CA2071479A1 - Megakaryocyte maturation factors - Google Patents

Abstract

Methods for the treatment of blood platelet disorders by factors which increase circulating platelet levels are disclosed. Also disclosed are methods for obtaining such factors and pharmaceutical compositions comprising platelet producing factors.

Description

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MEGAKARYOCYTE MATURATION FACTORS

The present invention relates to methods and pharmaceutical compositions for the production of blood platelet~. More specifically, the invention relates to treatment of platelet disorders using factors which increase the levels of circulating blood platelets. Also encompassed by the invention are pharmaceutical compositions of factors that promote platelet production.
Backaround of the Invention Pluripotent hematopoietic stem cells give rise to different types of terminally differentiated blood cells. The blood consists of red blood cells (erythrocytes), white blood cells (leucocytes) and platelets (thrombocytes). Platelets are derived from detached fragments of larger cells called megakaryocytes which reside predominantly in the bone marrow.
Platelets have a central role in blood clotting and wound healing.
Megakaryocytes undergo various stages of differentiation to produce mature platelets. A
pluripotent stem cell becomes committed to megakaryocyte development, then undergoes cellular and nuclear proliferation to generate a pool of megakaryocyte progenitor cells. These progenitor cells undergo endoduplication to form immature megakaryocytes, or megakaryoblasts, which are characterized by multilobulated, polyploid nuclei. The development of mature megakaryocytes from megakaryoblasts involves the formation of cytoplasmic granules containing platelet ~i; specific proteins. Mature megakaryocytes project cytoplasmic extensions, termed proplatelets, which fragment to produce mature platelets.

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Several purified factors promote megakaryocyte differentiation by stimulating the formation of mature megakaryocytes from megakaryocyte progenitor cells in vitro. These factors include granulocyte/macrophage colony stimulating factGr (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6) and megakaryocyte colony stimulating factor (Meg-CSF) (Hoffman et al. Blood Cells 1~, 75-86 (1987); Mazur et al. Exp. Hematol. 15, 1128-1133 (1987); McNiece et al.
Exp. Hematol. 1~, 807-810 (1987); Lu et al. Brit. J.
Hematol. 70, 149-156 (1988); Ishibashi et al. Proc.
Natl. Acad. Sci. U.S.A. 86, 5953-5957 (1989)). A factor referred to as megakaryocyte stimulating factor (MSF) has been described in U.S. Patent No. 4,894,440.
Purified MSF is involved in the cytoplasmic maturation ;of megakaryocytes as shown by its ability to stimulate in megakaryocytes the synthesis of platelet proteoglycans and platelet specific granule proteins such as platelet factor IV. Purified IL-6 has been reported to increase platelet levels i~ vivo (Ishibashi et al. Blood 74, 1241 (1989); Hill et al. J. Clin.
Invest. 85, 1242-1247 (1990)).
Thrombopoietic stimulating activity has been found in the plasma, serum and urine of thrombocytopenic patients and in the culture medium of human embryonic kidney (HEK) cells. This activity has been attributed ~- to thrombopoietin or thrombopoietic stimulating factor -~(TSF), a factor which is thought to be an important controlling element in megakaryocyte maturation (McDonald Ann. N.Y~ Acad. Sci. 509, 1-24 (1987)). TSF
from HEK cells has been purified (McDonald et al. J. ~ab Clin. Med. 106, 162-174 (1985)) but the corresponding activity from thrombocytopenic plasma has not been purified. The role of TSF in megakaryocyte development .. .
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has not yet been established. However, purified TSF
alone does not stimulate the formation of megakaryocytes from progenitor cells (Lu et al. Brit. J. Hematol. 70, 149-156 (1988)), suggesting that it plays a role in the later stages of megakaryocyte d ferentiation.
Inhibition and reversal of megakaryocyte differentiation and maturation have also been observed.
Platelet factor IV (PF-IV) and transforming growth factor (TGF)-~ block the development of megakaryocyte progenitor cells (Ishibashi et al. Blood 69, 1737-1741 (1987);
Gewirtz et al. J. Clin. Invest. ~, 1477-1486 (1989); Han et al. Blood 75, 1234-1239 (1990)). Various compounds that affect microtubule formation inhibit proplatelet formation (Leven et al. Blood 69, 1046-1052 (1987)). In addition, thrombin, a serum-derived serine esterase, reverses megakaryocyte maturation by stimulating the retraction of proplatelet extensions ~Radley et al., Thrombosis and Haemotosis ~, 732-736 (1987)).
Under the appropriate culture conditions, guinea piq megakaryocytes will differentiate in vitro and form long cytoplasmic extensions which are precursors to platelets (Leven et al., .supra; Handa~ama et al., Am. J. Vet. Res. 48, 1192-1146). These extensions, termed proplatelets, are observed to differentiate further into small anuclear cells the size of guinea pig platelets. Proplatelet formation represents an important event in the development of megakaryocytes to platelets. Factors influencing t~ 3 process will be important in the production of blood platelets.
~- As described above, a number of factors have been identified which stimulate various stages of megakaryocyte differentiation and maturation and promote increases in megakaryocyte number and size. However, no purified factors have been reported to stimulate further ' - : , , , -. .

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differentiation of mature megakaryocytes to proplatelet bearing cells. The identification and isolation of Eactors which stimulate the formation of proplatelets will be use~ul in the treatment of excessive bleeding resulting from platelet disorders.
The cytoplasm of mature megakaryocytes and platelets contains granules comprising proteoglycans and platelet specific proteins. Proteoglycans are highly acidic macromolecules having at least one glycosaminoglycan chain covalently attached to a protein core. A proteoglycan was purified from human platelets by monitoring uronic acid content of glycosaminoglycans and was found to contain four chondroitin sulfate chains attached to the protein core (Okayama et al. Biochem. J.
~, 73-81 (1986)). The purified human platelet proteoglycan protein core was sequenced (Perin et al.
Biochem. J. 255, 1007-1013 (1988); Alliel et al. FEBS
Letters 2~, 123-126 (1988)). The protein was 131 amino acids long and contained within it an 18 amino acid region having eight ser-gly repeats. Repeated ser-gly sequences had been observed in protein core regions of other proteoglycans and were predicted to be sites for glycosaminoglycan attachment. Serine residues at positions 67 and 69 of human platelet proteoglycan were thought to be modified with,chondroitin sulfate chains (Alliel et al., ~,U,E~). No biological activity of human , platelet proteoglycan was measured during or after purification.
, Genomic and cDNA sequences encoding the protein core of a secretory granule proteoglycan from the human promyelocytic leukemia cell line ~L-60 were disclosed in Stevens et al., PCT Publication No. WO
90/00606, and were also reported by Stellrecht et al.
(Nuc. Acids Res. 17, 7523 (1989)). Based upon these DNA
' 35 sequences, a protein having a molecular weight of 17,600 ' ,, : ` ' . ' ',' ' ,'' ' ' "' . ' ' ' ",' ' ` ~ ~

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was predicted which contained a 131 amino acid mature polypeptide and a 27 amino acid signal peptide. The mature human secretory granule proteoglycan had an a- o acid sequence identical to that reported for the human S platelet proteoglycan ~Alliel et al., suDra). The biological activity of human secretory granule proteoglycan was not disclosed in Stevens et al., suDra.
An object of the invention is a method for the treatment of excessive bleeding comprising the administration of factors that promote platelet production. A further object of this invention is the purification of factors that elevate proplatelet levels, thereby stimulating platelet formation. Another object of the invention is the production of pharmaceutical compositions comprising factors that promote platelet production.

Summary of the Invention The subject invention comprises methods for ~ increasing blood platelet levels and treating platelet ; disorders using factors invo -ed in megakaryocyte maturation and proplatelet formation. Megakaryocytes mature to form proplatelets which in turn undergo fragmentation and release platelets. Changes in proplatelet levels have a direct effect on the levels of blood platelets produced.
Factors of the invention which stimulate the ` production of proplatelets from megakaryocytes are referred to as megakaryocyte maturation factor~s) ~MMF).
These factors elevate blood platelet levels and are useful in the treatment of excessive bleeding. MMF used ;~ in treating platelet disorders may have some or all of the amino acid sequence of naturally-occurring MMF, may be the product of procaryotic or eucaryotic expression ~ .
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Megakaryocyte maturation factors may be used alone or in combination with other therapeutics for increasing blood platelet levels. Other factors that are useful in conjunction with MMF are stem cell factor (SCF), GM-CSF, G-CSF, IL-3, IL-6, Meg-CSF, MSF, and EPO.
The subject invention provides for a method of purifying factors which affect megakaryocyte maturation.
A method of purifying megakaryocyte maturation factors from MMF containing material comprises one or more steps of ion exchange chromatography.
A method for assaying a megakaryocyte maturation factor is also provided. The method comprises incubating MMF (either crude or purified) with megakaryocytes and monitoring the formation of proplatelets from megakaryocytes. MMF stlmulates production of proplatelets in this assay.
The subject invention further relates to pharmaceutically acceptable compositions of a purified ~ -and isolated megakaryocyte maturation factor. Also encompassed by the invention are pharmaceutically acceptable compositions of a megakaryocyte maturation factor further comprising pharmaceutically acceptable compositions of SCF, GM-CSF, G-CSF, IL-3, IL-6, Meg-CSF, MSF, and EPO.

B~ Description of the Drawinas Figure lA shows guinea pig megakaryocytes before proplatelet formation under Megacolor staining.
Figure lB shows guinea pig megakaryocytes after proplatelet formation under Wright Giemsa staining.
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Figure 2 shows the inhibition of p oplatelet formation in the in vitro assay by addition of human serum.
i Figure 3 shows inhibition of proplatelet formation by thrombin but not by trypsin, chymotryp~in, or thrombocytin.

Figure 4 shows the inhibition of proplatelet formation in the m vitro assay by prothrombin and thrombin.

Figure 5 shows the retraction of proplatelet formations in vitro induced by prothrombin and thrombin.
Figures 6A and 6B show the effect of : inactivating thrombin on inhibition of proplatelet formation and proplatelet retraction, respectively.

Figure 7 shows DEAE chromatography of human serum inhibitor of proplatelet formation and prothrombin.

Figure 8 shows Superose 6 chromatography of human serum inhibitor of proplatelet formation and prothrombin.

Figure 9 shows the conversion of prothrombin to thrombin by megakaryocytes, proplatelets and platelets.

Figures lOA and lOB show stimulation of ;~ proplatelet formation in the in vitro assay by guanidinium chloride and CHAPS lysates of human platelets.

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Figure 11 shows DEAE chromatography of human platelets lysed in the presence of guanidinium chloride.

Figure 12 shows Mono Q chromatography of MMF-III from a guanidinium chloride lysate of humanplatelets.

Figure 13 shows Superose 6 chromatography MMF-III from a guanidinium chloride lysate of human platelets.

Figure 14 shows C4 reverse phase HPLC of MMF-III from a guanidinium chloride lysate of human platelets.
Figure 15 shows DEAE chromatography of human platelets lysed in the presence of CHAPS buffer.

Figure 16 shows Mono Q chromatography of MMF-III from a CHAPS lysate of human platelets.

Figure 17 shows an analysis of MMF-IIIs from guanidinium chloride and CHAPS lysates by SDS-PAGE.

Figures 18A and 18B shows stimulation of proplatelet formation and inhibition of proplatelet retraction, respectively, by MMF1-131 in the in vitro assay in the presence of increasing thrombin concentration. -Figure 19 shows a comparison of MMF1-131 and ' ! `
MMF58-131 activity on proplatelet formation.

Figure 20 shows the activity of MMF1-131 after removal of chondroitin sulfate.

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~. WO92/06712 2071479 pCT/US91/07~7 g Eigure 21 shows platelet levels in mice receiving 4 ~g day or 20 ~g/day of MMF1-131.

Figure 22 shows platelet levels in mice receiving 2 ~g/day or 10 ~g/day of human recombinant IL-6.

Figure 23 shows platelet levels in mice receiving 20 ~g/day of MMF1-131, 2 ~g/day of IL-6 or a combination of 20 ~g/day of MMF1-131 and 2 ~g/day of IL-6.

petailed Description of the Invention The present invention relates to a class of megakaryocyte maturation factors (MMF) which stimulate megakaryocyte maturation and proplatelet formation, thereby elevating circulating platelet levels. Factors of the invention have a property of promoting the production of proplatelets from megaxaryocytes in vitro when an inhibitory factor is present. As described in Example 2, one such inhibitory factor is found in human serum.
MMF is obtained from a variety of sources including, but not limited to, human serum, urine, megakaryocytes and platelets. The presence of MMF
- activity in megakaryocytes and platelets is described in Example 7 and 8. However, any biological material that ; promotes proplatelet formation in vitro may be used as a source of MMF and the term "MMF containing material"
encompasses said bio~ogical material.
Factors that stimulate proplatelet formation have not been previously disclosed. Hematopoietic factors that promote megakaryocyte development such as G-CSF, GM-CSF, IL-3 and IL-6 were tested in the in vitro . . . ..
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assay and did not stimulate proplatelet formation in the presence of a serum inhibitor. The activity of these factors is therefore distinct from the activity of factors that are the subject of the present application.
A method for assaying a megakaryocyte maturation factor is also provided. The method is described in Example l and comprises incubating either crude or purified MMF with megakaryocytes and monitoring the formation of proplatelets from megakaryocytes. Said method is preferably carried out in the presence of an inhibitor of proplatelet formation. An inhibitor of proplatelet formation is present in human serum (see Example 2).
The present invention also relates to factors which inhibit megakaryocyte maturation and proplatelet formation and are herein referred to as megakaryocyte maturation inhibitors. Megakaryocyte maturation inhibitors have properties of blocking the spontaneous maturation of megakaryocytes to proplatelets and stimulating the retraction of proplatelet extensions vitro. Human serum inhibits megakaryocyte maturation (Example 2). The inhibitory activity present in human serum is shown to copurify with prothrombin, an enzymatically inactive precursor to thrombin ~Example 5). Although both purified prothrombin and thrombin inhibit proplatelet formation, thrombin has inhibitory activity at lower concentrations than prothrombin. It is shown that thrombin is the megakaryocyte maturation inhibitor present in human serum and that prothrombin in human serum is converted to thrombin in order to inhibit megakaryocyte maturation Isolated megakaryocytes also carry out the conversion of prothrombin to thrombin (Example 6).
, Factors other than thrombin and prothrombin that inhibit , : . :
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~, WO 92~06712 2 0 7 1 ~ 7 9 pCr/US91/07367 , proplatelet formation and induce retraction of proplatelets are readily detected in the Ln vitro assay.
A method for purifying MMF is also provided.
This method comprises the steps of lysing human platelets and subjecting the human platelet lysate to two steps of ion exchange chromatography (e.g., DEAE and Mono Q). Throughout the procedure, the presence of MMF
is detected by Ln vitro maturation of megakaryocytes to proplatelets in the presence of an inhibitor, either human serum or purifi~d thrombin. Platelets are lysed in the presence of either guanidinium chloride or CHAPS
buffer (Example 8) although other methods suitable for platelet lysis may also be used. As shown in Example 9, platelet lysates obtained by either method are subjected to DEAE chromatography and three distinct peaks of proplatelet formation activity are observed ~Figs. 11 and 15). The three peaks are designated MMF-I, MMF-II
and MMF-III depending upon the salt concentration required for elution from the column. In Fig. 15, fractions containing MMF-I are not assayed for proplatelet formation. The biological activities of MMF-I, MMF-II and MMF-III are summarized in Example 12.
MMF-III is further purified by Mono Q chromatography (Figs. 12 and 16).
Two different biologically active forms of MMF-III are purified from human platelets. As shown in Example 10, lysis of platelets in the presence of guanidinium chloride to inactivate platelet proteases results in purified MMF-III having an amino terminal sequence starting with Y-P-T-Q. Lysis of platelets in the presence of CHAPS buffer results in purified MMF-III
having an amino terminal sequence starting with R-I-F-P.
The sequence of 16 amino acids originating from the amino terminus of MMF-III from the-guanidinium chloride - 35 lysate is identical to the sequence of 16 amino acids 2~71~79 W092/06712 PCT/US91/07~7 originating from the amino terminus of a purified human platelet proteoglycan (Alliel et al., ~La; Perin et al., ~La). The complete 131 amino acid long sequence of human platelet proteoglycan (Alliel et al., ~L~) also contains within it a nine amino acid internal sequence extending from residues 58 to 67 which is identical to the first nine amino acids of MMF-III from a CHAPS lysate. MMF-III from the guanidinium chloride lysate is hereafter referred to as MMF1-131 and is identical to the human platelet proteoglycan. MMF-III
from the CHAPS lysate is a truncated form of human platelet proteoglycan representing the carboxy terminal half of the full-length protein and is hereafter ; referred to as MMF58-131. Purified MMF1-131 stimulates proplatelet formation in ~i~LQ in the presence of thrombin and blocks thrombin-induced retraction of proplatelets (Figs. 18A and 18B).
The ability of a factor having part or all of the amino acid sequence of human platelet proteoglycan to promote megakaryocyte maturation to proplatelets has not been disclosed previously. Tt has been suggested that human platelet proteoglycan may act as a carrier for delivery of platelet factor IV to sites of blood vessel injury or as an inhibitor of complement sub-component Clq (Okayama et al., S~L~; Perin et al., suDra). However, no Ln vitro or in ~i~Q biological activity of human platelet proteoglycan has been disclosed, nor has any therapeutic benefit resulting from the administration of human platelet proteoglycan been described.
The present invention also encompasses megakaryocyte maturation factors having part or all of the amino acid sequence of MMF1-131 and having the property of promoting proplatelet formation from mature megakaryocytes. The factors described herein include ; 35 biologically active peptide fragments and amino acid :
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variants of naturally-occurring MME1-131. Said biologically active peptides are generated by proteolysis of MMF1-131 either by the action of cellular proteases in sitU or by protease treatment of full-S length purified MMF1-131 to generate protein core fragments having the ability to stimulate proplatelet formation. For example, purified MMF58-131 has equivalent proplatelet formation activity, based upon amount of uronic acid, compared to full-length MMF1-131 (Example 11).
In a preferred embodiment, MMF is the product of procaryotic or eucaryotic expression of exogenous DNA, that is, MMF is preferably recombinant MMF.
Recombinant mouse MMF and human MMF1-131 are described in Example 13. Exogenous DNA is obtained from genomic or cDNA cloning or from gene synthesis. Expression of MMF is carried out in procaryotic (bacteria) or eucaryotic (yeast, plant, insect or mammalian cells) host cells.
Analogs of MMF are also provided. Such analogs are produced by the manipulation of DNA seq~ences encoding the protein core of MMF1-131 to produce deletions, additions, or subs_itutions of nucleotides within the coding sequence so as to generate altered amino acid sequences. Such analogs are prepared using published procedures known to those skilled in the art.
Purified MMF having a carbohydrate structure - different from that of naturally-occurring MMF is also encompassed by the invention. The presence of glycosaminoglycan side chains on MMF1-131 is essential for megakaryocyte maturation activity as indicated by the loss of this activity upon treatment of purified MMF1-131 with chondroitinase ABC to remove attached carbohydrate chains (Example 11). Variation in carbohydrate structure can give rise to MMF molecules :. .
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2~71~79 WO92/~712 PCT/US91/07367 di.ffering in overall charge which are termed isoforms.
Isoforms of MMF are separated from each other and purified by techniques such as isoelectric focusing or chromatofocusing which have been described in the art.
The invention also provides for chemically modified forms of MMF which may exhibit increased solubility, stability and/or circulating half-life compared to unmodified MMF. The covalent attachment of !
a water soluble polymer to MMF is an example of one such chemically modified form. The water soluble polymer may be polyethylene glycol or a copolymer of polyethylene glycol and polypropylene glycol and said polymer is unsubstituted or substituted at one end with an alkyl group. These and related modifications are described in U.S. Patent No. g,179,337 hereby incorporated by reference.
Antibodies specifically binding to purified MMF are also comprehended by the invention. Such antibodies are directed to multiple antigenic determinants (polyclonal) or are directed to a single determinant (monoclonal) and are prepared using procedures known to those skilled in the art. Polyclonal and monoclonal antibodies are raised to purified glycosylated or deglycosylated MMF1-131 and MMF58-131.
Antibodies to MMF may be used in affinity chromatography to selectively remove MMF from media, serum, or urine.
In addition, antibodies specifically binding to MMF so as to inhibit proplatelet formation In yitro may be used to treat conditions resulting from excessive platelet production by stabilizing or decreasing circulating platelet levels.
The invention provides for the use of MMF
alone or in combination with other therapy in the treatment of platelet disorders. The methods and compositions of the subject invention are useful in . .
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'~WO92/~712 2 0 7 1 `I 7 9 PCT~US91/~7~7 treating thrombocytopenia, a condition marked by subnormal platelet levels in the circulating blood and the most common cause of abnormal bleeding.
Thrombocytopenia results from three processes~
deficient platelet production; (2) accelerated platelet destruction; and (3) abnormal distribution of platelets within the body. A compilation of specific disorders related to thrombocytopenia is shown in Table 1 (see ! - :
Wintrobe et al.(1981) In Clinical Hematology, Eighth edition, pp. 1090-1127).

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2071~79 -Platelet Disorders I. Deficient Platelet Production A. ~yDoDlasia or suppression of meaakaryQ~y~es Chemical and physicaL;agents (ionizing radiation, antineoplastic drugs), aplastic anemia, congenital megakaryocytic hypoplasia -myelophthisic processes, some viral infections B. Ineffective thrombo~oiesis ::
Disorders due to deficiency of vitamin B12 or folic acid C. Disordered control mechanisms Deficiency of thrombopoietin, cyclic thrombocytopenia D. Miscellaneous Many hereditary forms II. Accelerated Platelet Destruction A. ~Due to immunoloaic ~rocesses Idiopathic Thrombocytopenia Purpura, drug-. induced antibodies, various hemolytic anemia, fetomaternal incompatibility, post-transfusion B. pue to nonimmunoloaic processes Kasabach-Merritt syndrome, thrombotic thrombocytopenic purpura, infections ~viral, - bacterial, protozoan), massive transfusions : 30 . III. Abnormal Platelet Distribution .,~ ' .
A. Disorders of the spleen B. Hypothermia anesthesia :~ 35 ''`' ' ................. . .

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,~:WO9~/~712 2 0 71 ~ 7 9 PCT/US91/07~7 Advantageous applications of the subject invention are to thrombocytopenia resulting from deficient platelet production and, in some cases, from accelerated platelet destruction. In instances where levels of mature megakaryocytes are normal but platelet levels are low, MMF is used alone to stimulate proplatelet formation leading to increased platelet production. In cases where depressed platelet levels result from low levels of megakaryocytes, MMF is used in combination with one or more additional hematopoietic factors such as stem cell factor (SCF), G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF, and EPO to elevate both megakaryocyte and platelet levels.
Deficient platelet production results from a number of processes. The most common are those that depopulate the stem cell or megakaryocyte compartments, such as marrow injury by myelosuppressive drugs or irradiation, aplastic anemia, congenital megakaryocytic hypoplasia or myelodysplastic syndrome. A purified factor termed stem cell factor (SCF) has the ability to stimulate the formation of early hematopoietic progenitor cells, including megakaryocyte progenitor ; cells. SCF is described in U.S. Patent Application Ser.
No. 573,616 hereby incorporated by reference. Patients suffering from thrombocytopenia as a result of depleted stem cell levels are treated by administration of a pharmaceutically effective amount of SCF in combination with a pharmaceutically effective amount of MMF.
Thrombocytopenia resulting from depleted megakaryocyte levels is treated by administration of a therapeutically effective amount G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF, or EPO in combination with a therapeutically effective amount of MMF.

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Deficient platelet production may also result from ineffective thrombopoiesis where levels of mature megakaryocytes are normal or even elevated but platelet production is insufficient, as in, for example, megaloblastic hematopoiesis. Under these conditions, a therapeutically effective amount of MMF alone is sufflcient to raise platelet levels. In addition, abnormalities related to thrombopoietic control, such as cyclic thrombocytopenia, are treated with a therapeutically effective amount of MMF.
Accelerated platelet destruction results in thrombocytopenia due to a more rapid rate of platelet turnover than platelet production by meqakaryocyte maturation. Disorders such as idiopathic thrombocytopenic purpura (ITP), which are characterized by accelerated platelet destruction via an autoimmune response, may show reduced rates of platelet production.
In these instances, ITP is treated with a therapeutically effective amount of MMF.
Also comprehended by the invention are pharmaceutical compositions comprising therapeutically effective amounts of MMF together with suitable diluents, adjuvants, solubilizers, preservatives and/or carriers. A therapeutically effective amount of MMF is that amount sufficient to elevate circulating platelet levels in a mammal. A therapeutically effective amount of MMF in a pharmaceutical composition can be determined by the ordinary artisan taking into account such variables as the half-life of MMF preparations, route of administration and the clinical condition being treated.
Pharmaceutical compositions of MMF include diluents of various buffers (e.g. Tris-HC1, acetate, phosphate) having a range of pH and ionic strength that is compatible with MMF, solubilizers (e.g., Tween, Polysorbate), preservatives, (e.g., Thimerosol, benzyl .~

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alcohol) and carriers (e.g., human serum albumin).
Compositions comprising MMF may be administered by any route appropriate to the condition being treated, for example, by continuous infusion, sustained release formulation, or injection. The preferred route will be apparent to one skilled in the art.
The invention also comprises compositions of MMF and one or more additional hematopoietic factors such as SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF, `
0 and EPO.
Megakaryocyte maturation inhibitors are used to stabilize or decrease blood platelet levels.
Excessive platelet concentrations can lead to extensive blood ~tting, a situation observed in deep venous thrombc~.s and in thrombosis associated with post-surgery recovery. Maturation inhibitors are used alone or in combination with other therapeutics as anti-coagulants. Heparin and aspirin are currently used in anti-coagulation therapy.
Th~ following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.

Assay for ProDlatelet Formation An in vitro assay for the formation of platelet precursors from megakaryocytes was developed based upon observations of Radley et al., ~eL~ and Leven et al., supra. Guinea pig megakaryocytes were purified from bone marrow as previously described (Leven et al., suDra). Approximately 5,000 megakaryocytes (counted using a hemocytometer) were placed into wells ~ .

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, W092/~7t2 2 0 7 1 ~ 7 9 PCT/US91/07~7 f' of flat-bottomed 96-well microtiter plates (Falcon) in 100 ~1 of Iscoves media (Gibco) supplemented with S0 ~M
2-mercaptoethanol and 100 ~g/ml heat inactivated bovine serum albumin ~Sigma). After 18 hours incubation at 37C in 7% CO2 the cells were fixed in 10 mM EDTA, 0.37%
formaldehyde and examined under bri`ght field microscopy for the number of cells in each;~,well that had developed proplatelet formations. The data are expressed as the number of proplatelet formations per well (PPF/well).
Under these conditions, guinea pig megakaryocytes elaborate cytoplasmic extensions (proplatelets) without any other additions to the media. Photomicrographs of developing megakaryocytes before and after incubation are shown in Figures lA and lB.

Serum Inhibition of Proplatelet Formation The addition of increasing volumes of human serum ~Gibco) to guinea pig megakaryocytes prepared and incubated in medium at 37C for 18 hours as described in Example 1 inhibited proplatelet formation ~n vitro ~Fig. 2). Human serum present at 0.03% or greater resulted in complete inhibition of proplatelet ; formation.
The inhibition of proplatelet formation by human serum in the i~ vitrQ assay was transient. As ` shown in Table 2, megakaryocytes incubated in 0.1% human serum do not develop proplatelet formations when incubated for 18 hours at 37C, but do so after 42 hours incubation at 37C.
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' ~- W092/06712 2 0 7 1 4 7 9 PCT/U591/07~7 Transient Inhibition of Proplatelet Formation by Human Serum PPF/well Cells cultured in: 18 hours 42 hours Human serum 0 189 media 371 322 ~' 10 Inhibition of Proplatelet Formation by Prothrombin and Thrombin Thrombin, a serum-derived serine protease, was tested for inhibition of proplatelet formation ~n vitro.
~ighly purified thrombin (Sigma) was an effective inhibitor of proplatelet formation at concentrations less than 10 pM and complete inhibition was observed at 25 pM (Fig. 3). The inhibitory effect of thrombin was specific and was not observed with equivalent concentrations of the serine proteases trypsin (human pancreatic from Calbiochem) or chymotrypsin ~human ;~ pancreatic from Calbiochem). Nanomolar levels of trypsin and chymotrypsin were lethal to megakaryocytes while similar levels of thrombin did not affect megakaryocyte viability even though differentiation was ~
30 blocked. In addition, thrombocytocin, a thrombin-like -serine protease from ~othro~s atrox venom (Kirby et al.
Biochemistry 1~, 3564-3570 (1979), obtained from Sigma) was tested for inhibition of proplatelet formation. No inhibition was observed and cell viability was maintained up to 2.8 nM.

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2071~79 WO92/~712 -PCT/US91/07~7 Thrombin activity was detected in a c:hromogenic assay using chromogenic substrates S-2238 ~Sigma) or Chromozyme-Pca (Boehringer Mannheim) as clescribed (Lottenberg et al.~-~ethods Enzymol. 80, 341-361 ~1981)). When thrombin:-was present in complex mixtures, the specificity of the reaction was confirmed by the addition of hirudin, a specific anti-thrombin reagent. Using this assay, thrombin was detected in lots of human serum that inhibited proplatelet formation, but the amount present was too low to account for the extent of inhibition that was observed ~see Fig.
2). However, prothrombin, the unprocessed precursor of thrombin, is reported to be present in human serum at 1-2 ~M ~Mann et al. Methods Enzymol. 80, 286-303 ~1981)).
Purified prothrombin inhibited proplatelet formation L~
vitro when added to 2-5 nM ~Fig. 4). Complete inhibition was observed at 5 nM.
As with human serum, proplatelet inhibition by either thrombin or prothrombin is transient. No proplatelet formations were seen after 18 hours incubation at 37C in the presence of either 0.35% human serum, 25 pM thrombin, or 5 nM prothrombin. However, by 42 hours the inhibition had been overcome (Table 3).

Transient Inhibition of Proplatelet Formation by Thrombin and Prothrombin PPF/well Inhibitor 18 hours 42 hours Human Serum 3 252 Thrombin 0 227 Prothrombin 0 163 None 229 312 . .
.

2071~79 `
WO92/~712 PCT/US91/0~7 In addition to blocking proplatelet formation, thrombin and prothrombin induced the dedifferentiation of proplatelets. Approximately 5000 guinea pig megakaryocytes were incubated a~ described in Example 1 to form proplatelets, thrombin or prothrombin was then added to 66 pM or 5.7 nM respectively, and the cultures were returned to 37C. The number of proplatelets remaining were counted at the times indicated in Fig. 5.

ExAMæLE 4 Effect of Inactivatin~ Thrombin on proplatelet Inhibition Functions Purified thrombin (12.5 ~g/ml) in 40 mM Tris, pH 8.0, 100 mM NaCl, 2 mM ^aCl2 and 150 ~g/ml bovine serum albumin was reactec 1th 4.2 mM final concentratlon of diisopropyl fluorophosphate (DFP, obtained from Sigma) for two hours at room temperature to inactivate serine esterase activity. After incubation, the DFP-reacted thrombin was dialyzed extensively against 40 mM Tris, pH 8.0, 100 mM NaCl ar;
2 mM CaCl2 before use. Inactivation of serine esterase activity was confirmed by the inability of DFP-reacted thrombin to use chromozyme-Pca as a substrate.
DFP-reacted thrombin was compared to unreacted thrombin for its ability to block proplatelet formation (Fig. 6A) and induce retraction of proplatelet extensions (Fig. 6B). Inactivate thrombin had 2% and 1.5~ of the activity of thrombin in preventing proplatelet formation and inducing proplatelet retraction, respectively.
In addition to chemical inactivation of thrombin, thrombin inhibition of proplatelet formation is prevented by the addition to the proplatelet assay of ' , .

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2071~7~ ` I
WO92/~712 PCT/US91/07~7 agents which neutralize thrombin or prevent conversion of prothrombin to thrombin. As shown in Table 4, addition of 2.5 mM EDTA, 0.04 unit,s/ml heparin, 0.04 units/ml hirudin, or 0.10 units/ml antithrombin III
: 5 allows proplatelet formation in ~lhrQ~

10Agents Which Neutralize Thrombin Prevent Inhibition of Proplatelet Formation Neutralizing PPF
: Ex~. Inhibitor Aaent well 1 None None 120 Human Serum None 2 Human Serum 2.5 mM EDTA 106 ':
20 2 None None 230 Human Serum, None 2 Human Serum 0.04 units/ml Heparin 214 ..
. . , 3 None None 162 ' 25 Human Serum None 0 Human Serum 0.04 units/ml Hirudin 143 ., .
., 4 None None 255 Human Serum None 24 Human Serum 0.10 units/ml , 278 `~ Antithrombin III
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:,', , ' . ' 2071~79 ;W092/~712 PCT/US91/07~7 Purification of Human Serum Inhibitor and Com~ariqon With Prothrombin The inhibitory activity present in human serum was purified by DEAE-Sepharose chromatography ~Fig. 7) and Superose 6 chromatography (Fig. 8). Prot~-ombin levels were measured by conversion of prothro~in to thrombin with snake venom prothrombinase (Owen et al.
Thrombosis Res. ~, 705-714 (1973), obtained from Sigma) and assayed as described in Example 3 for thrombin. 20 ml of human serum were dialyzed against 40 mM Tris-HCl, pH 8.0 and loaded at 2 ml/min onto a 300 ml bed volume DEAE-Sepharose column (5 cm x 15 cm) equilibrated with the same buffer. Proteins bound to the column were e:.~ted with a linear NaCl gradient from 0 to 1 M in the same buffer. As shown in Figure 7, the peak of proplatelet inhibitory activity coincided with the peak obtained by the prothrombin assay. Fractions corresponding to this peak were pooled, concentrated and ~;
loaded at 0.75 ml/min onto a Superose-6 gel filtration column equilibrated in 20 m~ Tris-HCl, 0.1 M NaCl, 0.01%
polyethylene glycol 600, pH 7Ø As shown in Figure 8, ; 25 the proplatelet inhibitory activity eluted as a single broad peak having a molecular weight slightly higher than bovine serum albumin and coinciding with the peak obtained by prothrombin assay.

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WO9~/067l2 207 1 ~7 9 PCT/U591/07~7 :
~ EXAMPLE 6 Conversion of prothrom~in to Thrombin by Meaakaryocytes s Prothrombin is biologically inert until it is enzymatically converted to thrombin. The ability of megakaryocytes to convert prothrombin to thrombin is shown in Fig. 9.
Megakaryocytes were prepared as described in Example 1. Megakaryocytes with proplatelet formations - (PPF-megs) were prepared by incubating megakaryocytes as described in Example 1. Platelets were isolated from guinea pig marrow by differential centrifugation; they remain in the supernatant after centrifugation at 500 x g for ten minutes and are pelleted at 1,500 x g. .
Prothrombin was added to the indicated number of guinea pig platelets, megakaryocytes ~megs) or megakaryocytes with proplatelet formations (PPF-megs) to 143 ~gJml final concentration and the cultures were incubated for one hour at 37C. The culture supernatants were recovered and assayed for thrombin using the chromophore S-2238 as described in Example 3. Thrombin was generated under these conditions only when cells and ;~ 25 prothrombin were both present. Megakaryocytes and megakaryocytes with proplatelet formations were equally effective at the conversion of prothrombin to thrombin while several hundred times more platelets than megakaryocytes were needed for the conversion.

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~Y~aL ~o-f Serum Inhibition by a Meaakaryocyte Factor 5 Either human serum at O . 035 %, thrombin at 25 pM,or prothrombin at 5 nM were incubated for 42 hours at 4C or 37C under conditions described for the ln vitro assay. Approximately 5,000 megakaryocytes were added and the number of proplatelets formed after 18 hours was determined. No proplatelet formation was observed (Table 9). However, when human serum, thrombin or prothrombin were first incubated with megakaryocytes for 42 hours at 37C and 50 ~l of the reaction supernatant was then transferred to fresh megakaryocyte cultures, extensive proplatelet formation occurred after 18 hours (Table 5). The inhibitory activity of human serum, thrombin and prothrombin had been neutralized by prior incubation with megakaryocytes.

Effect of Inhibitor Pretreatment on Proplatelet Formation Pretreatment of Inhibitor PPF/well 42 hrs, 37C, Tnh;hitor42 hrs. 4C 42 hrs. 37Cwith ~egs - Human Serum 0 62 327 . .
Thrombin 0 0 218 None 279 298 343 ' :
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~ - 28 -Megakaryocytes were incubated in medium as described in Example l in the absence of inhibitor for 42 hours at 37C. The conditioned medium was harvested, concentrated six-fold by centrifugation through a Centricon-lO membrane filter, and 50 ~l of the concentrated medium was incubated with an equal volume of fresh megakaryocytes and either human serum or thrombin for 18 hours at 37C. Proplatelet formation was observed when megakaryocyte conditioned medium was used, whereas inhibition occurred in the presence of unconditioned medium (Table 6~.

Stimulation of Proplatelet Formation by Megakaryocyie Conditioned Medium Megakaryocyte Inhibitor Conditioned Medium PPF/Well None No 20S
Human Serum No l5 Human Serum Yes 258 Thrombin No l8 25 Thrombin Yes 223 These experiments indicated that megakaryocytes produce and secrete soluble factors that : 30 neutralize or functionally override the inhibition of -.~ proplatelet formation by human serum or purified ` thrombin. These factors are referred to as megakaryocyte maturation factors (MMF).
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~ W092/067t2 2 0 7 1 ~ 7 ~ PCT/US91/07~7 Stimulation of ProDlatelet Formation by Human Platelet Lysate~
The presence of megakaryocyte maturation factors in platelets was determined by preparing human platelet lysates and assaying for in vitro proplatelet formation (Fig. 10).
Human platelets from normal donors were obtained in plateletpheresis packs containing approximately 3-4 x 1011 platelets in approximately 200 ml of platelet rich plasma ~PRP, obtained from HemaCare). Platelets were used within 24 hours of the - 15 draw. Apyrase (Sigma) was added directly to the blood bag to a final concentration of 2 units/ml and incubated at 37C for 20 minutes. PRP was transferred to 50 ml polypropylene tubes and centrifuged at 120 x g for 8 minutes at room temperature to remove contaminating blood cells. The supernatant was transferred to polycarbonate tubes and centrifuged at 1,500 x g for 20 minutes to pellet platelets.
For platelet lysis in CHAPS, the pellet was washed three times by centrifugation at 1,500 x g for 20 - 25 minutes at room temperature and resuspension in the following buffers: Wash 1, 280 ml of Tyrodes buffer (137 mM NaCl, 2.7 mM KCl, 12 mM NaHC03, 0.4 mM NaH2P04, lmM
MgCl2, 2 mM CaC].2, 5.5 mM dextrose, pH 7.35) . . .
supplemented with ~.4% human serum albumin and 2 units/ml Apyrase; Wash 2, 140 ml Tyrodes buffer and ` 2 units/ml Apyrase; Wash 3, 140 ml Tyrodes buffer -followed by the final centrifugation. Platelets were lysed in 5 mM 3-(3-cholamidopropyl)-dimethyl-ammonio-1-propanesulfonate (CHAPS, obtained from Calbiochem) at 1.6 x 101 platelets/ml for one hour on ice. The lysate . - , .
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2071~79 WO92/~12 PCT/US91/07367 was centrifuged at 2,200 x g and dialyzed against four changes of 90 mM Tris, pH~8 (4 liters each change). The platelet lysate was~c~arified by centrifugation at 150,000 x g for 60 minutes.
For platelet lysis in guanidinium chloride, the pellet was washed se~uentially in the following buffers: Wash 1, 280 ml Tyrodes buffer supplemented with 22 mM trisodium citrate, 0.4% human serum albumin and 2 units/ml apyrase, pH 6.5; Wash 2, 140 ml Tyrodes buffer supplemented with 22 mM trisodium citxate and 2 units/ml apyrase, pH 6.5; Wash 3, 140 ml Tyrodes buffer and 22 mM
trisodium citrate, pH 6.5 followed by the final centrifugation. Platelet pellets were solubilized in 6 M guanidinium chloride in 50 mM sodium acetate, 10 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride and 10 mM
6-amino hexanoic acid, pH 6.0 at 8 x 109 platelets/ml for 3 hours at 4C with gentle stirring. The solution was then dialyzed against four changes of 4 liters each of 40 mM Tris, pH 8, 1 mM PMSF, and clarified by 20 centrifugation at 150,000 x g for 60 minutes.

purification of Meaakaryocyte Maturation Factors From Human Platelets .`: -MMF from human platelets was pur.ified by thefollowing procedures. The presence of MMF during purification was detected by proplatelet formation j 30 Ln vitro in the presence of a maturation inhibitor.

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. Purification of MMF from guanidinium chloride extracted platelets. i A guanidinium chloride lysate of human platelets (160 ml containing 240 mg of protein) was prepared according to Example 8. The lysate was equilibrated with 90 mM Tris-HCl, pH 8.0 and loaded onto a 220 ml (2.6 cm x 40 cm) DEAE-Sepharose column at a flow rate of 1 ml/min. The column was washed with the Tris buffer and developed with a linear NaCl gradient from 0 to 1 M in the same buffer (total gradient volume was 800 ml). As shown in Figure 11, assay of column fractions for proplatelet formation activity revealed three distinct peaks, designated M~F-I, MMF-II and MMF-III, eluting at different NaCl concentrations.
The fractions corresponding to MMF-III were pooled and dialyzed against 5 mM sodium citrate, 0.01%
PEG 600, pH 5Ø The dialyzed pool (237 ml at 0.012 A2go/ml) was loaded onto a Mono-Q FPLC column (0.5 x 5 cm) equilibrated with 5 mM sodium citrate, 0.01% PEG
600, pH 5Ø The flow rate was adjusted to 0.5 ml/min.
After washing with the same buffer, the column was developed with a linear NaCl gradient from 0 to 1 M
~total gradient volume was 60 ml) followed by a 1 M NaCl wash. As shown in Figure 12, a broad peak corresponding to proplatelet formation corresponds with a peak of - absorbance at 280 nm. The fractions corresponding to this peak were combined and the resulting pool (18 ml at - 0.072 A2go/ml) were analyzed for purity, molecular weight, and amino acid sequence.
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B. Analysis of purified MMF from guanidinium chloride lysates.

2 ml of the Mono-Q pool was concentrated to 200 ~l by ultrafiltration using a Centricon-10 filtration device and loaded onto a Superose-6 gel filtration column (1 cm x 30 cm) at a flow rate of 0.5 ml/min in 40 mM Tris-HCl, O.lmM NaCl, 2 mM CaCl2, pH
8Ø As shown in Figure 13, a peak of activity corresponding to proplatelet formation activity coincides with a peak and shoulder measured by absorbance at 230 nm appearing in the void volume. This indicates that the MMF-III preparation in heterogeneous in size, but the different forms of MMF-III have similar levels of activity.
2 ml of the Mono-Q pool were dialyzed against 0.1~ trifluoroacetic acid (TFA) and concentrated to 200 ~l by ultrafiltration using a Centricon-10 filtration device. The concentrated sample was loaded onto a C4-reverse phase high pre~sure liquid chromatography column(0.46 x 25 cm Vydac C4 column 214TP54) in 0.1% TFA at ~- 0.75 ml/min and the column was developed with an acetonitrile gradient in 0.1% TFA. As shown in Figure 14, a broad peak of proplatelet formation activity coincided with a peak of absorbance at 214 nm, again indicating different forms of active MMF-III.

C. Purification of MMF from CHAPS lysates of human platelets.
A platelet lysate extracted with CHAPS buffer as described in Example 8 and equilibrated with 40 mM
; Tris-HCl, pH 8.0, was purified by DEAE-Sepharose and Mono-Q column chromatography as described above for the ; 35 guanidinium chloride extracted platelets. As shown in ' . . . . ' . ~ , . . . . ..
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E`igure 15, two distinct peaks of proplatelet formation activity designated MMF-II and MMF-III were obtained by ~EAE-Sepharose chromatography. A third peak of activity, designated MMF-I, is present in the flow-through fractions but was not assayed in thispreparation. The peak of activity around fraction 38 (corresponding to MMF-III from the guanidinium lysates) was pooled and applied to a Mono-Q column. As shown in Figure 16, proplatelet formation activity was eluted in a broad peak from fractions 28 to 36.

D. Analysis of purified MMF-IIIS by SDS-PAGE.

Aliquots of the Mono-Q pools from guanidinium chloride and CHAPS lysates were treated with 0.1 unit of chondroitinase ABC at room temperature for 24 hours in 40 mM Tris-HCl, pH 8Ø The samples were dried in a speed-vac and analyzed along with untreated samples on a 12.5% SDS-polyacrylamide gel (Fig. 17). Samples that had not undergone chondroitinase ABC treatment were not detected in the gel, suggesting that purified MMF-III
from CHAPS or guanidinium chloride lysates had a substantial amount of covalently attached carbohydrate that prevented entry into the gel. Chondroitinase-treated samples migrated as several bands on SDS-PAGE, ; suggesting that not all carbohydrate could be removed from the protein core even after exhaustive digestion.

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2071~73 W O 92/06712 PC~r/US91~07367 N-terminal Amino Acid Se~uences of MMF-IIL_~om Guanidinium Chloride and CHAPS Lysates of Human Platel~

MMF-III purified from guanidinium chloride and CHAPS lysates of human platelets were subjected to N-terminal sequencing using Applied Biosystems Models 470A and 973A sequencers with on-line PTH analysis using the manufacturer's high pressure liquid chromatography systems. Sequence assignments were made by comparison of the cycle to cycle chromatograms. The following sequences were assigned:
MMF-III from guanidinium chloride lysate:
Y-P-T-Q-R-A-R-Y-Q-W-V-R-X-N-P-D

MMF-III from CHAPS lysate:
R-I-F-P-L-S-E-D-Y
The N-terminal amino acid sequence determined for MMF-III from the guanidinium chloride lysate was identical to the N-terminal sequence of human platelet proteoglycan (Alliel et al., supra; Perin et al., supra). MMF-III from the guanidinium chloride lysate, ; which is identical to human platelet proteoglycan, is ~`~ referred to as MMF1-131.
The N-terminal sequence of MMF-III from the CHAPS lysate was identical to the sequence of amino acids 58 to 66 of human platelet proteoglycan (Alliel et al., ~L~; Perin et al., supra). MMF-III from the CHAPS
- lysate is identical to the carboxy terminal 64 amino - acid fragment of human platelet proteoglycan (and MMF1-131) and is referred to as MMF53-131.
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WO92/~712 PCT~US9~/07~7 A~Yi~Y of Purif;ed MMF1-131 and 58-131 MMF1-131 and MMF58-l3l were assayed for uronic acid content as described (Bitter and Muir, Anal.
Biochem. 4, 330-339 (1962)). Protein concentrations were determined by theoretical extinction coefficients based upon the amino acid sequence data of each form and amino acid yields obtained during sequencing. MMF58-13 had approximately 140 ~g uronic acid/~g protein and MMF1-131 had approximately 80-lO0 ~g uronic acid/~g protein.
MMF1-131 was assayed for its ability to prevent thrombin-induced inhibition of proplatelet formation ~Fig.18A). Purified thrombin was serially diluted in Iscoves media or in an MMF1-131 preparation, added to culture wells and incubated at 37C for 3 hours. MMF1-131 was present at 0.1 ~g/ml protein and 10 ~g/ml uronic acid and thrombin was present from 0.35-100 pM. Approximately 5000 megakaryocytes per well were added and the number of proplatelets in each well was counted after 18 hours.
MMF1-131 was assayed for its ability to prevent thrombin-induced proplatelet retraction ~Fig.
18B). Purified thrombin and an MMF1-131 preparation were distributed into culture wells as described above and incubated at 4C for 18 hours. The contents of the ' - wells were transferred to wells containing proplatelets and the number of proplatelets remaining were counted ; after 10 minutes.
MMF1-131 and MMF58-131 were added to the ln vitro proplatelet formation assay described in Example 1 at equivalent uronic acid concentrations and proplatelet formations were determined ~Fig. 19).

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MMF1-131 and MMF5~8 131 were equally active (per ~g of uronic acid) in this assay.
The role of attached carbohydrate ~chondroitin sulfate) in the biological activity of MMF-III was determined. MMF58-131 from DEAE chromatography was incubated in 40 mM Tris, 90 mM Na acetate, pH 8.0 in the presence or absence of 0.1 unit/ml chondroitinase ABC
(Boehringer Mannheim) for 18 hours at 37C. Treated MMF58-131 was exchanged into Iscoves media and added at up to 50$ of the volume the proplatelet assay. The results in Fig. 20 show that MMF53-131 treated with chondroitinase ABC lacks detectable proplatelet formation activity.

Pro~erties of Meaakaryocvte Maturation Factors .Separated bv DEAE ChromatoaraDhy Table 7 shows a comparison of the biological activities of MMF-I, MMF-II and MMF-III which were obtained by lysis of human platelets in CHAPS buffer as described in Example 8 and DEAE chromatography as described in Example 9.

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Clonina and Ex~ression of Mouse and Human ~MFl-131 Genes Except where noted, recombinant DNA procedures described in Maniatis et al. (Molecular ~lQn1n~, Cold Spring Harbor Laboratory, pp. 212-246 (1982)) were used.

A. Amplification and Cloning of the Mouse MMF cDNA.

RNA was purified from the murine cell line MC/9.5, a subclone of MC/9 (ATCC No. CRL 8306) using the cesium trifluoroacetate pelleting protocol (Okayama et al. Meth. Enzym. 154, 3-28 (1987)). Oligonucleotide primers M1-M4 were designed from the published cDNA
sequence of a mouse mast cell secretory granule proteoglycan (Avraham et al. Proc. Natl. Acad. Sci. USA
86, 3763-3767 (1989)) and synthesized on an Applied Biosystems DNA synthesizer.
First strand cDNA synthesis was derived from MC/9.5 RNA as template and the antisense primer 5'-CTGAATACATTGTTCCACATGG-3' (Ml) -whose sequence is complementary to a portion of the cDNA
sequence of mouse mast cell secretory granule proteoglycan at the 3' side of the protein coding region. cDNA synthesis was carried out with M-MLV
` reverse transcriptase using procedures supplied by the manufacturer (Bethesda Research Laboratories, Gaithersburg, MD).
First strand cDNA from about 60 ng of RNA was ; used as template for polymerase chain reaction (PCR) ; amplification (Saiki et al. Science 239, 487-491 (1988)) using the oligonucleotide primer 5'-CTAATCCAGAGGCTGAGTGGA-3' (M2) . , .
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a sense strand pr~mer positioned at the 5' side of the coding region. The product of this PCR amplification was further amplified using the nested primers 5'-GACGGATCCAAGCTTCCACCATGCAGGTTCCCGTCGGCA-3' (M3) and 5'-GTGAGTCGACAGAGACCGTCACATTCA-3' ~M9).
Primer M3 contains the sequence 5'-CCACC-3' immediately preceding the coding sequence for murine MMF-III, such a sequence having been shown previously to be optimal for translational efficiency (Kozak, Nuc. Acid Res. 1~, 8125-8148 (1987)).
The products of PCR amplification using primers M3 and M4 were digested with BamHI and SalI and ligated into pDSR~2, a derivative of vector pCD (Okavama et al., Mol. Cell. Biol. 3, 280-298 (1983)), yieldir.
the recombinant plasmid pDSR~2 (muMMF). The DNA
sequence of murine MMF insert was determined by the dideoxy method (Sanger et al. Proc. Natl. Acad. Sci., USA ~, 1934-1938 ~1977)). The sequence of murine MMF
was identical to that reported for the mouse mast cell ; secretory granule proteoglycan (Avraham et al., ~upra).
.
B. Amplification and Cloning of the Human1~131 cDNA.

RNA was purified from a human leukemic cell ~;
line (HEL, ATCC No. TIB 810) using procedures described j~ above. Oligonucleotide primers Hl-H4 were designed from the sequence of the human secretory granule proteoglycan (Stevens et al., supra).
- First strand cDNA synthesis was derived from HEL RNA as template and the human MMF antisense primer 5'-TGCTAACTAATTGCCTGGTGT-3' (Hl).
PCR amplification was performed with primers H1 and 5'-GAGAGCTAGACTAAGTTGGTCA-3' (H2).

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i The product of PCR was further amplified using the nested primers 5'-GAGGATCCAAGCTTCCATGATGCAGAAGCTAC-3' tH3) and 5'-GCCGTAGTCGACAACCTGGGAAAACCTCTT-3' (H4) which contain the restriction sites HindIII and SalI, respectively.
The product of PCR amplification using primers H3 and H4 were digested with HindIII and SalI and ligated into pDSRa2 as described above yielding the recombinant plasmid pDSRa2 (hUMMFl-l3l). The DNA
sequence of human MMF was determined by the dideoxy method (Sanger et al. supra) following irreversible denaturation of supercoiled DNA. The sequence of human MMF was identical to that reported for the human secretory granule proteoglycan (Stevens et al., sy~La)-C. Expression of murine and human MMF-III.

For expression of mouse and human MMF, plasmid pDSRa2 (huMMF-III) or pDSRa2 (muMMF) was transfected into COS cells by electroporation (Potter et al. Proc.
Natl. Acad. Sci. ~SA ~1, 7161-7165 (1984)) or into Chinese Hamster Ovary (CHO) cells by calcium phosphate 25 coprecipitation (Wigler et al. Cell 11, 223-232 (1977)).
Transfected COS cells were grown for 2-5 days at 37 in Dulbecco's modified essential medium (DMEM) supplemented with 1% fetal calf serum (FCS). Conditioned media is harvested and assayed for proplatelet formation in Yi~LQ
- 30 as described in Example 1. Transfected CHO cells were seeded at a low density (-105 cells/100 mm dish) and grown for 10-14 days at 37C in DMEM supplemented with nonessential amino acids and 10% dialyzed FCS. Colonies ` were picked or cells were treated with trypsin and transferred to fresh media for an additional 10-14 days.
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Conditioned media is harvested and assayed for proplatelet formation ~n vitro as described in E,xample 1. Transfected CHO cell cultures that stimulate proplatelet formation are then grown in the presence of methotrexate to amplify MMF expression.

Effect of MMF1-l3l on Blood Platelet Levels Experiments designed to determine the effects of administering MMF1-13l on circulating platelet levels were performed on female Balb/c mice (Charles River) 6-8 weeks old. All animals within an experiment were from age-matched litters.
MMF1-131 was purified from human platelets as described in Example 9. Human recombinant IL-6 was purified from CHO cell conditioned media. Mice were injected subcutaneously with 200 ~l of either MMF1-13 or IL-6 in 150 mM NaCl, 0.1% bovine serum albumin ~BSA) - two times per day at eight hour intervals for a total of ten injections. Three hours after the final injection, a 20 ~l blood sample was taken from each animal through a small incision in the lateral tail vein using calibrated microcapillary tubes. The samples were ~ diluted directly into a diluent required for analysis in `~ a Sysmex microcell counter F-800 (TOA Medical Electronics Co.). The resulting data were analyzed by Scheffe's F-test using that Statview 512+ software program. Data having significance at greater than 95%
are indicated by an asterisk.
MMFl-13l increased platelet levels when administered at 4 ~g/day or 20 ~g/day ~Fig. 21). A
statistically significant increase of 21% in platelet levels was observed when MMF1-131 was administered at a ' .' ~
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207~9 W092/06712 ` PCT/US91/07~7 dose of 20 ~g/day. IL-6 also increased platelet levels when administered at 2 ~g/day or l0 ~g/day with a statistically significant increase of 34% observed at a dosage of l0 ~g/day (Fig. 22). A combination of MMF1-l3l at 20 ~g/day and IL-6 at ~2-~g/day resulted in a 40% increase in platelet levels. -This increase is statistically significant compared to the levels obtained upon administration of only MMF1-131 at 20 ~g/day or only IL-6 at 2 ~g/day (Fig. 23). Under the conditions of the experiment, the doses of MMF1-131 and IL-6 used did not, by themselves, raise platelet levels to significantly higher levels. Other hematological parameters such as white and red blood cell counts and hematocrit were unaffected by MMF1-131 or IL-6 treatments.

* * *

While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations ` ~ which come within the scope of the invention as claimed.
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Claims (23)

WHAT IS CLAIMED IS:

1. A method for increasing blood platelet levels in a mammal comprising administering a therapeutically effective amount of a megakaryocyte maturation factor.

2. A method as in Claim 1 further comprising administering a therapeutically effective amount of SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF or EPO.

3. A method as in Claims 1 or 2 wherein the megakaryocyte maturation factor has part or all of the amino acid sequence of MMF1-131.

4. A method as in Claims 1 or 2 wherein the megakaryocyte maturation factor is the product of procaryotic or eucaryotic expression of an exogenous DNA sequence.

5. A composition comprising a therapeutically effective amount of a purified and isolated megakaryocyte maturation factor and one or more of a pharmaceutically acceptable adjuvant, diluent, solubilizer, preservative or carrier.

6. A composition as in Claim 5 further comprising a therapeutically effective amount of SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF or EPO.

7. A composition as in Claims 5 or 6 wherein the megakaryocyte maturation factor has part or all of the amino acid sequence of MMF1-131.

8. A composition as in Claims 5 or 6 wherein the megakaryocyte maturation factor is the product of procaryotic or eucaryotic expression of an exogenous DNA sequence.

9. A composition as in Claims 5 or 6 wherein the megakaryocyte maturation factor is covalently attached to a water-soluble polymer.

10. A composition as in Claim 9 wherein the polymer is selected from the group consisting of polyethylene glycol or copolymers of polyethylene glycol and polypropylene glycol, and said polymer is unsubstituted or substituted at one end with an alkyl group.

11. A method for assaying a megakaryocyte maturation factor comprising incubating MMF with megakaryocytes in a proplatelet formation assay and monitoring the response of the megakaryocytes to MMF.

12. A method according to Claim 11 wherein the MMF is derived from human serum, urine, megakaryocytes or platelets.

13. A method for the purification of a megakaryocyte maturation factor from MMF containing material comprising one or more steps of subjecting MMF
containing material to ion exchange chromatography.

14. A method as in Claim 13 wherein the MMF
containing material is human blood platelets.

15. A method for the treatment of thrombocytopenia in a mammal caused by ineffective thrombopoiesis or abnormal thrombopoietic control comprising administering a therapeutically effective amount of a megakaryocyte maturation factor.

16. A method as in Claim 15 wherein thrombocytopenia results from megaloblastic hematopoiesis or cyclic thrombocytopenia.

17. A method for the treatment of thrombocytopenia in a mammal caused by accelerated platelet destruction comprising administering a therapeutically effective amount of a megakaryocyte maturation factor.

18. A method as in Claim 17 wherein thrombocytopenia results from idiopathic thrombocytopenic purpura.

19. A method for the treatment of thrombocytopenia in a mammal caused by depopulation of stem cell or megakaryocyte compartments comprising administering a therapeutically effective amount of a megakaryocyte maturation factor and a therapeutically effective amount of SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF or EPO.

20. A method as in Claim 19 wherein thrombocytopenia results from myelosuppressive drugs or irradiation.

21. A method as in Claim 19 wherein thrombocytopenia results from aplastic anemia.

22. A method as in Claim 19 wherein thrombocytopenia results from congenital megakaryocytic hypoplasia.

23. A method as in Claim 19 wherein thrombocytopenia results from myelodysplastic syndrome.