted by alanine. The Solulin as used in the invention can comprise a mutation of the amino acid 3 7 Gin, which is substituted by an amino acid selected from the group consisting of Lys, Gly, lie, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a further aspect of the invention Solulin comprises a mutation of the amino acid 38 8 Met, which is substituted by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore, the invention relates to a Solulinwhich comprises a mutation of the amino acid 38 9 Phe, which can be substituted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In addition, in one embodiment of the invention Solulin comprises an insertion of a hydrophobic amino acid, preferably Ala between one of the following pairs of amino acids: a 6 Cys and 38 7 Gln, s 7 Gln and 8 Leu, 3 8 Leu and 38 9 Phe, as 9 Phe and 390 Cys (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Solulin can be modified in order to comprise an oxidised 3 8 Met residue (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore, it can comprise a mutation of the amino acid 376 Phe, which preferably is a Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a preferred aspect of the invention relates to a thrombomodulin analogue that essentially consists of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, which can comprise the sequence modifications of one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) as 9 Gln; b) 3 98 Asp; WO 2011/157283 PCT/EP2010/007632 - 29 c) " 0 Asp; d) 4 2 Asn; e) 0 8 Glu; f) 413 Tyr; g) 41 4 11e; h) 415 Leu, i) 417 Asp; j) 43 9 Asn. In one aspect of the invention the amino acids as listed from a) to j) are substituted by alanine. The TM analogue of the invention can also essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise the sequence modifications in one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 3 8 Met, with a modification other than a change into Leu b) 4 1 6 Asp; c) 4 23 Asp; d) 425 Asp; e) 426 Glu; f) 42 9 Asp; g) "4Leu; h)46'Asp. The amino acids as listed from a) to h) are preferably substituted by alanine. The invention further relates to TM analogues that essentially consists of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 38 7 Gln, which can be substituted by an amino acid selected from the group consisting of Lys, Gly, lIe, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention also relates to TM analogues that essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 388 Met, which can be substituted by an amino acid selected from the group WO 2011/157283 PCT/EP2010/007632 - 30 consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore, the invention relates to TM analogues that essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid as 9 Phe, which can be substituted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In addition, the invention relates to TM analogues that essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise an insertion of a hydrophobic amino acid, preferably Ala between one of the following pairs of amino acids: 386 Cys and 3 7 aGln, 38 7 Gln and 3 8 Met, 3 8 Met and as 9 Phe, 3 9 Phe and 390 Cys (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore the invention relates to TM analogues that essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise an oxidised 3 8 Met residue (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore the invention relates to TM analogues that essentially consist of the six EGF domains as given by residues 227 to 462 of SEQ ID NO:1, or residues 224 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 3 76 Phe, which preferably is a Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In one embodiment of the invention a TM analogue is claimed that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise modifications of one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 359 GIn; b) 398 Asp; c) 400Asp; WO 2011/157283 PCT/EP2010/007632 - 31 d) 40 2 Asn; e) 0 8 Glu; f) 413 Tyr; g) 41 4 11e; h) 415 Leu, i) 417 Asp; j) 4 39 Asn. The amino acids as listed from a) to j) can be substituted by alanine. In one embodiment of the invention a TM analogue is claimed that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise modifications of one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 3 8 Met, with a modification other than a change into Leu b) 4 1 6 Asp; c) 423 Asp; d) 425 Asp; e) 426 Glu; f) 429 Asp; g) * 0 Leu; h) "'Asp. The amino acids as listed from a) to h) can be substituted by alanine. The invention further relates to a thrombomodulin analogue that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of those fragments can comprise a mutation of the amino acid 38 7 Gln, which can be substituted by an amino acid selected from the group consisting of Lys, Gly, lie, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention also relates to a thrombomodulin analogue that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of the fragments can comprise a mutation of the amino acid s 8 aMet, which can be substituted by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, WO 2011/157283 PCT/EP2010/007632 - 32 Asp or Cys, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore, the invention relates to a thrombomodulin analogue that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 3 9 Phe, which can be substituted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which is deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In addition, the invention relates to a thrombomodulin analogue that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise an insertion of a hydrophobic amino acid, preferably Ala between one of the following pairs of amino acids: asCys and 38 rGln, saGln and s 8 aMet, asMet and s 9 Phe, 3 9 Phe and 3 90 Cys (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a further aspect of the invention a thrombomodulin analogue is claimed that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of the fragments can comprise an oxidised 3 8 Met residue (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a further aspect of the invention relates to a thrombomodulin analogue that essentially consists of the EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO:1, or residues 308 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 376 Phe, which preferably is a Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a preferred aspect of the invention a thrombomodulin analogue is claimed that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments comprise modifications of one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 3 9 Gln; b) 39 8 Asp; c) 40 0 Asp; WO 2011/157283 PCT/EP2010/007632 - 33 d) 4 2 Asn; e) 40 'Glu; f) 413 Tyr; g) 41 4 1le; h) 41 5 Leu, i) 4 17 Asp; j) 439 Asn. The amino acids as listed from a) to j) can be substituted by alanine. In one aspect of the invention a thrombomodulin analogue is claimed that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments comprise modifications of one or more, preferably one, of the subsequent amino acid positions (the numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1): a) 3 8 Met, with a modification other than a change into Leu b) 41 6 Asp; c) 4 23 Asp; d) 425 Asp; e) 426 Glu; f) 42 9 Asp; g) 44" 0 Leu; h) 46 1 Asp. The amino acids as listed from a) to h) are preferably substituted by alanine. The invention further relates to a thrombomodulin analogue that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 3 7 Gln, which can be substituted by an amino acid selected from the group consisting of Lys, Gly, lle, Trp, Tyr, Leu, Asn, Phe, Asp, Cys, or Pro, or which can be deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention also relates to a thrombomodulin analogue that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 3 8 Met, which can be substituted by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys, or which can be deleted (numbering is WO 2011/157283 PCT/EP2010/007632 - 34 related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). Furthermore, the invention relates to a thrombomodulin analogue that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 3 9 Phe, which can be substituted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro, or which can be deleted (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In addition the invention relates to a thrombomodulin analogue that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereby this thrombomodulin analogue comprises an insertion of a hydrophobic amino acid, preferably Ala between one of the following pairs of amino acids: 38 6 Cys and 38 7 Gln, 8 7 GIn and 3 8 Met, s 8 aMet and 38 9 Phe, as 9 Phe and 3 90 Cys (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). In a preferred aspect of the invention a thrombomodulin analogue is claimed that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise an oxidized 3 8 Met residue (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). The invention relates preferably to a thrombomodulin analogue that essentially consists of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO:1, or residues 330 to 459 of SEQ ID:NO 2, whereas each of these fragments can comprise a mutation of the amino acid 376 Phe, which preferably is a Phe376AIa mutation (numbering is related to the amino acid sequence of mature thrombomodulin depicted in SEQ ID No: 1). According to the invention the TM analogues according to SEQ ID NO:2 can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4. In yet another embodiment of the invention the TM analogues according to SEQ ID NO :4 can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4.

WO 2011/157283 PCT/EP2010/007632 - 35 In yet another embodiment of the invention the TM analogues according to SEQ ID NO 3 can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 2 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2 (amino acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 3 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3 (amino acid position 307 to 462 as numbered in SEQ ID NO:1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3 (amino acid position 333 to 462 as numbered in SEQ ID NO:1) whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 4 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4 (amino acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 4. No. First amino acid modification Second amino acid modification 1 Phe 376AIa Gln387Lys 2 Phe 376AIa Gln387Gly 3 Phe 376AIa Gin38711e 4 Phe 376AIa Gln387Trp 5 Phe 376AIa Gln387Tyr 6 Phe 376Ala Gln387Leu WO 2011/157283 PCT/EP2010/007632 - 36 7 Phe 376Ala Gln387Asn 8 Phe 376Ala Gln387Phe 9 Phe 376Ala Gln387Asp 10 Phe 376Ala Gln387Cys 11 Phe 376Ala Gln387Pro 12 Phe 376Ala Met388Ile 13 Phe 376Ala Met388Phe 14 Phe 376Ala Met388His 15 Phe 376AIa Met388Arg 16 Phe 376Ala Met388Pro 17 Phe 376Ala Met388Val 18 Phe 376Ala Met388Thr 19 Phe 376AIa Met388Ser 20 Phe 376Ala Met388AIa 21 Phe 376Ala Met388Trp 22 Phe 376Ala Met388Asn 23 Phe 376Ala Met388Lys 24 Phe 376Ala Met388Gly 25 Phe 376Ala Met388GIu 26 Phe 376Ala Met388Asp 27 Phe 376Ala Met388Cys 28 Phe 376AIa Phe389Ser 29 Phe 376Ala Phe389Arg 30 Phe 376Ala Phe389Lys 31 Phe 376Ala Phe389Met 32 Phe 376Ala Phe389Tyr 33 Phe 376Ala Phe389Gly 34 Phe 376Ala Phe389Cys 35 Phe 376Ala Phe389Pro 36 Phe 376Ala Deletion GIn387 37 Phe 376Ala Deletion Met388 38 Phe 376Ala Deletion Phe389 39 Phe 376AIa Ala insert 386/387 40 Phe 376Ala Ala insert 387/388 41 Phe 376Ala Ala insert 388/389 42 Phe 376Ala Ala insert 389/390 43 Phe 376Val Gln387Lys 44 Phe 376Val Gln387Gly 45 Phe 376Val Gln3871le WO 2011/157283 PCT/EP2010/007632 - 37 46 Phe 376Val GIn387Trp 47 Phe 376Val Gln387Tyr 48 Phe 376Val Gln387Leu 49 Phe 376Val Gln387Asn 50 Phe 376Val Gln387Phe 51 Phe 376Val GIn387Asp 52 Phe 376Val Gln387Cys 53 Phe 376Val Gln387Pro 54 Phe 376Val Met3881le 55 Phe 376Val Met388Phe 56 Phe 376Val Met388His 57 Phe 376Val Met388Arg 58 Phe 376Val Met388Pro 59 Phe 376Val Met388Val 60 Phe 376Val Met388Thr 61 Phe 376Val Met388Ser 62 Phe 376Val Met388Ala 63 Phe 376Val Met388Trp 64 Phe 376Val Met388Asn 65 Phe 376Val Met388Lys 66 Phe 376Val Met388GIy 67 Phe 376Val Met388GIu 68 Phe 376Val Met388Asp 69 Phe 376Val Met388Cys 70 Phe 376Val Phe389Ser 71 Phe 376Val Phe389Arg 72 Phe 376Val Phe389Lys 73 Phe 376Val Phe389Met 74 Phe 376Val Phe389Tyr 75 Phe 376Val Phe389Gly 76 Phe 376Val Phe389Cys 77 Phe 376Val Phe389Pro 78 Phe 376Val Deletion GIn387 79 Phe 376Val Deletion Met388 80 Phe 376Val Deletion Phe389 81 Phe 376Val Ala insert 386/387 82 Phe 376Val Ala insert 387/388 83 Phe 376Val Ala insert 388/389 84 Phe 376Val Ala insert 389/390 WO 2011/157283 PCT/EP2010/007632 - 38 85 Phe 376 Ile GIn387Lys 86 Phe 376 Ile Gln387GIy 87 Phe 376 Ile GIn387lie 88 Phe 376 Ile GIn387Trp 89 Phe 376 Ile GIn387Tyr 90 Phe 376 Ile Gin387Leu 91 Phe 376 Ile GIn387Asn 92 Phe 376 Ile GIn387Phe 93 Phe 376 Ile GIn387Asp 94 Phe 376 lie GIn387Cys 95 Phe 376 Ile GIn387Pro 96 Phe 376 Ile Met388lle 97 Phe 376 Ile Met388Phe 98 Phe 376 Ile Met388His 99 Phe 376 Ile Met388Arg 100 Phe 376 lie Met388Pro 101 Phe 376 lie Met388VaI 102 Phe 376 Ile Met388Thr 103 Phe 376 Ile Met388Ser 104 Phe 376 lie Met388AIa 105 Phe 376 Ile Met388Trp 106 Phe 376 Ile Met388Asn 107 Phe 376 Ile Met388Lys 108 Phe 376 lie Met388Gly 109 Phe 376 lie Met388Glu 110 Phe 376 Ile Met388Asp 111 Phe 376 Ile Met388Cys 112 Phe 376 Ile Phe389Ser 113 Phe 376 lie Phe389Arg 114 Phe 376 Ile Phe389Lys 115 Phe 376 Ile Phe389Met 116 Phe 376 Ile Phe389Tyr 117 Phe 376 lie Phe389Gly 118 Phe 376 Ile Phe389Cys 119 Phe 376 Ile Phe389Pro 120 Phe 376 Ile Deletion Gln387 121 Phe 376 lie Deletion Met388 122 Phe 376 Ile Deletion Phe389 123 Phe 376 Ile Ala insert 386/387 WO 2011/157283 PCT/EP2010/007632 -39 124 Phe 376 Ile Ala insert 387/388 125 Phe 376 Ile Ala insert 388/389 126 Phe 376 Ile Ala insert 389/390 127 Phe 376 Leu Gln387Lys 128 Phe 376 Leu Gln387Gly 129 Phe 376 Leu Gln38711e 130 Phe 376 Leu Gln387Trp 131 Phe 376 Leu GIn387Tyr 132 Phe 376 Leu Gln387Leu 133 Phe 376 Leu Gln387Asn 134 Phe 376 Leu GIn387Phe 135 Phe 376 Leu Gln387Asp 136 Phe 376 Leu Gln387Cys 137 Phe 376 Leu Gln387Pro 138 Phe 376 Leu Met3881le 139 Phe 376 Leu Met388Phe 140 Phe 376 Leu Met388His 141 Phe 376 Leu Met388Arg 142 Phe 376 Leu Met388Pro 143 Phe 376 Leu Met388Val 144 Phe 376 Leu Met388Thr 145 Phe 376 Leu Met388Ser 146 Phe 376 Leu Met388Ala 147 Phe 376 Leu Met388Trp 148 Phe 376 Leu Met388Asn 149 Phe 376 Leu Met388Lys 150 Phe 376 Leu Met388Gly 151 Phe 376 Leu Met388Glu 152 Phe 376 Leu Met388Asp 153 Phe 376 Leu Met388Cys 154 Phe376 Leu Phe389Ser 155 Phe 376 Leu Phe389Arg 156 Phe 376 Leu Phe389Lys 157 Phe 376 Leu Phe389Met 158 Phe 376 Leu Phe389Tyr 159 Phe 376 Leu Phe389Gly 160 Phe 376 Leu Phe389Cys 161 Phe 376 Leu Phe389Pro 162 Phe 376 Leu Deletion Gln387 WO 2011/157283 PCT/EP2010/007632 -40 163 Phe 376 Leu Deletion Met388 164 Phe 376 Leu Deletion Phe389 165 Phe 376 Leu Ala insert 386/387 166 Phe 376 Leu Ala insert 387/388 167 Phe 376 Leu Ala insert 388/389 168 Phe 376 Leu Ala insert 389/390 169 Phe 376Ala Asp416Ala 170 Phe 376Ala Asp423Ala 171 Phe 376Ala Asp425 Ala 172 Phe 376Ala Glu426Ala 173 Phe 376Ala Asp429Ala 174 Phe 376Ala Leu440Ala 175 Phe 376Ala Asp461Ala 176 Phe 376Ala Gln359Ala 177 Phe 376Ala Asp398Ala 178 Phe 376Ala Asp400Ala 179 Phe 376Ala Asn402Ala 180 Phe 376Ala Glu408Ala 181 Phe 376Ala Tyr413Ala 182 Phe 376Ala lle414Ala 183 Phe 376Ala Leu415Ala 184 Phe 376Ala Asp417Ala 185 Phe 376Ala Asn439Ala Table 4: First and second amino acid modifications According to the invention the TM analogues according to SEQ ID NO 2 can each contain the modifications (a "first, second and third amino acid modification") as depicted in table 5. Table 5 depicts an alanine substitution for Phe376 ("First amino acid modification"). In certain embodiments of the invention the "first amino acid modification" is constituted either by glycine, valine, leucine or isoleucine. These modifications are each combined with the "second amino acid modification" and "third amino acid modification" as given in table 5. These further embodiments of the invention are collectively summarised as "modifications as depicted in table 5. In yet another embodiment of the invention the TM analogues according to SEQ ID NO 4 can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 5.

WO 2011/157283 PCT/EP2010/007632 -41 In yet another embodiment of the invention the TM analogues according to SEQ ID NO 3 can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 5. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 2 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2 (amino acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 5. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 3 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 3 (amino acid position 333 to 462 as numbered in SEQ ID NO1) whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 5. In yet another thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 4 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 4 (amino acid position 333 to 462 as numbered in SEQ ID NO1), whereas each of these EGF domain containing fragments can contain two sequence modifications (a "first amino acid modification" and a "second amino acid modification"), which are depicted in table 5. No. 1st aa modification 2nd aa modification 3 rd aa modification 1 Phe 376Ala Asp416Ala Gln359Ala 2 Phe 376AIa Asp416Ala Asp398Ala 3 Phe 376AIa Asp416Ala Asp400Ala 4 Phe 376Ala Asp416Ala Asn402AIa 5 Phe 376Ala Asp416AIa Glu408Ala 6 Phe 376Ala Asp416AIa Tyr413Ala WO 2011/157283 PCT/EP2010/007632 -42 7 Phe 376AIa Asp4l6AIa Ile4l4Ala 8 Phe 376AIa Asp4l6AIa Leu4l5AIa 9 Phe 376AIa Asp4l6AIa Asp4l7AIa 10 Phe 376AIa Asp4l6AIa Asn439AIa 11 Phe 376AIa Asp4l6AIa Gln387Cys 12 Phe 376AIa Asp4l6AIa Gln387Pro 13 Phe 376AIa Asp4l6Ala Oxidated Met388 14 Phe 376AIa Asp4l6Ala Met388Asp 15 Phe 376AIa Asp4l6Ala Met388Cys 16 Phe 376Ala Asp4l6Ala Phe389Cys 17 Phe 376AIa Asp4l6AIa Phe389Pro 18 Phe 376Ala Asp4l6Ala Deletion Gln387 19 Phe 376AIa Asp4l6Ala Deletion Met388 20 Phe 376AIa Asp4l6AIa Deletion Phe389 21 Phe 376AIa Asp4l6AIa Ala insert 386/387 22 Phe 376Ala Asp4l6Ala Ala insert 387/388 23 Phe 376Ala Asp4l6AIa Ala insert 388/389 24 Phe 376Ala Asp4l6Ala Ala insert 389/390 25 Phe 376Ala Asp423Ala Gln359Ala 26 Phe 376AIa Asp423Ala Asp398AIa 27 Phe 376Ala Asp423Ala Asp400Ala 28 Phe 376AIa Asp423Ala Asn402Ala 29 Phe 376Ala Asp423Ala Glu408Ala 30 Phe 376Ala Asp423Ala Tyr4l3AIa 31 Phe 376Ala Asp423Ala lle414Ala 32 Phe 376Ala Asp423Ala Leu4l5Ala 33 Phe 376Ala Asp423Ala Asp41 7Ala 34 Phe 376Ala Asp423Ala Asn439Ala 35 Phe 376Ala Asp423Ala Gln387Cys 36 Phe 376Ala Asp423Ala Gln387Pro 37 Phe 376Ala Asp423AIa Oxidated Met388 38 Phe 376Ala Asp423Ala Met388Asp 39 Phe 376Ala Asp423Ala Met388Cys 40 Phe 376Ala Asp423Ala Phe389Cys 41 Phe 376Ala Asp423Ala Phe389Pro 42 Phe 376Ala Asp423Ala Deletion Gln387 43 Phe 376Ala Asp423Ala Deletion Met388 44 Phe 376Ala Asp423Ala Deletion Phe389 45 Phe 376Ala Asp423Ala Ala insert 386/387 WO 2011/157283 PCT/EP2010/007632 -43 46 Phe 376Ala Asp423Ala Ala insert 387/388 47 Phe 376Ala Asp423Ala Ala insert 388/389 48 Phe 376AIa Asp423Ala Ala insert 389/390 49 Phe 376Ala Asp425Ala Gln359Ala 50 Phe 376AIa Asp425Ala Asp398AIa 51 Phe 376Ala Asp425AIa Asp400Ala 52 Phe 376Ala Asp425Ala Asn402Ala 53 Phe 376Ala Asp425Ala Glu408Ala 54 Phe 376Ala Asp425Ala Tyr4l3Ala 55 Phe 376Ala Asp425Ala lle414Ala 56 Phe 376Ala Asp425Ala Leu4l5Ala 57 Phe 376Ala Asp425Ala Asp4l7Ala 58 Phe 376Ala Asp425Ala Asn439Ala 59 Phe 376Ala Asp425Ala Gln387Cys 60 Phe 376Ala Asp425Ala Gln387Pro 61 Phe 376AIa Asp425Ala Oxidated Met388 62 Phe 376AIa Asp425AIa Met388Asp 63 Phe 376AIa Asp425Ala Met388Cys 64 Phe 376AIa Asp425Ala Phe389Cys 65 Phe 376Ala Asp425Ala Phe389Pro 66 Phe 376Ala Asp425Ala Deletion GIn387 67 Phe 376Ala Asp425Ala Deletion Met388 68 Phe 376Ala Asp425Ala Deletion Phe389 69 Phe 376Ala Asp425Ala Ala insert 386/387 70 Phe 376Ala Asp425Ala Ala insert 387/388 71 Phe 376AIa Asp425Ala Ala insert 388/389 72 Phe 376Ala Asp425Ala Ala insert 389/390 73 Phe 376Ala Glu426Ala Gln359Ala 74 Phe 376Ala Glu426Ala Asp398Ala 75 Phe 376Ala Glu426Ala Asp400Ala 76 Phe 376Ala Glu426Ala Asn402Ala 77 Phe 376Ala Glu426Ala Glu408Ala 78 Phe 376AIa Glu426Ala Tyr4l3Ala 79 Phe 376Ala Glu426Ala ||e414Ala 80 Phe 376Ala Glu426Ala Leu4l5Ala 81 Phe 376Ala Glu426Ala Asp417Ala 82 Phe 376Ala Glu426Ala Asn439Ala 83 Phe 376Ala Glu426Ala Gin387Cys 84 Phe 376Ala Glu426Ala Gln387Pro WO 2011/157283 PCT/EP2010/007632 -44 85 Phe 376Ala Glu426Ala Oxidated Met388 86 Phe 376Ala Glu426Ala Met388Asp 87 Phe 376Ala Glu426Ala Met388Cys 88 Phe 376Ala Glu426Ala Phe389Cys 89 Phe 376Ala GIu426Ala Phe389Pro 90 Phe 376Ala Glu426Ala Deletion GIn387 91 Phe 376Ala GIu426Ala Deletion Met388 92 Phe 376Ala Glu426Ala Deletion Phe389 93 Phe 376Ala Glu426Ala Ala insert 386/387 94 Phe 376Ala Glu426Ala Ala insert 387/388 95 Phe 376Ala Glu426Ala Ala insert 388/389 96 Phe 376Ala Glu426Ala Ala insert 389/390 97 Phe 376Ala Asp429Ala GIn359Ala 98 Phe 376Ala Asp429Ala Asp398Ala 99 Phe 376Ala Asp429Ala Asp400Ala 100 Phe 376Ala Asp429Ala Asn402Ala 101 Phe 376Ala Asp429Ala Glu4O8Ala 102 Phe 376AIa Asp429Ala Tyr4l3Ala 103 Phe 376Ala Asp429Ala lIe4l4Ala 104 Phe 376Ala Asp429Ala Leu4l5Ala 105 Phe 376Ala Asp429Ala Asp4l7Ala 106 Phe 376Ala Asp429Ala Asn439Ala 107 Phe 376Ala Asp429Ala Gln387Cys 108 Phe 376AIa Asp429Ala GIn387Pro 109 Phe 376Ala Asp429Ala Oxidated Met388 110 Phe 376Ala Asp429Ala Met388Asp 111 Phe 376Ala Asp429Ala Met388Cys 112 Phe 376Ala Asp429Ala Phe389Cys 113 Phe 376Ala Asp429AIa Phe389Pro 114 Phe 376Ala Asp429Ala Deletion Gln387 115 Phe 376Ala Asp429Ala Deletion Met388 116 Phe 376Ala Asp429Ala Deletion Phe389 117 Phe 376Ala Asp429Ala Ala insert 386/387 118 Phe 376Ala Asp429AIa Ala insert 387/388 119 Phe 376AIa Asp429Ala Ala insert 388/389 120 Phe 376Ala Asp429Ala Ala insert 389/390 121 Phe 376Ala Leu440Ala Gln359Ala 122 Phe 376Ala Leu440Ala Asp398Ala 123 Phe 376Ala Leu440Ala Asp400Ala WO 2011/157283 PCT/EP2010/007632 -45 124 Phe 376AIa Leu440Ala Asn402Ala 125 Phe 376AIa Leu440Ala Glu408Ala 126 Phe 376AIa Leu440Ala Tyr4l3Ala 127 Phe 376Ala Leu440Ala lle414Ala 128 Phe 376Ala Leu440Ala Leu4l5Ala 129 Phe 376AIa Leu440Ala Asp4l7AIa 130 Phe 376AIa Leu440Ala Asn439Ala 131 Phe 376AIa Leu440Ala Gln387Cys 132 Phe 376Ala Leu440Ala GIn387Pro 133 Phe 376AIa Leu440Ala Oxidated Met388 134 Phe 376AIa Leu440Ala Met388Asp 135 Phe 376Ala Leu440Ala Met388Cys 136 Phe 376Ala Leu440Ala Phe389Cys 137 Phe 376Ala Leu440Ala Phe389Pro 138 Phe 376Ala Leu440Ala Deletion Gln387 139 Phe 376AIa Leu440Ala Deletion Met388 140 Phe 376Ala Leu440Ala Deletion Phe389 141 Phe 376Ala Leu440Ala Ala insert 386/387 142 Phe 376Ala Leu440Ala Ala insert 387/388 143 Phe 376Ala Leu440Ala Ala insert 388/389 144 Phe 376Ala Leu440Ala Ala insert 389/390 145 Phe 376Ala Asp461Ala Gln359Ala 146 Phe 376Ala Asp461Ala Asp398Ala 147 Phe 376Ala Asp461Ala Asp400Ala 148 Phe 376Ala Asp461Ala Asn402Ala 149 Phe 376Ala Asp461Ala Glu408Ala 150 Phe 376Ala Asp461Ala Tyr4l3Ala 151 Phe 376Ala Asp461Ala -le414Ala 152 Phe 376Ala Asp461Ala Leu4l5Ala 153 Phe 376Ala Asp461Ala Asp4l7Ala 154 Phe 376Ala Asp461Ala Asn439Ala 155 Phe 376Ala Asp461Ala Gln387Cys 156 Phe 376Ala Asp461Ala Gln387Pro 157 Phe 376Ala Asp461Ala Oxidated Met388 158 Phe 376Ala Asp461 Ala Met388Asp 159 Phe 376AIa Asp461 Ala Met388Cys 160 Phe 376Ala Asp461Ala Phe389Cys 161 Phe 376Ala Asp461Ala Phe389Pro 162 Phe 376Ala Asp461Ala Deletion Gln387 WO 2011/157283 PCT/EP2010/007632 - 46 163 Phe 376Ala Asp461Ala Deletion Met388 164 Phe 376AIa Asp461Ala Deletion Phe389 165 Phe 376Ala Asp461Ala Ala insert 386/387 166 Phe 376Ala Asp461Ala Ala insert 387/388 167 Phe 376Ala Asp461Ala Ala insert 388/389 168 Phe 376Ala Asp461Ala Ala insert 389/390 Table 5: First, second and third modifications WO 2011/157283 PCT/EP2010/007632 -47 The following tables gives an overview on the reduced cofactor activities that results from specific amino acid modifications of thrombomodulin analogues. aa modification Cofactor activity Normalized Source against F376A - 5% M388L Wang 2000, Fig. 4B (-65% TAFI act.) M388L -180% M388L W093/25675, Fig. 2B (-90% TaFI act.) wild type D398A -20% M388L W093/25675, Fig. 2B D400A -2% M388L W093/25675, Fig. 2B N402A -10% M388L W093/25675, Fig. 2B E408A -24% M388L W093/25675, Fig. 2B Y413A -2% M388L W093/25675, Fig. 2B 1414A -5% M388L W093/25675, Fig. 2B L415A -5% M388L W093/25675, Fig. 2B D416A -4% M388L W093/25675, Fig. 2B D417A -20% M388L W093/25675, Fig. 2B D423A -12% M388L W093/25675, Fig. 2C 1424A -2% M388L W093/25675, Fig. 2C E426A -2% M388L W093/25675, Fig. 2C N429A -10% M388L W093/25675, Fig. 2C N439A -2% M388L W093/25675, Fig. 2C L440A -25% M388L W093/25675, Fig. 2C D461A -9% M388L W093/25675, Fig. 2C Gln387Lys -30% wild type Clarke 1993, Figure 2 Gln387Gly -30% wild type Clarke 1993, Figure 2 Gln3871le -30% wild type Clarke 1993, Figure 2 Gln387Trp -28% wild type Clarke 1993, Figure 2 Gln387Tyr -30% wild type Clarke 1993, Figure 2 Gln387Leu -25% wild type Clarke 1993, Figure 2 Gln387Asn -25% wild type Clarke 1993, Figure 2 Gln387Phe -20% wild type Clarke 1993, Figure 2 Gln387Asp -18% wild type Clarke 1993, Figure 2 Gln387Cys 2% wild type Clarke 1993, Figure 2 Gln387Pro 0% wild type Clarke 1993, Figure 2 Met388lle 48% wild type Clarke 1993, Figure 2 Met388Phe 40% wild type Clarke 1993, Figure 2 Met388His 40% wild type Clarke 1993, Figure 2 WO 2011/157283 PCT/EP2010/007632 -48 Met388Arg 35% wild type Clarke 1993, Figure 2 Met388Pro 35% wild type Clarke 1993, Figure 2 Met388VaI 30% wild type Clarke 1993, Figure 2 Met388Thr 22% wild type Clarke 1993, Figure 2 Met388Ser 22% wild type Clarke 1993, Figure 2 Met388Ala 22% wild type Clarke 1993, Figure 2 Met388Trp 20% wild type Clarke 1993, Figure 2 Met388Asn 20% wild type Clarke 1993, Figure 2 Met388Lys 15% wild type Clarke 1993, Figure 2 Met388Gly 15% wild type Clarke 1993, Figure 2 Met388Glu 15% wild type Clarke 1993, Figure 2 Met388Asp 8% wild type Clarke 1993, Figure 2 Met388Cys 5% wild type Clarke 1993, Figure 2 Phe389Ser 5% wild type Clarke 1993, Figure 2 Phe389Arg 45% wild type Clarke 1993, Figure 2 Phe389Lys 45% wild type Clarke 1993, Figure 2 Phe389Met 45% wild type Clarke 1993, Figure 2 Phe389Tyr 45% wild type Clarke 1993, Figure 2 Phe389Gly 25% wild type Clarke 1993, Figure 2 Phe389Cys 15% wild type Clarke 1993, Figure 2 Phe389Pro 8% wild type Clarke 1993, Figure 2 Deletion GIn387 2% wild type Clarke 1993, Figure 3 Deletion Met388 4% wild type Clarke 1993, Figure 3 Deletion Phe389 5% wild type Clarke 1993, Figure 3 Ala insert 386/387 5% wild type Clarke 1993, Figure 3 Ala insert 387/388 3% wild type Clarke 1993, Figure 3 Ala insert 388/389 10% wild type Clarke 1993, Figure 3 Ala insert 389/390 5% wild type Clarke 1993, Figure 3 Table 6: First and second amino acid modifications Therapeutic treatment according to the invention In one aspect of the invention the thrombomodulin analogues as disclosed in here are used for treatment of coagulopathy with hyperfibrinolysis in patients, who possess anti factor Vill antibodies. These antibodies can inhibit factor VillI activity. In the typical case, they arise as alloantibodies during replacement therapy of haemophilia A patients. They can be responsible for the failure of FVIII replacement therapy in haemophilia A patients. Thus, according to one aspect of the invention the WO 2011/157283 PCT/EP2010/007632 -49 thrombomodulin analogues disclosed herein can be used as rescue medication for haemophilia patients who are non-responders for factor Vill. According to a further aspect of the invention, patients, who are deficient in factor Vill, are treated with a combination of factor VillI and the TM analogues of the invention. Factor VIII and the TM analogue can be administered either concomitantly or sequentially. The patients are treated preferably with recombinant factor Vill or a recombinant B-domain-deleted factor Vill molecule, more preferably Octocog-alfa or moroctocog-alfa. In one embodiment isolated human factor VIll can be used, e.g. Aafact@. According to the invention also haemophilia patients can be treated who had been treated with factor Vill in the past or are currently under factor VIll treatment. Accordingly these patients have a pharmaceutically effective factor VIll level. In a further aspect of the invention, the claimed thrombomodulin analogues can be used to screen haemophilia patients for the presence of factor Vill antibodies since the presence of factor Vill antibodies causes characteristic changes in the thromboelastogram (see Figure 11 A vs. Figure 12A). Embodiments of the invention In one aspect of the invention the use of a thrombomodulin analogue for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis is claimed, whereas said TM analogue is characterized by exhibiting at therapeutically effective dosages an antifibrinolytic effect. In a further aspect of the invention the use of a thrombomodulin analogue for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis is claimed, whereas the thrombomodulin analogue exhibits one or more of the following features: (i) a binding affinity towards thrombin that is decreased compared to the rabbit lung thrombomodulin, and/or a binding affinity towards thrombin with a kD value of more than 0.2 nM; and/or (ii) a reduced cofactor activity compared to cofactor activity of the TM analogue TMEM388L, (iii) an increased ratio of TAFI activation activity to cofactor activity as compared to the TM analogue TMEM388L.

WO 2011/157283 PCT/EP2010/007632 - 50 In a further aspect of the invention the use of the above described thrombomodulin analogues is claimed, whereas the coagulopathy with hyperfibrinolysis is selected from the group of diseases as follows: haemophilia A, haemophilia B, haemophilia C, von Willebrandt disease (vWD), acquired von Willebrandt disease, Factor X deficiency, parahemophilia, hereditary disorders of the clotting factors 1, 11, V, or VII, haemorrhagic disorder due to circulating anticoagulants or acquired coagulation deficiency. In a further aspect of the invention said thrombomodulin analogues can be used to treat one or more of the bleeding events selected from the group consisting of: intracranial or other CNS haemorrhage, bleeding in joints, microcapillaries, muscles, the gastrointestinal tract, the respiratory tract, the retroperitoneal space or soft tissues The intracranial bleeding event treated with the thrombomodulin analogue of the invention can be an intra-axial, an extra-axial or a subarachnoid haemorrhage (SAH) or an epidural or subdural haematoma. Preferably a patient with SAH is treated, more preferably a aneurismal bleeding after SAH is treated. In a preferred aspect of the invention the thrombomodulin analogue according to the invention is used to treat hyperfibrinolysis after physical trauma, preferably a CNS trauma. A physical trauma as defined herein refers to a body wound or shock produced by sudden physical injury, as from violence or accident. The physical trauma encompasses polytrauma, head injury, chest trauma, abdominal trauma, extremity trauma, facial trauma, genitourinary system trauma, pelvic trauma and soft tissue injury. In a further aspect of the invention the thrombomodulin analogue used for the treatment of coagulopathy with hyperfibrinolysis, is given in combination with a further fibrinolysis inhibitor. In particular a substance which corrects the normal adhesion of platelets can be used such as Etamsylate. Preferably an inhibitor of proteolytic enzymes can be used, which more preferably is an inhibitor of plasmin such as aprotinin. In even more preferred aspect of the invention the further antifibrinolytic drug blocks the lysine-binding site of plasmin, such as epsilon-aminocaproic acid or tranexamic acid. In a preferred aspect of the invention the patients which are treated with the thrombomodulin analogues have anti- factor Vill antibodies. In another aspect of the invention the patients which are treated with the thrombomodulin analogues are further treated with factor Vill, preferably recombinant factor Vill or a recombinant B-domain-deleted factor Vill molecule, more preferably Octocog-alfa or moroctocog-alfa.

WO 2011/157283 PCT/EP2010/007632 -51 In a preferred aspect of the invention the thrombomodulin analogues according SEQ ID NOs: 5 to 11 are given in the above described doses or dose ranges. In a further aspect of the invention the thrombomodulin analogue used for the treatment of coagulopathy with hyperfibrinolysis, is given in combination with factor VIII, preferably recombinant factor VIII a recombinant B-domain-deleted factor VIII molecule, more preferably octocog-alfa or moroctocog-alfa. In one aspect of the invention the thrombomodulin analogue is administered at the time of a bleeding episode of the coagulopathy with hyperfibrinolysis or in advance of an increased bleeding risk, e.g. a surgery or a tooth extraction. In a further aspect of the invention the thrombomodulin analogue is administered to patients that are refractory to blood/plasma transfusion or coagulation factor replacement therapy. In one aspect of the invention the thrombomodulin analogue is administered in multiple doses, preferably once daily, bidaily, or every third, fourth, fifth, sixth or seven days over a total time period of less than one week to four weeks, more preferably as chronic administration. In a preferred aspect of the invention the thrombomodulin analogues according SEQ ID NOs: 5 to 11 are administered according to the administration schemes described above. In a preferred aspect of the invention the thrombomodulin analogue is given as parenteral application, preferable as intravenous or subcutaneous application. In one aspect of the invention the thrombomodulin analogue is given in an amount to yield a plasma concentration in the subject to be treated of less than 5 nM/L, preferably of less than 3nM/L and more preferably of less than 1.5 nM/L. In a further aspect of the invention the thrombomodulin analogue is titrated so that the plasma concentration is between 0.1 nM/L and 5nM/L, preferably between 0.1 nM/L and 3 nM/L. In a preferred aspect of the invention the thrombomodulin analogues according SEQ ID NOs: 5 to 11 are used to yield the above disclosed plasma concentrations. In another aspect of the invention the thrombomodulin analogue is given to the subject to be treated in a dose between 0.1 pg/kg and 140 pg/kg, preferably in a dose between WO 2011/157283 PCT/EP2010/007632 - 52 0.5 pg/kg and 40 pg/kg, more preferably in a dose between 0.5 pg/kg and 4 pg/kg and specifically in a dose between 0.75 and pg/kg (kg refers to kg bodyweight of the subject to be treated). In a further aspect of the invention the thrombomodulin analogue is given in a dose of, 0.75, 1.5, 2.5 or 4.0 pg/kg which equals to a body weight adjusted dose of 0.6, 1, 3, or 4.0 mg/patient. In a further preferred aspect of the invention the thrombomodulin analogues according SEQ ID NOs: 5 to 11 are given in the above described doses or dose ranges. In a more preferred aspect of the invention the thrombomodulin analogue is a soluble TM analogue. In an even more preferred aspect of the invention the thrombomodulin analogue is a human soluble TM analogue. In one aspect of the invention said thrombomodulin analogue comprises at least one structural domain selected from the group containing EGF3, EGF4, EGF5, EGF6, preferably comprising the fragment EGF3-EGF6 and more preferably comprising the EGF domains 1-6. In a further aspect of the invention said thrombomodulin analogue consists of EGF domains EGF1 to EGF6, and more preferably consists of the EGF domains EGF3 to EGF6. In one aspect of the invention the thrombomodulin analogue has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1 or SEQ ID NO:3) and comprises one or more of the following modifications: a) removal of amino acids 1-3 b) M388L c) R456G d) H457Q e) S474A, and terminating at P490. In a further aspect of the invention the thrombomodulin analogue has an amino acid sequence which comprises a sequence of at least 85%, or at least 90% or 95% sequence identity with SEQ ID NO: 2.

WO 2011/157283 PCT/EP2010/007632 -53 In a preferred aspect of the invention the thrombomodulin analogue has an amino acid modification at one or more positions corresponding to natural sequence at (according to SEQ ID NO: 1 or SEQ ID NO:3): ab) 3 5 Asn; ae) as 9 GIn; af) 3'Gln; ag) 3 63Leu; ah) 3 4 Asn; ai) s 8 Tyr; aj) 371 val; ak) 37 4 Glu; al) 376 Phe; am) 3 4 His; an) ' 5 Arg; ba) 38 7 GIn; bb) a8 9 Phe; bc) 39 8 Asp; bd) 400Asp; be) 4o 2 Asn; bf) 403Thr; bg) 40 8 Glu; bh) 411 Glu; bi) 413 Tyr; bj) 41 4 1le; bk) 415 Leu; bl) 41 6 Asp; bm) 41 7 Asp; bn) 4 20 1 e; bo) 4 23 Asp; bp) 424 1ie; bq) 425 Asp; br) 426 Glu; ca) 428 Glu; cb) 429 Asp; cc) 432 Phe; cd) 4 34Ser; ce) 4 MVal; cf) 438 His; Cg) 43 9 Asp; ch) 44 0 Leu; ci) 44 3 Thr; WO 2011/157283 PCT/EP2010/007632 - 54 cj) 44 4 Phe; ck) " 5 Glu; ci) 456 Arg; cm) 45 8 lie; or cn) 46 'Asp. In a further aspect of the invention the thrombomodulin analogue has a modification of the phenylalanine at position 376 according to SEQ ID NO:1 or SEQ ID NO:3, preferably substituted with an aliphatic amino acid, more preferably substituted with glycine, alanine, valine, leucine, or isoleucine and most preferably substituted with alanine. In a further aspect of the invention the thrombomodulin analogue has a modification of one or more of the following amino acids according SEQ ID NO:1 or SEQ ID NO:3: a) 3 7 Gln; b) 3"Met; b) s 9 Phe, whereby the amino acids are deleted, inserted by one or more additional amino acids or preferably substituted. In a further aspect of the invention the thrombomodulin analogue is used in its oxidised form, preferably oxidised with chloramine T, hydrogen peroxide or sodium periodate. In a further aspect of the invention a thrombomodulin analogue is us used, whereas one or more of methionine residues within the TM analogue are oxidised, preferably the methionine residue at position 388 (according SEQ ID NO:1 or SEQ ID NO:3). In another aspect of the invention a method for screening for analogues of thrombomodulin suitable for the treatment of coagulopathy with hyperfibrinolysis is claimed, whereas the thrombomodulin exhibits one or more of the following features: (i) a reduced binding affinity towards thrombin, (ii) a reduced cofactor activity, (iii) an increased TAFI activation activity, comprising the steps of: a) making one or more amino acid substitution of the thrombomodulin sequence (SEQ ID NO:1 or SEQ ID NO:3), preferably of the amino acid positions listed in claim 15; b) comparing the modified analogue with a control molecule, preferable a rabbit lung TM or a soluble human TM analogue with regard to one or more of the following characteristics: ba) binding affinity to thrombin (KD value); WO 2011/157283 PCT/EP2010/007632 -55 bb) cofactor activity; bc) TAFI activation activity or TAFla potential; bd) ratio of TAFI activation activity and cofactor activity; be) effect of protein oxidation; bf) effect on clot lysis in time in an in vitro assay; or bg) effect in a coagulation-associated animal model. In another aspect of the invention a method of treating coagulopathy with hyperfibrinolysis is claimed, comprising administering a therapeutically effective amount of a thrombomodulin analogue according to any of the claims 1 to 20. In another aspect of the invention a thrombomodulin analogue according to SEQ ID NO:2 is claimed which has a modification of the phenylalanine at position 376 (numbering according to SEQ ID NO:1), preferably substituted with an aliphatic amino acid, more preferably substituted with glycine, alanine, valine, leucine, or isoleucine and most preferably substituted with alanine. In a preferred aspect of the invention a thrombomodulin analogue is us used as given in Figure 19 and depicted by the amino acid sequences SEQ ID NO:5 to SEQ ID NO:11. These preferred TM analogues are: 1. A TM fragment extending from aa 4 to aa 490 having the following amino acid exchanges: Phe376Ala, Met388Leu, Arg456Gly, His457Gln and Ser474Ala (equals SEQ ID NO: 5). 2. A TM fragment extending from aa 227 to aa 462 (= EGF1-6) having the following amino acid exchanges: Phe376Ala, Met388Leu, Arg456Gly and His457Gln (equals SEQ ID NO: 6). 3. A TM fragment extending from aa 333 to aa 462 having the following amino acid exchanges: Phe376Ala, Met388Leu, Arg456Gly and His457GIn (equals SEQ ID NO: 7). 4. A TM fragment extending from aa 227 to aa 462 having the following amino acid exchanges: Phe376Ala and Met388Leu (equals SEQ ID NO: 8). 5. A TM fragment extending from aa 333 to aa 462 having the following amino acid exchanges: Met388Ala, Arg456Gly and His457Gln (equals SEQ ID NO: 9).

WO 2011/157283 PCT/EP2010/007632 - 56 6. A TM fragment extending from aa 333 to aa 462 having the following amino acid exchanges: Met388Leu, Arg456Gly, His457Gln and Glu461Ala (equals SEQ ID NO: 10). 7. A TM fragment extending from aa 333 to aa 462 having the following amino acid exchanges: Phe376Ala, Met388Ala, Arg456Gly and His457Gln (equals SEQ ID NO: 11). Definitions As used in the context of the present invention the term "antifibrinolytic effect" shall refer to the ability of a thrombomodulin analogue to prolong the clot lysis time (as described in Example 1) compared to identical assay conditions without addition of the thrombomodulin analogue. The antifibrinolytic effect is due to a prevalence of the antifibrinolytic activity of the TM analogue compared to its profibrinolytic activity. As used herein the term "profibrinolytic effect" shall refer to the ability of a thrombomodulin analogue to significantly reduce the clot lysis time in an in vitro assay (as described in Example 1) compared to identical assay conditions without addition of the thrombomodulin analogue. The terms "significantly reduce" and "significantly prolong" as used herein refers to a prolongation or reduction of the clot lysis time that is significantly different from the basis value at the p= 0.1 level and/or refers to a prolongation or reduction that exceeds 10%, preferably 20%, more preferably 30% and most preferably 40%, 50%, 60%, 70%, 80/o 100%, 150% Or 200%. As used in the context of the present invention the words "treat," "treating" or "treatment" refer to using the TM analogues of the present invention or any composition comprising them to either prophylactically prevent a bleeding event, or to mitigate, ameliorate or stop a bleeding event. They encompass either curing or healing as well as mitigation, remission or prevention, unless otherwise explicitly mentioned. Also, as used herein, the word "patient" refers to a mammal, including a human. As used herein the term "coagulopathy with hyperfibrinolysis" shall refer to a coagulopathy as a disease affecting the coagulability of the blood, whereby a markedly increased fibrinolysis causes, aggravates or prolongs bleeding events. As used in the context of the present invention the term "thrombomodulin analogue" refers to both protein and peptides having the same characteristic biological activity as membrane-bound or soluble thrombomodulin. Biological activity is the ability to act as a WO 2011/157283 PCT/EP2010/007632 -57 receptor for thrombin and increase the activation of TAFI, or other biological activity associated with native thrombomodulin. The term "binding affinity" used herein refers to the strength of the affinity between the thrombomodulin analogue and thrombin and is described by the dissociation constant KD. The KD value for the binding affinity between thrombin and thrombomodulin may be determined by equilibrium methods, (e.g. enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g. BIACORE T M analysis), for example. The binding affinity is preferably analysed using a kinetics assay as described in Example 11 of the present invention. "KD" refers to the relative binding affinity between the TM analogue and thrombin. High KD values represent low binding affinity. The precise assays and means for determining KD are provided in example 11. The term "cofactor activity" as used herein refers to the ability of the thrombomodulin analogues to complex with thrombin and potentiate the ability of thrombin to activate protein C. The assay procedures used to measure cofactor activity are given in Example III of the present invention. The terms "TAFI activation activity" as used herein refers to the ability of the thrombomodulin analogues to complex with thrombin and potentiate the ability of thrombin to activate TAFI. The assay procedures used to measure TAFI activity is given in Example IV of the present invention. "Km" refers to the Michaelis constant and is derived in the standard way by measuring the rates of catalysis measured at different substrate concentrations. It is equal to the substrate concentration at which the reaction rate is half of its maximal value. The "Km" for the TM analogues of the present invention is determined by keeping thrombin concentrations at a constant level (e.g. 1 nM) and using saturation levels of TM (e.g. 100 nM or greater) depending on the KD. Reactions are carried out using increasing concentrations of protein C (e.g., 1-60 pM). Km and kcat are then determined using Lineweaver-Burke plotting or nonlinear regression analysis. "TME" refers to an analogue of TM consisting of the six EFG repeats (amino acids 227 to 462 according to SEQ ID NO:1 or SEQ ID NO:3). "TMEM388L" refers to an analogue of TM consisting of the six EFG repeats (aa 227 to 462) with a substitution of the native methionine at position 388 (based on SEQ ID NO:3) by an leucine residue.

WO 2011/157283 PCT/EP2010/007632 - 58 The term "therapeutically effective amount" is defined as the amount of active ingredient that will reduce the symptoms associated with coagulopathy with hyperfibrinolysis, such as bleeding events. "Therapeutically effective" also refers to any improvement in disorder severity, frequency or duration of incidence compared to no treatment. The term ,,sequence modification" as used in the context of the present invention relates to the modification of a primary amino acid sequence, in particular by amino acid substitution, deletion or insertion. Where not otherwise explicitly defined this term means the substitution of one amino acid by another amino acid, which substantially differs from the first amino acid in terms of its polarity, hydrophilic or hydrophobic property, acidic or basic property, size or aromaticity, respectively. Given the common classes of amino acids, namely classes of acidic, basic, polar, nonpolar, negatively charged, positively charged, aromatic and aliphatic amino acids, the concept of sequence modifications as of the invention preferably requires a substitution of one amino acid with another amino acid of a different class of amino acids. In a preferred aspect an amino acid is selected as a substituent which has an "opposite characteristic" of the amino acid to be substituted. The subsequent amino acid (aa) substitutions are particularly suggested: acidic aa vs. basic aa, polar aa vs. nonpolar aa, negatively charged aa vs. positively charged aa, aromatic aa vs. aliphatic aa. A particular embodiment of the sequence modification of the present invention is the substitution of an amino acid by an aliphatic amino acid such as glycin, alanine, valine, leucine and isoleucine, most preferred is the substitution with alanine. The sequence modification as of the invention can also include a substitution with a non-natural amino acid. A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. The term "non natural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally-occurring amino acids that are not naturally-encoded include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and 0 phosphotyrosine, ornithine, taurine, The sequence modification includes also an attachment of other groups to the amino acid. These groups include acetate, phosphate, various lipids and carbohydrates, which preferably changes the chemical nature of the amino acid. The sequence modification further includes oxidation or reduction of the respective amino acid, preferably of a Met or Cys residue.

WO 2011/157283 PCT/EP2010/007632 -59 PAGE LEFT INTENTIONALLY BLANK WO 2011/157283 PCT/EP2010/007632 -60 I. EXAMPLE Clot lysis assay in human plasma Using a model of in vitro clot lysis the ability of soluble thrombomodulin (Solulin) to decrease or increase the clot lysis time in mixtures of normal plasma and Factor VIII deficient plasma was tested. 1. Test system Within the plasma compositions the clotting was initiated in vitro by mixing thrombin (Factor Ila), calcium chloride and phosphatidylcholine/phosphatidylserine (PCPS) vesicles. Time course of coagulation and fibrinolysis were determined with a turbidity assay, and the "TAFla potential" using a functional assay. 2. Experimental procedures Materials. Thrombin and fibrinogen were prepared as described in Walker et al. (J.Biol. Chem. 1999; 274: 5201-5212) with one exception: for the fibrinogen preparation, the solution was made to 1.2% PEG-8000 instead of 2% PEG-8000 by the addition of 40% (w/v) PEG-8000 in water, subsequent to E-alanine precipitation. This change in protocol allowed for a greater yield of fibrinogen. QSY-FDPs (fibrin degradation products that are covalently attached to the quencher, QSY9 C5-maleimide) and TAFla standards used in the TAFla assay were prepared as described (Kim et al., 2008; Anal. Biochem 372: 32-40; Neill et al., 2004; Anal. Biochem. 330: 332-341) and recombinant human Pg (S741C) and the fluorescein derivative (51AF-Pg) were prepared as described by Horrevoets et al. (J. Biol. Chem 1997; 272: 2176-2182). S525C prothrombin was purified and fluorescently labelled with 5-iodoamidofluorescein (51AF) as previously described by Brufatto et al. (J. Biol. Chem. 2001; 276: 17663-17671). QSY9 C5-maleimide and 5-iodoamidofluorescein were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Plasmin was purchased from Haematologic Technologies Inc. (Essex Junction, VT, USA) and recombinant human soluble thrombomodulin (Solulin; sTM) was provided from Paion Deutschland GmbH (Aachen, Germany). Normal human pooled plasma (NP) was obtained from healthy donors at the blood bank in the Kingston General Hospital (KGH) in Kingston, Ontario, Canada, and FVIII-deficient plasma (FVIII-DP) was purchased from Affinity Biologicals, Inc. (Hamilton, ON, Canada). TAFI-deficient plasma (TDP) was prepared by affinity chromatography of normal plasma on a column of immobilized anti-human TAFI monoclonal antibody, as described by Schneider et al., (J. Biol. Chem. 2002; 277: 1021-1030). The plasmin inhibitor D-Val-Phe-Lys chloromethyl ketone (VFKck), the thrombin inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPAck) and potato tuber carboxypeptidase inhibitor (PTCI) were purchased from Calbiochem (San Diego, CA, USA). Tissue-type plasminogen activator (Activase; tPA) was purchased from the pharmacy at KGH (Kingston, ON, Canada). All other reagents were of analytical quality.

WO 2011/157283 PCT/EP2010/007632 -61 3. Methods. Clot lysis assays and the preparation of samples to determine the extent of TAFI activation. FVIII-DP was mixed with NP so that the final percentage of NP was 0, 1, 6, 10, 50 or 100% (0-100% NP). Before mixing, each plasma was diluted to an optical density of 32 and added to an equal volume of a solution containing 1.5 nM tPA, 40 pM PCPS and 20 mM CaC1 2 in the presence or absence of 20 nM thrombin (final concentrations: 0.75 nM tPA, 20 pM PCPS, 10 mM CaC1 2 , ±10 nM thrombin) and the samples were divided into multiple Eppendorf tubes and placed in a 37 0 C water bath. Clotting and lysis were stopped in these tubes at various time points by the addition of 10 pM PPAck and 10 pM VFKck to selectively inhibit thrombin and plasmin, respectively. The samples were mixed vigorously, then centrifuged for 30 s at 16 000 g (room temperature) and immediately placed on ice to prevent thermal inactivation of TAFla. The supernatant of each sample was serially diluted by 5-fold with TAFI-deficient plasma and TAFla was measured using a functional assay described by Kim et al. (Anal. Biochem 2008; 372: 32-40). Identical experiments were conducted in a covered, 96-well plate and the turbidity was monitored at 400 nm over time using a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) to determine the timing of coagulation and fibrinolysis. Similar experiments were conducted in the presence or absence of soluble thrombomodulin (0-100 nM) at 4 tPA concentrations (0.25, 0.75, 1.5 and 3 nM) to determine the effect of sTM on TAFI activation and lysis times. These experiments were also conducted in the presence of 5 pM PTCI to show the TAFIa dependent prolongation of lysis in normal and FVIII-deficient plasma. Determination of the time course of prothrombin activation in normal and FVIII deficient plasma. Normal and FVIII-deficient plasmas (0-100% NP) were supplemented with the prothrombin derivative (51AF-Il; 300 nM final) as well as 20 pM PCPS and 10 mM CaC1 2 in the presence of 10 nM thrombin to initiate clotting. These experiments were conducted in an opaque, plastic-covered 96-well plate. A SpectraMax GeminiXS (Molecular Devices, Sunnyvale, CA, USA) was used to monitor fluorescence intensities over time at 37 0 C with excitation and emission wavelengths of 495 nm and 535 nm, respectively, employing a 530-nm emission cut-off filter. Fluorescence was normalized (0-1) to reflect the baseline and maximal fluorescence, which correlates with full prothrombin activation. Determination of the TAFla potential. The area under the TAFla plots was chosen as a parameter to quantify the effect of TAFla over the course of the experiments. This parameter was designated the "TAFla WO 2011/157283 PCT/EP2010/007632 - 62 potential" by analogy with the "thrombin potential" defined by Hemker et al. (Thromb. Haemost. 1993; 85: 5-11). TAFla potential, like thrombin potential, is proportional to the amount of substrate cleaved and is explained mathematically, as follows: dS [TAFIa] [S], () where dS/dt is the rate of substrate consumption and S is the substrate. If S is constant (i.e. limited consumption of S), dS k d= -S /ft [TAFIa] (2) dS=-S k [TAFIa] di (3) For some interval 0 to t, S(t) ( dS =S [TAFla] d1 (4) S(O) 0 Realizing that the integral on the right in equation (4) is the area under the TAFla plot, AS(t) = -S -(area under curve) (5) Km AS(t) = -S- (TAFla Potential) (6) Km 4. Results Olot lysis time is increased by addition of normal plasma to FVIII-deficient plasma. Clotting was initiated with 10 nM Factor lla, 10 mM CaCl 2 and 20 pM PCPS vesicles to create a model where the clot structure is insensitive to the FVIII concentration. Because the clot structure is similar regardless of the FVIII concentration, the effect of FVIII on tPA-dependent (0.75 nM) clot lysis can be determined. Using this lysis model, lysis times increased as the percentage of normal plasma increased. Figure 1 shows the clot lysis profiles for FVIII-DP with 0 - 100% added normal plasma and the lysis times are summarized in Fig. 1 (inset). In FVIII-DP the lysis time is 37 min and can be increased by approximately 50% by the addition of normal plasma.

WO 2011/157283 PCT/EP2010/007632 - 63 10% normal plasma is sufficient to restore clot lysis in FVIII-DP. At 10% normal plasma the shortened lysis time associated with FVIII-DP has been corrected to that observed in normal plasma (see Fig. 1, inset). 50% of the TAFla potential is sufficient to restore clot lysis in FVIII-DP. TAFI activation was measured in normal, FVIII-deficient and mixed plasmas to quantify the effect of FVIII on the time course of activation. A functional assay was used to measure TAFla over the time course of clotting and lysis and the results are presented in Figure 2. When thrombin, calcium ions and PCPS were used to initiate clotting in FVIII-DP, approximately 30 pM TAFla was measured after 5 min. As the percentage of normal plasma increased so too did the peak concentration of TAFla. Although the lysis time was corrected by supplementing FVIII-DP with 10% normal plasma, this was not sufficient to fully correct TAFI activation. By calculating the area under the TAFla time course plots (Fig. 2A) it was determined that approximately the same TAFla potential (Fig. 2B) was achieved over the first 50 min in normal plasma and 50% normal plasma (16 800 pM mins and 14 100 pM mins, respectively) but FVIII-DP plasma mixed with 10% normal plasma had a TAFla potential of only 50% of the TAFla potential in normal plasma. There is a strong correlation between lysis time and TAFla potential. In order to quantify the relationship between lysis time and TAFI activation over the range 0-100% FVIII, log lysis time vs. log TAFla potential was plotted (Fig. 2B, inset). As expected, the data show a strong positive correlation between lysis time and TAFla potential in plasma containing 0-100% FVIII. The TAFI activation profile in Fig. 2A can be rationalized by analyzing prothrombin activation in plasma (Fig. 3) because thrombin is the activator of TAF. The general trend is that as the percentage of normal plasma increased, the rate of prothrombin activation also increased (which can be determined by examining the slope of the curve in Fig. 3). An exception occurs with normal plasma. In normal plasma the rate of prothrombin activation is lower than in FVIII-DP mixed with 50% normal plasma. While the rate is slower in normal plasma, prothrombin activation persists for about twice as long as in FVIII-DP mixed with 50% normal plasma. In every experiment, the timing of prothrombin activation corresponds well with TAFI activation. Normal plasma was also clotted using calcium ion and PCPS, without added thrombin. Calcium-induced coagulation does not occur immediately; it takes approximately 15 min for the clot to form in normal plasma. At this time, prothrombin activation enters the propagation phase and as a result, TAFI is activated. The extent and timing of TAFI activation with respect to clot formation is the same whether clotting is initiated in the presence or absence of added thrombin, which suggests that TAFI activation is a result of thrombin generated in situ and not of WO 2011/157283 PCT/EP2010/007632 -64 thrombin added to induce clotting. In the presence of thrombin there was a TAFla potential of 16,800 pM min compared with 14,150 pM min in the absence of thrombin. Soluble thrombomodulin prolongs clot lysis in normal and FVIII-deficient plasma. In normal plasma, peak TAFla levels and TAFla potential increased from 600 pM and 16 800 pM min, respectively, in the absence of sTM to approximately 6000 pM and 150,000 pM min, respectively, in the presence of 10 nM sTM. This increase in TAFI activation resulted in a 70% increase in the lysis time. The effect of 10 nM sTM on the relative prolongation of lysis in FVIII-DP was similar to normal plasma in that lysis was prolonged by 65% when FVIII-DP was clotted and lysed in the presence of sTM. In the presence of 10 nM sTM, 750 pM TAFla was present at peak TAFla concentration compared with 30 pM in the absence of sTM. In the time from clot initiation to the clot lysis time the TAFla potential was measured to be 12 800 pM min in the presence of 10 nM sTM compared with 600 pM min in the absence of sTM. The increase of clot lysis time in normal and FVIII-deficient plasma depends on tPA and sTM concentrations. The effect of TAFI activation on lysis time was analyzed over a range of tPA and sTM concentrations to determine if the lysis defect in FVIII-DP could be corrected by stimulating TAFI activation. The lysis times summarized in Fig. 4 are relative to lysis times from similar experiments containing PTCI, which is an inhibitor of TAFla. In the presence of PTCI, there is no functional TAFla so the relative lysis times presented in Fig. 4 are representative of TAFla-dependent prolongation of lysis. At the lowest concentration of tPA (0.25 nM), the maximal TAFla-dependent prolongation of lysis (2 fold) was observed when 1 nM sTM was added to normal plasma. Supplementing FVIII-DP with sTM caused a dose-dependent prolongation of the lysis time (Fig. 4). When 100 nM sTM was added to FVIII-DP the lysis time was fully corrected to that seen in normal plasma. As the tPA concentration increased, a higher concentration of sTM was required to get maximal TAFla-dependent prolongation of lysis. For example, when 1.5 nM tPA (Fig. 4) is present, 25 nM sTM is required to maximize the TAFla dependent prolongation of lysis in normal plasma and 100 nM sTM is required in FVIII DP. Also, as tPA is increased in these clot lysis experiments TAFla appears to have a much greater effect on lysis time (up to 5.2-fold at 1.5 nM tPA compared with 2.3-fold at 0.25 nM tPA). It appears that as the tPA concentration is increased, the concentration of sTM required to get any TAFla-dependent prolongation of lysis also increases. At 0.25 nM tPA, no sTM was required to get prolongation of lysis in normal plasma whereas 25 nM sTM was required to get prolongation of lysis when 3 nM tPA (Fig. 4) was added to normal plasma. In order to show how the actual lysis times are affected by tPA and sTM the lysis times in TAFla inhibited normal and FVIII-deficient plasma are presented in Table 1.

WO 2011/157283 PCT/EP2010/007632 -65 Thrombomodulin very substantially promotes TAFI activation and prolongs lysis in both normal and FVill-deficient plasma. In normal plasma TAFI activation is shown to be significantly increased in the presence of 10 nM sTM (*; 6000 pM TAFla at its peak level) compared to the absence of sTM (o; 600 pM TAFla; see Figure 5 A). The accompanying clot-lysis profile reveals that the addition of 1OnM sTM resulted in a 70% increase in the lysis time. In FVIII-DP supplemented with 1OnM sTM TAFla was measured to be 750 pM at its peak compared to 30 pM in the absence of sTM (see Figure 5 B). The increase in TAFI activation resulted in a 60% prolongation of lysis compared to FVIII-DP lacking sTM. II. EXAMPLE Analysis of binding affinity between thrombin and thrombomodulin Using a fluorescent kinetics assay the affinity expressed as a K 0 value was determined for the binding between thrombin and the thrombomodulin analogue. 1. Test system The affinity for the binding between thrombin and the thrombomodulin analogue was determined using a fluorescent kinetics assay and expressed as a Ko value. 2. Experimental procedures Materials. The human thrombin was isolated from plasma as described by Bajzar et al. (J. Biol. Chem. 1995; 270: 14477-14484). Recombinant soluble thrombomodulin (Solulin) was obtained from PAION Deutschland GmbH (Aachen, Germany). All other reagents were obtained from Sigma in analytical quality. Methods. Measurement of the Binding of Thrombin to Thrombomodulin and TAFI The binding of thrombin to thrombomodulin was measured as an equilibrium binding assay. A solution containing thrombin (20 nM), thrombomodulin (1.54 pM), and DAPA (20 nM, dansylarginine N-3-(ethyl-1,5-pentanediyl)amide, a fluorescent, reversible thrombin inhibitor) in 0.02 M Tris-HCI, 0.15 M NaCl, 5.0 mM CaC1 2 , 0.01% Tween 80, pH 7.4, was added in small successive aliquots to an otherwise identical solution that lacked thrombomodulin. The additions were performed in a cuvette fitted with a magnetic stirrer in the sample compartment of a Perkin-Elmer model LS50B spectrofluorimeter. Intensity values were continuously recorded with excitation and emission wavelengths of 280 and 545 nm, respectively. A 430-nm cut-off filter was used in the emission beam. The data were analyzed as follows. The intensity of WO 2011/157283 PCT/EP2010/007632 -66 fluorescence, /, was assumed to be the sum of intensities from thrombin-DAPA (T-D) and thrombin-thrombomodulin-DAPA (T-TM-D). That is, / = if[T-D] + i 2 -[T-TM-D], where i1 and i2 are the coefficients of fluorescence for T-D and T-TM-D (since excitation was at 280 nm, the emission from free DAPA was negligible). Because TM does not appreciably alter the Km for either protein C activation or TAFI activation (see Bajzar et al., 1996; J. Biol. Chem. 271: 16603-16608), it can be assumed that it does not alter the affinity of the thrombin-DAPA interaction. Thus [T-D] = (M + [T-D])/(1 + KDAPA/[DAPA]) and [T-TM-D] = ([T-TM] + [T-TM-D])/(1 + KDApA/[DAPA]), where KDAPA is the dissociation constant for the thrombin-DAPA interaction. Therefore, / = i1-([T] + [T-D])/(l + KDApA/[DAPA]) + i 2 ([T-TM] + [T-TM-D])/(l + KDAPA/[DAPA]). If f and b are defined as the fractions of thrombin, respectively, free and bound to thrombomodulin, and [T]o is the total concentration of thrombin, then f = ([T] + [T-D])/[To, b = ([T-TM]+[T-TM-D])/[T]o and f + b = 1. The fluorescence intensity then is given by / = i1-f[T]o/(1 + KDApA/[DAPA]) + i2-b[7]o/(1 + KDApA/[DAPA]). If lo is defined as the initial intensity when no thrombomodulin has been added, then f = 1 and lo = i1[T]o/(1 + KDApA/[DAPA]). Similarly, if /max is defined as the intensity upon saturation of thrombin with thrombomodulin, then b = 1 and /max = i 2 Mo/(1 + KDApA/[DAPA]). Thus, / = 1 0 -f+ /max-b. Substituting 1 - b for f then gives: / = lo + (/mx - 1 0 )-b or A/ = Almax-b. Normalizing to the initial intensity gives (A///o) = (Almax//o)-b. If DAPA binds T and T-TM with equal affinity, then TM binds T and T-D with equal affinity. Therefore, with KTm defined as the dissociation constant for the thrombin thrombomodulin interaction, [T][TM] = KT[T-TM]; [T-D][TM] = KTM[T-TM-D]; and ([T] + [T-D])-[TM] = KTM([T-TM] + [T-TM-D]). The last expression is identical to f-[TM] = KTm-b. Since f= 1 - b and [TM] = [TM]o - b-[T]o, where [TM]o is the total thrombomodulin concentration, the following equation is obtained: (1 - b)([TM]o b-[T]o) = KTm-b. This is a quadratic equation in b, which when solved and substituted in the expression above for (Al/ 0 ) gives the equation: (A//lo) = (A /max/o)-0.

5 -(Km + f]o + [TM]o - ((KTM + [T]o + [TM]o) 2 - 4-[T]o-[TM] 0

)

1 2 ). This latter equation expresses the relationship between fluorescence intensity values, the nominal concentrations of thrombomodulin and thrombin, the dissociation constant for the thrombin-thrombomodulin interaction, and the fluorescence intensity increment that signals the interaction of thrombomodulin with thrombin-DAPA. Intensity data were fit to the above equation by nonlinear regression analysis, with [TM]o as the independent variable and KTM and Almax as best-fit parameters.

WO 2011/157283 PCT/EP2010/007632 -67 3. Results Thrombin binds to soluble thrombomodulin with an affinity of KD= 23 ± 14nM. The binding of thrombin to soluble thrombomodulin was measured by perturbation of the fluorescence of DAPA. As depicted in Figure 6, the titration curve showed a increase of the relative fluorescence for the concentration range of soluble thrombomodulin between 0 and 75nM. The data analysis revealed that thrombin binding to soluble thrombomodulin was characterized by KD= 23 ± 14nM. Ill. EXAMPLE Analysis of cofactor activity for mutated thrombomodulin analogues Using a fluorescent kinetics assay the affinity expressed as a KD value was determined for the binding between thrombin and the thrombomodulin analogue. 1. Experimental procedures Materials and Methods. The ability of TM mutants to act as cofactor for thrombin-mediated activation of protein C was assayed directly in the shockates. Recombinant human protein C was from Dr. John McPherson, Genzyme Corp., Framingham, MA., and was purified as described (BioTechnology 1990; 8: 655-661). Twenty five pl of each shockate was mixed with equal volumes of recombinant human protein C (final concentration of 0.3 pM) and human alpha thrombin (Sigma Chemicals, St. Louis, MO) at a final concentration of 1 nM in a microtiter plate. All reagents used were diluted in 20 mM Tris, pH7.4/100 mM NaCI/3.75 mM CaCl 2 /0.1 % NaN 3 (wN) containing 5 mg/ml bovine serum albumin. Mixtures were incubated for 1 hr at 37*C and the reaction was terminated by addition of 25 pl of hirudin at 800 units/ml (Sigma Chemicals, St. Louis, MO). The amount of activated protein C was determined by addition of 100 pl of chromogenic substrate D valyl-L-leucyl-L-arginine-p-nitroanilide (S-2266) (1mM). The change is measured by the absorbance at 405 nm with time using a plate reader. Data is recorded as milliOD unit/min and determined for each sample by measuring the absorbance every 10 seconds for 15 minutes using a Molecular Devices plate reader. All assays contained triplicate shockate samples each of DH5 alpha cells transfected with either pSELECT-1 vector (no TM), pTHR211 (wild type) or pMJM57 (pTHR211 with methionine at 388 altered to leucine), as internal controls. Cofactor activities of TM mutants were expressed as mean of that obtained for pMJM57. Statistical Analysis.

WO 2011/157283 PCT/EP2010/007632 - 68 Each mutant was assayed for activity at least twice (three times for those mutants for which only two positive clones were isolated), and all the data were included in the determination of the significance of difference using Student t-Test. Coefficient of variation between plates was 16.7% (n=18). Western blot analysis E. coli shockates were run in 10% Tris-tricine SDS PAGE under reduced conditions according to the manufacturer's specifications (Novex Inc., San Diego, CA). Reduced and alkylated samples were prepared by boiling shockates in sample buffer (62.5 mM Tris, pH6.8, 2% SDS, 10% glycerol, 0.0025% bromophenol blue) containing 10 mM dithiothreitol for 10 minutes, followed by incubation with 50 mM iodoacetamide. Proteins were transferred to nitrocellulose filter in transfer buffer (192 mM glycine, 25 mM Tris, pH8.3, 20% methanol) at 4*C. The nitrocellulose filter was blocked with a blocking buffer (1% bovine serum albumin in 10 mM Tris, pH7.5, 0.9% NaCl, 0.05% NaN 3 ), and then incubated with mouse polyclonal antiserum (raised against reduced and alkylated EGF domain of human thrombomodulin) in the blocking buffer. After washing with a washing buffer (10 mM Tris, pH7.5, 0.9% NaCI, 0.05% NaN 3 , 0.05% Tween 20), the filter was incubated with biotinylated goat anti-mouse IgG antibodies in the blocking buffer containing 0.05% Tween 20. Proteins were detected using the Vectastain ABC solution (Vector Laboratories, Burlingame, CA) and ECL detection system (Amersham Corporation, Arlington Heights, IL) according to the manufacturer's specifications. IV. EXAMPLE Analysis of thrombomodulin analogues for TAFI and protein C activation Using a fluorescent kinetics assay the affinity expressed as a KD value was determined for the binding between thrombin and the thrombomodulin analogue. 1. Experimental procedures Proteins and Reagents. Truncated forms of thrombomodulin comprising Solulin (residues 4-490), TME (residues 227-462), TMEC-lop 3-6 (residues 333-462), and TMEi4-6 (residues 345-362) were prepared as described by Parkinson et al. (Biochem. Biophys. Res. Commun. 1992; 185: 567 - 576). Sf9 cells were transfected with the TM constructs, and the proteins were isolated from the media by a combination of chromatography procedures utilizing anion exchange, gel filtration, and thrombin affinity. Purity, assessed by SDS- WO 2011/157283 PCT/EP2010/007632 - 69 polyacrylamide gel electrophoresis and silver staining, was 95% or greater. Human plasma TAFI was isolated as described by Bajzar et al. (J. Biol. Chem. 1995; 270: 14477 - 14484). Human protein C and thrombin were prepared as described by Bajzar and Nesheim (J. Biol. Chem. 1993; 268: 8608 - 8616). The thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was synthesized as described by Nesheim et al. (Biochemistry 1979; 18: 996 - 1003). Point mutants resulting from alanine scanning were generated from the TMEM388L construct. Proteins were expressed in Escherichia coli. The procedures and preparation of periplasmic extracts have been described by Nagashima et al., (J. Biol. Chem. 1993; 268: 8608 - 8616). HEPES, the basic carboxypeptidase substrate hippuryl-arginine, cyanuric chloride, and 1,4-dioxane were obtained from Sigma. All other reagents were of analytical quality. Measurement of the rates of Protein C and TAFI activation with point mutants of thrombomodulin analogues. For the activation of TAFI, a 20-pl aliquot of each periplasmic extract was preincubated with thrombin (13 nM final) in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 for 5 min at room temperature. The mixtures were then incubated with purified recombinant TAFI (18 nM final) and a substrate, hippuryl-arginine (1.0 mM final), in a total volume of 60 pl for 60 min. The amount of activated TAFI was quantitated by measuring the hydrolysis of hippuryl-arginine to hippuric acid, followed by conversion of hippuric acid to a chromogen with 80 pl of phosphate buffer (0.2 M, pH 8.3) and 60 pl of 3% cyanuric acid in dioxane (w/v). After thorough mixing, absorbance of the clear supernatant was measured at 382 nm. The amount of thrombin-dependent activation of TAFI was calculated by subtracting the background absorbance produced in the absence of thrombin for each mutant. Activation of protein C by TMEM388L-alanine mutants was assayed as follows. All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI, pH7.4, 100 mM NaCl, 2.5 mM CaCl 2 , 0.5 % BSA). Samples and TM standards (0-1 nM) were incubated for 60 min in 60 pl total volume at 37 0 C in a 96-well plate with 0.5 pM protein C and 1 nM thrombin to generate APC before being quenched with 20 pl of hirudin (0.16 U/pl, 570 nM). The amount of APC formed was determined by monitoring the hydrolysis of S-2266 (100pi of 1 mM) at 1-min intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of activated protein C / min (37*C). All assays contained extracts of DH5a cells transfected with either pSelect-1 vector (no TME), wild-type TME(M388), or TME(M388L) as internal controls. Cofactor activities of TME(M388L) alanine mutants were expressed as percentages of the activity of TME(M388L). Each TM mutant was assayed for both protein C and TAFI activation in duplicate using three independent preparations of extracts. 2. Results WO 2011/157283 PCT/EP2010/007632 - 70 The results obtained with the TM mutants (Figure 7) indicate that five out of eigth mutants have a substantially reduced cofactor activation. From these five mutants four mutants show also a concomitant reduced activation activity of TAFI. Only the mutation at F376A resulted in a profound loss in protein C activation, but only in a modest reduction in TAFI activation. Intriguingly, the difference in importance of Phe 376 for TAFI and protein C activation suggests the requirements for thrombomodulin structure are more constrained when protein C is the substrate of the thrombin-thrombomodulin complex. V. EXAMPLE Analysis of Met-specific TM mutants for protein C activation with regard to oxidation. Using specific methionine mutants of thrombomodulin analogues the role of these residues for cofactor activation also with respect to protein oxidation was analysed using a protein C activation assay. 1. Experimental procedures Proteins and Reagents. Human recombinant protein C was from Genzyme Corp. (Boston, MA). Bovine thrombin was from Miles Laboratories Inc. (Dallas, TX). D-Val-Leu-L-Arg-p-nitroanilide was prepared as described by Glaser et al. (Prep. Biochem. 11975; 5: 333 - 348). Human alpha-thrombin (4,000 NIH U/mg), bovine serum albumin (fraction V) and chloramine T were from Sigma Chemical Co. (St. Louis, MO). Expression of TME (Sf9) All procedures were performed at 4*C. The DNA sequence encoding the six EGF-like repeats of TM (amino acids 227 - 462) was linked to the signal sequence of the insect protease, hypodermin A, and the hybrid gene placed under control of the polyhedron gene promoter in the baculovirus shuttle vector pTMHY101. Recombinant virus was generated using standard techniques. Mutant analogues described were prepared by use of a mutator site-specific mutagenesis kit (Stratagene, Inc., La Jolla, CA) and virus was prepared for expression in the baculovirus system by the same methods. Purification and oxidation with Chloramine T WO 2011/157283 PCT/EP2010/007632 - 71 Growth media containing secreted mutants of TME (Sf9) was clarified by centrifugation, lyophilized and redisolved in 1:10 volume of 0.2% NEM-Ac, pH 7, and 0.008% Tween 80. Aliquots were treated with either 5 pl of H 2 0 or 5 pl of 100 mM chloramine T; incubated for 20 min at room temperature; oxidant removed by dilution; desalted on NAP-5 columns (20 mM Tris-HCI, 0.1 M NaCI, 2.5 mM CaCl 2 , 5 mg/ml BSA, pH 7.4; Pharmacia Inc.); and assayed for activation of protein C as follows. Measurement of TM cofactor activity (APC assay) All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI, pH7.4, 100 mM NaCl, 2.5 mM CaC1 2 , 0.5 % BSA). Samples and TM standards (0-1 nM) were incubated for 60 min in 60 pl total volume at 37*C in a 96-well plate with 0.5 pM protein C and 1 nM thrombin to generate APC before being quenched with 20 pl of hirudin (0.16 U/pl, 570 nM). The amount of APC formed was determines by monitoring the hydrolysis of S-2266 (100 pl of 1 mM) at 1-min intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of activated protein C/min (37*C). 2. Results Reduced cofactor activity of TM by oxidation of Met388. Mutant and wild-type TME (Sf9) were expressed in insect cells, treated with chloramines T, assayed for cofactor activity and the results compared (Table 2). When TME is treated with an oxidant such as chloramine T it looses approx. 85% of its cofactor activity (see Table 2). Site-specific mutations of Met 291 and Mets 8 a demonstrate that inactivation of TME (Sf9) is due to oxidation of a single methionine. Derivatives that retain Meta8s were inactivated by chloramine T to a similar extent (>80%) whereas the Met388Leu mutant was resistant. Mutants in which Met 291 is replaced were active but were not resistant to oxidative inactivation. VI. EXAMPLE Analysis of TM analogues with mutations of the interdomain loop between EGF4 and EGF5 (Gin387, Met388, Phe389) Using specific mutants of thrombomodulin analogues the role of these residues and their oxidation was analysed using a protein C activation assay. 1. Experimental procedures Plasmid constructions. A thrombomodulin fragment consisting of only the EGF-like domains (TME) was expressed in E.coli as follows, DNA fragment coding for TME WO 2011/157283 PCT/EP2010/007632 - 72 (residues 227-462) of full length TM was obtained by polymerase chain reaction from human genomic DNA using primers 5' CCGGGATCCTCAACAGTCGGTGCCAATGTGGCG-3' and 5' CCGGGATCCTGCAGCGTGGAGAACGGCGGCTGC-3'. This fragment was placed under the control of a p-lactamase promoter and signal sequence in pKT279. An EcoRV-Bgill fragment of the resultant plasmid and a Scal-Sacl fragment of pGEM3zf containing the f1 origin of replication was then inserted into the pSelect-1 vector at the EcoRV-BamH/ and Scal-Sacl site, respectively, to construct E.coli expression plasmid pTHR21 1. Plasmids coding for TM mutants at position 387, 388, or 389 were constructed using a site-directed mutagenesis procedure described in the altered sites in vitor mutagenesis kits with a single stranded pTHR211 DNA template. Each primer of the site-specific mutation was confirmed by restriction analysis. To measure cofactor activity of the mutants, the individual E.coli cultures expressing mutant proteins were centrifuged, washed, and the cell pellets incubated (10 min, 4*C) in 20% sucrose, 300mM Tris-HCI, pH 8.0, 1 mM EDTA, 0.5mM MgCI 2 . Shockates were prepared by centrifugation of cell pellets treated with 0.5 mM MgC 2 (10 min, 4*C) and assayed in the APC assay. The data are an average of the results from each of three independent clones. Measurement of TM cofactor activity (APC assay) All samples and reagents were diluted in APC assay diluent (20 mM Tris-HCI, pH7.4, 100 mM NaCI, 2.5 mM CaC1 2 , 0.5 % BSA). Samples and TM standards (0-1 nM) were incubated for 60 min in 60 pl total volume at 37*C in a 96-well plate with 0.5 pM protein C and 1 nM thrombin to generate APC before being quenched with 20 pl of hirudin (0.16U/pi, 570 nM). The amount of APC formed was determined by monitoring the hydrolysis of S-2266 (100 pl of 1 mM) at 1-min intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, CA). 1 U of activity generates 1 pmol of activated protein C/min (37 0 C). 2. Results Reduced cofactor activity of TM by mutation of the interloop domain. Using the site-directed mutagenesis, TM mutants that have either an altered amino acid, a deletion or an insertion at positions 387, 388, or 389 were expressed (Figure 8). The cofactor activity of the TM mutants are an average obtained from three independent clones and are expressed as a percentage of the activity found for TME(Sf9)WT. Gel scans on the Western blots were performed using a polyclonal antibody against TM for all new mutants at position 388 and for selected mutants at position 387. These scans gave approximately equivalent amounts of TM, indicating that expression differences cannot account for the observed activity differences. In addition, in an independent substitution at position 387 (Figure 8A), 388 (Figure 8B), WO 2011/157283 PCT/EP2010/007632 -73 389 (Figure 8C), or insertions and deletions anywhere in the inter-domain loop (Figure 8D) result in analogues which generally are poorer cofactors in the APC assay then wildtype TME. Analogues where Glnsa 7 is replaced by Thr, Met or Ala retain >70% cofactor activity, but substitution with Glu reduces this to 58% of control, and all other amino acids result in >50% loss. Only the substitution of Mets 8 a with Leu results in a substantially higher cofactor activity (1.8-fold) than wildtype. All other substitutions of Meta 8 8 except Gin and Tyr resulted in >50% loss of cofactor activity. TM cofactor activity is less sensitive to amino acid replacement of Phe33 9 and nine of the point mutants at this position retain >70% of the activity found in the control. Pro or Cys substitution at any positions reduced the activity to >10% except for Met388Pro which retained 30% activity. Varying the length of the interdomain loop between EGF4 and EGF5 by either deleting individual amino acids or inserting an Ala into each of the four possible positions resulted in mutants with less than 10% of the activity of wild type TME. Vil. EXAMPLE Analysis of fibrinolysis in canine haemophilic plasma Using models of in vitro clot lysis and thrombelastography the ability of soluble thrombomodulin (Solulin) to decrease or increase the clot lysis time was tested in whole blood or plasma of dogs with haemophilia A. Materials and methods Clot Lysis assay The clot lysis assay was performed as described in Example I of the present invention. Thromboelastrography Thrombelastography was performed as described in Prasad et al., 2008; Blood 111: 672-679. Canine haemophilic whole blood (± Factor Vill neutralizing antibodies) (320pL) was added to channels of a Haemoscope TEG@ 5000 (Haemonetics Corp. Braintree, MA) containing a 40 pL solution of 90 nM thrombin, 9 nM tPA and 0 - 390 nM sTM. After mixing thoroughly, the pin was seated and coagulation and fibrinolysis was monitored continuously. The Haemoscope TEG@ 5000 allows for measurement of the clot time, clot kinetics, clot strength and clot stability (fibrinolysis) by measuring the torque on a wire which is connected to the clot through the pin. As a clot forms, the torque on the pin increases and is represented by an increase in the amplitude (output). Similarly, during fibrinolysis, degradation of the clot results in a decrease of the torque and a decrease in the amplitude.

WO 2011/157283 PCT/EP2010/007632 - 74 The inhibitory antibodies (inhibitors) developed by some of the dogs at the Queen's University haemophilic dog colony have been described previously by Giles et al., 1984; Blood 63:451-456 and by Tinlin et al., 1993; Thromb Haemost 69: 21-24. The haemophilic dog plasma with inhibitors was drawn from a dog with an inhibitor titre of >150 Bethesda Units (>5 B.U. is considered untreatable with Factor VIII replacement therapy). Results Solulin prolongs clot lysis even at a concentration of 500nM. In canine hemophilic plasma Solulin dose-dependently increased the clot lysis time with an approx. 2-fold prolongation in the presence of 25 nM Solulin, reaching a plateau of approx. 9-fold prolongation at 200 - 500 nM Solulin, which represents the highest dose tested (see Figure 9). Conclusion: The pronounced antifibrinolytic effect of Solulin in hemophilic plasma even at very high concentrations strongly argues for an efficacious and safe use of thrombomodulin analogues in Haemophilia. Solulin prolongs clot lysis in whole blood from a canine with haemophilia A. In the thromboelastogram the maximum amplitude of the torsion wire is a measure of the maximum clot strength. In these experiments (see Figure 11A) sequential clot formation and lysis was induced by transfering canine hemophilic whole blood into a cuvette containing 90 nM thrombin and 9 nM rt-PA. Solulin was present at concentrations of 0 - 390 nM. An amplitude of approx. ± 85 mm was observed in the absence of Solulin. Solulin at concentrations of 100 and 250 nM completely blocked clot lysis: Amplitudes were larger and remained unchanged for up to 80 to 90 minutes (end of experiment). A further increase of Solulin concentration to 390 nM was still associated with a larger amplitude and a slower clot lysis compared with control experiments (no Solulin) (Figure 11 A). In this experiment the area under the clot-lysis-curve (AUCL) can be used for quantification of the clot firmness, whereby Solulin at 100 and 250 nM increased this parameter by greater than 5-fold (Figure 11 B). Prolongation of clot lysis time in the same experiments is plotted in Fig. 11C. Lysis time was increased from 20 min in the absence of Solulin to 3 hours at 100 and 250 nM Solulin. Conclusion: The antifibrinolytic effect of Solulin shown by thrombelastrography is in line with the results of the above clot lysis assay. Furthermore Solulin appears to increase clot firmness.

WO 2011/157283 PCT/EP2010/007632 - 75 Solulin prolongs clot lysis in whole blood from a canine with haemophilia A in the presence of anti-Factor Vill antibodies. These thrombelastography experiments on blood known to contain a high titre of anti Factor Vill antibodies (> 150 Bethesda Units) were performed as described above: Sequential clot formation and lysis was induced by transfering canine haemophilic whole blood into a cuvette containing 90 nM thrombin and 9 nM rt-PA. Solulin was present at concentrations of 0 - 3510 nM. Without Solulin, an amplitude of approx. ± 20 mm was observed (see Figure 12A). Solulin dose dependently increased the amplitudes to approx. ±60 mm at concentrations of 100 and 250 nM, reaching an amplitude of approx. ±90 mm at 390 nM. Furthermore Solulin dose-dependently prolonged the clot-lysis time from approx. 18 min in the control to approx. 46 min at 390 nM Solulin (see Fig. 12A). Increased clot firmness is also indicated by a more than 10fold enhancement of the Area Under the Elasticity Curve (0 nM Solulin compared to 390 nM; see Fig. 12B). Conclusion: The virtually complete inhibition of Factor Vill activity by the anti-Factor Vill antibodies reduced but failed to suppress the ability of Solulin to prolong clot lysis. Viewed from a different angle, this experiment reveals that the presence of residual Factor VillI activity in haemophilic whole blood (absence of antibodies, see Fig. 1 1A) is sufficient to augment the efficacy of Solulin to prolong clot-lysis. This supports the use of the claimed thrombomodulin analogues in haemophilic patients treated with Factor Vill. Furthermore also patients, who suffer from haemophilia and still possess functional Factor Vill concentrations could be treated with the claimed thrombomodulin analogues. Finally also patients, who are lacking Factor VIll or show high titres of anti-Factor VillI antibodies are amenable for a treatment with the claimed thrombomodulin analogues. Vill. EXAMPLE Ratio of TAFI activation vs. protein C activation for different TM analogues Several TM analogue as depicted in Figure 19 and given in SEQ ID NO: 5-11 were generated. The solulin analog with a Phe 376Ala exchange was tested in vitro for protein C and TAFI activation in comparison with solulin. Materials and methods WO 2011/157283 PCT/EP2010/007632 - 76 Measurement of TM cofactor activity (APC assay) . A DNA fragment encoding Solulin with the selected mutation was synthesised and cloned into a pGAEx vector (a standard HEK293-derived vector). The pGAEx vector includes a standard promoter-, leader- and excretion sequence for HEK293 cells and a His-6-Tag at the C-terminus of the protein. HEK293 cells were transfected with the pGAEx-Solulin vector (transient transfection) and cultured in suspension in a 1 liter scale. Six days after transfection the protein was harvested by a Ni-HiTrap column (linear gradient from 20-500mM Imidazole in PBS, 500 mM NaCI). Target for purification was the His-6-Tag. After dialysis (16 h against PBS) the proteins were analysed. The protein was quantified by Coomassie blue staining of an SDS-PAGE. Thereafter, protein identity was verified by Western Blot (labelled anti-His-6 Antibodies). Measurement of TM cofactor (APC assay) and TAFI activity The TM analogues were tested in an assay buffer containing 0 to 2 pM TAFI or protein C, respectively, 0.5 nM thrombin, 5 mM calcium chloride and 25nM solulin or the solulin Phe376Ala analogue, respectively. The reactions were quenched with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) for TAFI experiments or hirudin for protein C experiments. TAFla and activated protein C activity was determined using N-(4 methoxyphenylazoformyl)-Arg-OH (AAFR) and pyroGlu-Pro-Arg-pNA-HCI (S-2366), respectively. The amount of TAFla or aPC generated in 10 minutes was determined by comparing the rate of substrate hydrolysis to a set of standards. Using the standards, we determined the kinetics of TAFI and protein C activation by lla-sTM. Results The exchange of Phe376 against Ala leads to an increased ratio of TAFI vs. protein C activation rates, the increase being more pronounced at lower substrate concentrations While Michaelis-Menten constants could be determined for the activation of TAFI (Fig. 15, upper panel), no such constants could be derived for protein C activation (Fig. 15, lower panel). The ratio of activation rates of TAFI and protein C (TAFI/protein C) by solulin and Phe376Ala at various substrate concentrations is illustrated in Fig. 16. The high TAFI/PC ratio of Solulin could be further improved by the amino acid exchange from Phe376 to Ala. Conclusion: WO 2011/157283 PCT/EP2010/007632 - 77 The increase in the TAFI/protein C ratio, which can be used as a measure of TAFI preference, occurred at relevant substrate (TAFI or protein C) concentrations, being most pronounced at the lowest concentration tested (250 nm), which is close to the physiological concentrations of both TAFI (- 75 - 220 nM) and protein C (- 70 nM) and corroborates the contention that both solulin and its variant are suitable for preferential TAFI activation. On the other hand, especially the protein C activation by Phe376Ala proved to be so slow that it must be considered unlikely that it could enhance protein C activation to any appreciable extent at physiological protein C concentrations (- 70 nM). Thus, the relevant effect of the additional mutation in Phe376Ala can be seen in sizable reductions of both the TAFI and protein C activating capabilities, which still allow TAFI activation, but practically preclude protein C activation under physiological conditions. Furthermore these results confirm the general importance of single amino acid mutations and in particular of Phe376Ala since the beneficial effect of this mutation is not only observed for a Solulin fragment extending from EGF1 to EGF6 but also for the full length soluble thrombomodulin including the N-terminal lectin-like domain, the six EGF-domains and the O-linked glycosylation domain. IX. EXAMPLE In vivo analysis of fibrinolysis in dog model of haemophilia Objective of the study Restitution of factor Vill (fVIll) is the mainstay of treatment of patients with haemophilia A. While providing effective treatment in the majority of cases, a sizable number of patients develop neutralizing anti-fVil antibodies, a problem which cannot be overcome so far with the available adjunctive therapeutic options. Other downsides of fVIlI therapy include complicated dose determination and the need for intravenous administration and regular monitoring. There has been increasing evidence that, besides the dominating failure to produce fVlll in sufficient quantities, also an unrestrained activity of tissue plasminogen activator (tPA) contributes to the condition. Normally, the growing and maturing clot is protected from tPA by activation of the Thrombin-Activatable Fibrinolysis Inhibitor (TAFI). This is achieved by a complex formed by thrombin, generated in the process of vascular lesion closure, and thrombomodulin, a protein constitutive to the endothelial cell membrane. The thrombin/thrombomodulin complex converts a plasma zymogen, TAFI, to its active form (TAFla), which acts by removing carboxyl-terminal lysines and arginines from clot WO 2011/157283 PCT/EP2010/007632 - 78 fibrin to impair the ability tPA to bind and, in this conformation, activate plasminogen. This control of tPA efficacy is faulty in Haemophilia A, due to the insufficient thrombin generation and probably also a decreased availablity of the zymogen, TAFI. Thus, one of the hallmarks of Hemophilia A is a premature clot lysis by tPA. In-vitro work has shown that Solulin is able to correct premature clot lysis in plasma from haemophiliacs, partially or entirely, dependent on the concentration of tPA. It is the aim of the present study to test whether the in vitro efficacy can be confirmed in vivo, using the well-established model of haemophilic dogs. Pilot experiments have ascertained that haemophilic dog plasma, like human plasma, allows Solulin to correct premature clot lysis, justifying the use of this animal model for this purpose. Study substance Identification: Solulin Physical state: clear colorless solution, pH 7.0 Dissolution buffer: sodium phosphate, 13.4; potassium chloride, 2.7; sodium chloride, 137 (all in mmol/l); mannitol, 5% Content of vial: 3 mg Solulin in 1 ml (3 mg/ml) Vial handling: Use ethanol to wipe the vial septum, then insert a sterile needle through the septum to draw the required volume. Store vials with residual volume in the refrigerator. Storage conditions and time: To be stored in the refrigerator at 2-6*C. Residual volumes can be kept in the refrigerator for 28 days. Within this period, they can be used for further experiments, but it is recommended to use separate vials for each dog. Preparation of dosing formulations The appropriate volume is taken from the original Solulin vial(s) and complemented with 0.9 % NaCI to achieve a dosing volume of 3 ml. The final dose formulations will be prepared freshly for each experimental day. Animals: Species and maintenance The study was conducted in dogs with haemophilia A (haemophilia A dogs) characterized as "cured" FVIII gene therapy dogs. After canine FVIII gene therapy, these dogs had undetectable levels of plasma FVIII (< 1%) but showed shortening of their whole blood clot times and did not experience further spontaneous bleeding episodes for at least.3 years after treatment (normal frequency approximately 5/yr). These dogs may be regarded as showing partial phenotypic correction after gene transfer. They best recapitulate the clinical picture documented in approximately 10% of severe haemophilia A patients (plasma FVIII < 1%) who rarely bleed. Such a profile WO 2011/157283 PCT/EP2010/007632 - 79 also mirrors the outcome of a low dose FVIII prophylaxis protocol. The age and body weight of the animals were approximately 3 years and 10-15 kgs, respectively. Except for the haemophilia trait, only healthy animals were included into the study. Experimental procedures Pilot study (Pharmacokinetics and in vitro efficacy) As a first step, the pharmacokinetics of Solulin was explored in haemophilic dogs together with pharmacodynamic effects on coagulation/fibrinolysis. Based on in vitro efficacy data available on dog plasma and pharmacokinetic modelling, Solulin was administered at a dose of 0.5 mg/kg in a volume of 3 mL by intravenous bolus injection. According to the results obtained in a first dog, a second dog was treated the same way or at an adapted dose of Solulin. Thereafter, a decision was made on initiation of the main study or further exploration of pharmacokinetics. Preparation of blood samples Two different sets of blood samples were taken from contralateral legs (see Table below). Full blood is harvested on citrate buffer (final volume: 1 part of 3.13 % sodium citrate mixed with 9 parts of blood) and processed immediately (see preparation below). Unless assayed in close temporal relationship as indicated for the individual splits, they are adequately labeled and stored at -80'C until further use. Samples for Time relative to dosing (min) TEG, whole blood clotting, Thrombin Generation PK,TAFla, PT/aPTT Potential Sample 1 Sample 2 Blood volume; Blood volume; drawn and splitted for whole drawn from contralateral blood assays and plasma leg and processed generation separately Predose 14 ml 1 ml* 0: Dosing 10 30 14 ml 1 ml* WO 2011/157283 PCT/EP2010/007632 - 80 60 14 ml 120 14 ml 1 ml* 480 14 ml * preceded by 1 ml, which is discarded Sample 1 The first milliliter to be discarded was taken at the times indicated in the table above, citrated, and split in five parts. 14 mls of citrated whole blood were portioned for split I (thrombelastography x 5 mls whole blood) and split 2 (whole blood clotting; 2 mis whole blood). The remaining volume was centrifuged at 1500 g for 15 minutes. The resulting supernatant was split into three portions (split 3, Solulin concentration, 0.5 ml plasma; split 4, activated TAFI, 0.5 ml plasma; split 5, PT/aPTT, 0.5 ml plasma and stored at -80*C until further use. Sample 2: Sample 2 (2 ml; the first ml. to be discarded) was taken from the leg contralateral to the sample 1 side and citrated. Sampling times are indicated in the table above. This sample is for determination of thrombin generation potential and needs special precautions. It is important to avoid contact activation, which means that the sample should be drawn with the widest needle possible (preferably not from a butterfly needle). The required volume of plasma is 0.5 ml. There is a detailed description of plasma preparation for thrombin generation potential: 1 st centrifugation step: o Put in the following centrifugation parameters: - Break power: 9 - Rotor velocity 3790 at r=156 mm - duration: 5 minutes. o Centrifuge the sample for 5 minutes at 2500 g (for the Hettich Rotina 35 R this is reached at the setting 3790 rpm at r=1 56 mm) o Use a plastic disposable Pasteur pipette to transfer the plasma supernatant to a labelled plastic centrifuge tube. Be careful not to suck up any cells from the buffycoat, leave 0.5 cm plasma on top of the buffy coat.

WO 2011/157283 PCT/EP2010/007632 -81 2 nd centrifugation step: o Apply the following centrifugation parameters: - Temperature: 18 'C - Centrifugation velocity: 11000 RPM at r=156 mm - Duration: 10 minutes o Centrifuge the plasma for 10 minutes at 10000 g. (for the Universal 30 RF this is reached at the setting 11000 rpm at r=1 56 mm) o Pour or pipette in one movement the content of the centrifuge tube into a labelled tube (to avoid mixing of sediment with supernatant). o When applicable, combine the content of several identical plasmas into one large plastic tube. Pharmacokinetic and pharmacodynamic assays Thrombelastography. Thrombelastography was performed in citrated whole blood. 340 pl of whole blood are added to channels of a Haemoscope TEG@ 5000 (Haemonetics Corp. Braintree, MA) containing a 20 pL solution of 1/15000 dilution (18X) of Innovin, 270mM CaCl 2 and 18nM tPA. After mixing thoroughly, the pin was seated and coagulation and fibrinolysis were monitored continuously. The Haemoscope TEG@ 5000 allows for measurement of the clot time, clot kinetics, clot strength and clot stability (fibrinolysis) by measuring the torque on a wire which is connected to the clot through the pin. Whole Blood Clotting Time: Whole blood clotting time was determined by incubating citrated whole blood at 37 0 C in a glass test tube with continuous monitoring for clot formation. Pharmacokinetics. Plasma concentrations were determined using an ELISA validated for dog plasma. This study will be performed under the responsibility of the sponsor. Results will be integrated in the final report. Activated TAF. TAFla was determined using the methods of Kim et al. (Anal Biochem. (2008), 372(1):32-40). Briefly, fibrin degradation products containing a covalently attached quencher moiety (QSY-FDP) bind to fluorescently labelled plasminogen (F Pg). When F-Pg is bound to QSY-FDP, the fluorescence is quenched. When TAFla is added, F-Pg binding sites are removed and F-Pg is released from QSY-FDP resulting in an increase in fluorescence. The rate of this fluorescence increase is proportional to the TAFla concentration. Using plasma spiked with known concentrations of TAFla a standard curve was developed and used to determine the TAFla concentration in plasma samples.

WO 2011/157283 PCT/EP2010/007632 - 82 Coagulation assays. Diluted PT (dPT) and aPTT assays have been performed. The dilute PT assay involve a 1/5 dilution of test platelet poor plasma. TriniCLOT PT HTF thromboplastin reagent was added to the plasma and then following a 180 sec incubation, the sample is re-calcified. The aPTT was performed with TriniCLOT Automated APTT Reagent. Again, a 180 sec incubation is performed and then the sample is re-calcified. All studies were performed on a Coag-A-Mate MAX (bioMerieux) automated coagulometer. Endogenous thrombin potential. Endogenous thrombin potential was determined using the Calibrated Automated Thrombogram (CAT) assay in a 96-well plate fluorimeter. The CAT assay is a proprietary method of CoagScope B.V. (Maastricht, The Netherlands). Briefly, the CAT assay measures the concentration of thrombin in clotting plasma over time. Citrated plasma is triggered with PPP reagent that contains Tissue Factor and phospholipids. The plasma is recalcified with another reagent containing Calcium Chloride and a fluorogenic Substrate (FluCa). This Substrate is converted by thrombin into a fluorophore, the fluorescence being followed in a fluorometer. Simultaneously another sample of the same plasma is measured in the presence of Thrombin Calibrator (a calibrated amount of thrombin activity). Comparing the results of these parallel measurements allows the calculation of thrombin concentration over time. Main study (Cuticle bleeding assay) Animal number, study drug and dose level Three haemophilic dogs were studied to test the effect of Solulin on blood coagulation and fibrinolyis. Solulin was administered by intravenous bolus injection at a dose selected according to the pilot study described under 8.1. Anesthesia and supportive medications Premedication: Hydrocortisone 100 mgs and Benadryl 50 mgs IV Maintenance: 1-2% Isoflurane Pain management post-surgery: Buprenorphine Time course and procedures The course of the experiment is visualized in the table below. 15 min after anaesthesia, a baseline cuticle test is performed, followed by a 15 min observation period. Thereafter, nominally 30 min post-anaesthesia, the dogs receive an intravenous bolus injection of Solulin, based on the actual body weight. After a 30 min exposure to Solulin, a second cuticle assay is performed on a contralateral paw. With the WO 2011/157283 PCT/EP2010/007632 - 83 subsequent observation period of 15 min, the total time of anaesthesia is approximately 75 min. For the cuticle bleeding test, the dogs are placed in the lateral recumbency position, and all hair around the nail bed is carefully removed by clipping around the.base of the claw to be used for the cuticle bleeding time assay. Silicone grease is applied to the claw to prevent blood tracking back underneath the nail. The apex of the cuticle is visualized, and the nail severed proximal to the dorsal nail groove using a spring loaded sliding blade guillotine clipper. The animal's paw is subsequently positioned over the edge of the operating table and blood from the severed cuticle allowed to fall freely. The number of blood drops in each of the subsequent 15 minutes is recorded and converted to a cumulative score for the 15 minutes. After 15 minutes of observation, if the cuticle is still bleeding, the site of injury is cauterized by topical application of silver nitrate. All assays are performed by the same experienced veterinary technologist. Results and Discussion Dogs with severe haemophilia A were studied in vivo. Led by the solulin concentrations of several hundred nM found effective in the in vitro experiments presented in example VII, a first doq was injected with a dose of 500 p/kg to see whether the improvement in the thrombelastogram observed after in vitro addition of solulin could be confirmed in vivo. Contrary to expectations, this dose was not only ineffectve, it even severely inhibited clot formation and strength (Fig. 17). This effect was manifest 30 min post administration and sustained at 2 hours. At the end of the day, the decrease in clot formation was still evident, but the inhibition seemed to slowly subside. As the 24 hour test was technically flawed, the experiment could be considered a failure. Even so, it was decided to draw a final sample at 48 hours to see whether the animal had recovered. This seemed to be the case, but, to some surprise, there seemed to be even a trend to a slight improvement over the baseline thrombelastogram. Encouraged by this notion, a further sample was investigated 72 hours post solulin and, much unexpected, a clear improvement in clot formation and strength was recognized. This led to the conclusion that much lower doses than anticipated from the in vitro studies were effective and even required, as larger doses establish higher plasma concentrations, which then would deteriorate clot properties, a hypothesis that was tested in a further severely haemophilic doq (Fig 18). The second dog received a much lower dose of solulin (10 pq/kg) than the first one. This experiment confirmed the hypothesis: A higher plasma concentration was WO 2011/157283 PCT/EP2010/007632 -84 ineffective at improving clot properties, whereas concentrations as low as 0.2 nM were able to improve it.

WO 2011/157283 PCT/EP2010/007632 - 85 Figure legends Fig. 1: Clot-lysis profiles and lysis times of factor VIII deficient plasma (FVIII-DP), normal plasma (NP) and FVIII-DP mixed with NP. Clot lysis profiles are shown for 0 ( ), 1 (----), 6 (- - -), 10 (---), 50 (---) and 100% (---) NP. From the clot-lysis profiles, the lysis time was determined by taking the time at which the clot has been degraded to one half of its highest optical density. In the inset, the lysis times are summarized, with the general trend being an increase in lysis time as the percentage of NP (and consequently amount of FVIII) is increased. The effect of adding NP on lysis time reaches a plateau at 10% NP. Fig. 2: Thrombin activatable fibrinolysis inhibitor (TAFI) activation in plasma containing various percentages of FVIII: (A) When FVIII-deficient plasma (FVIII-DP) is mixed with normal plasma (NP) TAFI activation is enhanced. In FVIII-DP only 30 pM TAFla was measured at its peak (-) compared with -600 pM TAFla in 50% NP (0) and 100%NP (A). These experiments were conducted in triplicate and the data represent the mean ± SE. The TAFla potential (B), defined here as the area under the time course of activation plot (A) from the time of clot initiation to the last time point, increases as the percentage of NP increases to a plateau at 50% NP. The TAFla potentials of 50% NP and 100% NP are similar (14,100 pM min and 16,800 pM min, respectively) despite the shape of their respective TAFI activation plots being quite different. The relationship between lysis time (Fig. 1, inset) and TAFla potential, as it relates to FVIII levels, is presented (Fig. 2B, inset) using a plot of log lysis time vs. log TAFla potential. As expected, the data show a strong positive correlation between lysis time and TAFla potential in plasma containing 0 - 100% FVIII. Fig. 3: Prothrombin activation in plasma containing various percentages of FVIII. The time course of prothrombin activation is shown for FVIIIDP mixed with 0 (-), 1 (0), 6 (A), 10 (0), 50 (0) and 100% NP (A). Generally, the rate of prothrombin activation increases as the percentage of NP increases. At 50% NP prothrombin activation occurs at a high rate (as determined by examining the slope of each plot) and appears to be over within 15 min, whereas 100% NP has a slower rate of prothrombin activation over a longer time period.

WO 2011/157283 PCT/EP2010/007632 - 86 Fig. 4 A-D: The effect of sTM on thrombin activatable fibrinolysis inhibitor (TAFI) activation in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP) at various concentrations of both sTM (0 - 100 nM) and tPA (0.25nM in Figure 4A, 0.75 nM in Figure 4B, 1.5 nM in Figure 4C, and 3 nM in Figure 4D). The TAFla-dependent defect in prolonging lysis in FVIII-DP is corrected by the addition of 100 nM sTM to plasma containing 0.25 nM tPA. As the concentration of tPA is increased only partial correction of the lysis defect is observed in FVIII-DP in the presence of 100 nM sTM. In these experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create a condition in which there is no functional TAFla. Therefore, any increase in lysis, as presented by the ratio lysis time/lysis time + PTCI is TAFla dependent. Fig. 5: TAFI activation and clot lysis profiles in normal plasma (NP) (A) and FVIII deficient plasma (FVIII-DP) (B) in the presence of 10 nM thrombomodulin (.) or without thrombomodulin (o). The accompanying clot-lysis profile is shown (-) and the clot lysis profile for no sTM is shown as a reference (---).These experiments were conducted in triplicate and the data represents the mean ± SE. Fig. 6: Thrombin binding to thrombomodulin. Binding of thrombin to thrombomodulin was determined by titrating 1.5 ml of a solution composed of thrombin (20 nM) and DAPA (20 nM) in 20 mM Tris-HCl, 150 mM NaCl, 5.0 mM Ca 2 , 0.01% Tween 80 with 1.54 pM thrombomodulin in an identical solution. Fluorescence intensity was measured (Xex = 280 nm, Xem = 545 nm). Fig. 7: Relative cofactor activities of point mutants in TAFI and protein C activation. Alanine-scanning mutagenesis was used to prepare point mutations in soluble thrombomodulin. Rates of protein C and TAFI activation (relative to the rate of activation with mutant TMEM388L) are shown for TAFI (solid bars) and protein C (hatched bars). Fig. 8: Mutations of the interdomain loop between EGF4 and EGF5. Three independent plasmids were constructed in E.coli for each mutant. Shockates were prepared, assayed for cofactor activity by the APC assay, and samples were analysed on Western blots (not shown). Activity values are the average from three separate clones. Panel A, substitution mutants at Glna3 7 ; panel B, substitution at Met386; panel C, substitution mutants at Phe389; panel D, deletions and alanine insertions in the interdomain loop. The activity measured for shockates from E.coli-transfected with the control plasmid, pSelect, lacking the TM insert is shown. See Clarke et al. (J. Biol. Chem. 1993; 268:6309-6315) for additional details.

WO 2011/157283 PCT/EP2010/007632 -87 Fig. 9: Schematic diagram of the pro- and antifibrinolytic effects of thrombomodulin (modified after Mosnier and Bouma, Arterioscler. Thomb. Vasc. Biol. 2006; 26: 2445 - 2453). The increase in clot lysis time at low TM concentrations is attributable to stimulation of TAFI activation and illustrates the antifibrinolytic activity of TM. At higher concentrations of the rabbit lung TM the clot lysis time decrease because of the activation of protein C and inhibition of TAFI activation; illustrating the profibrinolytic activity of rabbit lung TM (solid line). Note that above 15 nM the profibrinolytic activity of rabbit lung TM exceeds the antifibrinolytic activity resulting in an overall profibrinolytic effect. In contrast the soluble TM analogue shows only an antifibrinolytic effect (dashed line). Fig. 10: Relationship between TAFla-dependent Fold propagation of clot lysis and added Solulin. The dose-dependent increase in clot lysis time is attributable to stimulation of TAFI activation and illustrates the antifibrinolytic activity of TM. Fig. 11A: Solulin-dependent change in clot appearance in whole blood from a canine with haemophilia A (as measured by thrombelastrography) Fig. 11B: Solulin-dependent increase of clot firmness in whole blood from a canine with haemophilia A. The area under the clot-lysis curve was measured by thrombelastrography as a function of Solulin (sTM) concentration. Fig. 11C: Solulin-dependent dependent prolongation of lysis in whole blood from a canine with haemophilia A. The clot-lysis time as measured by thrombelastography as a function of Solulin (sTM) concentration. Fig. 12A: Solulin-dependent prolongation of lysis in whole blood from a canine with hemophilia A and inhibitory anti-factor Vill antibodies (as measured by thrombelastrography) Fig. 12B: Solulin-dependent increase of clot firmness in whole blood from a canine with haemophilia A in the presence of inhibitory anti-factor Vill antibodies. The area under the clot-lysis curve was measured by thrombelastrography as a function of Solulin (sTM) concentration. Fig. 13: Figure showing Table 1: Summary of the data used to construct Fig. 4, including the absolute lysis time in the presence of PTCI to enable determination of the lysis time under each condition. In all cases, the lysis time is expressed relative to that obtained in the presence of the TAFla inhibitor, PTCI. TAFI, thrombin activatable fibrinolysis inhibitor; PTCI, potato tuber carboxypeptidase inhibitor.

WO 2011/157283 PCT/EP2010/007632 - 88 Fig. 14: Figure showing Table 2: Chloramine T oxidation of site-specific mutant analogues of TME (Sf9). The results after chloramine T treatment were expressed as a percentage of the activity after control treatment. *Average of duplicate determinations and deviation from the mean. Fig. 15: Activation rates of TAFI (upper panel) and protein C (lower panel) by solulin and the solulin mutant, Phe376Ala (both present at 25 nM) at rising substrate concentrations. Fig. 16: Ratio of TAFI activation vs. protein C activation for Solulin (black bar) compared to a Solulin analogue with Phe376Ala mutation ((light grey bar) as determined at TM concentrations of 0.25, 0.5, 0.75, 1, 1.5 and 2 pM. The most right bars indicate the ratios combined from easurements at all concentrations. Fig. 17: Thrombelastography of blood samples from a dog with severe haemophilia A. Samples were taken at different times after intravenous injection of solulin at a dose of 500 pg/kg. Fig. 18: Bell-shaped concentration dependence of improvement of clot formation and stability in the blood from a severely haemophilic dog. Solulin was injected at a dose of 10 pg/kg and plasma samples were taken at the indicated times for determination of the thrombelastogram. At each thrombelastogram, time of sampling and plasma concentration of solulin are indicated. Fig. 19: Overview on TAFI-specific thrombomodulin analogues Table legends Table 1: Summary of the data used to construct Fig. 4, including the absolute lysis time in the presence of PTCI to enable determination of the lysis time under each condition. In all cases, the lysis time is expressed relative to that obtained in the presence of the TAFla inhibitor, PTCL. TAFI, thrombin activatable fibrinolysis inhibitor; PTCI, potato tuber carboxypeptidase inhibitor. Table 2: Chloramine T oxidation of site-specific mutant analogues of TME (Sf9) The results after chloramine T treatment were expressed as a percentage of the activity after control treatment. *Average of duplicate determinations and deviation from the mean.

Claims (34)

1. A thrombomodulin analogue with reduced cofactor activity upon binding to thrombin as compared to TMEM338L with a.) an amino acid sequence according to SEQ ID NO 2 or b) amino acid sequence according to SEQ ID NO 3 or c) an amino acid sequence according to SEQ ID NO 4 or d) with an amino acid sequence which has at least 90%, more preferred at least 95%, most preferred at least 98% identity to the amino acid sequences according to SEQ ID NO 2, SEQ ID:3 or SEQ ID NO:4 or e) a thrombomodulin fragment consisting essentially of the 6 EGF-like repeat domains of SEQ ID NO 2, SEQ ID:3 or SEQ ID NO:4 (amino acid position 227 to 462 as numbered in SEQ ID NO 1), the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2, SEQ ID:3 or SEQ ID NO:4 (amino acid position 307 to 462 as numbered in SEQ ID NO 1) or from the c-loop of the EGF-like repeat domain 3 to the EGF-like repeat domain 6 of SEQ ID NO 2, SEQ ID:3 or SEQ ID NO:4 (amino acid position 333 to 462 as numbered in SEQ ID NO1), whereas the phenylalanine in position 376 (as numbered according to SEQ ID NO1) is deleted or substituted by glycin, alanine, leucine, isoleucine.

2. A thrombomodulin according to claim 1 which further comprises a deletion or substitution of the glutamine residue at position 387 (as numbered in SEQ ID NO:1), whereas the substitution preferably is substituted with Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, lie, Tr, Tyr, Leu, Asn, Phe, Asp, Cys.

3. A thrombomodulin according to claim 1 or 2 which further comprises a deletion or substitution of the methionine residue at position 388 (as numbered in SEQ ID NO), whereas the methionine residue preferably is substituted with GIn, Tyr, lie, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys.

4. A thrombomodulin according to any of the above claims, which further comprises a deletion or substitution of the phenylalanine residue at position 389 (as numbered in SEQ ID NO:1), whereas the phenylalanine preferably is substituted with Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, lIe, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro.

5. A thrombomodulin according to any of the claims whereas the thrombomodulin comprises one or more first and a second amino acid modifications as depicted in table 4. WO 2011/157283 PCT/EP2010/007632 - 90

6. A thrombomodulin according to any of the claims whereas the thrombomodulin comprises one or more first, second and third amino acid modifications as depicted in table 5.

7. Use of a thrombomodulin analogue for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis, whereas a) TM analogue is defined as according to one of the above claims or b) said TM analogue is characterized by exhibiting at therapeutically effective dosages an antifibrinolytic effect.

8. Use according to claim 7 whereas the thrombomodulin analogue exhibits one or more of the following features: (i) a binding affinity towards thrombin that is decreased compared to the rabbit lung thrombomodulin, and/or a binding affinity towards thrombin with a kD value of more than 0.2 nM; and/or (ii) a reduced cofactor activity compared to cofactor activity of the TM analogue TMEM388L, (iii) an increased ratio of TAFI activation activity to cofactor activity as compared to the TM analogue TMEM388L.

9. Use according to claim 7 or 8, whereas the coagulopathy with hyperfibrinolysis is selected from the group of diseases as follows: haemophilia A, haemophilia B, haemophilia C, von Willebrandt disease (vWD), acquired von Willebrandt disease, Factor X deficiency, parahemophilia, hereditary disorders of the clotting factors I, 11, V, or VII, haemorrhagic disorder due to circulating anticoagulants or acquired coagulation deficiency.

10. Use according to any of the above claims whereas the thrombomodulin analogue is used to treat one or more of the bleeding events selected from the group consisting of: intracranial or other CNS haemorrhage, bleeding in joints, microcapillaries, muscles, the gastrointestinal tract, the respiratory tract, the retroperitoneal space or soft tissues.

11. Use according to any of the above claims, whereas the patients have anti- factor Vill antibodies.

12. Use according to any of the above claims, whereas the patients are treated with factor VillI, preferably recombinant factor Vill or a recombinant B-domain-deleted factor Vill molecule, more preferably Octocog-alfa or moroctocog-alfa. WO 2011/157283 PCT/EP2010/007632 -91

13. Use of a thrombomodulin analogue according to any of the above claims for the treatment of coagulopathy with hyperfibrinolysis, characterized in that the thrombomodulin analogue is given in combination with factor VIII, preferably recombinant factor VIII a recombinant B-domain-deleted factor VIlI molecule, more preferably octocog-alfa or moroctocog-alfa.

14. Use according to any of the above claims, whereas the patients are treated with factor VIII, preferably recombinant factor VIII, more preferably Octocog-alfa.

15. Use according to any of the above claims whereas the thrombomodulin analogue is administered at the time of a bleeding episode.

16. Use according to any of the above claims whereas the thrombomodulin analogue is administered in advance of an increased bleeding risk, e.g. a surgery or a tooth extraction.

17. Use according to any of the above claims whereas the thrombomodulin analogue is administered to patients that are refractory to blood/plasma transfusion or coagulation factor replacement therapy.

18. Use according to any of the above claims whereas the thrombomodulin analogue is administered in multiple doses, preferably once daily, bidaily, or every third, fourth, fifth, sixth or seven days over a total time period of less than one week to four weeks, more preferably as chronic administration.

19. Use according to any of the above claims whereas the thrombomodulin analogue is given as parenteral application, preferable as intravenous or subcutaneous application.

20. Use according to any of the above claims whereas the thrombomodulin analogue is a soluble TM analogue.

21. Use according to claim 20, whereas the thrombomodulin analogue is a human soluble TM analogue.

22. Use according to any of the above claims whereas said thrombomodulin analogue comprises at least one structural domain selected from the group containing EGF3, EGF4, EGF5, EGF6, preferably comprising the fragment EGF3-EGF6 and more preferably comprising the EGF domains 1-6. WO 2011/157283 PCT/EP2010/007632 - 92

23. Use according to any of the above claims whereas said thrombomodulin analogue consists of EGF domains EGF1 to EGF6, and more preferably consists of the EGF domains EGF3 to EGF6.

24. Use according to any of the above claims, whereas the thrombomodulin analogue has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (depicted in SEQ ID NO:1 or SEQ ID NO:3) and comprises one or more of the subsequent modifications: a) removal of amino acids 1-3 b) M388L c) R456G d) H457Q e) S474A, and terminating at P490.

25. Use according to any of the above claims, whereas the thrombomodulin analogue has an amino acid sequence which comprises a sequence of at least 85%, or at least 90% or 95% sequence identity with SEQ ID NO: 2.

26. Use according to any of the above claims whereas the thrombomodulin analogue has an amino acid modification at one or more positions corresponding to natural sequence at (according to SEQ ID NO: 1 or SEQ ID NO:3): aa) 34 9 Asp; bb) 3 5 Asn; ac) 5 7 Glu; ad) 3aTyr; ae) 5 9 GIn; af) se 1 Gln; ag) 3 3Leu; ah) 6 4 Asn; ai) e 8 Tyr; aj) 371 Val; ak) 374 Glu; al) 376 Phe; am) 38 4 His; an) 8 5 Arg; ba) a 7 Gln; bb) as 9 Phe; bc) 398 Asp; bd) 40OAsp; be) 40 2 Asn; bf) 403Thr; WO 2011/157283 PCT/EP2010/007632 - 93 bg) 40 8 Glu; bh) 41 1 Glu; bi) 413 Tyr; bj) 41 4 1le; bk) 415 Leu; bl) 4 16 Asp; bm) 4 1 7 Asp; bn) 42 0 1le; bo) 4 23 Asp; bp) 424 1le; bq) 4 25 Asp; br) 42 6 Glu; ca) 42 8 Glu; cb) 429 Asp; cc) 432 Phe; cd) 4 34Ser; ce) 4 36Val; cf) 4 3His; cg) 439 Asp; ch) 4 0 Leu; ci) 4 3 Thr; cj) 4"Phe; ck) " 5 Glu; cl) "5Arg; cm) 4"Ile; or cn) ** 1 Asp.

27. Use according to any of the above claims whereas the thrombomodulin analogue has a modification of the phenylalanine at position 376 according to SEQ ID NO:1 or SEQ ID NO:3, preferably substituted with an aliphatic amino acid, more preferably substituted with glycine, alanine, valine, leucine, or isoleucine and most preferably substituted with alanine.

28. Use according to any of the above claims whereas the thrombomodulin analogue has a modification of one or more of the following amino acids according SEQ ID NO:1 or SEQ ID NO:3:' a) 38 7 Gln; b) asMet; b)asPhe, whereby the amino acids are deleted, inserted by one or more additional amino acids or preferably substituted. WO 2011/157283 PCT/EP2010/007632 - 94

29. Use according to any of the above claims whereas the thrombomodulin analogue is used in its oxidised form, preferably oxidised with chloramine T, hydrogen peroxide or sodium periodate.

30. Use according claim 19, whereas one or more of methionine residues within the TM analogue are oxidised, preferably the methionine residue at position 388 (according SEQ ID NO:1 or SEQ ID NO:3).

31. A method for screening for analogues of thrombomodulin suitable for the treatment of coagulopathy with hyperfibrinolysis, where the thrombomodulin exhibits one or more of the following features: (i) a reduced binding affinity towards thrombin, (ii) a reduced cofactor activity, (iii) an increased TAFI activation activity, comprising the steps of: a) making one or more amino acid substitution of the thrombomodulin sequence (SEQ ID NO:1 or SEQ ID NO:3), preferably of the amino acid positions listed in claim 15; b) comparing the modified analogue with a control molecule, preferable a rabbit lung TM or a soluble human TM analogue with regard to one or more of the following characteristics: ba) binding affinity to thrombin (KD value); bb) cofactor activity; bc) TAFI activation activity or TAFla potential; bd) ratio of TAFI activation activity and cofactor activity; be) effect of protein oxidation; bf) effect on clot lysis in time in an in vitro assay; or bg) effect in a coagulation-associated animal model.

32. Method of treating coagulopathy with hyperfibrinolysis, comprising administering a therapeutically effective amount of a thrombomodulin analogue according to any of the claims 1 to 20.

33. Method of treating coagulopathy with hyperfibrinolysis, comprising administering a dose between 0.75 pg/kg and 140 pg/kg body weight of the subject to be treated of a thrombomodulin analogue according to any of the claims 1 to 20.

34. Pharmaceutical composition comprising a thrombomodulin analogue according to any of the claims 1 to 20 for treating coagulopathy with hyperfibrinolysis in a dose between 0.75 pg/kg and 140 pg/kg body weight of the subject to be treated.