PROTEINS AND DERIVATIVES THEREOF

Abstract: Authentic and modified human thrombomodulin produced by recombinant DNA techniques, DNA sequences coding for these molecules, pharmaceutical preparations containing such molecules and their use in human therapy and prophylaxis for the treatment and prevention of thrombic episodes.

Description: Proteins and derivatives thereof The present invention relates to authentic and modified human thrombomodulin produced by recombinant DNA techniques, DNA sequences coding for these molecules, pharmaceutical preparations containing such molecules and their use in human therapy and profylaxis for the treatment and prevention of thrombic episodes. BACKGROUND OF THE INVENTION Physiology of thrombomodulin The coagulation of blood is the classical example of chemical amplification. In coagulation a miniscule stimulus of key substances, such as platelets, factor XII and/or factor VII, can trigger off complete gelation of the blood. The amplification is mediated by a series of proteolytic activations, involving coagulations factors XII, IX, VIII, X, V and prothrombin, which leads to precipitation of fibrin in plasma and entrapment of the cellular elements. However, blood coagulation must be controlled by other interactions that counterbalance procoagulant impulses, and tune the inherently explosive cascade reaction to local physiological needs. Without these influences, initiated coagulation would run away and disseminated intravascular coagulation ensue. The endothelial cells that form the interior lining of blood vessels are one source of potent antithrombotic materials. Heparan sulfate on the endothelial surface greatly potentiates antithrombin III capable of inhibiting the clotting process at several levels . SUBSTITUT __ * l Tissue plasminogen activator, another blood fluidity regulatory principle, is released from endothelial cells after certain stimuli and initiates reactions to redissolve already formed fibrin fibers. A third example, to which this patent is devoted, is thrombomodulin. Thrombomodulin is a glycoprotein on the surface of endotheliu and certain other cells which contributes to the control of coagulation (W. Owen and C. Esmon, J.Biol.Chem. 256:5532-5535; N. Esmon et al., J.Biol.Chem. 257:859-864, 1982; see also L. Clouse and P. Comp, N. Engl.J.Med. 314:1298-1304, 1986)). It binds stoichiometrically to thrombin, the penultimate step in the coagulation cascade, and alters its proteolytic specificity. Normally, thrombin converts fibrinogen into insoluble fibrin. It also activates factors V and VIII to Va and Villa, respectively, and thereby enhances the coagulation by positive feedback. After binding to thrombomodulin a new activity becomes prominent: The thrombin-thrombo odulin complex activates Protein C. The activated Protein C (PCa) , in combination with protein S, degrades the activated cofactors Va and Villa, and thus turns coagulation off (R. arlar et al., Blood 59:1067- 1072, 1982). Activated Protein C also degrades the inhibitor of plasminogen activator (PAI) thereby facilitating fibrinolysis (V. van Hinsburgh et al., Blood 65:444-451, 1985). At the same time the interaction between thrombomodulin and thrombin apparently may cause thrombin to lose its ability to clot fibrinogen, activate factor V, and aggregate p.latelets (C. Esmon et al., J.Biol.Chem. 257:7944-7947, 1982, N. Esmon et al., J.Biol.Chem. 258:12238-12242, 1983). In short, the interaction with thrombomodulin switches thrombin from a procoagulant to an anticoagulant and fibrinolytic role. SUBSTITUTE SHEET The effect of thrombomodulin is dramatic, it accelerates the formation of PCa from 100 to 20,000 fold. Indeed, the thrombin-thrombomodulin complex is by far the most potent activator of protein C known, and probably the only or major one of physiological relevance. The negative feedback loop initiated by the thrombin-thrombomodulin complex and mediated by Protein C is of great importance in vivo. Humans with partial Protein C deficiency, presumably due to a heterozygous genetic defect, have recurrent episodes of intravascular thrombus formation (L. Clouse and P. Comp, ibid). The fate of patients with complete Protein C deficiency is even harsher. They usually die as infants from massive thrombotic events, unless treated with Protein C-rich concentrates. Thrombomodulin-initiated feedback thus appears to play an important regulatory role in the body's hemostatic mechanism. In accordance with this view, solubilized thrombomodulin has anticoagulant properties i vitro. A concentration of approximately 4 nM, kept in solution by detergent, has been reported to double the partial thromboplastin time of human plasma (S. Kurosawa and N. Aoki, Thromb.Res. 37:353-364, 1985). The thrombomodulin molecule Human thrombomodulin has been purified from placenta (H. Salem et al. , J.Biol.Chem. 259:12246-12251; S. Kurosawa and N. Aoki, _ibid. ) . Briefly, the procedures consisted of preparation of membranes, solubilization in a non-ionic detergent, and twice affinity chromatography on solid phase thrombin, whose protease activity had been SUBSTITUTE SHEET destroyed by diisopropylphosphofluridate. Both purifications included an additional step, either ion exchange chromatography or size exclusion chromatography. Thrombomodulin is an extraordinarily stable glycoprotein. Activity can be quantitatively recovered from samples that have been treated with 1% SDS, 8 M Urea, pH 2, pH 10, or even boiled for 10 minutes. However, treatment with mercaptoethanol or pepsin completely destroys activity. The molecule is very poorly soluble in the absence of detergents. Purified preparations of thrombomodulin show one dominating band in gelelectrophoresis. Biological activity can be recovered from the gels and coincide with the band. The electrophoretic mobility corresponds roughly to a molecular weight of 88-105,000 dalton, but estimates appears rather dependent on the gel system. The isoelectric point is low, approximately 4. An estimated aminoacid composition has been published (S. Kurosawa and N. Aoki, ibid. ) , but detailed characterization has been seriously hampered by paucity of material. Studies of thrombomodulin from other species largely corroborate the above description (N. Esmon et al., J.biol.Chem. 257:859-864, 1982) In addition, recent results may shed further light on the molecular properties of human thrombomodulin. A partial cDNA sequence of bovine thrombomodulin comprising the C-terminal half indicates that this portion has a certain resemblance to the LDL receptor (R. Jackman et al., Proc.Natl.Acad.Sci, USA 8_3:8834-8838, 1986). An approximately 240 amino acid region contains cysteine residues whose spacing would be consistent with the formation of six modules resembling epidermal growth factors (EGF) . There are two potential s ' ites for N-linked glycosylation in this region. It is also partly overlapped by a region of 56 amino acid rich in serine, threonine and proline residues, which in SUBSTITUTE SHEET analogy to the LDL receptor could carry O-linked sugar chains. Then follows a stretch of 24 hydrophobic amino acids probably representing a transmembrane region, and finally a 56 amino acid segment starting with multiple positively charged residues, which probably represents a short cytoplasmic domain. As described below, our results indicate that human and bovine thrombomodulin are at least partly homologous. Rabbit thrombomodulin was recently separated by ion-exchange into an acidic and a non-acidic fraction which differed in their anticoagulant properties (M.C. Bourin et al. , Proc.Natl.Acad.Sci . USA 83:5924-5928, 1986). Both fractions contained thrombin-cofactor activity, required for Protein C activation. The acidic fraction, in addition, prevented fibrinogen clotting by thrombin and accelerated thrombin neutralization by antithrombin in a heparin-like fashion. The two latter properties were abolished by the polycation Polybrene and by heparinase. These results would indicate that direct and indirect thrombin-neutralizing activities depend on secondary glycosylation of the peptide chain, while Protein C coactivator activity does not. In international patent application No. PCT JP86/00330 (WO 87/00050) mention is made of a compound isolated from human lung having thrombomodulin activity. Certain problems have till now seriously hampered the use of thrombomodulin as anticoagulant: 1) The molecule is only available in minimal quantities of limited purity from primary human material. 2) The molecule is only soluble .in the presence of detergents. These problems must be solved before thrombomodulin can be clinically useful. In addition, it would be desirable to increase the specificity of thrombomodulin by targeting the molecule to specific physiological, structures. SUBSTITUTE SHEET In this connection it is interesting to note that the presence of a soluble form of thrombomodulin has been indicated to exist (H. Ishii and P.W. Majerus, J.Clin.Invest. 7_6:2178-2181, 1985). The authors also speculate about the composition of the soluble thrombomodulin and mention the possibility that it may originate from synthesis of thrombomodulin without the membrane binding domain. These problems have now been overcome by the inventors of the present invention by use of DNA recombinant technology in expressing the gene for human thrombomodulin in suitable hosts, and by modifying said gene in order to eliminate inherent disadvantages in the naturally occurring human thrombomodulin and/or increase its utility by targetting. DEFINITIONS Prior to setting forth the invention it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereafter. Complementary DNA or cDNA: A DNA molecule or sequence which have been enzymatically synthesized from sequences present in a mRNA template. DNA Construct: A DNA molecule, or a clone of such a molecule, either single- or double-stranded, which may be isolated in partial form -from a naturally occurring gene or which has been modified to contain segments of DNA which are combined and juxtaposed in a manner which would not otherwise exist in nature. SUB S TITUTE SHEET Plasmid or Vector: A DNA construct containing genetic information which may provide for its replication when inserted into a host cell. A plasmid generally contains at least one gene sequence to be expressed in the host cell, as well as sequences encoding functions which facilitate such gene expression, including promoters and transcription initiation sites. It may be a linear or closed circular molecule. Joined: DNA sequences are said to be joined when the 5' and 3 ' ends of one sequence are attached by phosphodiester bonds to the 3' and 5' ends, respectively, of an adjacent sequence. Joining may be achieved by such methods as ligation of blunt or cohesive termini, by synthesis of joined sequences through cDNA cloning, or by removal or intervening sequences through a process of directed mutagenesis. Pre-pro region: An amino acid sequence which generally occurs at the amino termini of the precursors of certain proteins, and which is generally cleaved from the protein, at least in part, during secretion. The pre-pro region comprises, in part, sequences directing the protein into the secretory pathway of the cell. Domain or Module: A three-dimensional, self-assembling array of amino acids of a protein molecule, which contains structural elements necessary for a specific biological activity of that protein. For further definition of and further references to domains and modules with special relevance to coagulation and fibrinolysis, including fingers, growth factor regions, kringles, and vitamin K-dependent SUBSTITUTE SHEET gammacarboxylated regions see L. Banyai et al., FEBS Letters 163:37-41, 1983, and L. Patthy, Cell 43 :657-663, 1985. Thrombomodulin activity Thrombomodulin may have three separate activities. First, it binds to thrombin with high affinity. Secondly, it specifically endows the thrombin- thrombomodulin complex with the ability to activate Protein C. Thirdly, this binding may modify the reactivity of the complexed thrombin to a number of different components, including fibrinogen, platelets, and antithrombin. For the purpose of this invention we shall consider thrombomodulin activity equivalent to the first and second of these activities, and disregard the third type of activity. Consequently, thrombomodulin derivatives or generally thrombomodulins will be considered active if they possess protein C coactivator activity, regardless whether or not they modify other reactivities of the bound thrombin. Activation of Protein C Protein C is a two-chain glycoprotein with a molecular weight of approximately 57,000 dalton. One chain designated the light chain consists of an amino-terminal gammacarboxylated region, followed by two domains with homology to epidermal growth factor (EGF) . The other chain designated the heavy chain contains -a serine protease domain. Activation of protein C consists of cleavage of a single peptide bond, Arg-12 to Leu-13 in the heavy chain. It is this reaction whose catalysis by the thrombin- thrombomodulin complex can accelerate dramatically, typically between 100 and 20000 fold. Indeed, the SUBSTITUTE SHEET thrombin-thrombomodulin complex is by far the most potent activator of protein C that is known, and probably the only of physiological relevance. DESCRIPTION OF THE INVENTION In one aspect this invention relates to a group of compounds which in this specification has been designated thrombomodulins, comprising human thrombomodulin and derivatives thereof. The thrombomodulins of the invention all have certain features in common which have been alluded to above and will be specified in further detail below. It is foreseen that said thrombomodulins will be of use in the therapeutical control of coagulation. By increasing the amount of thrombomodulins in a patient it will be possible to increase the activation of Protein C, thereby potentiating the patient's anticoagulant capacity. It is anticipated that the thrombomodulin- derived anticoagulants of the invention will be superior to well known anticoagulants, such as heparin and Vitamin K antagonists. These well known anticoagulants interfere at several levels of the coagulation cascade and prevent normal coagulation mechanisms by inhibiting the synthesis or activity coagulation factors. Moreover, Vitamin K antagonists indavertently diminish the physiological anticoagulatory response of Protein C by blocking the formation of gammacarboxylated proteins indiscriminately, and thrombic episodes which typically manifest themselves as skin necrosis have occurred early in anti-Vitamin K treatment (A. Broekmans et al.., Thromb Haemost 49 :251, 1983) . SUBSTITUTE SHEET Thrombomodulins, in contrast, will reinforce natural anticoagulant and fibrinolytic mechanisms by limiting survival of PAI and activated factors Va and Villa, but leave the reservoir of coagulation factors in plasma largely intact. Spatial specificity will be high as the activity is dependent on local formation of thrombin, and systemic activity should thus be negligible. The anticoagulant activity of the thrombomodulins of the invention should not interfere directly with formation of the hemostatic plug, neither does it change the structure of the fibrin. Therefore fewer complications in the form of bleeding episodes are anticipated compared to the use of the traditional anticoagulant substances. Thrombomodulins will be particularly useful as anticoagulants in patients with increased bleeding risk or in patients where such a risk cannot be accepted. Such patients include those who have recently suffered from a stroke caused by a thrombus in a cerebral vessel, patients who recently have undergone surgery, or such that have a potential source of bleeding (tumor, ulcer etc). The invention will be disclosed in further detail below with reference to the drawings and the examples which in no way should be construed as limiting for the scope of the invention as defined in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS in the drawings Fig. 1 shows the sequence of a 60-mer DNA probe used for identifying human thrombomodulin cDNA clones, SUBSTITUTE SHEET Fig. 2 shows an alignment of DNA sequences with deduced amino acid sequences from bovine thrombomodulin (B) cDNA (nucleotides 850 to 1035) (Jackman- et al. , PNAS, USA 8_3: 8834-8838, 1986, Fig. 3) and from the corresponding region in human thrombomodulin (H) cDNA clone p2.1 (this invention). A strong homology (indicated by boxes) is observed in this part of the molecule, which contains the transmembrane region, Fig. 3 shows an- alignment of DNA sequences with deduced amino acid sequences from bovine thrombomodulin (B) cDNA (Jackman et al. , PNAS, USA 83_: 8834-8838, 1986, Fig. 3) and from the corresponding regions in the human thrombomodulin (H) cDNA clone p2.1 (this invention). Two regions (bovine nucleotide number 1 to 66 and 145 to 186) are compared, and identities between the two molecules are boxed, Fig. 4 shows a human thrombomodulin cDNA sequence from clone p2.1 (this invention). The sequence shows 365 bp corresponding to nucleotides 1754 to 2123 (369 bp) in the 3' untranslated part of the bovine cDNA sequence (Jackman et al., PNAS, USA 8J3: 8834-8838, 1986, Fig. 3), Fig. 5 shows a RNA blot analysis of human cell line A549 mRNA. Five micrograms of mRNA from two different preparations were separated on a 1% agarose gel, blotted to nitrocellulose, and hybridized with a nick-translated restriction fragment from plasmid p2.1. The nucleotide length of known DNAs is indicated, Fig. 6 shows the amino acid sequence of human tissue plasminogen activator with selected domains marked A, B, and C, SUBSTITUTE SHEET Fig. 7 shows the DNA sequence of the 3640 bp insert in p2.1. The amino acid sequence of human thrombomodulin, with its signal peptide at the amino terminus, is shown with all the amino acid residues given by their one-letter abbreviations, Fig. 8 shows the sequence of the BamHI insert of plasmid pBoel743-2-9-8. Regions constructed from synthetic oligonucleotides are underlined with horizontal arrows. The amino acid sequence of the encoded human tPA signal peptide and the human thrombomodulin mutant is given by the one-letter code for all amino acid residues. The position of some restriction endonuclease recognition sites are indicated, Fig. 9 shows the construction of the mammalian expression vector pBoel-TMl, and Fig. 10 shows the construction of the mammalian expression vector pBoel-TM2. DETAILED DESCRIPTION OF THE INVENTION In one of its main aspects this invention relates to a group of compounds having thrombomodulin activity as defined by a high affinity binding to thrombin, and the capacity of endowing a complex between such a compound and thrombin with the ability to activate Protein C, and comprising from the C-terminal part of the molecule two or more of the following structural elements: a) a short cytoplasmatic domain of approximately 56 amino acids, SUBSTITUTE SHEET b) a transmembrane region of approximately 24 amino acids, c) a region rich in serine, threonine and proline residues, d) a domain comprising at least two EGF domains, and e) an N-terminal where one or more of the elements a), b), and/or c) may be omitted or replaced by affinity imparting entities or entities enhancing the solubility in physiological solutions, or physiologically compatible derivatives thereof. In this aspect the present invention describes molecules with thrombomodulin activity, which have been imparted improved solubility characteristics, relative to authentic human thrombomodulin in physiological solutions. Other molecules of the invention have been imparted affinity for specific tissue structures occurring in vivo, or lend themselves to conjugation to surfaces or other molecules or entities. Such modified thrombomodulins contain part of the amino acid sequences of human thrombomodulin, but the C-terminal portion, including part or all of the hydrophobic membrane spanning region has been deleted or replaced by affinity imparting entities. One preferred site to terminate the human thrombomodulin sequence is at the start of the hydrophobic, membrane-spanning region, corresponding to His-12 to Gly-14 (bases 883-891) in figure 2 (corresponding to His-514 to Gly-516 (bases 1670-1678) in fig. 7). A second preferred site to terminate the human thrombomodulin sequence is at the C-terminal of the sequence of growth factor domains. SUBSTITUTE SHEET Further preferred embodiments comprise molecules containing only regions e) and d) where the number of EGF domains is equal to or greater than four. This indicates further preferred embodiments wherein the human thrombomodulin sequence is terminated at the N-terminal of any of the two first growth factor domains, and C-terminally at the start of the hydrophobic, membrane spanning region, or the C-terminal of any of growth factor domains 4, 5, or 6. The modified thrombomodulins may at the same time be fusion proteins, in that they have been provided with new desirable heterologous protein domains as N- or C-terminal extensions, said domains having desirable affinity to specific physiological structures, i.e. fibrin clots, membrane surfaces, receptor molecules, or extracellular matrix components. One preferred site to fuse the human thrombomodulin sequence to a suitable heterologous domain is at the start of the membrane spanning region, corresponding to His-12 to Gly-14 in figure 2. A second preferred site to fuse the human thrombomodulin to a suitable heterologous domain is at the C-terminal of the growth factor module sequence. Further preferred sites to fuse the human thrombomodulin to a suitable heterologous domain are at the N-terminal of any of the growth factor domains 1, 2, or 3, or at the C-terminal of any of the growth factor domains 4, 5, or 6. Domains with specific affinity for physiological structures, which domains, are suitable as fusion partners encompass growth factor modules, kringles, finger-modules, vitamin K-dependent calcium-binding gammacarboxylated regions, and antibody-derived, antigen-recognizing structures. M U , - f ώ=_.S~--~ ϊ~r - τ * ~ VT • - -- ES ' Preferred heterologous domains with affinity for physiological structures encompass the finger modules of tissue plasminogen activator and fibronectin, the growth factor modules of urokinase, tissue plasminogen activator, and protein C, the kringle module of tissue plasminogen activator and the first, fourth and fifth kringle module of plasminogen. In one specific embodiment of the present invention the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin, up to Ser-13 of figure 2, C-terminally extended to include the finger domain of tissue plasminogen activator (region A in fig. 6, encompassing amino acids 4 to 50). This would endow the molecule with affinity to fibrin. In a second specific embodiment of the present invention the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to Ser-13 of figure 2, C-terminally extended to include the second kringle domain (region B in fig. 6, encompassing amino acids 176 to 263) of tissue plasminogen activator. This would also endow the molecule with affinity to fibrin. In a third specific embodiment of the present invention the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to Ser-13 of figure 2, C-terminally extended to include the growth factor module of tissue plasminogen activator (region C in fig. 6, encompassing amino acid 50 to 87). In a fourth specific embodiment of the present invention the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to but not including the first cystein residue of its last growth factor module, C-terminally extended to include the growth factor module of tissue plasminogen activator starting at the first cystein residue in this module (amino acid 51 in figure 6) . TITUTE SHEET SUBS In a fifth specific embodiment of the invention the modified thrombomodulin consists of the four carboxyterminal epidermal growth factor domains together with the extracellular O-glycosylation rich domain N- terminally joined to the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence (fig. 8). A sixth specific embodiment corresponds to the fifth embodiment above, but excluding the extracellular O-glycosylation rich domain. Alternatively, the molecules may contain a C- teri inal extension including a free cysteine residue, or one or more lysine residues to facilitate covalent conjugation in vitro or simply a highly charged region, rich in lysine and arginine, or glutamate and aspartate, so as to mediate noncovalent adhesion to surfaces. In further embodiments of the invention the modified thrombomodulins may.consist of the N-terminal sequences of human thrombomodulin up to the cut off sites mentioned in the preceding paragraphs which N-terminal sequences through a spacer molecule are conjugated to affinity providing entities such as antibodies or fragments thereof. For a description of suitable spacer molecules and techniques see Tijssen "Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam, Holland, 1985, or Danish Patent Application No. 1107/87, which is hereby incorporated by reference. In a second main aspect the present invention provides for means to produce authentic as well as the aforementioned modified human thrombomodulins by expression of a cloned nucleic acid construct encoding all or parts of the complete human thrombomodulin molecule in a suitable cell. The thrombomodulin or relevant parts *$&& S thereof may be expressed alone or as fusions with other domains so as to facilitate production, or provide desired extra domains as expounded above. In one preferred embodiment the authentic or modified thrombomodulins are encoded in a cloned nucleic acid construct in a form including the natural regions of the human thrombomodulin gene which regions are responsible for the expression of such amino acids that are actively involved in the export of the natural protein from the cell, and which amino acids eventually completely or at least in part are cleaved from the protein. In a second preferred embodiment the authentic or modified thrombomodulins are encoded in a cloned nucleic acid construct in a pre-pro form including the pre-pro region of tissue plasminogen activator. In a third main aspect the present invention describes pharmaceutical compositions containing authentic or modified thrombomodulins, useful in the treatment of patients with a view to prevent or revert a thrombic condition. Due to the present invention it is now made possible to produce authentic human thrombomodulin as well as modified thrombomodulins through the use of recombinant DNA tehcnology using cDNA or genomic clones as starting material. Techniques for determining the complete sequence of DNA clones and to obtain new independent, but homologous clones once a partial sequence has been determined are well known in the art and described in publications such as Maniatis et al. "Molecular Cloning, A Laboratory Manual" Cold Spring Harbour Press, New York, N.Y., USA, which is hereby incorporated by reference. SUBSTITUTE SHEET The construction of DNA sequences encoding preferred terminated human thrombomodulins and of DNA sequences encoding fusions between parts of human thrombomodulins and preferred functional domains from other proteins are best carried out after the introduction of specific restriction enzyme recognition sites at specific points in the human thrombomodulin cDNA and in the cDNAs of the preferred domain-donor molecules. Introduction of restriction enzyme recognition sites at specific points in a DNA molecule can advantageously be obtained with the use of one of several well known, highly efficiency procedures for oligodeoxyribonucleotide directed site-specific mutagenesis (e.g. Y. Morinaga et al., BIO/TECHNOLOGY 2: 636-639, 1984). Generation of C- terminally deleted modified human thrombomodulins can advantageously be obtained through the use of oligonucleotide directed site-specific mutagenesis. One such preferred modified human thrombomodulin terminating at the start of the hydrophobic, membrane-spanning region, can be constructed by the specific introduction of a stop-codon at the position of the existing Gly-codon GGC (at base numbers 889 to 891 in fig. 2). In this same mutagenesis experiment an oligonucleotide could be used that also introduced a convenient restriction enzyme recognition site just 3' to the introduced stop-codon hereby facilitating further construction work with this shortened version of human thrombomodulin cDNA. The different parts of the human thrombomodulin cDNA and the different parts of other cDNAs from which specific domains are to be recruited can most conveniently be subcloned in small DNA vectors (such as pUCl9 , pBR322 and pGEM3) before mutagenesis. After appropriate mutagenesis to introduce new restriction sites at joining-positions, the cDNAs should be sequenced to confirm the mutated genotype. The mutated fragments can then be joined directly together by UBSTiTUTE SH T S ligation reactions in expression vectors or joined together through the use of short double stranded synthetic oligonucleotide adaptors. All these manipulations involve procedures well known in the art (T. Maniatis et al. , "Molecular cloning, A laboratory manual", Cold Spring Harbor Press, New York, - NY, USA, 1982) like ligation of DNA fragments and their cloning in different vectors for either further constructions or expression of the encoded mutated and fused proteins. Various host cells may be used to produce the proteins including mammalian cells, yeast, fungi and bacteria. However, cultured mammalian cells are preferred. One particularly preferred cell line is the BHK cell line tk-tsl3 (Waechter and Baserga, Proc .Natl.Acad.Sci. USA 79: 1106-1110, 1982). Methods for expressing cloned genes in each of these types of host are known in the art. For expression of modified thrombomodulins in cultured mammalian cells, expression vectors containing cloned thrombomodulin sequences are introduced into the cells by appropriate transfection techniques, such as calcium phosphate-mediated transfection (Graham and Van der Eb, Virology 52: 456-467, 1973; as modified by Wigler et al., Proc.Natl.Acad.Sci. , USA 77: 3567-3570, 1980). A DNA-calcium phosphate precipitate is formed, and this precipitate is applied to the cells. A portion of the cells take up the DNA and maintain it inside the cell for several days. A small fraction of the cells integrate the DNA into the genome of the host cell. These integrants are identified by cotransfection with a gene that confers a selectable phenotype (a selectable marker). A preferred selectable marker is the mouse dihydrofolate reductase (DHFR) gene, which imparts cellular resistance to the drug methotrexate (MTX) . After the host cells have taken up the Sl/B , © "* - DNA, drug selection is applied to select for a population of cells that are expressing the selectable marker in a stable fashion. Thrombomodulin active compounds produced by the transfected cells may be purified from the cell culture media by adsorption to an ion-exchange column or affinity chromatography. One preferred affinity column contains inactivated, immobilized thrombin (H. Salem et al. , J.Biol.Chem. 259: 12246-12251, 1984). Another technique which will be of advantage is by using monoclonal antibodies or fragments thereof directed against specific antigenic determinants on the desired thrombomodulin. These can be used as affinity reagents conjugated to a solid phase to provide efficient column purification. The raising of monoclonal antibodies, their conjugation to column matrices and the use of antibody affinity columns for isolation and purification are well known in the art. EXAMPLE 1 Preparation of messenger RNA from the human cell line A549 The human cell line A549 (American Type Culture Collection CCL 185) which was isolated from lung carcinomatous tissue (Giard et al. (1972) J. Natl. Cancer Inst. 51.:1417-1423 ) has been shown to express about 10,000 molecules of thrombomodulin per cell (ref) . A549 was used as a source for mRNA preparation. A549 was grown to a total cell number of 9.4 x 10 in RPMI 1640 containing 10% fetal calf serum and antibiotics. Total RNA was isolated by the quanidinium thiocyanate method (Chirgwin et al. (1979) Biochemistry 113:5293-5299) and purified by CsCl gradient centrifugation. A total of 950 μg RNA was obtained, and S UBSTITUTE SHEΞT mRNA was isolated by use of an oligo(dT)-cellulose column (Aviv & Leder (1972) PNAS 6_9:1408-1412 ) . Sixty-one micrograms of mRNA were obtained from 750 μg total RNA (one cycle) . After ethanol precipitaton, this preparation of mRNA was resuspended in 10 mM Tris HCL pH 7.5, 0.1 mM EDTA-Na_ at a final concentration of 1 μg/μl and stored at -80°C for subsequent use. The mRNA prepared as described was used for construction of a cDNA library and mRNA blot analysis. Construction of cDNA library from A549 mRNA A cDNA library was constructed by the method described by Okayama & Berg (Mol. Cell. Biol. 2_ :161-170 (1982); Mol. Cell. Biol. :28 °- 289 (1983)). E. coli K12 (MC1061) (Casadaban & Cohen C.J. Mol. Biol. 138:179-207) was used for transformation. MC1061 were grown in L-broth at 37°C to ODg 6Q = 0.5. Twenty ml were centrifuged, and the cell pellet was resuspended in 7 ml of ice-cold sterile 0.1 M CaCl~, incubated on ice for 30 minutes, centrifuged briefly, and finally kept in the cold room overnight. Ninety-five microliters of tranformation- competent E. coli MC1061 were added per 10 μl of cDNA preparation. The mixture was incubated on ice for 30 minutes, heat-shocked at 43.5°C for 45 seconds, and finally, after addition of L-broth, incubated at 37°C for 30 minutes. After resuspension, the cells were plated onto L-broth plates containing ampicillin (50 μg/ml) and grown for 8 hours at 37°C. A to_tal of 2.9x10 individual colonies could be obtained from this library. Screening of the A549 library for cDNA clones coding for human thrombomodulin. SUBSTI U sw«rττ 4 4x10 individual colonies were screened by standard colony hybridization technique using nitrocellulose filter (Maniatis et al., supra). A 60-mer oligonucleotide shown in figure 1 corresponding to the bovine thrombomodulin sequence covering nucleotides 532 to 591 as reported by Jackman et al. (1986) PNAS 83:8834- 8838) was synthesized (DNA synthesizer from Applied Biosystems, USA), labeled with 32P (using T. polynucleotide kinase and γ- 32P-ATP) and used for the screening. The hybridization solution contained 6xSSC, 5xDenhardt's solution, 0.05% SDS (Maniatis et al. ) and 10 cpm/ml. After low stringency wash, four colonies were selected for further analysis. Plasmid preparations were prepared, subjected to Hind III digestion, electrophoresed in 1% agarose, blotted onto nitrocellulse filters, and hybridized with the above 60-mer. One of the plasmids designated p2.1 gave a strong hybridization signal and was selected for further characterization. Characterization of p2.1. p2_l had a cDNA insert of approximately 3.8 kb. , and within this insert. partial DNA sequences (Maxam and Gilbert, Methods Enzymol. 65:499-560, 1980. Sanger et al., PNAS, USA 74:5463-5467, 1977) were obtained to further characterize and identify the clone. Fig. 2 and 3 show sequence homology between selected regions of the bovine (Jackman et al., PNAS, USA 83:8834-8838, 1986, Fig. 3) and the human cDNA as obtained from p2.1. Regions with amino acid identities are boxed-. From Fig. 2 it can be noted that within the 24 amino acid residues long putative transmembrane region in the bovine sequence (from Gly-14 (889) to Leu-37(960), only 2 amino acid substitutions are identified. SUBSTITUTE SHEET Fig. 4 shows a sequence from a 3' untranslated region of p2.1. In this part of the molecule well- conserved regions between the bovine and the human sequences can also be identified. In order to estimate the completeness of this clone the following experiment was performed. Blotting analysis of human thrombomodulin mRNA Five micrograms of human cell line A549 mRNA were separated together with denatured 32P-labelled DNA molecular weight markers (Phage lambda DNA digested with Hind III) by electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde (Maniatis et al. , "Molecular cloning: A laboratory Manual. Cold Spring Harbor laboratory Press " New York, N.Y., USA, 1982) . After electrophoresis, the gel was rinsed 2 x 10 min in 1 x SSC (1 x SSC is 0,150 M NaCl and 0.015 m sodium citrate (pH 7)) before blotting of the mRNA to a Gene Screen (TM) ' hybridization transfer membrane overnight in 10 x SSC. The blotted mRNA was baked to the membrane for 2 h at 80°C. Prehybridization (5h) and hybridization (20h) were done in 50% formamide; 0.1% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone; 5 x SSC, 1% SDS, and 0,5 mg/ml of heat denatured salmon sperm DNA at 42°C. As a hybridization probe a nick-translated p2.1 cDNA restriction fragment was used. After hybridization, the mRNA blot was washed successively in 2 x SSC for 2 x 5 min at room temperature, 2 x SSC, 0,5% SDS for 2 x 30 min at 65°C, and in 0,1 x SSC for 2 x 30 min at room temperature. In fig. 5 the autoradiography from this experiment is shown. Lanes 1 and 2 show the mRNAs from two different preparations, and lane 3 the DNA molecular weight markers. By this experiment an approximately 3,8 kb long human thrombomodulin mRNA was identified. S S- ».JM?SSm 5~S<&_>!TIT t l) , T | ar S &H- -E-.E- From the size of this human thrombomodulin mRNA it is concluded that p2.1 contains a full length or substan¬ tially full length human thrombomodulin cDNA insert. The complete sequence of the cDNA in the plasmid p2.1 was determined (Tabor, S. and Richardson, C.C. (1987) Proc. Natl. Acad. Sci USA, 84: 4767 - 4771.), and is presented in Fig- 7. In addition to the shown sequence, the plasmid contained stretches of G:C ho opolymer tails at the 5 1 end in addition to a poly(A) tail in the 3 1 end of the cDNA insert. EXAMPLE 2 Human Thrombomodulin mutants, Desirable human thrombomodulin (hTM) mutants should be expressed as soluble proteins without the membrane spanning region. To obtain such mutants, the ollowing steps were performed. p2.1 was digested with PstI, and a 870 bp fragment (nucleotide 952 to 1821 in Fig 7) was isolated and subcloned in pGEM3 (Promega Biotec) . A subclone with the Kpnl site in the hTM cDNA oriented towards the BamHI site of the multilinker in pGEM3 was designated pBoel743-2. The insert in this plasmid was mutagenized (Morinaga, Y. , et al. (1984) BIO/TECHNOLOGY, 2 z 636 - 639) with an oligodeoxyribonucleotide, NOR 570: 5 « (GATGGAGATGCCTATGGGATCCTAGGAGTGCACGAGCCCCACGGC) 3 ' All synthetic oligodeoxyribonucleotides described in this invention were synthezised on an Applied Bio- systems Inc. (USA) DNA Synthesizer, and purified on polyacrylamide gels. This mutation resulted in the introduction of a TAG translation stop codon at the position of the Glycine- 516 codon GGC. In addition to this change, the oligonu- i __. m m rn i t m . 1 cleotide introduced an ApaLI (GTGCAC) site just 5' to the new translation stop signal, an Ayrll (CCTAGG) site spanning the stop signal and a BamHI (GGATCC) site on its 3' side. A correct mutant pBoel743-2-9 was identified through colony hybridization and through mapping with appropriate restriction enzymes. In one desirable hTM derived mutant, the expressed protein contained only the four carboxyterminal epidermal growth factor homologous domains together with the extracellular O-glycosylation rich domain. This mutant was generated as follows. pBoel743-2-9 was digested with BamHI and Hindi, and a 0.53 kb Hindi/- BamHI fragment was isolated. This fragment was joined to synthetic DNA, which encoded the signal peptide from the human tissue type plasminogen activator (tPA) (Pennica et al., (1983) Nature, 301: 214 - 221) and a short adaptor sequence, in BamHI digested pUC13. A correct recombinant pBoel743-2-9-8 was identified with restriction enzyme digestions, and the synthetic region was sequenced. The 711 bp sequence of the tPA encoding region, the adaptor and the HindI/BamHI fragment of pBoel743-2-9-8 is shown in Fig 8. In this figure all synthetic DNA is indicated with horizontal arrows. Methods used for purification, annea- ling, cloning and ligation of the adaptor fragments to the 0.53 kb HincII/Bait-HI fragment were all standard techniques and well known to workers in the field of recombinant DNA technology (cf. Maniatis et al. supra) . pBoel743-2-9-8 was digested with BamHI, and a 0.71 kb fragment corresponding to the synthetic region was purified by agarose gel purification. This fragment was cloned in BamHI digested Zem219b (described in US patent application Serial Number 58061 filed 4 June 1987) . Zem219b is a mammalian expression vector, which expresses inserted cDNA under control of the mouse methalloth-ionein pro otor and the human growth hormone terminator. The SUBSTITUTE SHEET plasmid also carries a mouse DHFR (dihydrofolate reduc- tase) cDNA under control of SV40 regulatory elements to provide a selectable marker in transfected mammalian cells. A correct recombinant pBoel-TMl was isolated and characterized with restriction enzyme digestions. pBoel- TM1 was propagated in E. coli in large scale, and purified on CsCl/Ethidium Bromide gradients by ultracentrif gation. Another desirable hTM mutant should be expressed as a soluble protein without the region rich in O-glycosylation and without the membrane spanning region. To obtain such a mutant, the following steps were performed. pBoel743-2 was mutagenized (Fig 10) with an oligonucleotide, NOR 571: 5 • (CTCGCCAGAGCCGCTGGATCCCTAGTCCACCTTGCCGGA) 3 • This mutation resulted in the introduction of a TAG translation stop codon at the position of the Glycine-487 codon GGT. In addition to this change, the oligonucleotide introduced a BamHI (GGATCC) site just 3* to the transla¬ tion stop signal. A correct mutant pBoel743-2-10 was identified through colony hybridization, through mapping with restriction enzymes, and through DNA sequencing. pBoel743-2-10 was digested with Hindi and BamHI, and a 0.44 kb fragment was purified on a polyacrylamide gel. This DNA fragment was joined to a 0.18 kb BamHI/- Hindi fragment from pBoel743-2-9-8 in BamHI digested and alkaline phosphatase treated Zem219b to generate pBoel- TM2. The correct structure of this plasmid was checked with restriction enzyme digestions. pBoel-TM2 encodes a mutant hTM precursor in which the four carboxyterminal epidermal growth factor homologous domains can be secreted from a mammalian cell under control of the human tPA signal peptide. pBoel-TM2 was grown in large scale in E.coli to prepare enough pure plasmid for transfection of mammalian cells. EXAMPLE 3 Expression of TM mutants in mammalian cells. For expression of mutant hTM in cultured BHK cells (Syrian Hamster, thymidine kinase mutant line tk tsl3 , Waechter and Baserga, (1982), Proc. Natl. Acad. Sci. USA, 79: 1106 - 1110. American Type Culture Collection CRL 1632) expression vectors pBoel-TMl and pBoel-TM2 were introduced into the cells by the calcium phosphate mediated transfection procedure (Graham and Van der Eb, (1973), Virology, \_2 : 456 - 467). Transient expression. Forty-eight hours after transfection, each petri dish was washed with serum free Dulbeccos Modified Eagle Medium (containing 25 mM N-2-hydroxyethyl-piperazine N'-2- ethanesulfonic acid (HEPES) , pH 7.4, 10 mg/1 insulin, 0.2 % Bovine Serum Albumin) , and incubated in the same medium for 24 hrs. The used medium was harvested and assayed for TM activity in an assay described below. Selection of TM- utant-producing clones. Forty-eight hours after transfection, cells were trypsinized and diluted into medium containing 400 nM methotrexate (MTX) . After 10 to 12 days, individual colonies were cloned out and expanded separately. The expanded cultures were propagated for 24 hours in serum free medium as described above, and producer clones were identified using an assay for protein C coactivator activity. Protein C coactivator activity. Protein C activation was measured by a method modified from Bourin et.al. (Proc. Natl. Acad. Sci. US 8_3,5924 (1986)). Two hundred μl of serum free supernatant from transfected cultures were mixed with 40 μl Protein C and 10 μl thrombin ( final concentrations 1 μM and 20 nM, respectively) , and calcium chloride was added to a final concentration of 2 mM. The mixtures were then incubated at 37"C for 30 minutes and the reaction was stopped with 200 pmole Antithrombin III and 5 U heparin. The volume was adjusted to 650 μl with 50 mM Tris.HCl/0.1 M NaCl, pH 8.3 and 100 μl 1 mM S-2266 (D-Val-Leu-Arg-p_-nitroanilide, KabiVitrum) in the same buffer was added. Increasing absorbance at 405 nm due to Protein C activity was recorded. A standard curve was established using known amounts of protein C completely activated by thrombin in the presence of rabbit TM. The results of this test are shown in Table 1 below: SU STITUTE »Ϊ». a ^ lmZlZT Table 1 Protein C Coactivator Activity in Supernatants of Trans- fected Cultures. DNA transfeeted Culture type Protein C activation pmole/min None Transient 0.5 TM-1 Transient 1.7 TM-2 Transient 1.3 None Host cells 0.3 None Host cells 0.9 TM-1 Clone 1 1.5 TM-1 Clone 2 6.5 TM-2 Clone 1 3.8 TM-2 Clone 2 4.8 From the table it is clearly seen that the modified thrombomodulins of this invention have a Protein C co- activating activity. Inhibition of clotting activity. For inhibition of clotting activity 500 μl serum free tissue culture supernatant was dialyzed against 100 mM NaCl, 50 mM Tris.HCl, pH 7.4. One hundred microliter ' APTT reagent (Dade) was incubated with 100 μl standard citrate plasma for 3 min at 37°C. One hundred microliter dialyzed tissue culture supernatant was added and imme- diately thereafter 100 μl 30 mM calcium chloride. Coagulation was measured in seconds. The results of this test are shown in Table 2 below: sues* m (ξ*' - e-X M-nf Table 2 Anticoagulant Activity of Dialyzed Supernatants from Transfected Cultures. DNA transfected Culture type Clotting time seconds None Host cells 28 TM-1 Clone 1 35 TM-1 Clone 2 70 TM-2 Clone 1 56 TM-2 Clone 2 49 From Table 2 it is seen that the coagulation time for the supernatants containing the modified throm¬ bomodulins of this invention was considerably longer than for the control containing no thrombomodulin. Although the present invention has been disclosed and described in connection with specific embodiments thereof, it is to be construed as comprising such equiva¬ lents and modifications that come within the skill of the artisan, and the invention is further to be understood and interpreted in connection with the appended claims. SUBSTITUTE SHEET

Claims: CLAIMS 1. A protein having thrombomodulin activity as defined by a high affinity binding to thrombin, and the capacity of endowing a complex between said protein and thrombin with the ability to activate Protein C, and comprising from the C-terminal part of the molecule two or more of the following structural elements: a) a short cytoplasmatic domain of approximately 56 amino acids, b) a transmembrane region of approximately 24 amino acids, c) a region rich in serine, threonine and proline residues, d) a domain comprising at least two EGF domains, and e) an N-terminal where one or more of the elements a), b), and/or c) may be omited or replaced by affinity imparting entities or entities enhancing the solubility of said protein in physiological solutions, or physiologically compatible derivatives thereof. 2. The protein according to claim 1, wherein said structural elements a) and b) have been omited or replaced. SUBSTITUTE SHEET 3. The protein according to claim 1, wherein said structural elements a), b), and c) have been omitted or replaced. 4. The protein according to claim 1, 2 or 3, wherein said structural element d) comprises at least four EGF domains_ 5. The protein according to claim 1, 2, 3 or 4, wherein said structural element e) comprises the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence. 6. The protein according to claim 4, comprising from the C-terminal: c) an O-glycosylation rich domain d) at least four EGF domains, e) the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence. 7. The protein according to claim 5, comprising from the C-terminal: d) at least four EGF domains, e) the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence. SUB S TITUTE SHEET 8. The protein according to claim 6, comprising the following amino acid sequence: H D A H K R G L C C V L L L C < (Human tPA signal peptide) G A V F V S P S Q E I H A R F R R G A R Synthetic < S Y Q D V D D C I E P S P C P Q R C V . j > Synthetic adaptor < N T Q G G F E C H C Y P N Y D L V D G E C V E P V D P C F R A N C E Y Q C Q P L N Q T S Y L C V C A E G F A P I P H E P H R C Q M F C N Q T A C P A D C D P N T Q A S C E C P E G Y I L D D G F I C T D I D E C E N G G F C S G V C H N L P G T F E C I C G P D S A L A R H I G T D C D S G K V D G G D S G S G E P P P S P T P G S T L T P P A V G L V H S SUBS" TUTS SHEET 9. The protein according to claim 7, comprising the following amino acid sequence: H D A H K R G L C C V L L L C < (Human tPA signal peptide) G A V F V S P S Q E I H A R F R R G A R Synthetic < S Y Q D V D D C I L E P S P C P Q R C V -| > Synthetic adaptor < N T Q G G F E C H C Y P N Y D L V D G E C V E P V D P C F R A N C E Y Q C Q P L N Q T S Y L C V C A E G F A P I P H E P H R C Q M F C N Q T A C P A D C D P N T Q A S C E C P E G Y I L D D G F I C T D I D E C E N G G F C S G V C H N L P G T F E C I C G P D S A L A R H I G T D C D S G K V D SUBSTITUTE SHEET 10. The protein according to claim 2 or 3, wherein said replacing entity is an affinity imparting entity. 11. The protein according to claim 10, wherein said entity imparts affinity towards physiological structures such as fibrin clots, membrane surfaces, receptor molecules, or extracellular matrix elements, preferably fibrin clots. 12. The protein according to claim 11, wherein said entity is chosen from the group comprising growth factor modules, kringles, finger modules, vitamin K- dependent calcium-binding γ-carboxylated regions and antibody-derived structures. 13. The protein according to claim 12, wherein said entity is a growth factor, finger or kringle module originating from human tissue plasminogen actJ'-'.-tor. 14. The protein according to claim 10, wherein said entity imparts affinity towards the surfaces of artificial articles meant to be in contact with blood, such as articles designed to be inserted and/or implanted into the body of a mammal, such as artificial blood vessels. 15. The protein according to any of the claims 10 to 14, comprising a bifunctional spacer molecule for conjugating said affinity imparting entity to said structural elements d) or e). 16. The protein of any of the claims 1 to 15, substantially free of any contaminants of human origin. SUBSTITUTE SHEET 17. A DNA construct containing a nucleotide sequence consisting essentially of two or more of the following regions a) a first region encoding an N-terminal region, b) a second region positioned downstream of said first region, said second region encoding a number of EGF domains, said number being greater than one, c) a third region positioned downstream of said second region, said third region encoding for a domain rich in serine, threonine and proline residues, d) a fourth region positioned downstream of said third region, said fourth region encoding for a membrane spanni ■ Z .j-.in, and e) a fifth region positioned downstream of said fourth region, said fifth region encoding for a cytoplasmatic domain, where one or more of the regions e) , d) , and/or c) may be omitted or replaced by regions encoding for affinity imparting entities or domains encoding for entities enhancing the solubility of the encoded protein, said DNA construct coding for a protein which has substantially the same biological activity as human thrombomodulin as defined by a high affinity binding to thrombin and the capacity of endowing a complex between said coded protein and thrombin with the ability to activate Protein C. 18. The DNA construct of claim 17, wherein said regions d) and e) have been omitted or replaced. SUBSTITUTE 19. The DNA construct of claim 17, wherein said regions c), d) , e) have been omitted or replaced. 20. The DNA construct of claim 17, 18 or 19, wherein said region b) encodes for at least four EGF domains. 21. The DNA construct of claim 17, 18, 19or 20, wherein said region a) encodes for the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence. 22. The DNA construct of claim 20, containing a nucleotide sequence encoding for a) the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence, b) at least four EGF domains, and . c) an O-glycosylation rich domain. 23. The DNA construct of claim 21, containing a nucleotide sequence encoding for a) the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence, b) at least four EGF domains. SUBSTITUTE SHEET 24. The DNA construct of claim 22 containing the following nucleotide sequence: BamHI EcoRI Ncol . GGATCCGAATTCCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTG 60 I > Synthetic DNA (Human tPA signal peptide) Bglll TGGCGCCGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAG 120 Synthetic DNA < Sail . Xbal . . Hindi ATCTTACCAAGACGTCGACGACTGCATTCTAGAGCCCAGTCCGTGTCCGCAGCGCTGTGT 180 _|—.—> Synthetic 5' adaptor < TAACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTGGACGGCGA 240 -I • • • • • • GTGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCAGTGCCAGCCCCT 300 GAACCAAACTAGσrACCTCTG∞TCTGαϊCCGAGGGCTTCGCGCCCATTCCCCACGAGCC 360 GCACAGGTGCCAGATGTTTTGCAACCAGACTGCCTGTCCAGCCGACTGCGACCCCAACAC 420 CCAGGCTAGCrGTGAGTGCCCTGAAGGCTACATCCTGGAOTACCGTTTCATCTGCACGGA 480 CATCGA∞AGTGCGAAAACGGα^CTTCTGCTCCGGGGTGTGCCACAACCTCCCCGGT C 540 CTTCGAGTGCATCTGCGGGCCCGACTCGGCCCTTGCCCGCCACATTGGCACCGACTGTGA 600 CTCCGGCAAGGTGGACGGTGGCGACAGCGGCTCTGGCGAGCCCCCGCCCAGCCCGACGCC 660 ApaLI Sty I BamHI CGCCTCCACCTTGACTCCTCCGGCCGTGGGGCTCGTGCACTCCTAGGATCC 711 SUBSTITUTE SHEET 25 . The DNA construct of c laim 23 containing the following nucleotide sequence : BamHI EcoRI Ncol . . . . . GGATCCGAATTCCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTG 60 J > Synthetic DNA (Human tPA signal peptide) Bglll TGG∞CCGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAG 120 Synthetic DNA < Sail . Xbal . . Hindi ATCrrTACCAAGAOπ'CGACGACTGCATTCTAGAGCCCAGTCCGTGTCCGCAGCGCTGTGT 180 __ j > Synthetic 5' adaptor < • • • • • • TAACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTGGACGGCGA 240 GTGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCAGTGCCAGCCCCT 300 GAACCAAACTAGCTACCTCTGCGTCTGCGCCGAGGGCTTCGCGCCCATTCCCCACGAGCC 360 GCACAGGTGCCAGATGTTTTGCAACCAGACTGCCTGTCCAGCCGACTGCGACCCCAACAC 420 CCAGGCTAGCTGTGAGTGCCCTG AAGGCTACATCCTGG ACG ACGGTTTCATCTGCACGGA 480 CATCGACGAGTGCGAAMCGGC^GCTTCTGCTCCGGGGTGTGCCACAACCTCCCCGGTAC 540 CTTCGAGTGCATCTGCGGGCCCXΪACTCGGCCCTTGCCCGCCACATTGGCACCGACTGTGA 600 CTCCGGCAAGGTGGAC TAGGGATCC SUBSTITUTE SHEET 26. The DNA construct of claim 18 or 19, wherein said replacing region is a region encoding for an affinity imparting entity. 27. The DNA construct of claim 26, wherein said entity imparts affinity affinity towards physiological structures such as fibrin clots, membrane surfaces, receptor molecules, or extracellular matrix elements, preferably fibrin clots. 28. The DNA construct of claim 27, wherein said entity is chosen from the group comprising growth fn-ior modules, kringles, finger modules, vitamin K-dependent calcium-binding γ-carboxylated regions and antibody- derived structures. * _~ 29. The DNA construct of claim 28, wherein said entity is a growth factor, finger or kringle module originating from human tissue plasminogen activator. 30. The DNA construct of claim 18 or 19, wherein said entity imparts affinity towards the surfaces of artificial articles meant to be in contact with blood, such as articles designed to be inserted and/or implanted into the body of a mammal such as artificial blood vessels. 31. An expression vector capable of directing the expression of a protein having essentially the same biological activity as human thrombomodulin as defined by high affinity binding to thrombin and the capacity of endowing a complex between said protein and thrombin with the ability to activate Protein C, said vector including a promoter being operably linked to a DNA construct as defined in any of the claims 17 to 30. SUSSTϊTUTE SHEET 32. A cell containing a vector according to claim 31 33. A cell according to claim 32, which cell is a bacterial cell, funga " ! -ell, yeast cell or mammalian cell, preferably a mammalian cell. 34. A method of producing a protein which has substantially the same biological activity as human thrombomodulin as defined by high affinity binding to thrombin and the capacity of endowing a complex between said protein and thrombin with the ability to activate Protein C, comprising: a) inserting into cells a vector according to claim 31, b) growing said cells in an appropriate medium, and * c) isolating the protein product having thrombomodulin activity encoded by said vector and produced. by said cells. 35. The method of claim 34, wherein said cells are bacterial cells, yeast cells, fungal cells or mammalian cells, preferably mammalian cells. 36. A pharmaceutical preparation which contains at least one protein or a physiologically compatible salt or ester thereof according to any of the claims 1 to 16 in combination with a pharmaceutically acceptable carrier or excipient. SUBSTITUTE SHEET 37. The pharmaceutical preparation of claim 36 in the form of a physiological solution. ' 38. Use of a peptide according to any of the. claims 1 to 16 or a pharmaceutical preparation according to claim 36 or 37 for the therapy or profylaxis of thrombic events. 39. A method of treating thrombic events in mammals characterized in administering to a mammal an effective amount of at least one protein according to any of the claims 1 to 16 or a pharmaceutical preparation according to claim 36 or 37. 40. A method of preventing thrombic events in mammals characterized in administering to a mammal an effective amount of at least one protein according to any of the claims 1 to 16 or a pharmaceutical preparation according to claim 36 or 37. 41. Use of a peptide according to any of the claims 1 to 16 for coating articles designed for being in contact with blood and/or insertion and/or implantation into the body of a mammal. 42. An article designed for being in contact with blood and/or insertion and/or implantation into the body of a mammal, said article comprising a coating, the composition of said coating comprises a peptide according to any of the claims 1 to 16. SUBSTITUTE SHEET

Inventors: ESPER BOEL, NEXOE BJOERN ANDERSEN, ESPER, BOEL, NEXOE, BJOERN, ANDERSEN
Applicants: NOVO INDUSTRI AS, NOVO INDUSTRI A/S
Application year: 1988
Main classification: C12N15
IPC codes: A61K38, A61P7, C07K14, C12N5, C12N9, C12N15, C12P21, C12R1