SOLUBLE ANALOGS OF THROMBOMODULIN

Abstract: The present invention relates to the production and use of recombinant DNA technology, to manufacture soluble thrombomodulin analogs useful, e.g., in anti-thrombotic therapy. Novel proteins, nucleic acid sequences, vectors, pharmaceuticals and methods of inhibiting thrombotic activity are disclosed herein.

Description: SOLUBLE ANALOGS OF THROMBOMODULIN BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the production and use of recombinant DNA technology to manufacture soluble thrombomodulin analogs useful, e.g., in antithrombotic therapy. Novel proteins, nucleic acid sequences, vectors, pharmaceuticals and methods of inhibiting thrombotic activity are disclosed herein. Information Disclosure In the past decade, researchers and clinicians have intensified the search for improved antithrombotic compounds capable of preventing clot formation, as well as decreasing the chance of a reocclusion event after fibrinolytic therapy. Several disease states require treatment with an effective, safe anticoagulant. For example, anticoagulants are used for treatment of pulmonary embolism, to prevent deep venous thrombosis in patients undergoing prolonged bed rest or following certain surgical procedures, and for treatment of acute myocardial infarction with or without thrombolytic therapy. Currently, anticoagulant therapies rely on heparin or warfarin compounds, each of which has various drawbacks. (Prevention of Venous Thrombosis and Pulmonary Embolism. Consensus Development Conference Statement 1986, 6(2):1-23.) Heparin in particular has several other physiological effects that impinge on hemodynamics and hemostasis. Also, these treatments necessitate very stringent laboratory monitoring, as patients are at severe risk for bleeding episodes. In these therapies, it is difficult to determine the proper dose that will provide anticoagulation yet not cause excessive bleeding. Warfarin therapy often takes several days to become effective, and the effects require even longer to subside once the therapy is discontinued. Thrombomodulin, a 105 kDa cell membrane protein, has been shown to have anticoagulant properties. In humans, thrombomodulin is widely distributed on the endothelium of blood vessels and lymphatics of all organs except the central nervous system and helps modulate the balance between the formation and the dissolution of blood clots. Specifically, thrombomodulin controls the activity of thro bin, a central enzyme in the coagulation of blood. Thrombin's activities include conversion of fibrinogen to fibrin and stimulation of platelet activation and aggregation, ultimately resulting in blood clot formation. When thrombin is bound to thrombomodulin, however, the complex does not promote clot formation but instead acts as an anticoagulant by inhibiting the direct procoagulant actions of thrombin, and by accelerating the activation of Protein C. Activated Protein C is a potent anticoagulant enzyme which disrupts the coagulation cascade. (See, for example, N. Esmon et al. (1982), J. Biol. Chem. , 257:859-864; H. Salem et al. (1984), J. Biol. Che .. 2j>9:12246-51.) The DNA sequence encoding thrombomodulin has been isolated and sequenced both in its genomic form and as a cDNA molecule (R. Jackman ejt al. (1986) , Proc. Natl. Acad. Sci. USA.. 82:8834-38 and (1987), 84:6425- 29, both of which are incorporated herein by reference) and domains of thrombomodulin have been suggested (D. Wen et al. (1987), Biochemistry. £6:4350-57). Functional assignments for several domains have been proposed on the basis of comparison to the DNA sequence of other known proteins, such as the LDL receptor. One study has suggested that the fifth and sixth epidermal growth factor (EGF)-like domains (within the EGF-like domain) have the capacity to bind thrombin (S. Kurosawa et al. (1988) J. Biol. Chem.. 263:5993-5996) . Another study has suggested that EGF-like domains 4,5 and 6 are sufficient to give Protein C activating activity. (Zushi, et al., (1989) J.Biol.Chem. 264:10351.). Unfortunately, native thrombomodulin is generally unsuitable for antithrombotic therapy. The amounts needed for commercial application makes purification from autopsy or biopsy samples impractical, since it is estimated that only about 300 mg thrombomodulin are present in the total endothelium of a single person. Native thrombomodulin is membrane bound due to its inherent amino acid sequence. It is insoluble without detergent treatment, generally rendering it unsuitable for systemic therapies such as for anticoagulation. Moreover, it would be problematic to produce or purify from any source, including a genetically engineered cell. The Km of purified, detergent solubilized thrombomodulin for Protein C is only 5% that of the membrane bound protein, although the affinity is stronger when the isolated protein is incorporated into phospholipid vesicles (Galvin et al. (1987), J. Biol. Chem.. 257(5) :2199-2205) . It has been reported that native human thrombomodulin preparations are not very effective in blocking procoagulant activities of thrombin (Maruyama et al. (1985) , J. Clin. Invest.. 75:987-991). Soluble thrombomodulin-like molecules have been reported in human plasma and urine. These molecules have a greatly reduced ability to activate Protein C and may be degradation products of the cellular form of thrombomodulin (H. Ishii and Majerus, P., (1985), J. Clin. Invest. f 76:2178-81). These forms of thrombomodulin are present in such low amounts (approximately 0.8 mg/adult male), that they have proven difficult to characterize. More recently two groups have presented evidence that the levels of circulating thrombomodulin have been overestimated by a factor of 10 to 20 (Suzuki et aj_. , (1988), J. Biochem.. 104:628-632 and references therein). Elastase cleaved fragments of purified, native thrombomodulin have been described and partially sequenced (see Ishii, (1985), supra; Kurosawa et al., (1987), J. Biol. Chem.. 262:2206-2212; Kurosawa et al J. Biol. Chem.. (1988), 263:5593-5996; and Stearns et al., J. Biol. Chem.. 1989, 264:3352-3356). The elastase fragment and some smaller fragments retained the ability to enhance the activation of Protein C in vitro. Additional references include EP 290,419 and WO 88/05053, which discloses cDNA encoding the thrombomodulin protein. Analogs of thrombomodulin have been described in WO 88/05053 which discloses analogs with varying numbers of EGF domains. There exists a need for new compositions that exhibit the anticoagulant properties of thrombomodulin, are soluble in plasma, and are easily produced in large quantities. The present invention fulfills these and other needs. SUMMARY OF THE INVENTION This invention provides sequences of nucleic acid encoding a peptide selected from the group consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c ) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; where Y is selected from the sequence of amino acids provided in Table 2 ranging from amino acid 227 to amino acid 462, and where Q is selected from the sequence of amino acids provided in Table 2 ranging from amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 or amino acid 227 to amino acid 497. The sequences represented by Y and Q denote the epidermal growth factor-like domains of the native TM, the fourth, fifth and sixth EGF-like domains and the six EGF-like domains plus the O-linked glycosylation domain. Also provided are polynucleotide sequences encoding the peptides described above where the sequence is selected from the group consisting of: a) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X- GACTCCGGCAAGGTGGAC; b) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X-GACTCC; c) GGAGCCAGATCC-Z; and d) GCCGTCGTACCAAGATCC-Z; where X represents the nucleic acid bases numbered 879 to 1586, and where Z represents the nucleic acid bases numbered 879 to 1586, 1251 to 1586 or 879 to 1690 all numbers refer to bases depicted in Table 2 and vectors comprising such nucleotide sequences. Further provided are substantially pure protein compositions selected from the peptides described above. Such compositions may be those wherein the peptide is a species of the peptide which exhibits about the expected molecular weight for said peptide under non-reducing chromatography. By "exhibits about the expected molecular weight" it is meant that the peptide appears as a band on a chromatographic gel within about 10% of the predicted molecular weight of the peptide based on its structure (exclusive of any glycosylation that might be present) . They may also be in a dry and salt-free form, a lyophilized powder, and/or essentially free of detergents. In another embodiment, the compositions may comprise one of the described peptides chemically linked to a fibrinolytic enzyme, preferably where the chemical linkage arises from a covalent bond between the enzyme and an amino acid residue of the peptide. The fibrinolytic enzyme in such a complex is preferably a streptokinase molecule bound to a plasminogen molecule or t-PA. Pharmaceutical compositions having antithrombotic activity comprising a sterile preparation of a unit dose of thrombomodulin-like protein and having an amino acid sequence described above are disclosed, as well as methods for controlling thrombotic activity in a mammal by administering an effective amount of such compositions. Further disclosed are compositions which comprise a biocompatible polymer having a surface which has peptides bound thereto selected from the group of peptides described above and methods for inhibiting blood clotting induced by polymers implanted into a living mammal wherein a thrombomodulin-like protein is bonded to the polymer and the polymer is implanted into the mammal. This invention additionally dislcoses an isolated DNA fragment containing a DNA sequence coding for multi-functional soluble human thrombomodulin (TM) analog, said analog consisting essentially of amino acid residues 350 to 462 or 390 to 462 or 227 to 462 of native TM and a targeting component. The targeting component is a sequence of amino acids capable of binding to fibrin and preferably also imparting fibrinolytic activity to the bifunctional TM analog such as the amino acid residues 4 - 530 of human tissue plasminogen activator as depicted in Table 9. These DNA sequences are preferably operably linked to an expression control sequence and transfected into a cell. The analogs are preferably operably linked to a DNA sequence coding for a fusion protein comprising: a first amino acid sequence comprising a thrombomodulin fragment without a stop transfer domain; and a second amino acid sequence comprising a targeting component; wherein the bifunctional TM analog is secreted from the host. Preferred sequences of TM used in these analogs are as follows: delta 1-389, delta 463-557 TM; delta 1-226, delta 498-557 TM; delta 1-226, delta 463- 557 TM; and delta 1-349, delta 463-557. Where the delta regions are missing and the numbers refer to the amino acid sequence of thrombomodulin depicted in Table 2. The bifunctional analog may have fibrinolytic activity resulting from the fusion with t-PA as described above. The invention further provides for methods of suppressing coagulation in a patient comprising administering to the patient an amount of soluble TM analog effective to suppress further coagulation, wherein the analog comprises TM amino acids as described above. The invention also provides for treating a patient suffering form acute myocardial infarction caused by a thrombus, said method comprising administering to the patient an amount of bifunctional soluble TM analog effective to suppress further coagulation and lyse the thrombus, wherein the bifunctional TM analog comprises TM amino acids as described above wherein said TM amino acid sequence is fused at the C-terminus or the N-terminus to amino acids 4-530 of human t-PA. The invention also provides for a bifunctional TM analog capable of binding thrombin with substantially the same affinity for thrombin as native thrombomodulin, said analog comprising native thrombomodulin without a functional stop transfer domain, wherein said bifunctional analog is further characterized by the ability to bind fibrin and cleave plasminogen to plasmin. In particular there is provided the analog which consists essentially of the amino acid sequence set forth in Table 9. There is also provided, TM analogs wherein said analogs consist essentially of the signal sequence of t-PA covalently attached to the amino terminus of the 6 EGF-like domain of thrombomodulin and the 6 EGF-like domain of thrombomodulin covalently attached to the amino terminus of t-PA. This invention further relates to soluble thrombomodulin analogs and methods for making such analogs. More specifically these processes for making soluble thrombomodulin analogs comprising the steps of: culturing a host cell transformed with a DNA sequence encoding the analog; and collecting analogs secreted by the host cell; wherein the analogs lack a stop transfer sequence functional in the host cell. The host cells are preferably eukaryote cells such as yeast or mammalian cells. Of the soluble analogs it is preferred that the analog be derived from an isolated DNA fragment containing a DNA sequence coding for a soluble human thrombomodulin analog, capable of being secreted by a host cell transfected with said DNA fragment, wherein said DNA sequence encodes a polypeptide, comprising amino acid resides 227-462 of native TM as depicted in Table 2. The preferred analogs have been describe above for the bifunctional fusion analogs and the analogs. Recombinant cells producing such analogs, the protein analogs themselves, pharmaceutical preparations and methods of using the analogs are also disclosed herein. The analogs preferably retain substantially the same or increased affinity to thrombin as native thrombomodulin. Solubility is preferably achieved by deleting the stop transfer domain entirely or in part. BRIEF DESCRIPTION OF THE DRAWINGS Drawing 1 presents a schematic description of the role of thrombomodulin as an anti-coagulant molecule. Drawing 2A and 2B schematically illustrate analogs disclosed herein. Drawing 3 illustrates two of the synthetic oligonucleotides used in Example 1 and the plasmid pUC19pcrTM7. Drawing 4 illustrates eukaryotic expression plasmid pTM108 as described in Example 2. Drawing 5 illustrates the construction of transient mammalian expression vector pTHR13 from pTHR5 as described in Example 3. Drawing 6 illustrates the construction of baculovirus transfer vector pTMHYlOl as described in Example 4. Drawing 7 illustrates vectors used to produce fusion proteins that have both antithrombotic and fibrinolytic activity as described in Example 9. Drawing 8 schematically illustrates a multifunctional molecule comprising a fibrinolytic exzyme (such as lys-plasminogen streptokinase complex or t-PA) covalently conjugated to a TM analog. Drawing 9 illustrates an elution and activity profile produced during the separation of TM analog 6h- 227/462 varient forms using anion exchange chro atography. Drawing 10A shows a graph comparing the Protein C activating activity of native rabbit thrombomodulin and TM analog 6h/227-462. Drawing 10B shows a dose response curve for TM analog 6h/227-462 in the Protein C activation assay. Drawing 11A illustrates the activity of TM 6h/227-462 in the APTT. Drawing 11B shows the activity of TM analog 6h/227-462 compared to antithrombin III and heparin in the activated partial thromboplastin time assay. Drawing 12 shows a graph depicting the prolongation of clotting time by TM analog 6h/227-462 measured in three assays; APTT, TCT and PT. Drawing 13 shows the direct inhibition of thrombin mediated conversion of fibrinogen to fibrin by TM analog 6h/227-462. DETAILED DESCRIPTION In accordance with the present invention, novel compositions are provided which exhibit substantially all of the properties of native thrombomodulin except that they are made in a soluble form that is secreted from the producer cell. Also provided are methods for the production of these compositions. These soluble thrombomodulin analogs can be produced economically and are easily purified and administered. A variety of therapeutic uses are anticipated, particularly with respect to anticoagulant and/or antithrombotic therapies. In order to fully appreciate the invention, the following detailed description is set forth. "Thrombomodulin" or "TM", as used herein, refers to a protein with the biological activities of mammalian thrombomodulin typically found on endothelial cell surfaces and capable of acting as a receptor for thrombin (N. Esmon, (1987) Seminars in Thrombosis and Hemostasis. 13(4) :454-463 and European Patent Application No. 86904354.7, both incorporated herein by reference) . A DNA sequence encoding the full-length native human thrombomodulin protein has been isolated (European Patent Application No. 88870079.6, which is incorporated herein by reference) . The cDNA sequence encodes a 60.3 kDa protein of 575 amino acids, which includes a signal sequence of about 18 amino acids. Thrombomodulin within this definition includes natural allelic variations that may exist between individuals. The sequences for rabbit, bovine, mouse and human thrombomodulin exhibit a high degree of homology with one another. By analogy with other proteins, the structure of thrombomodulin can be presumptively divided into domains. The term "domain" refers to a discrete amino acid sequence that can be associated with a particular function or characteristic. The full length thrombomodulin gene encodes a precursor peptide containing the following domains: Approximate Amino Acid Position Domain -18—l Signal peptide 1-226 N-terminal domain 227-462 6 EGF-like domains 463-497 O-linked Glycosylation 498-521 Stop Transfer Sequence 522-557 Cytoplasmic domain See C. S. Yost et al. (1983) Cell. 3.4:759-766 and D. Wen et al. (1987) Biochemistry. 26:4350-4357, both incorporated herein by reference. In comparison to native TM, the TM analogs of the present invention have been modified to embrace selected specific regions of the native protein, that is (i) EGF-like domains 4, 5, and 6, (ii) all six of the six EGF-like domains EGF-like domains or (iii) the six EGF-like domains plus the O-linked glycosylation domain. Table 1 and Drawings 2A and 2B provide the specific analogs to which this invention is drawn. The two peptides 11/6 and 11/2 refer to peptides which are similar to the elastase cleaved TM. Elastase is an endopeptidase which cleaves at small neutral amino acids. The 11/6 refers to the number of amino acid residues present at the amino and carboxy termini respectively, which are outside the 227-462 region of the 6 EGF-like domains. This peptide is analogous to the rabbit TM elastase fragment in the number of residues which are outside this region. The second TM analog is the 11/2 peptide. The 11 refers to the number of residues on the amino terminus which are beyond the 227 residue and the 2 refers to the 2 amino acids residing beyond the 462 residue. The 11/2 analogue is believed to be the elastase fragment of human TM. Other species contain various regions of the native thrombomodulin protein. Due to post- translational processing, these species have additional amino acid residues which originate from the signal sequence. For example, the species 4t/227-462, 4t/227- 462:227-462, 4t/350-462, and 4t/227-497 all have four additional amino acids from the t-PA signal sequence. TM analogs 6h/227-462, 6h/227:462:227-462, 6h/350-462, and 6h/227-497 all have six additional amino acids from the hypodermin A signal sequence. These TM analogs are comprised of one or more of the EGF regions and/or the O-linked glycosylation domain. The specific regions embraced by these species are listed in the table below. Additional species are shown in Table 1 and Drawings 2A and 2B. X/227-462 EGFs 1-6 X/227-462:227-462 EGFs 1-6 + EGFs 1-6 X/350-462 EGFs 4,5,& 6 X/227-497 EGFS 1-6 + O-linked Gly. X is the number of amino acids derived from the signal sequence. Another aspect of the present invention describes multifunctional proteins, proteins in which the TM analog is joined with sequences corresponding to other proteins that impart biological characteristics to the TM analog not normally associated with native thrombomodulin. These multifunctional proteins are refered to as TM heterologs and are composed of a first functionality that is associated with native thrombomodulin, thrombin binding for example, and a second functionality that is heterologouε, ie. is a functionality from a source other than native thrombomodulin. The second functionality may effect localization of the TM heterolog so as to modify its affinity for specific tissue structures occurring in vivo, such as cell surfaces or fibrin clots. The heterologous peptide sequence may provide an additional biological activity, such as a proteolytic activity. A preferred proteolytic activity is the enzymatic cleavage of plasminogen to plasmin. The targeting components of this invention are designated as targeting to fibrin. It is preferred that the target also have fibrolytic activity. The multifunctional TM heterologs disclosed herein may be fusion proteins, wherein they have been provided with heterologous protein domains by N- and/or C-terminal extensions of the original TM sequences. Additionally, the multifunctional TM heterolog may be a chemical conjugate to an heterologous protein through a spacer molecule. Ruger et al, (1987), Proc. Natl. Acad. Sci. USA 84:7659-7662, and Smith and Cassels (1988), Fibrinolysis 2:189-195 have described chemical linkages between t-PA and other molecules. These molecules have an altered affinity for cell surfaces, or enhanced affinity for fibrin. Preferred heterologous protein domains may be encoded by nucleotide sequences isolated from tissue plasminogen activator or pro-urokinase. Suitable t-PA sequences may include, in addition to the entire protein: Domain Amino Acids Kringle 1, Kringle 2, Protease 95-530 Kringle 2, Protease 180-530 Protease 279-530 A. Chain 4-278 See L. Patthy, (1985) Cell. 41:657-663 for description of t-PA domains. For a heterologous domain isolated from human urokinase, amino acids 1-411 would be preferred (Jacobs et at. (1985) DNA. 4.:139-146. Human Glu-plasminogen and plasmin derivatives may also find use as heterologous domains which provide enhanced fibrinolytic activity. Amino acids 1-791 of Glu- plasminogen have such a utility. F. J. Castellino, Methods in Enzvmoloαy JLQ:365-378 Academic Press (1981). A particularly preferred embodiment of a heterologous domain of the present invention includes amino acids 4- 530 of human tissue plasminogen activator (t-PA) . In one aspect of the invention, the TM analogs described are secreted from the eukaryotic cells in which they are produced. As used herein, a "soluble TM analog" is a TM analog which is soluble in an aqueous solution and can be secreted by a cell. For pharmacological administration, the soluble TM analog may optionally be combined with phospholipid vesicles, detergents or other similar compounds well known to those skilled in the art of pharmacological formulation. The TM analogs of the present invention are soluble in the blood stream, making the analogs useful in various anticoagulant and other therapies. In general, the TM analogs of the present invention are active in one or more functional assays that measure aspects of clotting. These can include the activation of Protein C, by acting as a co-factor of thrombin, the inhibition of the conversion of fibrinogen to fibrin by thrombin (thereby substantially inhibiting blood coagulating activity) or the inhibition of thrombin mediated platelet activation and aggregation. The latter two assays can be run on an automatic coagulation timer according to the manufacturer's specifications; Medical Laboratory Automation Inc. distributed by American Scientific Products, McGaw Park, Illinois. (See also H. H. Salem et al. (1984) J. Biol. Chem.. 259:12246-12251, which is incorporated herein by reference) . Multifunctional proteins will also be active in assays that measure the activities not normally associated with TM. For instance, the fibrinolytic activity of fusion proteins combining TM analog sequences with, for example, t-PA can be measured in any of several assays, such as the fibrin plate assay described by Haverkatet and Brakman, (1975) Proσ. in Chem. Fibrin. Thromb. 1:151-159. Nucleic acid sequences encoding these proteins may be used to transform cells. These sequences, most typically DNA, may or may not contain an associated signal sequence. The resulting transformed or transfected cells can be cultured to readily produce these proteins in large quantities. The nucleic acid sequences described are operable and useful in a number of host cells which are adapted to tissue culture. Typically, the cells are eukaryotic cells, preferably human, that can grow rapidly in standard media preparations. Prokaryotic or yeast cells may also be suitable for the use of the described invention. This invention embraces molecular genetic manipulations that can be achieved in a variety of known ways. The recombinant cells, plasmids, and DNA sequences of the present invention provide a means to produce pharmaceutically useful compounds wherein the compound, secreted from recombinant cells, is a soluble derivative of thrombomodulin. The detailed description which follows discloses alternative methods for producing soluble thrombomodulin analogs from DNA sequences encoding thrombomodulin, and is followed by specific examples of preferred methods. Use of Thrombomodulin Analogs as an Anticoagulant/ Antithrombotic. The underlying pathology of thrombotic disorders is that a clot forms in response to a stimulus such as, for example, a damaged vessel wall. This stimulus triggers the coagulation cascade generating thrombin, which has the ability to convert fibrinogen to fibrin, the matrix of the clot. Soluble thrombomodulin analogs administered systemically will protect against thrombus formation because they will inhibit the generation of thrombin, via the activated Protein C system, and/or inhibit the action of thrombin on fibrinogen without disturbing other coagulation parameters. Thus, the use of soluble thrombomodulin analogs will be both safe and effective at preventing unwanted thrombus formation. The effect of thrombomodulin can be overcome by the large amounts of thrombin generated by a serious injury to vessels allowing a hemostatic plug to form. Diseases in which thrombus formation plays a significant etiological role include myocardial infarction, disseminated intravascular coagulation, deep vein thrombosis, pulmonary embolism, septic shock, acute respiratory distress syndrome, unstable angina and other arterial and venous occlusive conditions. In all of these, as well as in other diseases in which thrombus formation is pathological, soluble thrombomodulin analogs alone or in combination with thrombolytics are useful for treatment, either to cure the disease or to prevent its progression to a more severe state. Soluble thrombomodulin analogs also provide a safe and effective anticoagulant, for example, in patients receiving bioprostheses such as heart valves or patients requiring extracorporeal circulation. These compounds may replace heparin and warfarin in the treatment of, for example, pulmonary embolism or acute myocardial infarction. In particular these compounds would find a role in the prevention of deep vein thrombosis (DVT) , for instance after surgery. The formation of blood clots in the leg is itself a non-fatal condition but is very closely tied to the development of pulmonary embolism (PE) . PE is difficult to diagnose and can be fatal. Despite the investigation and clinical use of several prophylactic regimens, DVT and the resulting PE remain a significant problem in many patient populations and particularly in patients undergoing orthopedic surgery. (Prevention of Venous Thrombosis and Pulmonary Embolism. Consensus Development Conference Statement 1986, 6(2):1-23.). Existing prophylactic treatments such as heparin, warfarin and dextran typically reduce the incidence of DVT in orthopedic surgery patients from more than 50% in patients at risk receiving no prophylaxis to 25-30% among treated patients. There are serious side effects associated with these compounds, primarily bleeding complications. Daily laboratory tests and adjustments in dosage are required to minimize bleeding episodes while retaining some efficacy. It has been suggested that other inhibitors of thrombin such as antithrombin III may be useful to prevent DVT. Commercially available human ATIII, however, is purified from human plasma and carries with it the possibility of potentially serious contamination, such as from infectious viral particles. In addition, while ATIII inhibits thrombin directly, it does not enhance the activation of Protein C. The ability to activate Protein C is a decided improvement in an anticoagulant therapeutic. Based on the shortcomings of existing prophylactics, an antithrombotic which is effective at preventing DVT without predisposing the patient to bleeding or other complications could make a significant impact on patient recovery and well-being. Angioplasty is a procedure frequently used for restoring patency in occluded arteries. Although patency may be restored, this procedure often damages the endothelial lining of the artery, and blood clots begin to form as a result. Soluble thrombomodulin analogs administered in conjunction with angioplasty will prevent this deleterious side effect. Many acute thrombotic and embolic diseases are currently treated with fibrinolytic therapy in order to remove the thrombus. The condition that has been most widely investigated is acute myocardial infarction (heart attack) . Agents currently in use for treating acute myocardial infarction include streptokinase, tissue plasminogen activator and urokinase. Use of these agents can lead to serious bleeding complications. Patients who have had a thrombus removed by fibrinolytic therapy and in whom the blood flow has been restored frequently reocclude the affected vessel, i.e., a clot reforms. Attempts have been made to prevent reocclusion by increasing the dose or duration of treatment with a thrombolytic agent, but the incidence of bleeding then increases. Thus the therapeutic index for these drugs is narrow. The use of soluble thrombomodulin analogs provides protection against reocclusion, and its specific action is local rather than systemic, i.e., where thrombin is being generated or being released from a clot. Therefore, when used in combination with a thrombolytic agent, whose dose can then be decreased, the risk of bleeding can be substantially reduced. The TM heterologs containing additional domains that impart fibrinolytic activity in combination with antithrombotic activity will provide additional and superior utilities over currently available compounds. These multifunctional proteins direct the soluble TM heterolog to the site of the fibrin clot. The fibrinolytic activity conferred upon the compound by the heterologous domain provides a superior thrombolytic agent. As the clot is lysed by the fibrinolytic action of the, for example, t-PA domain(s), the TM domain(s) are inherently located precisely where needed to bind thrombin and inhibit any further growth of the clot matrix. This thrombin may be either newly generated by the coagulation pathway or released from the dissolving clot. The therapeutically effective dose of the multifunctional protein will be less than the doses of each molecule administered individually, reducing any concerns about the broader systemic action of either the TM analog or the t-PA and any consequential undesirable side effects. Administration of soluble thrombomodulin analogs would be by a bolus intravenous injection, by a constant intravenous infusion or by a combination of both routes. Also, soluble thrombomodulin mixed with appropriate excipients may be taken into the circulation from an intramuscular site. As used herein, a therapeutically effective dose is defined as that level of TM analog required to prevent formation of pathological clots. Systemic treatment with thrombomodulin analogs can be monitored by determining the activated partial thromboplastin time (APTT) on serial samples of blood taken from the patient. The coagulation time observed in this assay is prolonged when a sufficient level of TM analog is achieved in the circulation. However, this is a systemic measurement of efficacy, and perhaps a dose that is effective at the site of a clot would not be effective in prolonging the APTT. Dosing levels and regimens can be adjusted so that an adequate concentration of thrombomodulin is maintained as measured by, for example, the APTT assay or the Protein C activation assay. In contrast to full length thrombomodulin, the analogs of this invention should offer an improved pharmaceutical. It is anticipated that these analogs will offer superior characteristics from a manufacturing perspective, a pharmaceutical perspective or both. General Methods Generally, the definitions of nomenclature and descriptions of general laboratory procedures used in this application can be found in T. Maniatis et al. Molecular Cloning. A Laboratory Manual. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The manual is hereinafter referred to as Maniatis and is hereby incorporated by reference. All enzymes were used according to the manufacturer's instructions. Libraries are constructed in bacteriophage lambda vectors. Phage are packaged in vitro, as described in Maniatis. Recombinant phage are analyzed by plaque hybridization as described in Benton and Davis, (1977) Science. 196:180-182. Colony hybridization is carried out as generally described in M. Grunstein et al. (1975) Proc. Natl. Acad. Sci. USA.. 22:3961-3965. Nucleotide sizes are given in either kilobases (kb) or base pairs (bp) . These are estimates derived from agarose or acrylamide gel electrophoresis or from published DNA sequences. Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981) Tetrahedron Letts.. 22(20) :1859-1862 using an automated synthesizer, as described in D.R. Needham-VanDevanter et al. (1984) Nucleic Acids Res.. 12:6159-6168. Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in J.D. Pearson and F.E. Regnier, (1983) J. Chrom.. 255:137-149. The sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of A.M. Maxam et al. (1980) Methods in Enzvmology. .65:499-560. The sequence can be confirmed after the assembly of the oligonucleotide fragments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double-stranded templates of R.B. Wallace et aJL. (1981) Gene. 16 21-26. Southern Blot hybridization techniques were carried out according to Southern et al. (1975) J. Mol. Biol.. 98:503. This invention relates to cloning and use of genes for expression vectors in eukaryotic or prokaryotic cells. Intermediate vectors are cloned for amplification in prokaryotes such as £. coli. Bacillus or Streptomvces. Most preferred is E_ coli because that organism is easy to culture and more fully understood than other species of prokaryotes. The Maniatis manual contains methodology sufficient to conduct all subsequently described clonings in E. coli. Strain MH-1 is preferred unless otherwise stated. All E. coli strains are grown on Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids. Strains with resistance to antibiotics were maintained at the drug concentrations described in Maniatis. Transformations were performed according to the method described by D.A. Morrison, (1977) J. Bact.. 131:349-351 or by J.E. Clark-Curtiss and R. Curtiss, (1983) Methods in Enzvmology f 101:347-362. Eds. R. Wu et al. , Academic Press, New York. Representative vectors include pBR322 and the pUC series which are available from commercial sources. Definitions For purposes of the present invention the following terms are defined below. The term "vector" refers to viral expression systems, autonomous self-replicating circular DNA (plasmids) , and includes both the expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector," this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s) . Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome. The term "promoter" is a region of DNA involved in binding the RNA poly erase to initiate transcriptio . The term "operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The term "control sequence" refers to a DNA sequence or sequences which are capable, when properly ligated to a desired coding sequence, of affecting its expression in hosts compatible with such sequences. Such control sequences include at least promoters in both prokaryotic and eukaryotic hosts, and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be identified. As used herein, "control sequences" simply refers to whatever DNA sequence may be useful to result in expression in the particular host used. The term "free of native glycosylation" refers to material which is altered in its polysaccharide composition from that naturally produced by the human body. A "substantially pure composition" of the peptides described herein are those which migrate largely as distinct bands on reducing gels in standard electrophoresis techniques never mind. It is preferably referring to homogeneous compositions having less than about 10% contamination and most preferably less than 3% contamination with peptides other than that desired. Gene Synthesis Publication of the DNA sequence encoding human thrombomodulin facilitates the preparation of genes encoding soluble thrombomodulin analogs. The full length gene is used as a starting point to construct DNA sequences encoding soluble thrombomodulin analogs. The analogs of the present invention are soluble derivatives which lack a stop transfer sequence. Further, these analogs are secreted from eukaryotic cells which have been transfected or transformed with plasmids containing genes which encode these polypeptides. Methods for making modifications, such as amino acid substitutions, deletions, or the addition of signal sequences to cloned genes are known. The full length gene for thrombomodulin can be prepared by several methods. Human genomic libraries are commercially available. Oligonucleotide probes, specific to the thrombomodulin gene, can be synthesized using the published gene sequence. Methods for screening genomic libraries with oligonucleotide probes are known. The publication of the gene sequence for thrombomodulin demonstrates that there are no introns within the coding region. Thus a genomic clone provides the necessary starting material to construct an expression plasmid for thrombomodulin using known methods. A thrombomodulin encoding DNA fragment can be retrieved by taking advantage of restriction endonuclease sites which have been identified in regions which flank or are internal to the gene. (R.W. Jackman et al. (1987) Proc. Natl. Acad. Sci. USA.. £4:6425-6429) . Alternatively, the full length gene is obtained from a cDNA bank. Messenger RNA prepared from endothelial cells provides suitable starting material for the preparation of cDNA. A cDNA molecule containing the gene encoding thrombomodulin is identified as described above. Methods for making cDNA banks are well known (See Maniatis) . Synthetic oligonucleotides can be used to construct TM genes. This is done using a series of over-lapping oligonucleotides usually 40-120 bp in length, representing both the sense and non-sense strands of the gene. These DNA fragments can be annealed, ligated and cloned. An alternative and preferred method combines the use of synthetic oligonucleotide primers with polymerase extension on a mRNA or DNA template. This polymerase chain reaction (PCR) method amplifies the desired nucleotide sequence. Restriction endonuclease sites can be incorporated into the primers. U.S. Patents 4,683,195 and 4,683,202 describe this method. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector. Genes encoding soluble thrombomodulin analogs may be constructed using the gene encoding full length thrombomodulin as a starting material. Alternatively, analogs can be constructed from synthetic oligonucleotides or by use of the polymerase chain reaction on either a DNA or RNA template to produce a pure fragment of the desired gene which can then be cloned. Combinations of these methods can be used in order to obtain the DNA sequences that are desired. Alterations in the natural gene sequence can be introduced by the techniques of in vitro mutagenesis or by use of the polymerase chain reaction with primers that have been designed to incorporate appropriate mutations. For soluble thrombomodulin analogs constructed entirely from synthetic oligonucleotides, DNA codons may be selected to minimize secondary structure that would otherwise interfere with transcription and translation, without altering the native amino acid sequence (H. Grosjean and W. Fiers, (1982) Gene, 18:199-209). Further it may be desirable to alter the sequence of the native gene encoding thrombomodulin to incorporate unique restriction sites. Such restriction sites find use in construction of a vector capable of expressing a gene encoding a soluble thrombomodulin analog. It may also be useful to design particular codon usage for efficiency in expression by a particular host cell. Genes encoding soluble thrombomodulin analogs may have more than one copy of a native domain. By way of illustration and not limitation, a soluble thrombomodulin analog of the present invention may have more than one copy of the 6 EGF-like domains. Such a molecule may have enhanced utility in a pharmaceutical product since this region binds thrombin. The soluble TM analogs described herein are secreted when expressed in eukaryotic cell culture. Secretion may be obtained by the use of the native signal sequence of the thrombomodulin gene. Alternatively, genes encoding the soluble thrombomodulin analogs of the present invention may be ligated in proper reading frame to a signal sequence other than that corresponding to the native thrombomodulin gene. For example, the signal sequence of t-PA, (see commonly assigned co-pending USSN 074,083 filed July 16, 1987 incorporated herein by reference) or of hypodermin A or B (see commonly assigned co- pending U.S. Serial No. 148,749, filed January 27, 1989 incorporated hereby by reference) can be linked to the polypeptide. In the preferred embodiment of the present invention, use is made of the signal sequence of t-PA which contains the second intron of the human t-PA gene. The inclusion of the intron enhances the productivity of the adjacent structural gene (see commonly assigned co-pending USSN #003,611 filed January 14, 1987 incorporated herein by reference). With the analogs of this invention, that portion of the gene encoding the entire carboxyl terminal region of thrombomodulin is deleted. Therefore, it is necessary to add a stop codon so that translation will be terminated at the desired position. Alternatively, a stop codon can be provided by the desired expression plasmid. Additionally a polyadenylation sequence is necessary to ensure proper processing of the mRNA in eukaryotic cells encoding the soluble thrombomodulin analog. Also, it may be necessary to provide an initiation codon, if one is not present, for expression of the soluble TM analog. Such sequences may be provided from the native gene or by the expression plasmid. The TM analogs described may be encoded by genes wherein at least one N-terminal or C-terminal domain is deleted from the naturally occurring human DNA sequence encoding TM, and replaced with a DNA sequence encoding a fibrinolytic enzyme. T-PA and pro- urokinase are fibrinolytic enzymes useful for such an analog. It may be desirable to replace both an N- terminal and a C-terminal domain of the native thrombomodulin with heterologous gene sequences. For example, in one embodiment of the present invention the gene encoding full length TM is modified so that the signal sequence and N-terminal domains, comprising amino acids -18 through 226 are deleted and replaced with DNA encoding the signal sequence of t-PA (amino acids -32 to -1 according to the numbering system in Table 6) . In another embodiment, the TM analog described above may be further modified by the use of DNA encoding amino acids 4 to 530 of t-PA to replace the O-linked glycosylation, stop transfer, and cytoplasmic domains of native thrombomodulin (amino acids 463-557) . Such a recombinant DNA molecule can be transfected into a host cell, thus providing a multifunctional protein capable of binding fibrin, activating Protein C, and converting plasminogen to plasmin. A preferred source of the t-PA gene can be obtained by isolating the t-PA gene from an £. coli culture (strain MH-1) on deposit with American Type Culture Collection (ATCC) in Bethesda, Maryland having Accession No. 67,443. Standard cloning techniques are sufficient to obtain the t-PA plasmid and to insert heterologous domains, as desired, into genes encoding TM analogs. The use of a heterologous domain to impart fibrinolytic activity to a TM analog may be desirable for other TM analogs described herein, and the use of the gene encoding pro-urokinase would have analogous utility in creating a multifunctional protein. The thrombomodulin analogs of this invention are described by their amino acid sequences and by their DNA sequence, it being understood that the analogs include their biological equivalents such that this invention includes minor substitutions and deletions of amino acids that have substantially little impact on the biological properties of the analogs. It should also be understood that alternative sequences could be used to express soluble thrombomodulin analogs in various host cells. Furthermore, due to the degeneracy of the genetic code, equivalent codons may be substituted to encode the same polypeptide sequence. Cloning Vectors Cloning vectors suitable for replication and integration in prokaryotes or eukaryotes and containing transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of soluble thrombomodulin analogs are described herein. The vectors are comprised of expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems. Expression of Soluble Thrombomodulin Analogs in Prokaryotic Cells In addition to the use of cloning methods in E. coli for amplification of cloned sequences it may be desirable to express soluble TM analogs in prokaryotes. It is possible to recover a therapeutically functional protein from E. coli transformed with an expression plasmids encoding a soluble thrombomodulin analog. See Example 12. Methods for the expression of cloned genes in bacteria are well known. To obtain high level expression of a cloned gene, such as a DNA sequence encoding a soluble thrombomodulin analog in a prokaryotic system, it is essential to construct expression vectors which contain, at the minimum, a strong promoter to direct mRNA transcription termination. Examples of regulatory regions suitable for this purpose are the promoter and operator region of the £. coli /S-galactosidase gene, the E. coli tryptophan biosynthetic pathway, or the leftward promoter from the phage lambda. The inclusion of selection markers in DNA vectors transformed in E. coli are useful. Examples of such markers include the genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. See Maniatis for details concerning selection markers and promoters for use in E. coli. in the described embodiment of this invention pUC19 is used as a vector for the subcloning and amplification of desired gene sequences. Expression of Soluble Thrombomodulin Analogs in Eukaryotic Cells It is expected that those of skill in the art are knowledgeable in the expression systems chosen for expression of the desired TM analog and no attempt to describe in detail the various methods known for the expression of proteins in eukaryotes will be made. The DNA sequence encoding a soluble TM analog can be ligated to various expression vectors for use in transforming host cell cultures. The vectors typically contain marker genes and gene sequences to initiate transcription and translation of the soluble thrombomodulin analog gene. The vectors preferably contain a marker gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase, metallothionein, hygromycin, or neomycin phosphotransferase. The nuclear polyhedral viral protein from Autographa californica is useful to screen transfected insect cell lines from Spodoptera frugiperda and Bombvx mori to identify recombinants. For yeast, Leu-2, Ura-3, Trp-1, and His-3 are known selectable markers (Gene (1979) 8.:17-24). There are numerous other markers, both known and unknown, which embody the above scientific principles, all of which would be useful as markers to detect those eukaryotic cells transfected with the vectors embraced by this invention. In the examples provided below, hygromycin is included as a eukaryotic selection marker in CHL-1 cells. Of the higher eukaryotic cell systems useful for the expression of soluble TM analogs, there are numerous cell systems to select from. Illustrative examples of mammalian cell lines include RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines. Cells suitable for use in this invention are commercially available from the American Type Culture Collection. Illustrative insect cell lines include Spodoptera frugiperda (fall Armyworm) and Bombvx mori (silkworm) . As indicated above, the expression vector, ex. plasmid, which is used to transform the host cell, preferably contains gene sequences to initiate the transcription and sequences to control the translation of the soluble TM analog protein gene sequence. These sequences are referred to as expression control sequences. When the host cell is of insect or mammalian origin, illustrative expression control sequences include but are not limited to the following: the retroviral long terminal repeat promoters ((1982) Nature. 297:479-483) . SV40 promoter ((1983) Science. 222:524-527. thymidine kinase promoter (J. Banerji et al. (1982) Cell. 27:299-308), or the beta-globin promoter (P.A. Luciw et al. (1983) Cell. .33.:705-716) . The recipient vector nucleic acid containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable. This segment is ligated to a DNA sequence encoding a soluble TM analog by means well known in the art. For expression of heterologous proteins in yeast, the following promoters are useful for expression: GAL1,10 ((1984) Mol. and Cell. Biol.. 4.:1440-48); ADH2 ((1983) J. Biol. Chem. 258:2674-2682): and PH05 ((1982) EMBO J.. .6:675-680). When higher animal host cells are employed, polyadenylation or transcription termination sequences need to be incorporated into the vector. An example of a polyadenylation sequence is the polyadenylation sequence from SV40, which may also function as a transcription terminator. Genes incorporated into the appropriate vectors can be used to direct synthesis of proteins in either transient expression systems or in stable clones. In the former case yields are low, but the experiments are quick. In the latter case it takes more time to isolate high producing clones. Different vectors may be used for the two different types of experiments. In particular, in the case of transient expression, sequences may be included within the plasmid that allow the plasmid to replicate to a high copy number within the cell. These sequences may be derived from virus such as SV40 (e.g. C. Doyle et al. (1985) J. Cell Biol.. 100:704-714) or from chromosomal replicating sequences such as urine autonomous replicating sequences (Weidle et a_l. (1988) Gene. 71:427-437). The vector for use in transient expression should also contain a strong promoter such as the SV40 early promoter (e.g. , A. van Zonnenfeld et al. (1987) Proc. Natl. Acad. Sci. USA.. 83:4670- 4674) to control transcription of the gene of interest. While transient expression provides a rapid method for assay of gene products, the plasmid DNA is not incorporated into the host cell chromosome. Thus, use of transient expression vectors does not provide stable transfected cell lines. A description of a plasmid suitable for transient expression is provided by A. Aruffo & B. Seed, (1987) Proc. Natl. Acad. Sci. USA.. 84:8573-8577. The disclosed embodiment makes .use of CHL-1 cells. -These are derived from RPMI 7932 melanoma cells, a readily available human cell line. The CHL-1 cell line has been deposited with the ATCC according to the conditions of the Budapest Treaty and has been assigned #CRL 9446, deposited June 18, 1987. The soluble TM analogs may be expressed in yeast cells. The expression of heterologous proteins in yeast is well known. F. Sherman et al. , Methods in Yeast Genetics. Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods which have utility for producing soluble thrombomodulin analogs in yeast. Soluble TM analogs may alternatively be produced in the insect cell lines described above using the baculovirus system. This system has been described by V.A. Luckow and M.D. Summers (1988) Bio/Technology. j5:47-55. Generally, this expression system provides for a level of expression higher than that provided by most mammalian systems. The baculovirus infects the host insect cells, replicates its genome through numerous cycles, and then produces large amounts of polyhedron crystals. The polyhedron gene can be replaced with a TM analog gene. The polyhedron promoter will then make large amounts of analog protein following infection of the culture host cell and replication of the baculovirus genome. The non- secreted gene product is harvested from the host 3-7 days post infection. Alternatively, the soluble TM protein may be secreted from the cells if appropriate signal sequences are present on the protein. The host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, DEAE-dextran technique, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, electroporation and microinjection of the DNA directly into the cells. See, B. Perbal, "Practical Guide to Molecular Cloning," 2nd edition, John Wiley & Sons, New York and Wigler, et al. (1987) Cell. 16:777-785. Culturing Cells It is preferred that the host cell is capable of rapid cell culture and able to appropriately glycosylate expressed gene products. Cells known to be suitable for dense growth in tissue culture are particularly desirable and a variety of invertebrate or vertebrate cells have been employed in the art, both normal and transformed cell lines. The transfected cells are grown up by means well known in the art. For examples, see Biochemical Methods in Cell Culture and Virology, Kuchler, R. J. , Dowden, Hutchinson and Ross, Inc. (1977) . The expression products are harvested from the cell medium in those systems where the protein is secreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or enzymatic means, which are well known in the art. Purification of Soluble TM Analogs The present invention provides soluble TM analogs which are secreted by cultured recombinant eukaryotic cells. The analogs are produced in serum- free or serum supplemented media and are secreted intact. If prokaryotic cells are used, the TM analogs may be deposited intracellularly. The analogs may be glycosylated or non-glycosylated. Following the growth of the recombinant cells and concomitant secretion of TM analogs into the culture media, this "conditioned media" is harvested. The conditioned media is then clarified by centrifugation or filtration to remove cells and cell debris. The proteins contained in the clarified media are concentrated by adsorption to any suitable resin such as, for example, Q Sepharose or metal chelators, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other means known in the art may be equally suitable. Further purification of the soluble TM analogs can be accomplished in the manner described in Galvin, J. B. , et al. (1987) J. Biol. Chem.. 262:2199-2205 and Salem, H.H. et al. (1984) J. Biol. Chem.. 259:12246-12251 and in the manner described in the embodiment disclosed herein. The purification of soluble TM analogs secreted by cultured cells may require the additional use of, for example, affinity chromatography, ion exchange chro atography, sizing chromatography or other protein purification techniques. Recombinant TM analogs may be produced in multiple conformational forms which are detectable under nonreducing chromatographic conditions. Removal of the higher molecular weight species and those species having a low specific activity is desirable and is achieved by a variety of chromatographic techniques including anion exchange or size exclusion chromatography. Recombinant TM analogs may be concentrated by pressure dialysis and buffer exchanged directly into volatile buffers (e.g., N-ethylmorpholine (NEM) , ammonium bicarbonate, ammonium acetate, and pyridine acetate) . In addition, samples can be directly freeze- dried from such volatile buffers resulting in a stable protein powder devoid of salt and detergents. In addition, freeze-dried samples of recombinant analogs can be efficiently resolubilized before use in buffers compatible with infusion (e.g., phosphate buffered saline) . Other suitable buffers might include hydrochloride, hydrobromide, sulphate acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate. Chemically Linking the Soluble TM Analogs to Fibrinolytic Enzymes Blocking the active site of fibrinolytic enzymes can improve their therapeutic ratio. The p- anisoyl derivative of human lys-plasminogen activator complex (a complex between plasminogen and streptokinase also called APSAC or Eminase™ (Beecham Research Labs., Ltd., Middlesex, U.K.)), for example, has demonstrated clinical advantages when compared to streptokinase alone, such as a longer half-life. The acylation of the active site of the lys-plasminogen streptokinase complex leaves the fibrin binding site free, thus the complex retains the ability to bind to an unwanted thrombus. (Smith, RAG, Dupe, RJ, English, PD, & Geari, J. , (1981) Nature 290:505-507). The active fibrinolytic complex is produced in situ after slow deacylation by hydrolysis. The same technique can be used to chemically link other molecules, such as a soluble TM analog, to the active site of a fibrinolytic enzyme through free amine groups on the analog. The peptides disclosed herein can be chemically linked to a fibrinolytic enzyme such as lys-plasminogen streptokinase, t-PA or the like through a covalent bond between the enzyme, and an amino acid residue within the peptide. In one embodiment, lys-plasminogen streptokinase complex can be linked specifically and reversibly to a TM analog by using a heterobifunctional inverse substrate of plasmin (see, for example, Drawing 8) . In a second embodiment, the fibrinolytic enzyme linked to the TM analog can be tissue plasminogen activator (t-PA) . The acyl-enzyme is prepared according to the method described in Smith, RAG & Cassels, (1988), Fibrinolysis. 2:198-195. Following removal of excess acylating agent by gel filtration, the acyl-enzyme is reacted with thiolated TM analog. The acylation reaction is highly specific for the active center serine residue in either the plasminogen or the t-PA molecules. Thiolated TM is preferably produced using analogs having only one free amine group, 11/2, 4t-227/462 or 6h-227/462, which is at the N-terminus of the molecule. Thiolated TM analog is produced by reacting the analog with 2-iminothiolane as described in Smith (1988) , supra. The thiolated TM analog is then reacted with the acylated fibrinolytic enzyme (lys-plasminogen streptokinase complex or t-PA) producing TM>SK:Plg or TM>t-PA, which can then be further purified by high performance gel permeation chromatography or other purification means as described in Smith (1988) , supra. The activity of the TM analog or the fibrinolytic enzyme can be individually assayed by methods described herein. Hydrolysis of the acyl- enzyme conjugate can be monitored on SDS gels and by release of fibrinolytic activity. Chemical linkages are used to produce acyl- enzyme pro-drugs. This approach provides a reversible TM>fibrinolytic conjugate with the combined properties of both molecules, thus providing a route to thrombus specificity, via the lys-plasminogen streptokinase complex or t-PA, with a kinetic approach to the control of bleeding. The TM>fibrinolytic acyl-enzyme pro-drug acts as a relatively slowly cleared reservoir of fibrinolytic activity. Thus, the half-life of the fibrinolytic and the clot specificity of the TM analog is increased and the clearance rates and systemic effects of both are decreased. Evaluation of TM Analogs Thrombomodulin activity can be detected in a variety of assays that depend on alterations in the action of thrombin. The ability of thrombomodulin or its soluble analogs to accelerate the thrombin catalyzed activation of Protein C can be measured in the assay described by Salem et al. (above) and Galvin et al. In brief, this assay consists of two steps. The first step is the incubation of the test thrombomodulin or TM analog with thrombin and Protein C under defined conditions. In the second step, the thrombin is inactivated with hirudin or antithro bin III and heparin, and the activity of the activated Protein C is determined by use of a chromogenic substrate, whereby the chromophore is released by the proteolytic activity of activated Protein C. This assay is carried out with purified reagents. Alternatively the effect of a TM analog can be measured using plasma in clotting time assays such as the activated partial thromboplastin time (APTT) , thrombin clotting time (TCT) and/or prothrombin time (PT) . These assays distinguish between different mechanisms of coagulation inhibition. Inhibition in any one of these assays demonstrates the presence of a systemic anticoagulant state. The APTT measures the effect of all of the components involved in coagulation except platelets. Factor VII and calcium ions while the TCT specifically measures the inhibition of thrombin. The PT measures prothrombin-activity at constant lipoprotein and calcium concentrations. These assays are used to identify soluble TM analogs that are able to bind thrombin and to activate Protein C in both purified systems and in a plasma milieu. Further assays are then used to evaluate soluble TM analogs having enhanced properties by assaying them for their ability to inhibit thrombin induced fibrin formation, thrombin induced platelet activiation/aggregation and thrombin induced inactivation of protein S, as well as thrombin catalyzed activation of Factors V, VIII and XIII. One can also measure the ability of the analog to enhance the inactivation of thrombin by antithrombin III and heparin cofactor II. Soluble TM analogs which have utility as therapeutic agents are able to activate Protein C in physiological concentrations of calcium ion. Formulation and Use of Thrombomodulin Analogs Soluble thrombomodulin analogs described herein may be prepared in a lyophilized or liquid formulation. The material is to be provided in a concentration suitable for pharmaceutical use as either an injectable or intravenous preparation. These compounds can be administered alone or as mixtures with other physiologically acceptable active materials, such as one-chain t-PA, or inactive materials, or with suitable carriers such as, for example, water or normal saline. These compounds can be administered parenterally, for example, by injection. Injection can be subcutaneous, intravenous or intramuscular. These compounds are administered in pharmaceutically effective amounts and often as pharmaceutically acceptable salts, such as acid addition salts. Such salts can include, e.g., hydrochloride, hydrobromide, phosphate, sulphate, acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate, among others. The analogs described herein may display enhanced in vivo activity by incorporation into micelles. Methods for incorporation into ionic detergent micelles or phospholipid micelles are known. An antithrombotic agent can be prepared using the soluble TM analogs described herein and can consist of a completely purified thrombomodulin analog alone or in combination with a thrombolytic agent as described above. Compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutic applications such as, e.g., the inhibition of blood clot formation. Thus, these compounds can find use as therapeutic agents in the treatment of various circulatory disorders, such as, for example, coronary or pulmonary embolism, strokes, as well as the prevention of reocclusion following thrombolytic therapy, and these compounds have utility in the cessation of further enlargement of a clot during an infarction incident. Further, the compounds disclosed can be useful for treatment of systemic coagulation disorders such as DIC, which is often associated with septicemia, certain cancers and toxemia of pregnancy. These compounds can be administered to mammals for veterinary use, such as with domestic animals, and for clinical use in humans in a manner similar to other therapeutic agents, that is, in a physiologically acceptable carrier. In general, the administration dosage will range from about 0.0001 to 100 mg/kg, and more usually 0.001 to 0.1 mg/kg of the host body weight. These dosages can be administered by constant infusion over an extended period of time, until a desired circulating level has been attained, or preferably as a bolus injection. Coating of Biomaterials with TM Analogs The use of altered prosthetic endovascular or cardiovascular devices anywhere in the circulation system results in the formation of thrombus, a blood- derived mass as a pathological consequence of activating hemostatic mechanisms under variable flow conditions. Typically, thrombogenesis in association with prosthetic endovascular or cardiovascular devices includes the following sequence: (a) exposure of the surface to circulating blood; (b) platelet adherence, aggregation and release of platelet components; (c) thrombin generation and fibrin formation; (d) thrombin dissolution which requires plasmin generation and fibrinolysis. In general, when blood contacts an artificial surface, the surface will rapidly acquire a layer of absorbed plasma proteins which will mediate subsequent thrombotic events. This series of events also follows when blood is circulated through an extracorporeal device, such as a heart/lung machine. It has been desirable to introduce various coatings onto the polymeric surfaces of such blood- contacting devices to promote thromboresistance. Thrombomodulin represents a new class of molecule suitable for creating a thromboresistant surface. It is especially suitable as such a surface since it has no known inhibitors and will be available to function in this capacity for extended periods of time. The TM analogs described herein are particularly advantageous for this purpose as they are closely related to the protein fragment which is derived when full length TM is digested with porcine pancreatic elastase. The long-term stability of immobilized proteins is of paramount importance. Thus, the smaller, proteolytically resistant TM analog is more advantageous than the full length molecule which can be proteolysed by enzymes in the blood, resulting in the potential loss of active component from the biomaterial surface. The TM analogs will be particularly preferable over the use of the full length molecule, inter alia, during periods of physiological stress, e.g., inflammation, where potent white cell proteases, including leukocyte elastase, have access to the biomaterial surface. The TM analogs may be used to coat polymers used in a wide variety of biological applications including, but not limited to, arteriovenous shunts, intravascular shunts (eg., umbilical, angiographic) , vascular grafts, heart valves, artificial joints, pacemakers, left ventricle assist devices, and the like. The TM analogs are bonded to a biocompatible polymer. Biocompatible polymers may be any suitable polymeric biomaterial or combination thereof known and used in the art for biological application such as polyurethanes, silicone elastomers, hydrogels (e.g., poly(hydroxyethyl methacrylate) , polyesters, polyethers, polyvinyl alcohol, and the like. The TM analog may be bonded to coat the polymer material following activation of the biopolymer. Activation methods are known in the art and may utilize amino, carboxyl, hydroxyl or sulfhydryl functions on the compound to be coated. Activation may be achieved through a variety of known mono- and/or bi- functional reagents, including, but not limited to, glutaraldehyde, carbodiimide activated COOH, isocyanate, cyanuric acid, or hydrosuccinimide esters. Spacer arms, known in the art, may optionally be used. The single amine function at the N-terminus of some of the TM analogs disclosed herein, such as 11/2, 4t/227-462 and 6h/227-462 provide a highly selective means to covalently couple this protein in a highly oriented array to activated biomaterial surfaces. Once the biocompatible polymer has been coated, it may be implanted in a mammal as necessary according to the teaching in the art for the procedure at hand or used in any device that contacts blood where the blood must remain anticoagulated. The following examples are offered by way of illustration and not by way of limitation. EXAMPLES In all discussions of sequences the convention in the art is that the terms 5' and 3' are used to indicate direction on the coding strand of a gene and its surrounding sequences. Unless otherwise stated, all single stranded DNA illustrated in this document represents the coding strand of the gene. Example 1. Derivation of Oligonucleotides Encoding Thrombomodulin Analogs a. Isolation of genes encoding amino acids 227- 462. Human DNA was used to isolate a gene encoding the 6 EGF-like domains of thrombomodulin corresponding to amino acids 227-462. This DNA was isolated from fetal liver according to the method of Blin, N and D. W. Stafford, (1976) Nucleic Acids Res. 2:2303. The DNA was then used as a template in a polymerase chain reaction with synthetically derived primers selected to embrace the desired regions (see Drawings 2 and 3, and Tables 1 and 3) . The following steps provide a means to obtain a DNA insert encoding amino acids (aa) 227-462 and uses primers #1033 and #1034 (see Drawing 3) . It is understood that by modifying the procedures set forth below by using alternative primers, other TM analogs may be obtained. The sequence of the #1033 and #1034 primers correspond to the 5' and 3' ends of the desired domain; but they have been modified so that they contain a BamHI site. A termination codon (TGA) was introduced following base 1586. The polymerase chain reaction was run under the conditions described by Saiki, et al. , (1988) Science. 320:1350-1354 except that the initial temperature of annealing was 37 β C. After 10 cycles, the annealing temperature was raised to 45 β C for the remaining 30 cycles. An aliquot of the reaction products was separated on a 5% polyacrylamide gel and visualized by ethidium bromide staining. A band of the predicted size (700 bp) could clearly be seen. To confirm the identity of the insert, one can optionally sequence this band or hybridize it to an internal probe. b. Isolation of genes encoding other regions of TM. The polymerase chain reaction as herein described was used in the same manner to isolate additional fragments of thrombomodulin corresponding to the regions listed in Table 1 and shown schematically in Drawing 2A. In particular, these regions embrace one or more of the EGF-like domains and the O-linked glycosylation dormain. The sequences of the primers selected to produce the desired regions are shown in Table 3. c. Cloning plasmids cotaining the soluble TM analog genes. i. pUC19pcrTM7 The remainder of the polymerase chain reaction mixture described in part (a.) above was restricted with BamHI, separated on a 5% polyacrylamide gel, and the 700 bp band was excised and eluted. It was ligated to pUC19 that had been restricted with BamHI and the new plasmid was transformed into F ± . coli strain DH5α. White colonies that grew on a medium containing ampicillin and 5-bromo-4-chloro-3-indolyl-3- D-galactoside were picked onto a grid and hybridized by the Grunstein-Hogness technique with a synthetically derived gene corresponding to aa 283-352 of TM that had been cut out of a cloning plasmid (pTM2.1) with EcoRI and Hindlll before labelling with 32 P by random priming (Boehringer Mannheim) . After exposing the filters to X-ray film the one colony that hybridized to the pTM2.1 probe (pUC19pcrTM7) was grown up. DNA was extracted and was analyzed by restriction with either BamHI or Bglll. The restriction digests were separated on an agarose gel and the ethidium bromide stained bands confirmed that there was a 700 bp insert and that it had the correct restriction map for the 6 EGF-like domains of thrombomodulin. This gel was also transferred to nitrocellulose and analyzed by hybridization with the 32 P labelled insert from pTM2.1. The 700 bp insert released after digestion with BamHI hybridized to this probe as did the 486 bp Bglll fragment proving that indeed this clone contained the 6 EGF-like domains of thrombomodulin. Verification that no mutations had been introduced into pUC19pcrTM7 during the polymerase chain reaction and cloning procedures was obtained by sequencing the insert, (see Drawing 4) ii. Cloning plasmids containing other TM analog genes Other cloning plasmids, such as pTM309 and pTM323 were constructed using methods similar to those described in (i.) Plasmid pTM309 contains amino acids 350-462 of native TM (EGF-like domains 4,5,& 6) and pTM323 contains amino acids 227-497 (EGF-like domains 1-6 + O-linked glycosylation domain) . Additional plasmids were constructed that contain other TM analog gene sequences. (See Drawing 2A and Table 1) . Example 2. Production of Human TM Analogs in Mammalian Cells. a. Mammalian expression vectors for TM analogs This example provides a mammalian expression vector comprising the analog genes of Example 1. Drawing 4 provides an overview of the construction of one of these vectors. The genes are operably linked to the signal sequence of human tissue plasminogen activator. The expression plasmid, pPA124, contains a promoter contained within the three copies of the long terminal repeats derived from Harvey Sarcoma virus for the expression of cloned genes. This plasmid was derived from pPA119, and pSC672, both described in detail in co-pending USSN #074,083, filed July 16, 1987, incorporated herein by reference. A Bglll - Bell fragment containing the SV40 polyadenylation region was isolated from pSC672. This fragment was cloned into pPA119 which had been digested with Bglll and Bell. In the resulting plasmid, pPA124, both the Bglll and Bell sites remained intact. Plasmid pPA124 contains the t- PA signal sequence adjacent to an appropriate restriction site and this signal sequence also contains the second intron of the human t-PA gene. The gene encoding the soluble TM analog was removed from pUC19pcrTM7 by treatment with BamHI and ligated to pPA124 that had been treated with Bglll. Transformants were screened for the presence of the insert in the correct orientation, that is in which the t-PA signal sequence was linked to the 5' end of the thrombomodulin insert encoding an open reading frame. This plasmid, pTMlOl, was then digested with Clal and ligated to a Clal fragment containing the dhfr gene under the control of the SV40 promoter. The Clal fragment is described in WO88/02411 at page 26. Transformants were screened for the presence of this dhfr cassette and then the orientation relative to the plasmid was determined by restriction mapping (pTM103) . Plasmid pTM103, containing the dhfr sequence in the divergent direction to the thrombomodulin sequence, was treated with Bell and a DNA fragment encoding a gene providing hygromycin resistance on a BamHI fragment was ligated into the plasmid. Clones were selected, after transformation into £. coli strain DH5α, by their ability to grow on plates containing both ampicillin and hygromycin B. The orientation of the hygromycin B gene relative to the plasmid was determined by restriction mapping. One plasmid, pTM108, in which the hygromycin B gene lies in the opposite orientation to the TM gene, was grown up in culture. This plasmid has the sequences encoding the TM analog under the control of the triple LTR promoter, with both a gene that confers hygromycin B resistance and one that encodes dhfr present on the plasmid. The thrombomodulin sequence was linked to the tissue plasminogen activator signal sequence, ensuring its secretion. (See Table 6) b. Transfection, selection and amplification of stable mammalian clones. For the transfection, 10 μg of pTM108 was mixed with Lipofectin reagent (Bethesda Research Laboratories) and added to a monolayer of 10 5 CHL-1 host cells in 6-well plates. Forty-eight hours after transfection, a known number of cells were plated onto selective media. Resistance to hygromycin B was used as the selection marker. CHL-1 cells transfected with the bacterial hygromycin B gene can survive growth in 0.3 mg/ml hygromycin B. The transfection or selection frequency was 2/10 3 and was determined as the number of colonies arising after selection, divided by the total number of cells plated. The culture supernatant was shown to contain 1.5 U/ml TM activity after 24 hours in contact with the cells. A population of cells resistant to the first selection conditions were then subjected to a second round of selective pressure. Either lOOnM or 500nM methotrexate (MTX) was added to the growth medium to select for transfectants that expressed the dhfr gene. Only clones which had amplified the dhfr gene would be able to grow in this high level of MTX. In the process of gene amplification, other plasmid sequences will be co-amplified with the dhfr gene and thus lead to increased gene expression of the non-selectable gene as well. Resistant clones were apparent after 5 to 6 weeks. Individual clones resistant to these levels of MTX were isolated and assayed. A culture after selection in lOOnM MTX was shown to produce 4.9-14.7 U per ml of protein C activating activity (see below) . A pooled population was plated into a ten-fold greater concentration of MTX (lμM or 5μM) . Clones were again recovered from this selection step and assayed. At each step clones were shown to produce and secrete TM analog into the culture medium. Example 3. Transient Expression of TM Analogs in Mammalian Cells a. Construction of Transient Mammalian Expression Vectors. Two vectors suitable for use in transient expression experiments were constructed from commercially available plasmids. i. pPA133 Plasmid pPA133 is a modification of plasmid pLl (Okayma, H. and Berg P., (1983) Mol. Cell Biol. 3:280-289) purchased from Pharmacia LKB Biochemical (Piscataway, NJ) . This plasmid, PLl, carries the SV40 origin of replication the early region transcription promoter and mRNA splicing sequences. It was used to construct a plasmid containing a polylinker sequence that adds a unique series of restriction sites, the t- PA signal sequence under the control of the SV40 promoter and the SV40 origin or replication. The mRNA splicing sites were removed. Construction of this plasmid, pPA129, is described in detail in co-pending, commonly assigned patent application USSN 345,372, incorporated herein by reference. To create pPA133, pPA129 was digested with Bglll and another polylinker sequence was inserted immediately downstream from the t-PA signal sequence. This polylinker creates a plasmid with a unique series of restriction sites (Bglll, Notl, SnaBI and Spll) . ii. pTHR5 Plasmid pTHR5 is a modification of plasmid pCDM8 purchased from Invitrogen (San Diego, CA) and carries the cytomegalovirus immediate early promoter. Plasmid pPA133 was digested with Xhol and Notl to release the t-PA signal sequence which was isolated and cloned into pCDM8 that had been digested with Xhol and Notl so that the t-PA signal sequence is under the control of the cytomegalovirus promoter. (See Drawing 5) . b. Transient Expression Plasmids Containing TM Analog Genes. The cloning plasmids containing TM analog gene sequences described in Example l were digested with BamHI and/or Bglll and/or Notl. The TM gene sequences were isolated and ligated into either pPA133 or pTHR5 immediately adjacent to the t-PA signal sequence to insure that the transiently expressed peptides would be secreted into the cell culture medium. A model plasmid, pTHR13, is shown in Drawing 5. Table 2 lists other TM analog gene containing plasmids and their parent plasmids. Plasmid pTHR13 was made by digesting cloning plasmid pTM301 with BamHI and Bglll and ligating the TM analog gene fragment into the Bglll site of pTHR5. pTHR13 contains the gene sequence coding for the 6 EGF-like domains of thrombomodulin operably linked to the t-PA signal sequence. These plasmids were transfected into SV40 transformed African Green Monkey Kidney Cells (COS-1) by the Lipofectin technique. In controls, COS-1 cells were either transfected with the parental plasmids or mock transfected with phosphate buffered saline (PBS) . Protein C activation assays were done using conditioned cell culture medium to determine the presence of TM analog. Table 7 depicts a list of transient expression plasmids. Example 4. Production of human thrombomodulin analogs in insect cells infected with a baculovirus expression vector. Autographa California nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human thrombomodulin (TM) . By using specifically constructed plasmids, the protein coding sequence for selected portions of human thrombomodulin were linked to the signal sequence of hypodermin A. The hybrid hypodermin A-TM analog genes were inserted downstream of the polyhedrin transcription signal, and the TM- polyhedrin hybrid genes were transferred by homologous recombination to an infectious AcNPV expression vector. In this virus the wild type AcNPV polyhedrin gene has been replaced with the TM analog gene in the recombinant virus. Spodoptera frugiperda (sf9) cells were then infected for human TM analog production. a. Construction of AcNPV Transfer Vectors i. Vectors with the Hypodermin A signal seguence: pHYl and pSC716. Two oligo ers, COD#1198 and C0D#1199 were synthesized, see Table 6. These oligomers contain the Hypodermin A signal sequence, a translation initiation codon, a Bglll cloning site, a BamHI 5' overhand and a Kpnl 3' overhang. COD#1198 and C0D#1199 were annealed and cloned into pSC654, a pUC19 derivative, creating pHYl. See Drawing 6. Plasmid pHYl was restricted with BamHI and EcoRI, releasing the hypodermin A signal sequence. This sequence was then ligated to pSC714 to create the vector pSC716. Plasmid pSC714 is a derivative of pVL1393, obtained from Summers, et al. The only difference between the two is that in pSC714, the Bglll site has been destroyed. ii. Construction of a vector in which the hypodermin A signal sequence is linked to a gene encoding TM amino acids 227-462. The BamHI fragment from pUC19pcrTM7 was cloned into the Bglll site of pHYl and the orientation was chosen such that the hypodermin A signal sequence was adjacent to amino acid 227. This plasmid is PHY101. iii. Construction of the AcNPV transfer vector: pTMHYlOl. Plasmid pHYlOl was treated with BamHI/EcoRI which releases the Hypodermin A signal sequence linked to the TM analog coding sequence. Shuttle vector pVL1393 contains a partially deleted AcNPV polyhedrin gene and unique BamHI and EcoRI cloning sites. The BamHI/EcoRI fragment from pHYlOl was inserted downstream of the polyhedrin promoter, thus creating a plasmid, pTMHYlOl, in which the hybrid gene was under the control of the polyhedrin promoter. This plasmid is shown in Drawing 6. iv. Construction of other AcNPV transfer vectors Transfer plasmids containing other TM analog gene sequences were constructed using a strategy similar to that outlined above. Fragments from the cloning plasmids described in Example 1 above were cloned into pSC716 in frame so that the TM analog gene sequence was fused to the hypodermin A signal sequence. See Table 8 for a list of transfer vectors and the gene sequences contained therein. b. Construction of AcNPV Transfer Vectors PTMHY102 and pTMHY103. A new signal sequence is synthesized that contains a Ball cloning site, a BamHI 5 1 overhang and a Kpnl 3' overhang. The synthetic fragments are annealed and cloned into pSC654 as described above creating pHY2. New genes consisting of human analog 11/6 or human analog 11/2 are isolated by using human DNA in the polymerase chain reaction as described in Example 1. The primers introduce a Ball site at the 5' end and a termination codon and an EcoRI site at the 3' end (see Table 3). After treatment with Ball and EcoRI, the genes are cloned into pHY2, which has also been treated with Ball and EcoRI. The EcoRI site is a unique site in the original pSC654 vector used to make pHY2. The new plasmids pHY102 and pHY103 contain the signal sequence of hypodermin A fused to genes that encode analog 11/6 and analog 11/2, respectively. Plasmids pHY102 and pHY103 are treated with BamHI and EcoRI and the fragment released is cloned into pVL1393 at its unique BamHI and EcoRI sites creating plasmids pTMHY102 and pTMHY103 in which the hybrid genes are under the control of the polyhedrin promoter. c. Transferring Genes into the AcNPV Genome. Plasmids pTMHYlOl, pTHR22, etc. containing a gene encoding the Hypodermin A signal sequence and a TM analog coding sequence, were transferred to the AcNPV genome by in vivo recombination. A calcium phosphate precipitation technique modified for insect cells was used according to Summers and Smith. Briefly, the individual transfer vectors and AcNPV DNA were cotransfected into sf9 cells basically as follows: a T25 flask was seeded with 2xl0 6 cells. The cells were allowed to attach for one hour at room temperature. Then one microgram of transfer vector and 1 μg AcNPV DNA coprecipitated in calcium phosphate were incubated with SF9 cells for 4 hrs. TMN-FH media supplemented with 10% FBS was then replenished. A stock virus was created. The genes contained on plasmids pTMHY102 and pTMHY103 are transferred into the AcNPV genome in the same manner as described above. d. Detection and Purification of Recombinant Viruses. Plaque assays were performed to detect recombinant viruses. The transfection stocks were diluted to 10 "4 , 10 ~5 , 10 "6 on day 7 post transfection. A control vector in which ,9-galactosidase is under the control of the polyhedrin promoter was also included in the experiment. The results showed that the control vector assayed with X-gal gave an average of 2% recombinant blue plaques. There were approximately 2% occlusion negative recombinant TM plaques observed. Occlusion negative plaques from the cells transfected with the TM transfer vectors were picked and replated at 10 "1 , 10 "2 and 10 "3 dilution of plaque assay. On day 7, the plates showed 100% pure occlusion negative recombinant plaques. A single colony for each of the desired TM analogs, (6h/227-462, 6h/227-462:227-462, 6h/350-462, 6h/227-497, etc.) was selected for further work. e. Production of Soluble Thrombomodulin Analog, i. Production of TM activity by the cotransfeetion stocks. 2xl0 6 sf9 cells were infected with 1 ml of the transfection stock as described above. On day 3 the cells were pelleted and resuspended in serum free Excell 400 medium (JR Scientific) for another three days. Supernatant was collected and, when assayed, showed the presence of TM activity. ii. Growth of viral stocks. T25 flasks were seeded at a density of 1.5xl0 6 sf9 cells per 5 ml. An isolated recombinant plaque was picked (see above) and used to infect the sf9 cells in TMN-FH media supplemented with 10% FBS for viral stocks. On day 3, supematants from the cultures were collected and were shown to contain TM activity. iii. Production of TM activity by recombinant AcNPV 6h-227/462 Five 30 ml shaker flasks were seeded with 2xl0 7 cells and infected with 2 ml of the above-prepared recombinant viral stock. A spinner flask was also infected with one of the virus stocks prepared above. On day 4 the supernatant was collected for assay. The results showed that insect cells infected with recombinants AcNPV 6h-227/ 62 (and the others) were secreting thrombomodulin activity. iv. Production of TM activity by other recombinants The methods described above for cotransfection and production of viral stocks were used to produce infected insect cells which secrete other TM analogs including 6h/(227-462)2, 6h/350-462, 6h/227-497, etc. Example 5. TM Analog; t-PA Chimaeric Proteins The t-PA gene is used for the assembly of genes encoding chimaeric fusions of a fibrinolytic enzyme with thrombomodulin polypeptide sequences. The t-PA gene used for this purpose was isolated from plasmid pPA003, which is on deposit with the ATCC, accession No. 67293. Plasmid pPA003 is described in detail in co-pending, commonly assigned, patent application USSN 003,611 and is incorporated herein by reference. Tables 7 and 8 depict a list of mammalian expression plasmids and baculovirus transfer plasmids containing thrombomodulin analog genes fused with the t-PA gene. a. TM Heterologs Synthesized in Mammalian Cells. i. TM Analogs fused to the N-terminus of t-PA The 3' ends of the TM analog genes described in the previous examples all have termination codons which must be removed in order to create chimaeric proteins containing thrombomodulin gene sequences fused to the N-terminus of t-PA. Primers for use in a PCR were synthesized so that when incorporated into the DNA sequences of the TM analogs the termination codons at the 3' ends were deleted and a Bglll site was introduced. The 5' ends of the TM genes remained unchanged. These primers were used in a series of PCRε that synthesized variant fragments of the TM analog genes described in Example 1. These new TM analog genes were ligated into either of the two transient expression vectors described in Example 3, pPA133 or pTHR5, so that the TM analog gene was fused to the t-PA signal sequence and followed by a Bglll site. These plasmids were treated with Bglll and a gene encoding amino acids 4 to 530 of mature t-PA was inserted. This fragment was generated by PCR using the primers shown in table 3. The plasmids that result contain a gene that encodes a fusion protein consisting of a signal sequence from t-PA followed by the TM analog which is fused to mature t-PA under control of either the SV40 promoter or the cytomegalovirus promoter. See Drawing 7. ii. TM Analogs fused to the C-terminus of t-PA The termination codon at the 3' end of the t- PA gene was deleted using the same method described above. Two primers were synthesized for the PCR that introduced a new Bglll site immediately 3' to the proline at amino acid 530, which also deletes the termination codon, and creates a BamHI site at amino acid 4 in place of Bglll site found in the native t-PA gene sequence. The t-PA gene used as a template for the PCR was isolated from plasmid pPA509. This plasmid is on deposit with the ATCC, accession #67443, and is described in detail in co-pending, commonly assigned, patent application USSN 074,083, incorporated herein by reference. The new t-PA gene fragment was inserted into the Bglll site of pTHR5 so that the t-PA gene is fused to the t-PA signal sequence, creating plasmid pTHR16. See Drawing 7. Thrombomodulin analog gene sequences, digested with Bglll and/or Bam HI were inserted into pTHR16 at the Bglll site creating plasmids that encode soluble secreted fusion proteins whose N-terminus is the natural N-terminus of t-PA fused at the natural C- terminus of t-PA to the N-terminus of the TM analog gene. Schematic examples of these fusion peptides are shown in Drawing 2B. The DNA and amino acid sequence of t-PA fused with the 6 EGF-like domains of TM is shown in Table 9. b. TM Heterologs Synthesized in Insect Cells TM heterolog gene sequences for expression in insect cells were constructed by methods similar to those described above. For example, a plasmid for producing one TM heterolog in which the 6 EGFs were operably linked to the N-terminus of t-PA was constructed by digesting plasmid pTHR6 with Nhel and Notl and inserting the gene fragment into pTHRlO after digestion with the same enzymes. pTHR6 is a transient expression vector containing the 6 EGFs fused to t-PA for expression in mammalian cells. pTHRlO is a baculovirus transfer vector containing gene sequences for the 6 EGF-like domains. The resulting plasmid, pTHR25, contains gene sequences for the 6 EGF-like domains and t-PA in a baculovirus transfer vector. In another example where the 6 EGF-like domains were fused to the C-terminus of t-PA, a Smal- Notl fragment removed from pTHR17 and containing the 6 EGF domains was ligated into the Smal-Notl sites of PTHR12. Example 6. Purification of TM Analog 6h/227-462 TM analog 6h/227-462 was purified from conditioned media from cells infected with AcNPV- 6h/227- 62 by removal of cell debris, followed by five chromatography steps: 1) Q Sepharose, 2) thrombin affinity, 3) gel filtration, 4) anion exchange, and 5) a second gel filtration step. Substantially pure 6h/227-462 (>95%) is obtained after the second chromatography step (thrombin affinity) , however, when analyzed on SDS-PAGE gels, multiple active forms of analog 6h 227-462 were observed. These variant forms were separated from each other using anion exchange with Mono Q resin. The gel filtration steps effect an exchange of buffers. All chromatography steps were performed at 4" C. a. Materials Some of the chromatographic resins were purchased from commerical sources. Q Sepharose and Sephadex G25 were purchased from Sigma (St. Louis, MO) , and Mono Q 5/5TM from Pharmacia LKB (Piscataway, NJ) . DFP-thrombin agarose was prepared approximately as follows: 360 mg of bovine thrombin in 100 ml of 20 mM Na phosphate, pH 7.5 was added to approximately 100 ml of a 50% Affigel 10 resin slurry and mixed overnight at 4*C. The Affigel 10 was prepared for use as described by the manufacturer and equilibrated with the load buffer. Residual active esters were blocked by the addition of 100 ml of 0.1M glycine (pH 5.6) for one hour at 4'C. The gel was then equilibrated with 30 mM Tris-HCl, 2M NaCl, pH 7.5, and 20 μl of DFP was added to give a final concentration of about lmM DFP. After 16 hrs of mixing at 4 # C an additional 6 μl of DFP was added and mixing continued for 4 additional hours. The resin was then washed with 20 mM Tris-HCl, 2 M NaCl pH 7.5 and stored at 4'C. Thrombin activity was measured using the Kabi S-2238 substrate and indicated that >86% of the thrombin was removed from the solution, and presumably coupled to the resin, giving a final concentration of about 6 mg of thrombin per ml of resin. The enzymatic activity of the DFP treated resin was <1% of the starting activity. b. Production of pure 6h/227-462. Conditioned media was harvested and clarified by cross-flow filtration on a Microgon 0.3 ft 2 hollow fiber filter unit at about pH 6.0, then diluted by 50% with ultra-pure water. The pH was adjusted from about 6.0 to about pH 5.2 with glacial acetic acid. The adjusted media was then loaded onto a column of Q Sepharose resin at a flow rate of about 110-150 ml/min. The column had previously been equilibrated with about 4 column volumes of wash buffer 1 (117 mM Na acetate, 0.02% NaN 3 pH 5.0). After loading, the column was washed with wash buffer 1 followed by wash buffer 2 (25mM Na acetate, O.lMNaCl, pH5.0) then the TM analog was eluted with wash buffer 2 containing 0.3 M NaCl, pH 5.0. This column matrix can be reused by stripping with wash buffer 2 + 2.0 M NaCl. Column fractions containing activity as measured in the Protein C activation assay (see Example 11) were pooled and diluted 30% with of 0.3 M NaCl, 20 mM Tris-HCl, 0.5 mM CaCl 2 , 0.02% NaN., pH 7.5. The pH of the diluate was measured and adjusted to about 7.5 with NaOH. The ionic strength of the pool was about the ionic strength of a solution of 0.3 M NaCl. This adjusted pool was loaded overnight by gravity onto a thrombin agarose column pre-equilibrated with the same buffer used to dilute the Q Sepharose pool. The column was washed with diluent buffer, and the TM analog was removed from the matrix with 1.5 M GuHCl, 2.0 M NaCl, 20 mM Tris HC1, 1 mM Na EDTA, 0.02% NaN_, pH 7.5. c. Evaluation of Purity SDS Polyacrylamide Gel Electrophoresis was performed by the method of Laemmli using 3.3% acrylamide in the stacking and 12.5% acrylamide in the running gel. Nonreduced samples were diluted in Laemmli sample solubilization buffer (50 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, and .01% bromphenol blue) and loaded directly onto the gel. Reduced samples were incubated for 7 minutes at 100 # C in sample solubilization buffer containing 10 mM dithiothreitol. Following incubation, iodoacetamide was added to a final concentration of 50 mM, and the sample incubated an additional 5 minutes at room temperature, before loading on the gel. Pharmacia LMW Calibration Kit protein standards were used for MW markers, and the gels were silver stained. Under reduced-alkylated conditions, a single broad band was seen at about 53 kDa, while under non- reducing conditions, a doublet was seen at about 31 and 36 kDa, as well as low levels of a higher MW material (having an apparent molecular weight of about 52 kDa) . Both bands in the doublet were active as shown by elution of the bands from an SDS gel, followed by a Protein C activation assay. The 52 kDa material seen in non-reduced gels, which was estimated at 10% of the total protein by gel scanning, also appeared to be derived from active thrombomodulin as shown by the activity of the eluted high bands in the Protein C activation assay. Thus all material visualized on the non-reduced gel had TM activity. The lower MW bands seen in non-reduced gels were the predominant form or species of the isolated protein. The preferred species of peptide is one having a higher TM activity being free of other less active thrombomodulin-like peptides. This preferred species corresponds to the slower migrating of the two lower MW bands. Such peptide species were purified below to provide under non- reducing chromatography a single species having the expected apparent molecular weight predicted for the particular peptide. The TM analog 6h/227-462 gene has two potential N-linked glycosylation sites at amino acids 364 and 391 and two potential O-linked glycosylation sites at amino acids 319 and 393. The thrombin affinity purified product was shown to be glycosylated by treatment with N-glycanase which resulted in an apparent decrease in MW of about 2000 daltons. d. Amino Acid Composition and Sequencing. i. Sample Preparation. Since the expected product has 36 Cys residues in 18 disulfide bridges, samples were reduced and alkylated prior to N-terminal analysis. Guanidine in a Tris buffer was added to 1.5 ml of product (about 3435 pmoles, 90 μg) to give a final concentration of 4M guanidine, 0.1 M Tris-HCl, 1 mM EDTA, pH 8.5 in a volume of 3.0 ml. The resultant solution was reduced with a 43 fold molar excess of DTT over disulfide (2.67 μmoles, 0.89 μM final concentration) for 3 hr at 37*C, then alkylated for 60 minutes at 37*C by the addition of a 2.6 fold molar excess of iodoacetic acid over DTT (6.94 μmoles, 2.3 μM final concentration). The sample was purified using a Waters Delta PakC-4 reverse phase HPLC with a linear gradient of water + 0.1% TFA to 75% CH.CN + 0.1% TFA. Only a single protein peak was recovered following the reduction and alkylation protocol. Aliquots were dried down in a Savant evaporator for sequencing and amino acid analysis. ii. Amino Acid Analysis. The amino acid composition was determined as described above in the determination of specific activity starting at the hydrolysis step. The amino acid composition found for the protein agrees well with what was expected (see Table 4) . The exceptions are tryptophane and cysteine, which are not accurately measured by the methods used here, iii. Sequencing. Amino acid sequence analysis was performed on an Applied Biosystems Protein/Peptide sequencer (Model 477 connected to a 900A data module or a Model #470A connected to a chart recorder) . The phenylthiohydantoin (PTH) amino acids were identified on-line with a 120A Applied Biosystems PTH Analyzer by reverse phase HPLC using a Brownlee PTM-C18 cartridge (2.1 x 222 mm). The reduced-alkylated sample purified as described above gave the following sequence: Ala-Val-Val- Pro-Arg(5)-Ser-Cys-Ser-Val-Glu(10)-Asn- Gly-Gly-Cys/Glu-Glu(15)-His-Ala-Cys/Glu-Asn-Ala(20)- 1le-Pro-Gly-Ala-Pro(25)-Arg-Cys/Glu-Gln-xxx-Pro(30)- Ala-Gly-Ala-yyy-Leu(35)-Gln-Ala-Asp-Glu/Gly. sequence found gives an excellent fit with that expected for the cloned material. In several positions (i.e., 14,18,27), it was not unexpectedly impossible to distinguish between an assignment of Cys or Glu, while in position 29, identified as xxx, a clear signal for Cys was not evident. Also, due to the sequence Ala-Ala (33-34), the second Ala (yyy) could not be unequivocally assigned. Otherwise, all residues were as predicted from the DNA sequence. e. Separation of variant TM analog species, i. Buffer exchange The substantially pure, active TM analog 6h/227-462 was applied to a Sephadex G25 column and recovered in 0.2% N-ethylmorpholine acetate (NEM) pH 7.0. This step removes GuHCl and NaCl. ii. Anion Exchange Chromatography As noted above, more than one form of TM analog is detected in thrombin-affinity purified material run on an SDS-PAGE gel under non-reducing conditions. A method was developed to resolve these variants. TM analog 6h/227-462 collected from the Sephadex G25 column was applied to a Mono Q column (Pharmacia, 10 micron particles, quarternary amine) pre-equilibrated with 0.2% N-ethylmorpholine (NEM) . pH7.0. After washing with this buffer the various forms were separated using a gradient of 0 to 0.4 M NaCl. The elution and activity profiles are shown in Drawing 9. Samples of each fraction were evaluated on an SDS-PAGE gel under non-reducing conditions. Three distinct bands with slightly different mobilities could be seen on the stained gel. Fractions containing peptides with like mobilities were pooled (A = fxns 32- 35, B = fxns 40-44, C = fxns 70-71) and then assayed for total protein content and for activity in the Protein C activation assay. The specific activities are listed in the table below. No inactive peptides were detectable in any of the fractions. Test Material Specific Activity .U/mg) Mono Q Load 166,000 + 12,000 fxns 32 - 35 416,000 + 19,000 fxns 40 - 44 262,000 + 4,000 fxns 70 - 71 67,600 + 5,000 Example 10. Purification of TM Analog 4t/227-462 Conditioned cell culture media was collected and the ionic strength adjusted to be equivalent to about the ionic strength of 0.3 M NaCl using 1 M NaCl. The adjusted media was loaded directly onto a thrombin affinity column that had previously been washed with 0.3 M NaCl, 20 mM Tris HC1, 0.5 mM CaCl 2 and 0.02% NaN3, pH7.5 (wash buffer). After loading, the column was washed with wash buffer, then the TM analog was removed from the column matrix with 1.5 M GuHCl, 2.0 M NaCl, 20 mM Tris, 1 mM Na EDTA and 0.02% NaN 3 . A peak containing TM activity was collected and applied to a Sephadex G25 column and washed through with 0.2% NEM to effect a buffer exchange (removal of NaCl and GuHCl) . The active fractions were pooled and applied to a column of Mono Q 5/5 (Pharmacia, LKB) in 0.2% NEM, pH 7.0. The TM analog was selectively eluted with a gradient of 0 to 0.6 M NaCl in 0.2% NEM pH7.0. Fractions containing TM activity were pooled and samples run on SDS-PAGE gels under both reducing and non-reducing conditions. The TM analog appeared to be about 90-95% pure. The remaining high molecular weight contaminating material was removed by molecular exclusion chromatography. The activity containing fractions from the Mono Q column were lyophilized to dryness and resuspended in (380 ul) of 1.0% NEM, pH 7.0 then applied to a Superose 12 TM (Pharmacia, Piscataway, NJ) column at 0.5 ml/min. The active material collected from this chromatography step was estimated to be 99% pure TM analog by silver stained SDS-PAGE gel analysis. Example 7. Assays for Thrombomodulin Analogs, a. Materials Rabbit thrombomodulin, hirudin and human Protein C were supplied by American Diagnostica. Human thrombin is available from a variety of noncommercial and commercial sources. Bovine thrombin was purchased from Miles Labs, Dallas, Texas. D-valyl-L-leucyl-L- arginine-p-nitroanilide (S-2266) and D-Phe-Pip-Arg-p- nitroanilide (S-2238) were purchased from Kabi Diagnostica. Bovine serum albumin (fraction V) , citrated human plasma, and APTT reagent were purchased from Sigma Chemicals. Microtiter plates were supplied by Corning (#25861-96) . All other reagents were of the highest grade available. b. Methods and Results. i. Protein C Activation Assay (Chromogenic) This assay was performed by mixing 20 μl each of the following proteins in a microtiter plate: thrombomodulin sample (unknown or standard) , thrombin (3 nM) , and Protein C (1.5 μM) . The assay diluent for each protein was 20 mM Tris-HCl, 0.1 M NaCl, 2.5 mM CaCl 2 , 5 mg/ml BSA, pH 7.4. The wells were incubated for 2 hours at 37'C, after which Protein C activation was terminated by the addition of 20 μl of hirudin (0.16 unit/μl, 370 nM) in assay diluent and incubation for an additional 10 minutes. The amount of activated Protein C formed was detected by adding 100 μl of 1.0 mM S-2266 (in assay diluent), and continuing to incubate the plate at 37 # C. The absorbance at 405nm in each well was read every 10 seconds for 30 minutes, using a Molecular Devices plate reader. The absorbance data was stored, and the change in absorbance per second (slope) in each well was calculated. The change in absorbance per second is proportional to pmole/ml of activated Protein C. This ratio was determined empirically using varying concentrations of totally activated Protein C. Samples containing 100% activated Protein C were generated by mixing Protein C at 0 to 1.5 μM with 60 nM rabbit TM and 30 nM thrombin, incubating for 0 to 4 hours, adding hirudin and measuring S2266 activity as above. Conditions under which 100% of the Protein C was activated were defined as those in which the S2266 activity (A405/sec) reached a plateau. A unit of activity is defined as 1 pmole of activated Protein C generated per l/min under the reagent conditions defined above. The response of both purified rabbit TM and conditioned medium containing analog 6h/227-462 in this assay is shown in Drawing 10A. In Drawing 10B the enhancement of protein C activation by purified TM analog is depicted. In some cases the activity values reported were calculated using rabbit thrombomodulin as a standard. By using amino acid analysis to deduce protein mass, it has been determined that 1 nmole of TM analog 6h-227/462 has activity equivalent to 1 nmole of rabbit thrombomodulin. Other TM analogs showed Protein C activation enhancing activity including 4t/227-462, 4t/227- 462:227-462, PA:227-462(mammalian cells) , 6h/227- 462:PA, 6h/227-462:227-462, PA:227-462(insect cells) , 6h/350-462, and 6h/227-497. ii. Determination of Specific Activity of TM Analog 6h/227-462. TM analog samples were dialyzed exhaustively against 0.2% ethylmorpholine acetate, pH 7.5, and assayed for activity and amino acid composition (no activity was lost due to the dialysis) . Samples were prepared for analysis by dialysing samples into 0.2% N- ethylmorpholine-acetate, 0.02% NP-40, pM 7.5 using a 6000 Spectropor membrane and dried down in hydrolysis tubes. Hydrolysis was performed in evacuated tubes in a vapor phase at 110*C for 22 hours using 6 N constant boiling HC1 containing 1% phenol. The amino acid analysis was performed after samples were dried down and derivatized with phenylisothiocyanate (PITC) . They were analyzed by reversed phase HPLC on a 4μm alkyl binding (C18) silica column with detection at 254 nm. The chromatogram was developed by a gradient from Eluant A (131 mM NaOAc, pH 6.4, 0.047% TEA, 1 pm EDTA) to Eluant B (60% CH 3 CN in water) . Pierce Protein hydrolysate is used as a standard and norleucine is incorporated as an internal control. Nmoles protein from the amino acid composition were converted to ng using a molecular weight value of 26,200 for the TM analog 6h-227/462 peptide. Samples had an activity in the chromogenic assay of 0.25 units per ng of protein, or 250,000 units/mg. iii. Inhibition of the Activated Partial Thromboplastin Time (APTT) . The formation of a clot from citrated plasma is triggered by the addition of brain cephalin in ellagic acid ("APTT reagent") , and calcium ion. The time required for the clot to form is reproducible and increases proportionally with the addition of thrombomodulin. Reagents for the APTT are incubated at 37*c. before mixing, except for the citrated plasma, which is kept at 4*C. The reaction was carried out as follows: 100 μl of Sigma Citrated Plasma was added to a plastic cuvette (Sarstedt #67.742), incubated at 37'C for 1 in; 100 μl of Sigma APTT reagent was added and the mixture incubated for 2 min at 37*C; 100 μl of test sample (or control buffer) and 100 μl 25 mM CaCl, were added and the cuvette was immediately placed in a Hewlett-Packard 8451A spectrophotometer equipped with a circulating water bath to keep the cuvette at 37*C during reading. The absorbance due to light scattering at 320 nm was measured every 0.5 seconds, from 15 to 120 seconds, timed from the addition of CaCl_. A plot of absorbance vs. time yields a sigmoidal curve, with the clotting time defined as the time at which the slope is the steepest, corresponding to the inflection point of the curve. A dilution curve was generated for pure analog 6h/227-462 in the APTT assay (see Drawing 11A) (μg amounts shown were in a 100 μl sample volume) . The APTT was prolonged in a dose dependent fashion. Purified 6h/227-462 was compared to heparin and antithrombin III in the APTT assay. As seen in Drawing 11B, TM analog 6h/227-462 showed a greater ability to prolong the APTT at lower amounts than either heparin or AT III. The quantities of heparin and AT II used in this experiment are not effective doses in vivo suggesting that a TM analog will be more effective than either. iv. Inhibition of thrombin clotting time (TCT) and prothrombin reaction (PT) . Both the PT and TCT are determined using the Hewlett-Packard 8452 A diode-array spectrophotometer used for the APTT. For the PT reaction, 90 ul of either TM analog 6h/227-462 or PBS was added to 20 ul thromboplastin and 90 ul 25 mM CaCl in a cuvette. The mixture was incubated for 1 minute at 37 C, then 100 ul of citrated plasma was added. After loading the cuvette into the spectrophotometer,the absorbance due to light scattering at 320 nm was measured every 0.5 seconds, from 15 to 120 seconds, timed from the addition of the plasma. A plot of absorbance vs. time yields a sigmoidal curve, with the clotting time defined as the time at which the slope is the steepest, corresponding to the inflection point of the curve. The TCT is evaluated in the same manner. The initial reaction mixture contains 100 ul citrated plasma, 25 ul of 100 mM CaCl 2 and 162.5 ul of either PBS or TM analog. After 1 minute, 12.5 ul of thombin is added. The clotting time is measured as described above. Drawing 12 shows that the TM analog 6h/227- 462 prolongs both the PT and the TCT. v. Direct anticoagulant activity - Inhibition of thrombin catalyzed conversion of fibrinogen to fibrin. Thrombin and varying amounts of TM analog 6h/227-462 were incubated for 2 minutes at 37 C in microtitre plate wells. The total initial reaction volume was 50 ul PBS +7.5 mM CaCl 2 and 90 nM thrombin. After initial incubation, 100 ul of 3.75 mg/ml human fibrinogen was added per well, and the thrombin induced formation of fibrin was followed by measuring the change in absorbance at 405 nm in a Molecular Devices Vmax spectrophotometer (Molecular Devices, Menlo Park, CA) . The end-point of the assay was the time at which 50% of the final absorbance was reached. Residual thrombin activity was determined by reference to a thrombin standard curve, which linearly relates the reciprocal of the thrombin concentration to the clotting time. As can be seen in Drawing 13, TM analog 6h/227-462 inhibited the ability of thrombin to convert fibrinogen to fibrin in a dose dependant manner. vi. Inhibition of platelet activation and aggregation. The effects of TM analog 6h/227-462 on thrombin activation of platelets was tested by the methods of Esmon, et al, (1983) J. Biol. Chem. 258:12238-12242. When evaluated using this assay, TM analog 6h/227-462 inhibited the thrombin mediated activation and aggregation of platelets. vii. Fibrinolysis - Fibrin plate assay. The fibrinolytic activity of TM heterologs, fusions of TM analogs and t-PA, was evaluated using zonal clearing on plasminogen-enriched fibrin plates as described by Haverkatet and Brakman, (1975) Pro . in Chem. Fibrin. Thromb. 1:151-159. TM heterologs PA:227- 462(mammalian cells), PA:350-462(mammalian cells), 6h/227-462:PA, and PA:227-462(insect cells) all exhibited fibrinolytic activity using this assay. viii. Inhibition of Factor XIII activation. TM analog 6h/227-462 inhibited the activation of Factor XIII by thrombin as measured by the method of Polgar, et al., (1987) Thromb. Haemostas. 58:140. ix. Additional measures of TM antithrombotic activity. 1) TM analog's inhibition of activation of Factor V by thrombin is measured by the method described by Esmon et al., J. Bio. Chem.. (1982), 257:7944-7947. 2) Inhibition of the TM analog thrombin complex by antithrombin III and heparin cofactor II is measured as described by Jakubowεki et al., 1986. 3) TM analog's inhibition of the inactivation of protein S by thrombin is measured by the method described by Thompson & Salem, J. Clin. Invest.. (1986), 78(1):13-17. ix. Additional measures of TM antithrombotic activity. c. Activity of TM Analog In Vivo The in vivo activity of 6h/227-462 was tested in a mouse model of disseminated intravascular coagulation (Kumada et al (1987), Blood 71(3) :728-733) . Three groups of six mice each were anaesthetised and injected with 37.5 units of thrombin via the tail vein. In mice receiving no other treatment this caused pulmonary embolism rapidly leading to death. Two groups were pretreated by injection with TM analog 6h/227-462 (3 min. prior) at lx or 2x the concentration of thrombin. Groups treated with 6h/227- 62 were protected from lethal effects of thrombin. The surviving mice in the control group (thrombin alone) were comatose for 30 minutes whereas mice in the pretreatment groups appeared normal immediately on recovery from the anaesthetic. Example 8. fPCTula ipn gnfl y? - a. Concentration. Methods were developed to concentrate the TM analog 6h/227-462 in a variety of solutions under pressure without significant losses in activity. A number of aliquots of purified sample were concentrated with an A icon 8050 stirred-cell concentrator equipped with YM-10 membrane operated at 40 psi nitrogen gas at 20*C. i. A 56 ml sample containing TM analog (774 U/ml) in 20 mM Tris-HCl, 1.5 M Guanidine-HCl, 2 M NaCl, 1 mM EDTA, with 0.09% NP-40, pH 7.5 was buffer exchanged 2 times with 15 volumes of 0.2% N- ethylmorpholine-acetate (17.4 mM) , pH 7.5 containing 0.02% azide. The final concentration in 2.3 ml was estimated at (24,108 U/ml) and the recovery of Protein C activating activity was 99%. ii. A solution of the TM analog (544 U/ml) in 20 mM Tris-HCl, 1.0 M NaCl. 0.1 mM EDTA, 0.02% NP- 40, pH 7.5 was concentrated 5-fold without buffer exchange to give a 95% recovery of the original activity. iii. A 10 ml solution of the TM fragment (544 U/ml) in 20 mM Tris-HCl, 1.0 M NaCl, 0.1 mM EDTA, 0.02% NP-40, pH 7.5 was buffer exchanged 3 times with 10 volumes of the N-ethylmorpholine buffer to a final concentration of 1,509 U/ml (2.0 ml) with a 60% recovery of the original activity. iv. A sample containing 1,960 U/ml TM in 2M NaCl, 1.5 M guanidine-HCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, containing no NP-40, was concentrated 10-fold using an Amicon Centricon 10 microconcentrator device. Recovery of activity was >90%. In addition, the TM analog can be concentrated by drying and resolubilization as described below. b. Dialysis. The stability of the TM analog during dialysis was demonstrated. Dialysis was performed using 12,000-14,000 molecular weight cutoff dialysis tubing at 4'C. Purified TM analog (1,921 U/ml) in 2 M NaCl, 1.5 M guanidine-HCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, was extensively dialyzed against 0.2% N-ethylmorpholine (NEM) (pH 8.8). No loss in activity was detected although the protein was diluted by a factor of 1.2 (final concentration was 1,661 U/ml). A solution containing 450 U/ml of TM analog in 20 M Tris-HCl, pH 7.5 containing 2 M NaCl, 1.5 M guanidine, 1.0 mM EDTA + 0.02% NP-40 was dialyzed against the N-ethylmorpholine acetate buffer at pH 7.5 with no NP-40. Recovery of activity was 85%. c. Stability during freezing and thawing. The stability of the TM analog after freezing and thawing under various conditions was evaluated. A solution of TM analog in 20 mM Tris-HCl, 1.0 M NaCl, 0.1 mM EDTA, 0.02% NP-40, pH 7.5 was used as the starting material. Each of four aliquots were concentrated 5-fold and three were also buffer exchanged 3 x with 10 volumes of the solvents listed below. Each were then put through 5 freeze-thaw cycles, freezing in a dry ice: MeOH bath and thawing at room temperature. 1) 5-fold concentration of initial solution concentration 2) 5-fold concentration after buffer exchange into 0.2 M Tris-HCl, 0.02% azide, pH 7.5 3) 5-fold concentration after buffer exchange into 0.2 M Tris-HCl, 0.2 octylglucoside, 0.02% azide, pH 7.5 4) 5-fold concentration after buffer exchange into 0.2% N-ethylmorpholineacetate (17.4 mM) , 0.02% azide, pH 7.5. All samples showed >95% recovery of activity following the freeze-thaw cycles. d. Drying and Resolubilization. Four equal aliquots of TM analog in 0.5 ml solutions of 0.02% NEM (pH 8.8) were prepared in Eppendorf tubes, and to two of these aliquots NP-40 was added to a final concentration of 0.02%. The aliquots were taken to dryness in a Savant microconcentrator. Either 50 μl of PBS (phosphate buffered saline) or of 0.2% NEM (pH 8.8) were used to resuspend each sample overnight at 4*C. The Protein C activity assay was carried out on all samples. The results are provided in Table 5. In another experiment, a sample to purified TM analog 6h/227-462 was buffer exchanged on Sephades G25 into PBS then diluted 5-fold with pure water. One ml samples, containing 0.22 mg of TM analog protein, were lyophilized, and one was then resuspended in 0.2 ml water, a final concentration of 1.1 mg/ml. Greater that 95% of the activity was recovered. The remaining lyophilized protein was stored for several week at -70 degrees C with no loss of activity upon resuspension. These experiments demonstrate that: a) The TM analog can be efficiently dialysed from the thrombin eluent buffer into the volatile buffer N- ethylmorpholine at pH 8.8 in the absence of detergent, b) TM analog samples in the NEM buffer can be freeze- dried and stored in the absence of salts, detergents or other stabilizers, c) samples of the TM analog can be resolubilized in either NEM or PBS with or without NP- 40 resulting in little or no loss of biological activity. Other salts that are of use include hydrobromide, sulfate, acetate, citrate, malate, borate, lactate, glycine, glutamate and aspartate, among others, and d) samples can be effectively concentrated by this procedure, to at least l.lmg/ml. Example 9 Expression of TM Analog Activity in E. coli. A prokaryotic expression vector was contructed for the expression of a TM analog in E_j_ coli. The parent plasmid was pRIT5, purchased from Amersham, which contains a gene coding for Protein A under the control of the Protein A promoter and having the Protein A signal sequence. This plasmid was digested with BamHI and a BamHI fragment from plasmid pUC19pcrTM7 was inserted. The BamHI fragment carries the gene coding for the 6 EGF-like domains of native thrombomodulin. The resulting plasmid, pTHR8 was transformed into E coli. A culture was grown up and a partial purification of the TM analog protein was done as described by Amersham using an IgG Sepharose column. This protein was active in the Protein C activation assay. Table 1 Name Primers (CQP #). amino acids 5' ?' PTM301 1034 1411 aa 227—462 pTM302 1034 1408 aa 227—344 pTM312 1034 1433 aa 227—344 pTM303 1034 1209 aa 227—462 pTM304 1034 1412 aa 227—421 PTM314 1034 1435 aa 227—421 PTM305 1034 1410 aa 227—386 pTM315 1034 1434 aa 227—386 pTM306 1292 1411 aa 427—462 pTM307 1293 1410 aa 351—386 pTM317 1293 1434 aa 351—386 pTM308 1293 1412 aa 351—421 pTM318 1293 1435 aa 351—421 pTM309 1293 1411 aa 351—462 pTM310 1294 1412 aa 390—421 pTM320 1294 1435 aa 390—421 pTM311 1294 1411 aa 390—462 pTM322 1034 1481 aa 227—498 pTM323 1293 1481 aa 351—498 8 aa 497 Stop Transfer Sequence • I VaJ sSe:r-GlyI-euI_euIleGly^ GΦGC_ATTC 3GCTI 9α^^ 1690 TTOT0Gϊ>0∞GC ) GQ^ aa 557 TGCT_AO_AAAG(_^^ So o o o I Table 3. Synthetic Oligonucleotide Primers COD #1292 aa 427 CysGluAsnGlyGlyPhe 5'ATCGGATCCTGCGAAAACGGCGGCTCC primer/coding seqence BamHI COD #1293 aa 350 CysPheArgAlaAsnCys 5'GTGGGATCCTGCTTCAGAGCCAACTGC primer/coding sequence BamHI COD # 1294 aa 390 CysAsnGlnThrAlaCys 5'CAGGGATCCTGCACCCAGACTGCCTGT primer/coding sequence BamHI COD #1408 aa 339 LeuValAspGlyGluCys 5' (CTGGTGGACGGCGAGTGT) coding sequence GACCACCTGCCGCTCACACACCGCCGGCGCCT primer sequence Notl COD #1409 aa 456 ArgHisIleGlyThrAspCys 5' (CGCCACATTGGCACCGACTGT) coding sequence GCGGTGTAACCGTGGCTGACATCTCGCCGGCGTAG primer sequence Notl COD #1410 aa 381 HisGluProHisArgCys 5' (CACGAGCCGCACGGACGT) coding sequence GTGCTCGGCGTGTCCACGGTCTCGCCGGCGTT primer sequence Notl Table 3 contd. COD #1411 aa 456 ArgHisIleGlyThrAspCysSTOP 5' (CGCCACATTGGCACCGACTGTTGA) coding sequence GCGGTGTAACCGTGGCTGACAACTCGCCGGCGT primer sequence Notl COD #1412 aa 416 AspAspGlyPhelleCys 5' (GACGACGGTTTCATCTGC) coding sequence CTGCTGCCAAAAGGATACGCGCGGCCGGCTG primer sequence Notl COD #1433 aa 339 LeuValAspGlyGluCysSTOP 5' (CTGGTGGACGGCGAGTGTTGA) coding sequence GACCACCTGCCGCTCACAATCCGCCGGCGCCT primer sequence Notl COD #1434 aa 381 HisGluProHisArgCysSTOP 5' (CACGAGCCGCACGGACGTTGA) coding sequence GTGCTCGGCGTGTCCACGATCCGCCGGCGTT primer sequence Notl COD #1435 aa 416 AspAspGlyPhelleCysSTOP 5' (GACGACGGTTTCATCTGCTGA) coding sequence CTGCTGCCAAAGGATACGATCCGCCGGCGGCTG primer sequence Notl COD #1480 aa 462 CysAspSerGlyCysValAspSTOP 5 » (TGTGACTCCGGCAAGGTGGACTGA) coding sequence ACACTGAGGCCGTTCCACCTGACTCTTAAGCT primer sequence EcoRI Table 3 contd. COD #1479 aa 459 GlyThrAspCysAspSerSTOP 5' (GGCACCGACTGTGACTCCTGA) coding sequence CCGTGGCTGACACTGAGGACTCTTAAGCAG EcoRI COD #1478 aa 216 HisTrpAlaArgGluAlaPro 5'CCATGGCCACTGGGCCAGCGAGGCGCCG primer/coding sequence Ball COD #1481 aa 490 ProAlaValGlyLeuValHisSerSTOP 5' (CCGGCCGTGGGGCTCGTGCATTCGTGA) coding sequence GGCCGGCACCCCGAGCACGTAAGCACTCGCCGGCGGTA primer seq. Notl Oligomer primers for 6 EGF-like domains COD# 1289 - 3' aa 456 aa 462 ArgHisIleGlyThrAspCys (CGCCACATTGGCACCGACTGTAGATCTGGC) coding sequence GCGGTGTAACCGTGGCTGACATCTAGACCG primer sequence Bglll Oligomer primers for t-PA COD #1302 - 3 » aa 524 aa 530 IleArgAspAsnMetArgPro (ATTCGTGACAACATGCGTCCGAGATCTGGA) coding sequence TAAGCACTGTTGTACGCAGGCTCTAGACCT primer sequence Bglll ATTCGTGACAACATGCGTCCGTGATCTGGA native sequence COD #1305 - 5' aa 2 aa 11 AlaArgSerTyrGlnVallleCysArgAsp GCGGGATCCTACCAAGTGATCTGCAGAGAT primer/coding sequence BamHI GCCAGATCTTACCAAGTGATCTGCAGAGAT native sequence Table 4. Amino Acid Composition Results Amino Acid Expected Found ASX 30 28.6 GLX 30 28.7 SER 13 12.4 GLY 21 19.2 HIS 8 8.0 ARG 9 9.3 THR 10 9.9 ALA 20 19.2 PRO 22 21.5 VAL 11 9.5 MET 2 2.0 ILE 8 7.7 LEU 10 11.0 PHE 8 7.8 LYS 0 0.1 TYR 7 6.8 Table 5. Activity of Thrombomodulin Analogs Following Drying. Sample U/ml Uθ/ml % Recovery Dialyzed starting material 1661 8.88 _-_.--— PBS solub. 17277 92.4 104 PBS (NP-40) solub. 16635 88.9 100 NEM solub. 13568 72.5 82 NEM (+NP-40) solub. 17939 95.9 108 Table 6 Signal Sequences t-PA Signal Sequence -32 aa MetAspAlaMetLysArgGlyLeuCysCysValLeuLeuLeuCysGlyAlaValPhe ATGGATGCAATG7-AGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTC ACCrTACGTTACTTCTCTCCCGAGACGACACACGACGACGACACACCTCGTCAGAAG -13 aa -1 ) +1 ValSerProSerGlu GluIleHisAlaAroPheArgArgGlvAlaArq GTTTCGCCCAGCCAG|INTRON A]GAAATCCATGCCCGATTCAGAAGAGGAGCCAGA CAAAGCGGGTCGGTC CTTTAGGTACGGGCTAAGTCTTCTCCTCGGTCT +4 Ser TCC AGG Hypodermin λ Signal Sequence - pHYl MetLeuLysPheVallleLeuLeuCysSerIleAlaTyrVal COD #1198 GATCATGCTCAAGTTTGTTATTTTATTGTGCAGTATTGCCTATGTT BamHI TACGAGTTCAAACAATAAAATAACACGTCATAACGGATACAA PheGlyAlaValValProArgSerProArg TTCGGTGCCGTCGTACCAAGATCTCCCCGG AAGCCACGGCAGCATGGTTCTAGAGGGGCCCATGG COD #1199 Bglll Kpnl Hypodermin λ Signal Sequence - pHY2 MetLeuLysPheVallleLeuLeuCysSerIleAlaTyrVal COD #1482 GATCATGCTCAAGTTTGTTATTTTATTGTGCAGTATTGCCTATGTT BamHI TACGAGTTCAAACAATAAAATAACACGTCATAACGGATACAA PheGly Kpnl TTCGGCCAGGTAC AAGCCGGTC Ball COD #1483 Table 7. Transient Expression Plasmids Vector Parent TM Gene TM Analog pTHRl PPA133 aa 227-462(-) 4t/227-462a pTHR7 pTHR5 aa 227-462 (-) 4t/227-462b pTHR13 pTHR5 aa 227-462(+) 4t/227-462(+) pTHR19 pTHR5 aa 350-462 4t/350-462 pTHR20 pTHR5 aa 227-462 (-) + 4t/227-462:227-462 227-462(+) pTHR21 pTHR5 aa 227-497 4t/227-497 pTHR30 pTHR5 aa 227-421 4t/227-421 pTHR31 pTHR5 aa 350-421 4t/350-421 pTHR32 pTHR5 aa 390-462 4t/390-462 pTHR33 pTHR5 aa 350-386 4t/350-386 pTHR34 pTHR5 aa 390-421 4t/390-421 pTHR35 pTHR5 aa 427-462 4t/427-462 pTHR43 pTHR5 aa 227-343 4t/227-343 pTHR44 pTHR5 aa 227-386 4t/227-386 TM Heteroloσ pTHR4 pPA133 aa 227-462(-) 227-462:PA a pTHR6 pTHR5 aa 227-462 (-) 227-462:PA b pTHR17 pTHR5 aa 227-462 (+) PA:227-462 pTHR18 pTHR5 aa 350-462 PA:350-462 +/- indicates presence or absence of a stop codon at the 3' end of the TM gene Table 8. Vector Parent M Gene TM Analog pTHRlO pSC716 aa 227-462(+) 6h/227-462 pTHRll pSC716 aa 227-462(-) + 227-462(+) 6h/(227-462)2 pTHR22 pSC716 aa 350-462 6h/350-462 pTHR23 pSC716 aa 227-497 6h/227-497 pTHR45 pSC716 aa 350-421 6h/350-421 pTHR46 pSC716 aa 350-386 6h/350-386 pTHR47 pSC716 aa 390-421 6h/390-421 pTHR48 pSC716 aa 427-462 6h/427-462 pTMHY102 pHY2 aa 216-468 11/6 pTMHY103 pHY2 aa 216-464 11/2 TM Heterolog pTHR24 pSC716 aa 227-462(-) 6h/227-462:PA pTHR25 pSC716 aa 227-462(+) PA:227-462 +/- indicates presence or absence of a stop codon at the 3 ' end of the TM gene - M β o 6 EGF-LIKE DOMAINS OF THROMBOMODULIN FUSED TO THE C-TERMINUS OF MATURE TISSUE PLASMINOGEN ACTIVATOR cleavage sites for Mature t-PA I I t-PA aa +1 +4 GlyAlaArgSerTyrGlnVallleCys GGAGCCAGATCCTACCAAGTGATCTGC CCTCGGTCTAGGATGGTTCACTAGACG Bgl ll/Bam HI 10 ArgAspGluLysThrGlnMetlleTyrGlnGlnHisGlnSerTrpLeuArgProValLeu AGAGATGAAAAAACGCAGATGATATACCAGCAACATCAGTCATGGCTGCGCCCTGTGCTC TCTCTACTTTTTTGCGTCTACTATATGGTCGTTGTAGTCAGTACCGACGCGGGACACGAG 30 ArgSerAsnArgValGluTyrCysTrpCysAsnSerGlyArgAlaGlnCysHisSerVal AGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCAGGGCACAGTGCCACTCAGTG TCTTCGTTGGCCCACCTTATAACGACCACGTTGTCACCGTCCCGTGTCACGGTGAGTCAC 50 ProValLysSerCysSerGluProArgCysPheAsnGlyGlyThrCysGlnGlnAlaLeu CCTGTCAAAAGTTGCAGCGAGCCAAGGTGTTTCAACGGGGGCACCTGCCAGCAGGCCCTG GGACAGTTTTCAACGTCGCTCGGTTCCACAAAGTTGCCCCCGTGGACGGTCGTCCGGGAC 70 TyrPheSerAspPheValCysGlnCysProGluGlyPheAlaGlyLysCysCysGluIle TACTTCTCAGATTTCGTGTGCCAGTGCCCCGAAGGATTTGCTGGGAAGTGCTGTGAAATA ATGAAGAGTCTAAAGCACACGGTCACGGGGCTTCCTAAACGACCCTTCACGACACTTTAT 90 AspThrArgAlaThrCysTyrGluAspGlnGlylleSerTyrArgGlyThrTrpSerThr GATACCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTACAGGGGCACGTGGAGCACA CTATGGTCCCGGTGCACGATGCTCCTGGTCCCGTAGTCGATGTCCCCGTGCACCTCGTGT 10 Table 9 Cαntd. AlaGluSerGlyAlaGluCysThrAsnTrpAsnSerSerAlaLeuAlaGlnLysProTyr GCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCGCGTTGGCCCAGAAGCCCTAC CGCCTCTCACCGCGGCTCACGTGGTTGACCTTGTCGTCGCGCAACCGGGTCTTCGGGATG 130 SerGlyArgArgProAspAlalleArgLeuGlyLeuGlyAsnHisAsnTyrCysArgAsn AGCGGGCGGAGGCCAGACGCCATCAGGCTGGGCCTGGGGAACCACAACTACTGCAGAAAC TCGCCCGCCTCCGGTCTGCGGTAGTCCGACCCGGACCCCTTGGTGTTGATGACGTCTTTG 150 ProAspArgAspSerLysProTrpCysTyrValPheLysAlaGlyLysTyrSerSerGlu CCAGATCGAGACTCAAAGCCCTGGTGCTACGTCTTTAAGGCGGGGAAGTACAGCTCAGAG GGTCTAGCTCTGAGTTTCGGGACCACGATGCAGAAATTCCGCCCCTTCATGTCGAGTCTC 170 PheCysSerThrProAlaCysSerGluGlyAsnSerAspCysTyrPheGlyAsnGlySer TTCTGCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGCTACTTTGGGAATGGGTCA AAGACGTCGTGGGGACGGACGAGACTCCCTTTGTCACTGACGATGAAACCCTTACCCAGT 190 AlaTyrArgGlyThrGlnSerLeuThrGluSerGlyAlaSerCysLeuProTrpAsnSer GCCTACCGTGGCACGCAGAGCCTCACCGAGTCGGGTGCCTCCTGCCTCCCGTGGAATTCC CGGATGGCACCGTGCGTCTCGGAGTGGCTCAGCCCACGGAGGACGGAGGGCACCTTAAGG 210 MetlleLeuIleGlyLysValTyrThrAlaGlnAsnProSerAlaGlnAlaLeuGlyLeu ATGATCCTGATAGGCAAGGTTTACACAGCACAGAACCCCAGTGCCCAGGCACTGGGCCTG TACTAGGACTATCCGTTCCAAATGTGTCGTGTCTTGGGGTCACGGGTCCGTGACCCGGAC 230 GlyLysHisAsnTyrCysArgAsnProAspGlyAspAlaLysProTrpCysHisValLeu GGCAAACATAATTACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTG CCGTTTGTATTAATGACGGCCTTAGGACTACCCCTACGGTTCGGGACCACGGTGCACGAC 250 LysAsnArgArgLeuThrTrpGluTyrCysAspValProSerCysSerThrCysGlyLeu AAGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCTGCGGCCTG TTCTTGGCGTCCGACTGCACCCTCATGACACTACACGGGAGGACGAGGTGGACGCCGGAC 270 Table 9 Cαntd. ArgGlnTyrSerGlnProGlnPheArglleLysGlyGlyLeuPheAlaAspIleAlaSer AGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGCTCTTCGCCGACATCGCCTCC TCTGTCATGTCGGTCGGAGTCAAAGCGTAGTTTCCTCCCGAGAAGCGGCTGTAGCGGAGG 290 HisProTrpGlnAlaAlallePheAlaLysHisArgArgSerProGlyGluArgPheLeu CACCCCTGGCAGGCTGCCATCTTTGCCAAGCACAGGAGGTCGCCCGGAGAGCGGTTCCTG GTGGGGACCGTCCGACGGTAGAAACGGTTCGTGTCCTCCAGCGGGCCTCTCGCCAAGGAC 310 CysGlyGlylleLeuIleSerSerCysTrpIleLeuSerAlaAlaHisCysPheGlnGlu TGCGGGGGCATACTCATCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAG ACGCCCCCGTATGAGTAGTCGAGGACGACCTAAGAGAGACGGCGGGTGACGAAGGTCCTC 330 ArgPheProProHisHisLeuThrVallleLeuGlyArgThrTyrArgValValProGly AGGTTTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGGTCCCTGGC TCCAAAGGCGGGGTGGTGGACTGCCACTAGAACCCGTCTTGTATGGCCCACCAGGGACCG 350 GluGluGluGlnLysPheGluValGluLysTyrlleValHisLysGluPheAspAspAsp GAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCCATAAGGAATTCGATGATGAC CTCCTCCTCGTCTTTAAACTTCAGCTTTTTATGTAACAGGTATTCCTTAAGCTACTACTG 370 ThrTyrAspAsnAspIleAlaLeuLeuGlnLeuLysSerAspSerSerArgCysAlaGln ACTTACGACAATGACATTGCGCTGCTGCAGCTGAAATCGGATTCGTCCCGCTGTGCCCAG TGAATGCTGTTACTGTAACGCGACGACGTCGACTTTAGCCTAAGCAGGGCGACACGGGTC 390 GluSerSerValValArgThrValCysLeuProProAlaAspLeuGlnLeuProAspTrp GAGAGCAGCGTGGTCCGCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGG CTCTCGTCGCACCAGGCGTGACACACGGAAGGGGGCCGCCTGGACGTCGACGGCCTGACC 410 ThrGluCysGluLeuSerGlyTyrGlyLysHisGluAlaLeuSerProPheTyrSerGlu ACGGAGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCTATTCGGAG TGCCTCACACTCGAGAGGCCGATGCCGTTCGTACTCCGGAACAGAGGAAAGATAAGCCTC ._,. Oable 9 Cαntd. 430 ArgLeuLysGluAlaHisValArgLeuTyrProSerSerArgCysThrSerGlnHisLeu CGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCCGCTGCACATCACAACATTTA GCCGACTTCCTCCGAGTACAGTCTGACATGGGTAGGTCGGCGACGTGTAGTGTTGTAAAT 450 LeuAsnArgThrValThrAspAsnMetLeuCysAlaGlyAspThrArgSerGlyGlyPro CTTAACAGAACAGTCACCGACAACATGCTGTGTGCTGGAGACACTCGGAGCGGCGGGCCC GAATTGTCTTGTCAGTGGCTGTTGTACGACACACGACCTCTGTGAGCCTCGCCGCCCGGG 470 GlnAlaAsnLeuHisAspAlaCysGlnGlyAspSerGlyGlyProLeuValCysLeuAsn CAGGCAAACTTGCACGACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAAC GTCCGTTTGAACGTGCTGCGGACGGTCCCGCTAAGCCCTCCGGGGGACCACACAGACTTG 490 AspGlyArgMetThrLeuValGlyllelleSerTrpGlyLeuGlyCysGlyGlnLysAsp GATGGCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGACAGAAGGAT CTACCGGCGTACTGAAACCACCCGTAGTAGTCGACCCCGGACCCGACACCTGTCTTCCTA 510 ValProGlyValTyrThrLysValThrAsnTyrLeuAspTrpIleArgAspAsnMetArg GTCCCGGGTGTGTACACAAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGA CAGGGCCCACACATGTGTTTCCAATGGTTGATGGATCTGACCTAAGCACTGTTGTACGCT t-PA TM aa 530 aa 227 ProARGSERCysSerValGluAsnGlyGlyCysGluHisAlaCysAsnAlalleProGly CCGAGATCCTGCAGCGTGGAGAACGGCGGCTGCGAGCACGCGTGCAATGCGATCCCTGGG GGCTCTAGGACGTCGCACCTCTTGCCGCCGACGCTCGTGCGCACGTTACGCTAGGGACCC Bgl ll/Bam HI TM 244 AlaProArgCysGlnCysProAlaGlyAlaAlaLeuGlnAlaAspGlyArgSerCysThr GCTCCCCGCTGCCAGTGCCCAGCCGGCGCCGCCCTGCAGGCAGACGGGCGCTCCTGCACC CGAGGGGCGACGGTCACGGGTCGGCCGCGGCGGGACGTCCGTCTGCCCGCGAGGACGTGG 264 AlaSerAlaThrGlnSerCysAsnAspLeuCysGluHisPheCysValProAsnProAsp GCATCCGCGACGCAGTCCTGCAACGACCTCTGCGAGCACTTCTGCGTTCCCAACCCCGAC CGTAGGCGCTGCGTCAGGACGTTGCTGGAGACGCTCGTGAAGACGCAAGGGTTGGGGCTG 284 Table 9 Cont . GlnProGlySerTyrSerCysMetCysGluThrGlyTyrArgLeuAlaAlaAspGlnHis CAGCCGGGCTCCTACTCGTGCATGTGCGAGACCGGCTACCGGCTGGCGGCCGACCAACAC GTCGGCCCGAGGATGAGCACGTACACGCTCTGGCCGATGGCCGACCGCCGGCTGGTTGTG 304 ArgCysGluAspValAspAspCysIleLeuGluProSerProCysProGlnArgCysVal CGGTGCGAGGACGTGGATGACTGCATACTGGAGCCCAGTCCGTGTCCGCAGCGCTGTGTC GCCACGCTCCTGCACCTACTGACGTATGACCTCGGGTCAGGCACAGGCGTCGCGACACAG 324 AsnThrGlnGlyGlyPheGluCysHisCysTyrProAsnTyrAspLeuValAspGlyGlu AACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTGGACGGCGAG TTGTGTGTCCCACCGAAGCTCACGGTGACGATGGGATTGATGCTGGACCACCTGCCGCTC 344 CysValGluProValAspProCysPheArgAlaAsnCysGluTyrGlnCysGlnProLeu TGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCAGTGCCAGCCCCTG ACACACCTCGGGCACCTGGGCACGAAGTCTCGGTTGACGCTCATGGTCACGGTCGGGGAC 364 AsnGlnThrSerTyrLeuCysValCysAlaGluGlyPheAlaProIleProHisGluPro AACCAAACTAGCTACCTCTGCGTCTGCGCCGAGGGCTTCGCGCCCATTCCCCACGAGCCG TTGGTTTGATCGATGGAGACGCAGACGCGGCTCCCGAAGCGCGGGTAAGGGGTGCTCGGC 384 HisArgCysGlnMetPheCysAsnGlnThrAlaCysProAlaAspCysAspProAsnThr CACAGGTGCCAGATGTTTTGCAACCAGACTGCCTGTCCAGCCGACTGCGACCCCAACACC GTGTCCACGGTCTACAAAACGTTGGTCTGACGGACAGGTCGGCTGACGCTGGGGTTGTGG 404 GlnAlaSerCysGluCysProGluGlyTyrlleLeuAspAspGlyPhelleCysThrAsp CAGGCTAGCTGTGAGTGCCCTGAAGGCTACATCCTGGACGACGGTTTCATCTGCACGGAC GTCCGATCGACACTCACGGGACTTCCGATGTAGGACCTGCTGCCAAAGTAGACGTGCCTG 424 IleAspGluCysGluAsnGlyGlyPheCysSerGlyValCysHisAsnLeuProGlyThr ATCGACGAGTGCGAAAACGGCGGCTTCTGCTCCGGGGTGTGCCACAACCTCCCCGGTACC TAGCTGCTCACGCTTTTGCCGCCGAAGACGAGGCCCCACACGGTGTTGGAGGGGCCATGG Table 9 Contd. TM 444 aa 462 PheGluCysIleCysGlyProAspSerAlaLeuAlaArgHisIleGlyThr GCCCGCCACATTGGCACCGACTGTTTCGAGTGCATCTGCGGGCCCGACTCG CGGGCGGTGTAACCGTGGCTGACAAAGCTCACGTAGACGCCCGGGCTGAGC AspCysOP GCCCTTTGAGGATCT CGGGAAACTCCTAGA Bam Hl Bgl II

Claims: WHAT IS CLAIMED IS: 1. A sequence of nucleic acid bases encoding a peptide selected from the group consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 2. A polynucleotide sequence encoding the peptides of claim 1 where the sequence is selected from the group consisting of: a) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X- GACTCCGGCAAGGTGGAC; b) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X- GACTCC; C) GGAGCCAGATCC-Z; and d) GCCGTCGTACCAAGATCC-Z; where X represents the nucleic acid bases numbered 879 to 1586, and Z represents the nucleic acid bases selected from the group numbered 879 to 1586, 1251 to 1586 or 879 to 1690 wherein all numbers refer to bases as provided in Table 2. 3. A polynucleotide sequence according to claim 2 having a sequence of CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X-GACTCCGGCAAGGTGGAC. 4. A polynucleotide sequence according to claim 2 having a sequence of CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X-GACTCC. 5. A polynucleotide sequence according to claim .2 having a sequence of GGAGCCAGATCC-Z. 6. A polynucleotide sequence according to claim 2 having a sequence of GCCGTCGTACCAAGATCC-Z. 7. A recombinant vector comprising a sequence of nucleic acids encoding a peptide selected from the group consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 8. A vector of claim 7 where the sequence of nucleic acid is selected from the group consisting of: a) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X- GACTCCGGCAAGGTGGAC; b) CACTGGGCCAGGGAGGCGCCGGGCGCTTGGGAC-X-GACTCC; c) GGAGCCAGATCC-Z; and d) GCCGTCGTACCAAGATCC-Z; where X represents the nucleic acid bases numbered 879 to 1586, and Z represents the nucleic acid bases selected from the group numbered 879 to 1586, 1251 to 1586 or 879 to 1690 wherein all numbers refer to bases as provided in Table 2. 9. A substantially pure protein composition selected from a peptide of the group consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c ) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 10. A peptide of claim 9 having an amino acid sequence of: his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp. 11. A peptide of claim 9 having an amino acid sequence of his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser. 12. A peptide of claim 9 having an amino acid sequence of gly.ala.arg.ser. - Q. 13. A peptide of claim 9 having an amino acid sequence of ala.val.val.pro.arg.ser - Q. 14. A composition comprising a peptide of claim 9 wherein said peptide is a species of said peptide which exhibits about the expected molecular weight for said peptide under non-reducing chromatography. 15. A composition of claim 9, wherein the peptide is chemically linked to a fibrinolytic enzyme. 16. A composition of claim 15, wherein said chemical linkage arises from a covalent bond between the enzyme and an amino acid residue within the peptide. 17. A composition of claim 15, wherein said enzyme is a streptokinase molecule bound to a plasminogen molecule and wherein the peptide is bound to the active site of the plasminogen molecule. 18. A composition of claim 15 wherein the fibrinolytic enzyme is t-PA. 19. A composition of claim 9 wherein the composition is in a dry and salt-free form. 20. A pharmaceutical composition having antithrombotic activity comprising a sterile preparation of a unit dose of thrombomodulin-like protein free of contaminants of human origin and having an amino acid sequence selected from the group consisting of: a) hiε.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 21. A pharmaceutical composition of claim 20 having an amino acid sequence of his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp. 22. A pharmaceutical composition of claim 20 having an amino acid sequence of his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser. 23. A pharmaceutical composition of claim 20 having an amino acid sequence of gly.ala.arg.ser. - Q. 24. A pharmaceutical composition of claim 20 having an amino acid sequence of ala.val.val.pro.arg.ser. - Q. 25. A method of controlling thrombotic activity in a mammal by administering an effective amount of a sterile aqueous solution of a thrombomodulin-like protein, free of contaminants of human origin and having an amino acid sequence selected from the group consisting of: a) hiε.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 26. A composition comprising a biocompatible polymer having a surface wherein the surface has bound thereto peptides selected from the group consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.aεp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 27. A method for inhibiting blood clotting induced by polymers implanted into a living mammal, said method comprises the bonding of a thrombomodulin- like protein to the polymer and implanting the polymer into the mammal, said protein is selected from the group of peptides consisting of: a) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser.gly.lys.val.asp; b) his.trp.ala.arg.glu.ala.pro.gly.ala.trp.asp. - Y - asp.ser; c) gly.ala.arg.ser. - Q; and d) ala.val.val.pro.arg.ser. - Q; wherein Y is the sequence of amino acids ranging from amino acid 227 to amino acid 462; and Q is the sequence of amino acids selected from the group consisting of amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 and amino acid 227 to amino acid 497 wherein all numbers refer to amino acids as provided in Table 2. 28. An isolated DNA fragment containing a DNA sequence coding for multi-functional soluble human thrombomodulin (TM) analog, said analog consisting essentially of amino acid residues 350 - 462 of native TM and a targeting component. 29. An isolated DNA fragment encoding a bifunctional TM analog according to Claim 28, consisting essentially of amino acid residues: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 30. A DNA fragment according to Claim 28 wherein said targeting component comprises a polypeptide sequence which imparts fibrinolytic activity to the bifunctional TM analog. 31. An isolated DNA fragment according to Claim 28, wherein said targeting component comprises amino acid residues 4 - 530 of human tissue plasminogen activator as depicted in Table 9. 32. A DNA sequence according to Claim 28 operably linked to an expression control sequence. 33. A cell transfected with a DNA sequence according to Claim 32. 34. A method of producing a bifunctional soluble TM analog comprising culturing a host transformed with at least one recombinant DNA segment having an expression control sequence operably linked to a DNA sequence coding for a fusion protein comprising: a first amino acid sequence comprising a thrombomodulin fragment without a stop transfer domain; and a second amino acid sequence comprising a targeting component; wherein the bifunctional TM analog is secreted from the host. 35. A method according to Claim 34 wherein said first amino acid sequence is selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 36. A method according to Claim 35, wherein the bifunctional analog has fibrinolytic activity. 37. A method according to Claim 36, where said targeting component consists essentially of amino acids 4 -530 of human t-PA. 38. A method of suppressing coagulation in a patient comprising administering to the patient an amount of soluble TM analog effective to suppress coagulation, wherein the analog comprises TM amino acids selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 39. A method of treating a patient suffering form acute myocardial infarction caused by a thrombus, said method comprising administering to the patient an amount of bifunctional soluble TM analog effective to suppress further coagulation and lyse the thrombus, wherein the bifunctional TM analog comprises TM amino acids selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM. delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. wherein said TM amino acid sequence is fused at the C-terminus or the N-terminus to amino acids 4- 530 of human t-PA. 40. A method according to Claim 39, wherein the TM analog retains substantially the same or greater affinity for thrombin as native thrombomdulin. 41. A bifunctional TM analog capable of < binding thrombin with substantially the same affinity or greater as native thrombomodulin, said analog comprising native thrombomodulin without a functional stop transfer domain, wherein said bifunctional analog is characterized by the ability to bind fibrin and cleave plasminogen to plasmin. 42. A TM analog according to Claim 41, wherein said analog consists essentially of the amino acid sequence set forth in Drawing 9. 43. A TM analog according to Claim 41, wherein said analog consists essentially of the signal sequence of t-PA covalently attached to the amino terminus of the 6 EGF-like domain of thrombomodulin. 44. A TM analog according to Claim 41, wherein said analog consists essentially of the 6 EGF- like domain of thrombomodulin covalently attached to the amino terminus of . t-PA. 45. A process for making soluble thrombomodulin analogs, said method comprising the steps of: culturing a host cell transformed with a DNA sequence encoding the analog; and collecting analogs secreted by the host cell; wherein the analogs lack a stop transfer sequence functional in the host cell. 46. A method according to Claim 45, wherein the host cell is a eukaryotic cell. 47. An isolated DNA fragment containing a DNA sequence coding for a soluble human thrombomodulin analog, capable of being secreted by a host cell transfected with said DNA fragment, wherein said DNA sequence encodes a polypeptide, comprising amino acid resides 390-462 of native TM. 48. An isolated DNA fragment according to Claim 47 wherein said polypeptide is selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 49. A DNA sequence according to Claim 47 operably linked to an expression control sequence. 50. A cell transfected with a DNA sequence according to Claim 49. 51. A method of producing a soluble thrombomodulin (TM) analog comprising culturing a host transformed with at least one recombinant DNA segment, the segment comprising an expression control sequence operably linked to a DNA sequence coding for TM analog without a functional stop transfer domain, wherein the TM analog is secreted from the host and selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 52. A method according to Claim 51, wherein the TM analog DNA sequence comprises a signal sequence. 53. A method according to Claim 51, wherein the host is a eukaryotic cell line. 54. A method of suppressing coagulation in a patient comprising administering to the patient an amount of soluble thrombomodulin (TM) analog effective to suppress coagulation, wherein the TM analog is selected from the group consisting of: delta 1 - 389, delta 463 - 557 TM; delta 1 - 226, delta 498 - 557 TM; delta 1 - 226, delta 463 - 557 TM; and delta 1 - 349, delta 463 - 557. 55. A method according to Claim 54 wherein the TM analog retains substantially the same or increased affinity to thrombin as native thrombomoduli . 56. A soluble thrombomodulin analog capable of binding thrombin with substantially the same affinity as native thrombomodulin, said analog comprising native thrombomodulin without a functional stop transfer domain. 57. An analog according to Claim 56, wherein said analog lacks any amino acid from the stop transfer domain of native thrombomodulin. 58. A method of activating anticoagulant activities of thrombin comprising complexing the thrombin with a soluble thrombomodulin analog of Claim 56. 59. A method of treating a patient requiring anticoagulant therapy, said treatment comprising administering a therapeutically effective dose of the soluble TM analog of Claim 56. 60. A method of mitigating the risk of reocclusion in a patient recovering from myocardial infarction comprising administering a therapeutically effective dose of the soluble TM analog of Claim 56. 61. A method of prophylactically treating a patient at risk from suffering deep vein thrombus said method comprising administering to the patient a therapeutically effective dose of the soluble TM analog of Claim 56.

Inventors: GLASER CHARLES, MORSER MICHAEL JOHN, GLASER, CHARLES, MORSER, MICHAEL, JOHN
Applicants: CODON
Application year: 1990
Main classification: C12P21
IPC codes: A61K38, A61P7, C07K14, C07K19, C12N1, C12N9, C12P21, C12R1