PLATELET AGGREGATION INHIBITORS

Abstract: A platelet aggregation inhibitor useful as an antithrombotic is provided which consists of a synthetic cyclic pentapeptide containing the tripeptide sequence Lys-Gly-Asp and a thioether linkage in the cycle. A prefered platelet aggregation inhibitor is represented by formula (I), where: R1 and R9 are OH; R2, R4, R5, R6, R7, R8 and R14 are hydrogen; R3 and R4 are joined to form a pyrrolidine ring; X is sulfur; m is 1 and n is 3.

Description: PLATELET AGGREGATION INHIBITORS Field of the Invention The present invention relates to inhibitors of platelet aggregation. Specifically, the invention is directed to peptides comprising the tripeptide sequence -Lys-Gly-Asp- capable of acting as antagonists of the final common pathway of platelet aggregation and that act as potent antithrombotics. The invention further relates to therapeutic applications of these inhibitors in diseases for which blocking platelet aggregation and intracellular adhesion is indicated. Background of the Invention Platelets are particles found in whole blood known to participate in thrombus formation and blood coagulation. A membrane spanning glycoprotein receptor, GP li Wa. is present on the surface of platelets and is known to be involved in the coagulation process. GP lib I Ha is a non-covalent, calcium ion dependent heterodimer complex composed of alpha and beta subunits (Jennings, et al., J. Biol. Chem. (1982) 257, 10458) capable of binding protein ligands. This glycoprotein receptor contributes to normal platelet function through interactions with protein ligands containing the tripeptide amino acid sequence Arg-Gly-Asp (RGD). One protein ligand known to be important for thrombus formation and containing the RGD sequence is fibrinogen. Fibrinogen contains two RGD sequences located at Aα95-97 and Aα572-574 (Doolittle, R. F., Watt, K. W. K., Co lltrell, B. A., Strong, D. D. and Riley, M. (1979) Nature 280, 464-468) that have been shown to interact with the GP H Hla receptor (Hawiger.e. al., Biochemistry, 28, 2909-2914 (1989). A third region of fibrinogen, corresponding to the final 12 residues ,400-411 , of the gamma chain carboxy terminus and having the sequence HHLGGAKQAGD V, has also been demonstrated to bind with GP lib Ilia (Kloczewiak, M., Timmons, S., Lukas, T. J., and Hawiger, J. (1984) Biochemistry 23, 1767- 1774). Evidence for the involvement of both the RGD and gamma chain regions in binding with GP ll llia is largely derived from binding and inhibition data and from studies with synthetic RGD peptides (Gartner, T. K. and Bennett, J. S. (1985) J. Biol. Chem. 260, 11891- 11894; Plow, E. F., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G. A., and Ginsberg, . H. (1985) Proc. Nat. Acad. Sci. USA 82, 8057-8061 ; Haverstick, D. M., Cowan, J. F., Yamada, K. M., and Santoro, S. (1985) Blood 66, 946-952; and D'Souza, S. E., Ginsberg, M. H., Lam, S.- C. T., and Plow, E. F. (1988) J. Biol. Chem. 263, 3943-3951) and gamma chain carboxy terminus analogs (Kloczewiak, ., Timmons, S., Bednarek, M. A., Sakon, M., and Hawiger, J. (1989) Biochemistry 28, 2915-2919). In this latter strdy, all amino acid replacements in the gamma chain carboxy terminal dodecapeptide reduced inhibitory activity of the analog except replacement of Ala 408 (ie. immediately preceding GD) with Arg which increased inhibitory potency 6-fold (see also Timmons, et al.., Biochemistry 28 2919-2923 [1989]). The interaction of GP li Ilia with fibrinogen is stimulated by certain factors released or exposed when a blood vessel is injured. Multiple factors, including a variety of physiologic stimuli and soluble mediators, initiate platelet activation via several pathways. These pathways have, as a common final step, the activation of the GP lib Efl a receptor on the platelet surface and its subsequent binding to fibrinogen followed by aggregation and thrombus formation. By virtue of these interactions GP lib Wa is an important component of the platelet aggregation system (Pytela et al., Science (1986) 231 , 1559). Therefore, inhibition of the interaction of GP lib IHa with Arg-Giy -Asp containing proteins such as fibrinogen is one way of modulating thrombus formation. An inhibitor which prevents this binding interaction would antagonize platelet activation by any stimulus and therefore would have important antithrombotic properties. It is known, however, that proteins and peptides containing the RGD sequence are also recognized as ligands for a number of other cell adhesion receptors in addition to GP lib lll a . These cell adhesion receptors comprise a family of heterodimeric protein receptors known as the integrins (Ginsberg, M. H., Loftus, J. C, and Plow, E. F. (1988) Thrombosis and Haemostasis 59, 1-6; and Hynes, R. O. (1988) Cell 48, 549-554). Among the other receptors shown to bind RGD containing ligands are the vitr onectin receptors ( VnR) and the fibronectin receptors (FnR) (Pytela etal. (1985) Proc. Natl. Acad. Sci., USA 82, 5766-5770; Pytela etal. (1985) Cell 40, 191-198; and Sanchez-Madrid etal. (1983) J. Exp. Med. 158, 1785-1803). Furthermore, it is believed that other integrin receptors may be discovered that also interact with RGD containing ligands. Thus, it is believed that a particularly useful antithrombotic would be one that specifically inhibited the interaction between RGD containing proteins and the platelet GP llbWa receptor while not effecting the interaction between the other integrins and their endogenous ligands. Many common human disorders are characteristically associated with a hyperthrombotic state leading to intravascular thrombi and emboii. These are a major cause of medical morbidity, leading to infarction, stroke and phlebitis and of mortality from stroke and pulmonary and cardiac emboii. Patients with atherosclerosis are predisposed to arterial thromboembolic phenomena for a variety of reasons. Atherosclerotic plaques form niduses for platelet plugs and thrombii that lead to vascular narrowing and occlusion, resulting in myocardial and cerebral ischemic disease. This may happen spontaneously or following procedures such as angioplasty or endarteroectomy. Thrombii that break off and are released into the circulation cause infarction of different organs, especially the brain, extremities, heart and kidneys. In addition to being involved in arterial thrombosis, platelets may also play a role in venous thrombosis. A large percentage of such patients have no antecedent risk factors and develop venous thrombophlebitis and subsequent pulmonary emboii without a known cause. Other patients who form venous thrombi have underlying diseases known to predispose to these syndromes. Some of these patients may have genetic or acquired deficiencies of factors that normally prevent hypercoagulability, such as antithrombin-3. Others have mechanical obstructions to venous flow, such as tumor masses, that lead to low flow states and thrombosis. Patients with malignancy have a high incidence of thrombotic phenomena for unclear reasons. Antithrombotic therapy in this situation with currently available agents is dangerous and often ineffective. It is also known that patients whose blood flows over artificial surfaces, such as prosthetic synthetic cardiac valves or through extr acorporeal perfusion devices, are at risk for the development of platelet plugs, thrombii and emboii. For example, it is standard practice with patients having artificial cardiac valves to be continuously anti-co agulated. However, in all instances, platelet activation and emboii formation may still occur despite adequate anticoagulation treatment. Thus, a large category of patients, including those with atherosclerosis, coronary artery disease, artificial heart valves, cancer, and a history of stroke, phlebitis, or pulmonary emboii, are candidates for limited or chronic antithrombotic therapy. The number of available therapeutic agents is limited and these, for the most part, act by inhibiting or reducing levels of circulating clotting factors. These agents are frequently not effective against the patient's underlying hematologic problem, which often concerns an increased propensity for platelet aggregation and adhesion. They also cause the patient to be susceptible to abnormal bleeding. Available antiplatelet agents, such as aspirin, inhibit only part of the platelet activation process and are therefore often inadequate for therapy. An agent which effectively inhibits the final common pathway of platelet activation, namely fibrinogen binding to the GP lib Ilia receptor, should accordingly be useful in a large group of disorders characterized by a hyperthrombotic state as described above. The present invention contemplates such an agent which is a new composition, namely a polypeptide that may consist in part of natural amino acids and in part of unnatural amino acids as well as non- peptidyl portions. This new composition is believed to interfere with the interaction of Arg-Gly- Asp containing peptides, particularly fibrinogen, with the GP lib Hla complex thereby preventing platelet aggregation. Platelet aggregation has been identified as an early step in the formation of platelet plugs, emboii and thrombii in the circulatory system which in turn have been shown to play an active role in cardiovascular complications and disease. Inhibition of fibrinogen binding to the GP lib Hla complex has been shown to be an effective antithrombotic treatment in animals (H. K. Gold, et al., Circulation (1988) 77, 670-677; T. Yasuda, et al., J. Clin. Invest. (1988) 81, 1284-1291 ; B. S. Coller, etal., Blood (1986) 68, 783-786.) A number of synthetic peptides, including cyclic disulfides, have been disclosed as inhibitors of fibrinogen binding to platelets all of which contain the Arg-Gly-Asp sequence. See U.S. Patent 4,683,291 ; WO89/05150; EPO 0 319 506 A2; EPO 0 341 915 A2; Plow et al., Proc. Natl. Acad. Sci. USA (1985) 82, 8057-8061 ; Ruggeri etal., Proc. Natl. Acad. Sci. USA (1986) 83, 5708-5712; Haverstick etal., Blood (1985) 66, 946-952; Plow etal., Stood (1987) 70, 110-115; F. El F. Ali, etal., Proc. Eleventh Amer. Peptide Symp. (1990) 94-96; M. Pierschbacher and E. Ruoslahti, J. Biol. Chem. (1987) 262, 17294-17298; and references cited in the above publications. These Arg-Gly-Asp containing peptides are belived to act as competitive inhibitiors, out competing fibrinogen for the GPIIbHIa receptor. Synthetic peptides in which one or more of the RGD amino acid residues has been replaced with another amino acid or analog have also been described. EPO 0368486 A2 discloses a Arg-Tyr-Asp-21 mer that is about 10-fold less active in a platelet aggregation assay than the corresponding Arg-GIy-Asp-21 mer. Ali, et al., Peptides : Chemistry, Structure and Biology, Proceedings of the 11 th American Peptide Symposium, p.94-96, Rivier and Marshall eds. ESCOM, Leiden (1990) describe modifications to the sequence Arg-Gly-Asp-Ser in a platelet aggregation assay. Substitution of Lys for Arg in this sequence as well as most other subsfitutfons greatly decreased potency. Similariy, for cyclic RGD peptides, modification of the Arg residue (N,N'-Et2° ' uaπ ) produced a 10-fold lower potency. Garsky, etal., Proc. Natl. Acad. Sci. USA 864022-4026 (1989) describe a potent platelet aggregation inhibitor from the venom of the saw-scaled viper Echis carinatus . These arthors report the inhibitor is a 49 amino acid protein containing the sequence -Arg 24 -Gly -Asp- having an and that replacing Arg 24 with omithine (Orn) produces a mutant inhibitor, [Orn 2 lEchistatin, having 3-fold lower potency. Tetrapeptides containing the Orn- Gly-Asp sequence are also described having 10- to 50-fold lower potency. A search of the Dayhoff data base revealed 2193 occurences of the sequence KGD (compare 2026 for RGD). Of proteins containing the KGD sequence, placental anticoagulant protein (PAP) and two other members of the lipocortin family, are reported to have anticoagulant activity (Funakoshi, etal.. Biochemistry 265572-5578 (1987). However, these proteins are not reported to inhibit the final common pathway of platelet activation by inhibition of binding of fibrinogen with the GP HbHIa receptor, rather anticoagulant activity is ascribed to inhibition of prothrombin activation as well as inhibition of phospholipase A2 and subsequent prostaglandin release. Several synthetic cyclic peptides containing the thioether linkage have been synthesized. Gero etal., Biochem. Biophys. Res. Comm. (1984) 120, 840-845 describe a pseudohexapeptide analog of so mato statin where the group [CH2-S] is substituted for a peptide bond. Similarly, Edwards etal., Biochem. Biophys. Res. Comm. (1986) 136, 730-736 compare the biological activity of linear and cyclic enkephalin pseudopeptide analogs containing the thiomethyiene ether linkage. Other enkephalin related pseudopeptides and macrocycles containing the [CH2-S] substitution for peptides have been described, Spatola et al., Biopolymers (1986) 25, 229-244 and Spatola et al elrahedron (1988) 44, 821-833. None of these references disclose a peptide containing the amino acid sequence KGD having high platelet aggregation inhibition activity. None of these references disclose a peptide having high specificity for the GP HbHIa receptor relative to other integrin receptors. Finally, none of the references describe a small cyclic peptide stable to ring opening containing either -Lys-Gly-Asp- or -Orn-Gly -Asp- having a higher inhibition potency for fibrinogen GP H HIa ELISA than for vitronectin vitronectin receptor ELISA or for fibronectin/fibronectin receptor ELISA. Accordingly, it is an object of this invention to produce a peptide having high platelet aggregation inhibition activity. It is a further object of this invention to provide peptides having high specificity for the GP HbHIa receptor. It is still a further object to produce small cyclic peptides that are stable to ring opening having the above described properties. It is still a further object of this invention to provide a platelet aggregation inhibitor exhibiting diminished in vivo side effects such increased bleeding times and optionally to provide such inhibitors with increased lifetime These and other objects of this invention will be apparent from consideration of the invention as a whole. Summary of the Invention The objects of this invention are accomplished by providing a polypeptide containing the sequence Xaa-Gly-Asp having high specificity for the GP HbHIa receptor relative to the other integrin receptors, where Xaa is Omithine (Orn) or Lysine (Lys). Preferably the peptide contains the sequence Lys-Gly-Asp and contains fewer than about 345 amino acid residues. Also prefereably the peptide is cyclic, having from 5-10 amino acids in the cycle. Most preferably, the cyclic peptide has 5 amino acids forming the ring of the cycle. More preferably, the ring of the cyclic peptide contains from about 17 to about 18 atoms, most preferably 18 atoms. A particulariy preferred compound of the instant invention is a polypeptide having the structure Xaa ! -Xaa 2 -Gly-Asp-Xaa 3 -R i — z — i wherein Xaa is a D or L α-amino acid linked to Z through the α amino group; Xaa2 is Orn or Lys; Xaa3 is a D or L α-amino acid linked through the side chain to Z; Z is an amide bond, disulfide, COCH2S, COCH2SO, or COCH(C6H5)S; and R is OH or NH2. Also preferably, the ring will contain a D amino acid most preferably linked to the Lys of the tripeptide sequence. The invention in its broad aspects relates to peptide derivatives which are useful as inhibitors of platelet function mediated by the GP lib Hla receptor and for the prevention of thrombus formation. Preferred compounds of this invention are represented by Formula I: wherein R-| and Rg are the same or different and are selected from hydroxy, C-i-Cβ alkoxy, C3-C12 alkenoxy, C6-C12 arytoxy, di-C-j-Cβ alkylamino-Cι-C8-alkoxy, acylamiπo-Ci -Cβ-alkoxy selected from the group acetylaminoethoxy, nicotinoylaminoethoxy, and succinamidoethoxy, pivaloyloxyethoxy, C6-C12 aryl-Ci-Cδ-alkoxy where the aryl group is unsubstituted or substituted with one or more of the groups nitro, halo (F, Cl, Br, I), Cι-C4-alkoxy, and amino, hydroxy-C2-C8-alkoxy, dihydroxy-C3-C8-alkoxy, and NR10R11 wherein R10 and Ri 1 are the same or different and are hydrogen, Cι-C8-alkyl, C3- Cδ-alkenyl, C6-Ci2-aryJ where the aryl group is unsubstituted or substituted with one or more of the groups nitro, halo (F, Cl, Br, I), C-|-C4-alkoxy, and amino, Cβ-Ci 2-aryl- Ci-Cβ-alky! where the aryl group is unsubstituted or substituted by one or more of the groups nitro, halo (F, Cl, Br,l), C-|-C4-alkoxy, and amino; R2> R3. Rδ. R 6. R 7. R 8 are the same or different and are selected from hydrogen, C6-C12 aryl where the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), Ci-Cβ alkyl, halo-Cι- Cβ alkyl, C-t-Cβ-alkoxy, amino, phenyloxy, phenyl, acetamido, benzamido, di-C-|-Cδ alkylamino, C1-C8 alkylamino, C6-C12 aroyl, C1-C8 alkanoyl, and hydroxy-Ci-Cβ alkyl, C1-C12 alkyl either substituted or unsubstituted, branched or straight chain where the substituents are selected from halo (F, Cl, Br, I), C1-C8 alkoxy, C6-C12 aryloxy where the aryl group is unsubstituted or substituted by 5 one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), C-i-Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di-Ci -Cs alkylamino, C1 -Cβ alkylamino, C6-C12 aroyl, and C-|-Cβ alkanoyl, isothioureido, 1 0 C3-C7 cycloalkyl, ureido, amino, Ci-Cδ alkylamino, di-C-|-C8 alkylamino, 1 5 hydroxy, amino-C2-C8 alkylthio, amino-C2-Ce alkoxy, acetamido, benzamido wherein the phenyl ring is unsubstituted or substituted by one 20 or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), C1 -Cβ alkyl, Ci-Cs-alkoxy, amino, phenyloxy, acetamido, benzamido, di- C1-C8 alkylamino, C-|-Ce alkylamino, C6-C12 aroyl, and Ci-Cδ alkanoyl, C6-C12 arylamino wherein the aryl group is unsubstituted or substituted 25 by one or more of the groups nitro, hydroxy, halo, C1 -Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di- C-i-Cβ alkylamino, C-|-Cβ alkylamino, C6-C12 aroyl, and C-|- Cβ alkanoyl, guanidino, 30 phthalimido, mercapto, C-|-Cδ alkylthio, i C6-Ci2 arylthio, carboxy, v 35 carboxamide, carbo-Ci-Cδ alkoxy, C6-C12 aryl wherein the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo, C-|-Cβ alkyl, C-|-C8-alkoxy, amino, phenyloxy, acetamido, benzamido, di- C-i-Cβ alkylamino, Cf-Cβ alkylamino, hydroxy-Ci-Cβ alkyl, C6-C12 aroyl, and Ci-Cβ alkanoyl, and aromatic heterocycle wherein the heterocyclic groups have 5-10 ring atoms and contain up to two O, N, or S heteroatoms; R2 and R3, R5 and RQ > or R7 and Re may optionally and independently be joined together to form a carbocyclic or heterocyclic ring of from four to seven atoms where the heteroatoms are selected from O, S or NR12 where R12 is selected from hydrogen, C-| -Cβ-alkyl, C3-C8-alkenyl, C6-Ci2-aryl, C6-C-f2-aryl-Cι- Cβ-alkyl, Ci-Cβ alkanoyl, and C6-C12 aroyl, R4 is selected from hydrogen, Ci-Cδ alkyl, C3-Cιo cycloalkyl, C6-Ci2 aryl, and C6-C12 aryl-Ci -Cβ-alkyl; R2 or R3 may be optionally joined with R4 to form a piperidine, pyrrolidine or thiazolidiπe ring; R14 is selected from hydrogen, C1 -Cβ-alkyl, C3-Cβ-alkenyl, C6-C12-aryl, and C6-C12 aryl-Ci -Cβ- alkyl; X is selected from an O or S atom, an S atom bearing one or two O atoms, NR13 wherein R13 is hydrogen, C-i-Cβ-alkyi, C3-Cβ-alkenyl, Cβ-Ci 2-aryl, C6-Ci2-aryl-Ci -Cβ-alkyl, Ct-Cβ alkanoyl, and C6-C12 aroyl, and C6-Ci2 aryl, C-|-Cβ alkanoyl, (CH2)k where k is an integer from 0 to 5; n is an integer from 1 to 6; m is an integer from 0 to 4; and pharmaceutically acceptable salts thereof. As used herein and unless specified otherwise: alkyl, alkenyl and alkynyl denote straight and branched hydrocarbon chains having single, double and triple bonds, respectively; C6-C12 aryl groups denote unsubstituted aromatic ring or fused rings such as, for example, phenyl or naphthyl; hetero denotes the heteroatoms O, N, or S; aromatic heterocyclic groups have 5-10 ring atoms and contain up to four heteroatoms; halogen or halo denote F, Cl Br, or I atoms; alkoxy denotes an alkyl group attached to O. Examples of Ci-Cβ alkyl or C2-C6 alkenyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, isopentyl, hexyl, vinyl, ally I, butenyl and the like; examples of C3-Cιo-cycloalkyl groups include cyclopropyl, cyctopentyl, cyctohexyl, and the like; aromatic heterocyclic groups include but are not limited to pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazolyl, thiazoiyl, quinolinyl and isoquinolinyl. The present invention includes a method of making the compounds of Formula I. The present invention also includes a method for reducing platelet aggregation in a mammal. This method involves administering a therapeutically effective amount of the compounds of the present invention alone or in combination with a pharmacologically acceptable carrier. This general method may also be applied to treat a mammal having an increased propensity for thrombus formation. Additionally, the present invention is directed to compositions of matter for reducing platelet aggregation in a mammal; treating a mammal having an increased propensity for thrombus formation; or inhibiting binding of a ligand to GP lib Hla in a mammal; wherein each of these compositions contains as an active ingredient one or more of the cyclic peptides defined in Formula I. Detailed Description of the Invention The instant invention is the result of the surprising discovery that KGD containing polypeptide are potent inhibitors of platelet aggregation and in the Fg/GP HbHIa ELISA. The most potent KGD-containing inhibitors are small cyclic peptides and thus these peptides are preferred. It has also been discovered that the instant KGD peptides are weak inhibitors of the fibronectin (Fn)/fibronectin receptor (FnR) interaction and of the vitronectin ( Vn) vitronectin receptor VnR interaction. In comparing inhibition of various KGD-containing polypeptides it has been found that inhibition potency, as measured by IC50, is from 10-500 fold higher (i.e. a lower IC50) in the Fg/GPIIbHIa ELISA than in the Vn/VnR ELISA. Thus, the instant polypeptides exhibit high specificity for the GPIIbHIa receptor. It is contemplated that this high specificity will reduce the number and severity of side effects for these antithrombotic compounds. It is believed these compounds will exhibit high platelet aggregation inhibition without substantially increasing bleeding time in a mammal. Polypeptides of this invention can be made by chemical synthesis or by employing recombinant technology. These methods are known in the art. Chemical synthesis, especially solid phase synthesis, is prefered for short (eg. less than 50 residues) polypeptides or those containing unnatural or unusual amino acids such as; D-Tyr, Omithine, amino adipic acid, and the like. Recombinant procedures are prefered for longer polypeptides or for mutant or variant peptides containing the KGD sequence. When recombinant procedures are selected, a synthetic gene may be constructed de novo or a natural gene may be mutanigized by, for example, casette mutagenisis. Set forth below are exemplary general recombinant procedures. General Recombinant Procedures. From a purified protein and its amino acid sequence, a KGD-containing protein may be produced using recombinant DNA techniques. These techniques contemplate, in simplified form, taking the gene, either natural or synthetic, for the protein; inserting it into an appropriate vector; inserting the vector into an appropriate host cell; culturing the host cell to cause expression of the gene; and purifying the protein produced thereby. Somewhat more particularly, the DNA sequence encoding a KGD-containing protein is cloned and manipulated so that it may be expressed in a convenient host. DNA encoding parent polypeptides can be obtained from a genomic library, from cDNA derived from mRNA from cells expressing the protein, or by synthetically constructing the DNA sequence (Sambrook, J., Fritsch, E.F., and Maniatis, T., (1989), Molecular Cloning (2d ed.), Cold Springs Harbor Laboratory, N.Y.). The parent DNA is then inserted into an appropriate plasmid or vector which is used to transform a host cell. In general, plasmid vectors containing replication and control sequences which are derived from species compatible with the host cell are used in connection with those hosts. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells. For example, E * cadi may be transformed using pBR322, a plasmid derived from an £ coli species (Mandel, M. et al. (1970) J. Mol. Biol.53, 154). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides easy means for selection. Other vectors include different features such as different promoters, which are often important in expression. For example, plasmids pKK223-3, pDR?20, and pPL-lambda represent expression vectors with the tac, trp, or P|_ promoters that are currently available (Pharmacia Biotechnology). A preferred vector is pB0475. This vector contains origins of replication for phage and £.fiθli which allow it to be shuttled between such hosts, thereby facilitating both mutagenesis and expression (Cunningham, B., et al. (1989), Science 243, 1330-1336; Wells, J. and Cunningham, B., co-pending application WO 90/04788, published 3 May 1990. Other preferred vectors are pR1T5 and pRlT2T (Pharmacia Biotechnology). These vectors contain appropriate promoters followed by the Z domain of protein A, altowing genes inserted into the vectors to be expressed as fusion proteins. Further discussion of these vectors may be found below. Other preferred vectors can be constructed using standard techniques by combining the relevant traits of the vectors described above. Relevant traits include the promoter, the ribosome binding site, the decorsin or omatin gene or gene fusion (the Z domain of protein A and decorsin or omatin and its linker), the antibiotic resistance markers, and the appropriate origins of replication. The host cell may be prokaryotic or eukaryotic. Prokaryotes are preferred for cloning and expressing DNA sequences to produce parent polypeptides, segment substituted polypeptides, residue-substituted polypeptides and polypeptide variants. For example, £. K12 strain 294 (ATCC No.31446) may be used as E. coli B. £,221 X1776 (ATCC No. 31537), and £. C2li c600 and cδOOhf I, E ^ W3110 (F-, gamma-, prototrophic /ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonellajyphimurium or Serratia marcesans, and various pseudo monas species. The preferred prokaryote is J ≤ W3110 (ATCC 27325). When expressed by prokaryotes the polypeptides typically contain an N-terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein. These examples are, of course, intended to be illustrative rather than limiting. In addition to prokaryotes, eukaryotic organisms, such as yeast cultures, or cells derived from multicellular organisms may be used. In principle, any such cell culture is workable. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a reproducible procedure (Ηssue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines. Gene Fusions. A variation on the above procedures contemplates the use of gene fusions, wherein the gene encoding the desired protein is associated, in the vector, with a gene encoding another protein or a gragment of another protein. This results in the desired protein - here, a KGD-containing protein - being produced by the host cell as a fusion with another protein. The "other" protein is often a protein or peptide which can be secreted by the cell, making it possible to isolate and purify the desired protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the desired protein remains inside the cell. Alternatively, the fusion protein can be expressed intracellulariy. It is useful to use fusion proteins that are highly expressed. The use of gene fusions, though not essential, can facilitate the expression of heterologous proteins in E. coli as well as the subsequent purification of those gene products (Harris, T. J. R. (1983) in Genetic Engineering, Williamson, R., Ed., Academic, London, Vol. 4, p. 127; Uhlen, M., Moks, To. (1989) Methods Enzymol. (in press)). Protein A fusions are often used because the binding of protein A, or more specifically the Z domain of protein A, to IgG provides an "affinity handle" for the purification of the fused protein. It has also been shown that many heterologous proteins are degraded when expressed directly in £_2βli. but are stable when expressed as fusion proteins (Marston, F. A. O., (1986) Biochem J. 240, 1). A KGD-containing protein expressed a fusion protein may be properly folded or require folding to obtain the native structure. The properly folded fusion protein may be active and useful as a GP lib Hla antagonist and inhibitor of platelet aggregation. More preferred would be the correctly folded" native" protein that is obtained from the fusion protein by methods known in the art. Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an asn and giy. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5' end of the KGD- containing protein gene. Alternatively, one can employ proteolytic cleavage of fusion proteins, which has been recently reviewed (Carter, P. (1990) in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch, M. R., Willson, R. C, Painton, C. C, and Builder, S. E., eds., American Chemical Society Symposium Series No. 427, Ch 13, 181-193). Proteases such Factor Xa, thrombin, subtilisin and mutants, and a number of other have been successfully used to cleave fusion proteins. Typically, a peptide linker that is amenable to cleavage by the protease used is inserted between the "other" protein (e.g., the Z domain of protein A) and the KGD-contaning protein of interest. Using recombinant DNA methodology, the nucleotide base pairs encoding the linker are inserted between the genes or gene fragments coding for the other proteins. Proteolytic cleavage of the partially purified fusion protein containing the correct linker can then be earned out on either the native fusion protein, or the reduced or denatured fusion protein. The protein may or may not be properly folded when expressed as a fusion protein. Also, the specific peptide linker containing the cleavage site may or may not be accessible to the protease. These factors determine whether the fusion protein must be denatured and refolded, and if so, whether these procedures are employed before or after cleavage. When denaturing and refolding are needed, typically the protein is treated with a chaotrope, such a guanidine HCI, and is then treated with a redox buffer, containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the protein of interest is refolded to its native structure. General Chemical Synthetic Procedures When peptides are not prepared using recombinant DNA technology, they are preferably prepared using solid-phase synthesis, such as that generally described by Merrifield, J. Am. Chem. Soc. (1963) 85, 2149, although other equivalent chemical syntheses known in the art are employable as previously mentioned. Solid-phase synthesis is initiated from the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin. Such a starting material can be prepared by attaching an α-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a BHA resin or MBHA resin. The preparation of the hydroxymethyl resin is described by Bodansky etal., Chem. Ind. (London) (1966) 38,1597-1598. Chloromethylated resins are commercially available from BioRad Laboratories, Richmond, CA and from Lab. Systems, Inc. The preparatton of such a resin is described by Stewart etal., "Solid Phase Peptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1 , pp. 1-6. BHA and MBHA resin supports are commercially available and are generally used only when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus. The amino acids are coupled to the peptide chain using techniques well known in the art for the formation of peptide bonds. One method involves converting the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N- terminal amino group of the peptide fragment. For example, the amino acid can be converted to a mixed anhydride by reaction of a protected amino acid with ethylchloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate, pivaloyl chloride or like acid chlorides. Alternatively, the amino acid can be converted to an active ester such as a 2,4,5- trichlorophenyl ester, a pentachlorophenyl ester, a pentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimide ester, or an ester formed from 1-hydroxybenzotriazole. Another coupling method involves use of a suitable coupling agent such as N,N'- dicyclohexylcarbodiimide or N,N'-diisopropyl-carbodiimide. Other appropriate coupling agents, apparent to those skilled in the art, are disclosed in E. Gross & J. Meienhofer, The Peptides: Analysis, Structure, Biology, Vol. I: Major Methods of Peptide Bond Formation (Academic Press, New York, 1979). It should be recognized that the α-amino group of each amino acid employed in the peptide synthesis must be protected during the coupling reaction to prevent side reactions involving there active α-amino function. It should also be recognized that certain amino acids contain reactive side-chain functional groups (e.g. sulfhydryl, amino, carboxyl, and hydroxyl) and that such functional groups must also be protected with suitable protecting groups to prevent a chemical reactton from occurring at that site during both the initial and subsequent coupling steps. Suitable protecting groups, known in the art, are described in E. Gross & J. Meienhofer, The Peptides: Analysis, Structure, Biology, Vol.3: Protection of Functional Groups in Peptide Synthesis (Academic Press, New York, 1981). In the selection of a particular side-chain protecting group to be used in synthesizing the peptides, the following general rules are followed. An α-amino protecting group (a) must render the α-amino function inert under the conditions employed in the coupling reaction, (b) must be readily removable after the coupling reaction under conditions that will not remove side-chain protecting groups and will not alter the structure of the peptide fragment, and (c) must eliminate the possibility of racemization upon activation immediately prior to coupling. A side-chain protecting group (a) must render the side chain functional group inert under the conditions employed in the coupling reaction, (b) must be stable under the conditions employed in removing the α-amino protecting group, and (c) must be readily removable upon completion of the desired amino acid peptide under reaction conditions that will not alter the structure of the peptide chain. It will be apparent to those skilled in the art that the protecting groups known to be useful for peptide synthesis will vary in reactivity with the agents employed for their removal. For example, certain protecting groups such as triphenylmethyl and 2-(p- biphenylyl)isopropyioxycarbonyl are very labile and can be cleaved under mild acid conditions. Other protecting groups, such as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl. adamantyl- oxycarbonyl, and p-methoxybenzyloxycarbonyl are less labile and require moderately strong acids, such as trifluoroacetic, hydrochloric, or boron trifiuoride in acetic acid, for their removal. Still other protecting groups, such as benzytoxycarbonyl (CBZ or Z), hatobenzytoxycarbonyl, p- nitrobenzyloxycarbonyl cyctoalkytoxycarbonyl, and isopropyloxycarbonyl, are even less labile and require stronger acids, such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetate in trifiuor oacetic acid, for their removal. Among the classes of useful amino acid protecting groups are included: (1) for an α-amiπo group, (a) aromatic urethane-type protecting groups, such as fluorenyimethyloxycarbonyl (FMOC) CBZ, and substituted CBZ, such as, e.g., p- chlorobenzyloxycarbonyl, p-6-nitrobenzytoxycarbonyl, p-bromobenzyloxycarbonyl, and p-methoxybenzytoxycarbonyl, o-chlorobenzytoxycarbonyl, 2,4- dichtorobenzytoxycarbonyl, 2,6-dichlorobenzytoxycarbonyl, and the like; (b) aliphatic urethane-type protecting groups, such as BOC, t-amytoxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like; (c) cycloalkyl urethane-type protecting groups, such as cyctopentytoxycarbonyl, adamantytoxycarbonyl, and cyctohexyloxycarbonyl; and d) allyloxycarbonyl. The preferred α-amino protecting groups are BOC or FMOC. (2) for the side chain amino group present in Lys, protection maybe by any of the groups mentioned above in (1) such as BOC, p-chlorobenzyloxycarbonyl, etc. (3) for the guank ino group of Arg, protection may be by nitro, tosyl, CBZ, adamantytoxycarbonyl, 2,2,5,7,8-pentamethytohroman-6-sulfonyl or 2,3,6 -trimethyl-4- methoxyphenylsulfonyl, or BOC. (4) for the hydroxyl group of Ser, Thr, or Tyr, protection maybe, for example, by C1-C4 alkyl, such as t-butyi; benzyl (BZL); substituted BZL, such as p-methoxybenzyl, p-πitrobenzyi, p-chlorobenzyl, o-chtorobenzyl, and 2,6-dichtorobenzyl. (5) for the carboxyl group of Asp or Glu, protection may be, for example, by esterification using groups such as BZL, t- butyl, cyctohexyl, cyctopentyl, and the like. (6) for the imidazole nitrogen of His, the tosyl moiety is suitably employed. (7) for the phenolic hydroxyl group of Tyr, a protecting group such as tetrahydropyranyl, tert- butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl, and 2,6- dichtorobenzyl are suitably employed. The preferred protecting group is 2,6- dichlorobenzyl. (8) for the side chain amino group of Asn or Gin, xanthyl (Xan) is preferably employed. (9) for Met, the amino acid is preferably left unprotected. (10) for the thio group of Cys, p-methoxybenzyl is typically employed. The C-terminal amino acid, e.g., Lys, is protected at the N-amino position by an appropriately selected protecting group, in the case of Lys, BOC. The BOC-Lys-OH can be first coupled to the benzyhydrylamine or chloromethylated resin according to the procedure set forth in Horiki et al., Chemistry Letters, (1978)165-168 or using isopropylcarbodiimide at about 25°C for 2 hours with stirring. Following the coupling of the BOC-protected amino acid to the resin support, the α-amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The deprotection is carried out at a temperature between about 0°C and room temperature. Other standard cleaving reagents, such as HCI in dioxane, and conditions for removal of specific α-amino protecting groups are described in Schroder & Lubke, supra, Chapter I, pp. 72-75. After removal of the α-amino protecting group, the remaining α-amino and side-chain protected amino acids are coupled step within the desired order. As an alternative to adding each amino acid separately in the synthesis, some may be coupled to one another prior to addition to the solid-phase synthesizer. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N'-dicyclohexyl carbodiimide or diisopropylcarbodiimide. Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in excess, and the coupling is suitably carried out in a medium of dimethylformamide (DMF) or CH 2 CI 2 or mixtures thereof. If incomplete coupling occurs, the coupling procedure is repeated before removal of the N-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis may be monitored. A preferred method of monitoring the synthesis is by the ninhydrin reaction, as described by Kaiser etal., Anal. Biochem, (1970) 34, 595. The coupling reactions can be performed automatically using well known methods, for example, a Biosearch 9500 Peptide Synthesizer. Upon completion of the desired peptide sequence, the protected peptide must be cleaved from the resin support, and all protecting groups must be removed. The cleavage reaction and removal of the protecting groups is suitably accomplished simultaneously or stepwise. When the resin support is a chloro-methylated polystyrene resin, the bond anchoring the peptide to the resin is an ester linkage formed between the free carboxyl group of the C-terminal residue and one of the many chloromethyl groups present on the resin matrix. It will be appreciated that the anchoring bond can be cleaved by reagents that are known to be capable of breaking an ester linkage and of penetrating the resin matrix. One especially convenient method is by treatment with liquid anhydrous hydrogen fluoride. This reagent not only will cleave the peptide from the resin but also will remove all protecting groups. Hence, use of this reagent will directly afford the fully deprotected peptide. When the chloromethylated resin is used hydrogen fluoride treatment results in the formation of the free peptide acids. When the benzhydrylamiπe resin is used, hydrogen fluoride treatment results directly in the free peptide amines. Reaction with hydrogen fluoride in the presence of anisole and dimethylsulfide at O 0 C for one hour will simultaneously remove the side-chain protecting groups and release the peptide from the resin. When it is desired to cleave the peptide without removing protecting groups, the protected peptide-resin can undergo methanolysis to yield the protected peptide in which the C-terminal carboxyl group is methylated. The methyl ester is then hydrolyzed under mild alkaline conditions to give the free C-terminal carboxyl group. The protecting groups on the peptide chain then are removed by treatment with a strong acid, such as liquid hydrogen fluoride. A particularly useful technique for methanolysis is that of Moore etal., Peptides, Proc. Fifth Amer. Pept. Symp., M. Goodman and J. Meienhofer, Eds., (John Wiley, N.Y., 1977), p. 518-521 , in which the protected peptide-resin is treated with methanol and potassium cyanide in the presence of crown ether. Another method for cleaving the protected peptide from the resin when the chloromethylated resin is employed is by ammonolysis or by treatment with hydrazine. If desired, the resulting C-terminal amide or hydrazide can be hydrolyzed to the free C-terminal carboxyl moiety, and the protecting groups can be removed conventionally. It will also be recognized that the protecting group present on the N-terminal α-amino group may be removed preferentially either before or after the protected peptide is cleaved from the support. Purification of the polypeptides of the invention is typically achieved using conventional procedures such as preparative HPLC (including reversed phase HPLC) or other known chromatographic techniques such as gel permeation, ton exchange, partition chromatography, affinity chromotography (including monoclonal antibody columns) or countercurrent distribution. Polypeptide chains are polymerized by crosslinking monomer chains with polyfunctional crosslinking agents, including compound 1 , either directly or indirectly through multifunctional polymers. Ordinarily, two substantially identical polypeptides are crosslinked at their C or N termini using a bifunctional crosslinking agent. The agent is used to crosslink the terminal amino and/or carboxyl groups. Generally, both terminal carboxyl groups or both terminal amino groups are crosslinked to one another, although by selectton of the appropriate crosslinking agent the alpha amino of one polypeptide is crosslinked to the terminal carboxyl group of the other polypeptide. Preferably, the polypeptides are substituted at their C-termini with cysteine. Under conditions well known in the art a disulfide bond can be formed between the terminal cysteines, thereby crosslinking the polypeptide chains. For example, disulfide bridges are conveniently formed by metal-catalyzed oxidation of the free cysteines or by nucleophilic substitution of a suitably modified cysteine residue. Selectton of the crosslinking agent will depend upon the identities of there active side chains of the amino acids present in the polypeptides. For example, disulfide crosslinking would not be preferred if cysteine was present in the polypeptide at additional sites other than the C-terminus. Also within the scope hereof are peptides crosslinked with methylene bridges. Suitable crosslinking sites on the peptides, aside from the N-terminal amino and C- terminal carboxyl groups, include epsiloπ amino groups found on lysine residues, as well as amino, imino, carboxyl, sulf hydryi and hydroxyl groups located on the side chains of internal residues of the peptides or residues introduced into flanking sequences. Crosslinking through externally added crosslinking agents is suitably achieved, e.g., using any of a number of reagents familiar to those skilled in the art, for example, via carbodiimide treatment of the polypeptide. Other examples of suitable multifunctional (ordinarily bifunctional) crosslinking agents include 1 ,1-bis(diazoacetyl)-2-phenylethane; glutarakJehyde; N-hydroxysuccinimide esters (Bragg and Hou, Arch. Biochem. Biophvs. (1975) 167, 3II-32I; Anjaneyla and Staros, Int. J. Pep. Pro. Res. (1987) 30, 117-124), such as esters with 4-azidosalicylic acid; homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithtobis (succinimidyl-propionate) and dimethyladipimidate dihydrochloride (Zahn, Agnew. Chem. (1955) 67, 561-572; Golden and Harrison, Biochemistry (1982) 21, 3862-3866); bifunctional maleimides such as bis-N-maleimido-l,8-octane; disuccinimidyl suberate (Novick etal., J. Biol. Chem. (1987) 262, 8483-8487), t ≥(sulfosuccinimidyl) suberate (Lee and Conrad, J. Immunol. (1985) 134, 518-525); heterobifunctional crosslinking reagents (Lomants and Fairbanks, Arch. Biochem. Biophys. (1976)167, 311-321; Anjaneyula and Staros, ≤uβia; Partis etal., J. Pro.Chem. (1983) 2, 263-277; Wettman et al., BioTechniques, (1983)1, 148- 152; Yoshtake et al., J. Biochem. (1982) 92, 1423-1424), including those with an N- hydroxysuccinimide moiety at one end and a maleimido group on the other end; succinimidyl 4-(N-maleimidomethyI) cyclohexane - 1 - carboxylate (SMCC) (Mahan etal. Anal. Biochem. (1987)162, 163-170); sulfo-SMCC (Hashida et al., J. Applied Biochem. (1984) 6, 56-63); m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); sulfo-MBS; succinimidyl 4-(p- maleimidophenyl) butyrate (SMPB); sulfo-SMPB; N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochtoride (EDC); and N-hydroxysulfosuccinimide. Crosslinking agents such as methyl-3-[(p-azido-phenyl)dithio] propioimidate yield photoactivatable intermediates which are capable of forming crosslinks in the presence of light. If necessary, sensitive residues such as the side chains of the diargininyl group are protected during crosslinking and the protecting groups removed thereafter. Polymers capable of multiple crosslinking serve as indirect crosslinking agents. For example, cyanogen bromide activated carbohydrates and the systems described in U.S. patents 3,959,080; 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; 4,055,635 and 4,330,440 are suitably modified for crosslinking the peptides herein. Crosslinking to amino groups of the peptides is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde). Also useful are succinimidyl active esters, activated dithiocarbonate PEG, and 2,4,5 -trichlorophenyl-chloroformate- or p-nitrophenyl- chloroformate-activated PEG. Carboxyl groups are derivatized by coupling PEG-amine using carbodiimide. Ordinarily, however, the crosslinking agent is not a multifunctional polymer but instead is a small molecule being less than about 500 in MW. The peptides of this invention also may be conformationally stabilized by cyclization. The peptides ordinarily are cyclized by covalently bonding the N and C-terminal domains of one peptide to the corresponding domain of another peptide of this invention so as to form cyclooligomers containing two or more iterated peptide sequences, each internal peptide having substantially the same sequence. Further, cyclized peptides (whether cyclooligomers or cylcomonomers) are crosslinked to form 1-3 cyclic structures having from 2 to 6 peptides comprised therein. The peptides preferably are not covalently bonded through α-amino and main chain carboxyl groups (head to tail), but rather are cross-linked through the side chains of residues located in the N and C-terminal domains. The linking sites thus generally will be between the side chains of the residues. The cyclic structures of the present invention will have the general formula: wherein A and B represent the peptides of this invention and are the same or different. A and B are single peptides or head-to-tail polymers of two or more of such peptides. C represents one or more bonds or crosslinking moieties. Many suitable methods per se are known for preparing mono-or poly-cyclized peptides as contemplated herein. Lys/Asp cyclization has been accomplished using Nα-Boc-amino acids on solid-phase support with Fmoc/9-fluorenylmethyl (OFm) side-chain protection for Lys/Asp; the process is completed by piperidine treatment followed by cyclization. Glu and Lys side chains also have been crosslinked in preparing cyclic or bicyclic peptides: the peptide is synthesized by solid phase chemistry on a p-methylbenzhydrylamine resin. The peptide is cleaved from the resin and depr otected. The cyclic peptide is formed using diphenylphosphorylazide in diluted methylformamide. For an altemative procedure, see Schiller etal., Peptide Protein Res. (1985) 25, 171-177. See also U.S.Patent 4,547,489. Disulfide crosslinked or cyclized peptides are generated by conventional methods. The method of Pelton etal., {J. Med. Chem. (1986) 29, 2370-2375) is suitable, except that a greater proportion of cyclooligomers are produced by conducting there action in more concentrated solutions than the dilute reaction mixture described by Pelton et al., for the production of cyctomonomers. The same chemistry is useful for synthesis of dimers or cyclooligomers or cyctomonomers. Also useful are thiomethylene bridges {Tetrahedron Letters (1984) 25, 2067-2068). See also Cody et al., J. Med. Chem. (1985) 28, 583. The desired cyclic or polymeric peptides are purified by gel filtration followed by reversed-phase high pressure liquid chromatography or other conventional procedures. The peptides are sterile filtered and formulated into conventional pharmacologically acceptable vehicles. Specific Chemical Synthetic Procedures The products of Formula I and the preferred substituents can be made by using one of the methods depicted below or by other methods known in the art (see e.g., Spatola et al., Tetrahedron (1988) 44, 821 -833, and references cited therein). The definitions of the substituent groups are the same as for Formula I except where noted. METHOD A Support 1. Cleave R1 2. Cyclize IV A peptide derivative bound to a polymer support, depicted by intermediate II, may be prepared by sequential coupling of individual amino acid derivatives by standard techniques. (Merrifield, R. B., J. Am. Chem. Soc. (1963) 85, 2149-2154; Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis (1984), Pierce Chemical Co., Rockford, IL and additional references cited in the above publications). When the tetrapeptide derivative II is obtained, the terminal amino group is acylated with a suitable carboxylic acid derivative III. The acylation to yield IV may be accomplished using a number of standard methods which require activation of the carboxylic acid group of III. For example, activation may be obtained by the addition of an equimolar amount of dicyctohexylca od iimfcte or related carbodiimide reagent. If desired an additive such as 1-hydroxybenztriazole or N-hydroxysuccinimide may be incorporated. Alternatively, the carboxyl group may be activated by conversion to a halo derivative. For example, the chtoride may be obtained by treatment of the acid with thionyl chtoride or oxalyl chtoride in a compatible solvent such as dichtoromethane, toluene, or ethylene dichloride if desired. The substituent W is chosen such that it is readily displaceable by the group X. Suitable substituents W are, for example, halo atoms such as bromine or iodine or activated oxygen functions such as methanesulfonyloxy or p-toluensulfonytoxy and related sulfonic acid esters. Cyclization to the resin bound intermediate V may be accomplished by selectively exposing the nucleophilic group X by removal of R-|6 and altowing X to react such that it displaces group W with formation of a new chemical bond. For example, if X is a sulfur or oxygen atom and Ri β is a triphenylmethyl group, then R-iβ may be selectively cleaved from X using a very dilute solution of a strong acid such as trifluoroacetic acid in a solvent compatible with the polymer resin. EExamples of resin compatible solvents are dimethylacetamk e, dimethylformamide or dichtoromethane and the like. The end result of the cleavage process is replacement of the R-f β group with a hydrogen atom. After cleavage of R-jβ. the resin bound peptide derivative V (R-jβ = H) is allowed to react in a suitable solvent such as dimethylacetamide until cyclization is complete. If desired, a base such as N-methylmorpholine may be incorporated into the reactton. Other protecting groups in the peptide molecule IV must be stable to the reactton conditions chosen to form V. For example, Rg may be a group which affords an ester such as methoxy, ethoxy, benzyloxy, t-butyloxy and the like or an amide or substituted amide. R15 may be a protecting group such as ten 1 -butyloxycarbonyl. Final cleavage of the cyclized peptide product from the polymer resin may be accomplished in a variety of ways dependent upon the type of resin used and the chemical linkage between the cyclized peptide and the resin. If, for example, the resin is derived from a polymerized p-alkoxybenzyl alcohol derivative, then cleavage of the peptide-resin linkage may be carried out using a strong acid such as trifluoroacetic acid. If desired, additives such as phenol, anisoie and ethanedithtol may be added to the reactton. The groups Rg and R15 may be chosen, if desired, to also be cleavable concurrently with cleavage of the cyclized peptide from the polymer resin. Examples of such chemical groups are Rg = t-butyloxy, cleavage of which yields Rg = OH and R15 = t-butytoxycarbonyl, cleavage of which affords R15 = H. The crude product thus obtained may be further purified using chromatographic or other methods of chemical purification to obtain I. Further derivatization of I may be earned out if desired. For example, if X is S, treatment of I with a stoichiometric amount of an oxidizing agent such as 3-chloroperoxybenzoto acid or similar agent will produce the suifoxide derivative where X is SO. Use of an excess amount of oxidant will afford the sulfone derivative where X is SO2. METHOD B Cyclize VI Alternatively, the linear peptide derivative IV, prepared as described above in Method A, may be cleaved from the resin prior to cyclization to yield VI. For example, if IV is synthesized on a polystyrene resin the cleavage can be accomplished using liquid hydrogen flouride. The groups Rg, R15 and R16 may, if desired, be cleaved concurrently under these conditions. If concurrent cleavage is desired, then examples of suitable substituents Rg are t- butyloxy, benzyloxy or cyclohexyloxy, R15 is t-butyloxycarbonyl and R16 is triphenylmethyl or p-methylbenzyl if X is either O or S, or t-butoxycarbonyl if X is NR13. Cleavage of these groups would result in Rg being OH and R15 and 16 being hydrogen. The peptide derivative VI may then be cyclized in solution in the presence of a weak base such as ammonium hydroxide. The group W is as described in Method A. The resulting crude I may then be purified as described above in Method A. The purified I may be further transformed as described in Method A. Additionally and if desired, when X is NR13 and R13 is hydrogen, I may be acylated with, for example, acetyl chloride, acetic anhydride or benzoyl chtoride, methanesulfonyl chtoride or p-toluenesuKonyl chtoride and the like. Method C Intermediate VI may be prepared by the sequential coupling of amino acid derivatives in solution without the use of polymer resin or other solid supports. The methods useful for solution phase peptide synthesis are well documented in the chemical literature and are known to those skilled in the art (Houben-Weyl, Methoden der Organischen Chemie, 4th Edn., Vol. 15, Georg Thieme Verlag, Stuttgart 1974). The attached substituents R-j, Rg, R15 and Ri 6 may be chosen such that they are transformable concurrently or sequentially as described in Methods A and B above. Cyclization of of VI wherein Rιβ is H under conditions described above in Method B will provide compounds of Formula I. The starting materials required for the processes described herein are known in the literature or can be prepared using known methods and known starting materials. Isomeric Products In products of Formula I carbon atoms bonded to four nonidentical substituents are asymmetric. Accordingly, the compounds may exist as diastereoisomers, enanttomers or mixtures thereof. The syntheses described above may employ racemates, enanttomers or diastereomers as starting materials or intermediates. Diastereomeric products resulting from such syntheses may be separated by chromatographic or crystallization methods. Likewise, enantiomeric product mixtures may be separated using the same techniques or by other methods known in the art. Each of the asymmetric carbon atoms, when present in compounds of Formula I, may be in one of two configurations (R or S) and both are within the scope of the present invention. The carbon atoms bearing the (CH2)n sidechain and the (CH2)m sfctechain are generally preferred to have the S configuration. The carbon atom bearing the substituents R2 and R3 is generally preferred to have a configuratton corresponding to that of a D amino acid. The configuratton may be assigned R or S depending on the chemical compositton of R2 and R3. The compounds described in this invention may be isolated as the free acid or base or converted to salts of various inorganic and organic acids and bases. Such salts are within the scope of this invention. .Examples of such salts include ammonium, metal salts like sodium, potassium, calcium and magnesium; salts with organic bases like dtoyctohexylamine, N-methyl- D-glucamine and the like; and salts with amino acids like arginine or lysine. Salts with inorganic and organic acids may be likewise prepared, for example, using hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, methanesuifonto, malic, maleic, fumaric and the like. Non- toxic and physiologically compatible salts are particulariy useful although other less desirable salts may have use in the processes of isolation and purification. A number of methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, reactton of the free acid or free base form of a compound of Formula I with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble; or in a solvent like water after which the solvent is removed by evaporation, distillation or freeze drying. Alternatively, the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt or one salt form of the product may be converted to another using the same general process. The compounds described in the present invention inhibit the binding of fibrinogen to its receptor on platelets, GP lib Hl a , and thus prevent the aggregation of platelets and the formation of platelet plugs, emboii and thrombii in the circulatory system in mammals. Thromboembolic disorders have been shown to be directly related to the susceptibility of blood platelets to aggregate. Mammals exposed to medical procedures such as angioplasty and thrombolytic therapy are particularly susceptible to thrombus formation. The compounds of the present invention can be used to inhibit thrombus formation following angioplasty. They may also be used in combination with thrombolytic agents such as tissue plasminogen activator and its derivatives (US patents 4,752,603; 4,766,075; 4,777,043; EP 199,574; EP 0238,304; EP 228,862; EP 297,860; PCT WO89/04368; PCT WO89/00197), streptokinase and its derivatives, or urokinase and its derivatives to prevent arterial reocclusion following thrombolytic therapy. When used in combination with the above thrombolytic agents, the compounds of the present invention may be administered prior to, simultaneously with, or subsequent to the antithro mbolytic agent. Mammals exposed to renal dialysis, blood oxygenation, cardiac catheterization and similar medical procedures as well as mammals fitted with certain prosthetic devices are also susceptible to thromboembolic disorders. Physiologic conditions, with or without known cause may also lead to thromboembolic disorders. Thus, the compounds described herein are useful in treating thromboembolic disorders in mammals. The compounds described herein may also be used as adjuncts to anticoagulant therapy, for example in combination with aspirin, heparin or warfarin and other anticoagulant agents. The application of the compounds described herein for these and related disorders will be apparent to those skilled in the art. Platelet Inhibition Assays The evaluation of inhibitors of the fibrinogen-platelet interaction is guided by in vitro receptor binding assays and in vitro platelet aggregation inhibition assays. In-vitro biological activity of the compounds of Formula I was monitored using a modified fibrinogen-GP lib Hla ELISA based on the method of Nachman and Leung (J. Clin. Invest. (1982) 69, 263-269) which measures the inhibition of fibrinogen binding to purified human platelet GP lib Hla receptor. Human fibrinogen was prepared by the method of Lipinska, etal. (J. Lab. Clin. Med. (1974) 84, 509-516). Platelet GP lib Hla was prepared by the method of Fitzgerald, et al. {Anal. Btochem. (1985) 151, 169-177. Microtiter plates are coated with fibrinogen (10 μg/ml) and then blocked with TACTS buffer containing 0.5% bovine serum albumin (BSA). (TACTS buffer contains 20mM Tris.HCI, pH 7.5, 0.02% sodium azide, 2 mM calcium chtoride, 0.05% Tween 20, 150 mM sodium chtoride.) The plate is washed with phosphate buffered saline (PBS) containing 0.01% Tween 20 and the sample to be determined added, followed by addition of solubilized GP lib Hla receptor (40 μg/ml) in TACTS, 0.5% BSA. After incubatton, the plate is washed and 1 μg/ml of murine anti-platelet monoclonal antibody AP3 (P. J. Newman etal. Blood (1985) 65, 227-232) is added. After another wash a goat anti-mouse IgG conjugated to horseradish peroxidase is added. A final wash is performed and developing reagent buffer (10 mg o-phenylenediamine dihydrochloride, 0.0212% hydrogen peroxide, 0.22 mM citrate, 50 mM phosphate, pH 5.0) is added and then incubated until cotor devetops. The reactton is stopped with 1 N suifuric acid and the absorbance at 492 nm is recorded. In addition to the GP lib Hla ELISA assay, platelet aggregation assays may be performed in human platelet rich plasma (PRP). Fifty millil ' rters of whole human blood (9 parts) is drawn on 3.6% sodium citrate (1 part) from a donor who has not taken aspirin or related medications for at least two weeks. The blood is centrifuged at 160 x g for 10 min at 22° C and then altowed to stand for 5 min after which the PRP is decanted. Platelet poor plasma (PPP) is isolated from the remaining blood after centrifugation at 2000 x g for 25 min. T e platelet count of the PRP was adjusted to ca.300,000 per microliter with PPP. A 225 μL aliquot of PRP plus 25 μL of either a dilution of the test sample or a control (PBS) is incubated for 5 min in a Chrono-log Whole Blood Aggregometer at 25 * C. An aggregating agent (collagen, 1 mg/ml; U46619, 100 ng/ml; or ADP, 8 μM) is added and the platelet aggregation recorded. In the management of thromboembolic disorders the compounds of this invention may be utilized in compositions such as tablets, capsules or elixers for oral administration; suppositories for rectal administration; sterile solutions or suspensions for injectable administration, and the like. Animals in need of treatment using compounds of this invention can be administered dosages that will provide optimal efficacy. The dose and method of administration will vary from animal to animal and be dependent upon such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. Dosage Formulations Dosage formulations of the cyclic polypeptides of the present invention are prepared for storage or administration by mixing the the cyclic polypeptide having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poiyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sobitol; counterions such as sodium and/or nonionic surfactants such as Tween, Piuronics or polyethyleneglycol. Dosage formulations of the cyclic polypeptides of the present invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes such as 0.2 microne membranes. Cyclic polypeptide formulations ordinarily will be stored in lyophilized form or as an aqueous solution. The pH of the cyclic polypeptide preparations typically will be between 3 and 11 , more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of cyclic polypeptide salts. While the preferred route of administration is by hypodermic injection needle, other methods of administration are also anticipated such as suppositories, aerosols, oral dosage formulations and topical formulations such as ointments, drops and dermal patches. Therapeutic cyclic polypeptide formulations generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by hypodermic injection needle. Therapeutically effective dosages may be determined by either in vitro or in vivo methods. One method of evaluating therapeutically effective dosages consists of taking the cyclic polypeptide cyclo-S-acetyl-Gly-Lys-Gly -Asp-Cys-OH and determining a 50% inhibitory concentration (IC50) of inhibiting fibrinogen binding to the GP lib Hla platelet receptor. Similarly, in a platelet aggregation assay using the same cyclic peptide, the IC50 is measured. Based upon such in vitro assay techniques, a therapeutically effective dosage range may be determined. For each particular cyclic polypeptide of the present invention, individual determinations may be made to determine the optimal dosage required. The range of therapeutically effective dosages will naturally be influenced by the route of administration. For injection by hypodermic needle it may be assumed the dosage is delivered into the body's fluids. For other routes of administration, the absorption efficiency must be individually determined for each cyclic polypeptide by methods well known in pharmacology. The range of therapeutic dosages is from about 0.001 nM to 1.0 mM, more preferably from 0.1 nM to 100 μM, and most preferably from 1.0 nM to 50 μM. Typical formulation of compounds of Formula I as pharmaceutical compositions are discussed below. About 0.5 to 500 mg of a compound or mixture of compounds of Formula I, as the free acid or base form or as a pharmaceutically acceptable salt, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., as called for by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained. Typical adjuvants which may be incorporated into tablets, capsules and the like are a binder such as acacia, com starch or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent like com starch or alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose or lactose; a flavoring agent such as peppermint, wintergreen or cherry. When the dosage form is a capsule, in addition to the above materials it may also contain a liquid carrier such as a fatty oil. Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit. A syrup or elixer may contain the active compound, a sweetener such as sucrose, preservatives like propyl paraben, a cotoring agent and a flavoring agent such as cherry. Sterile composittons for injectton can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice. EXAMPLES In the following Examples, common α-amino acids may be described by the standard three letter amino acid code when referring to intermediates and final products. By common α- amino acids is meant those amino acids incorporated into proteins under mRNA direction. Standard abbreviattons are listed in The Merck Index, 10th Edition, pp Misc-2 - Misc-3. Unless otherwise designated the common α-amino acids have the natural or "L"- configuratton at the alpha carbon atom. If the code is preceded by a "D" this signifies the opposite enantiomer of the common α-amino acid. Modified or unusual α-amino acids such as norleucine (Nle) and omithine (Om) are designated as described in U.S. Patent and Trademark Office Official Gazette 1114TMOG, May 15, 1990. If the product or intermediate name is preceded by "cyclo" this shall be taken to mean that the peptide has been cyclized, e.g. compounds of Formula I or V. Example 1 Bromoacetvl-Glv-Lvs-Glv-AsD-Cvs-OH The title compound is prepared in protected form by standard solid phase peptide synthesis on 2% cross-linked polystyrene resin (Merrifield resin). Treatment of the resin bound intermediate with liquid hydrogen fluoride induces concomitant cleavage of the protecting groups from the title compound as well as cleavage of the peptide from the resin. The crude peptide is purified by reverse phase high performance liquid chromatography (HPLC) using a 4.6 mm x 250 mm column containing 10 micron, 300 Angstrom pore size C-18 packing. The elution of the column is with an acetontrile/0.1% aqueous trifluoroacetic acid gradient going from 0% - 40% acetonitrile linearly over 80 minutes. The title compound elutes at 14 minutes. Example 2 Cvclo-S-acetvl-Glv-Lvs-Glv-AsD-Cvs-OH The compound prepared in Example 1 is dissolved in deionized water (1mg/ml) and the pH of the solution is adjusted to 7.0-8.5 with ammonium hydroxide. After stirring for 4 hr at ambient temperature the reactton solution is acidified to pH 3.0 - 3.5 with trifluoroacetic acid and then lyophilized. The resulting crude product is purified by HPLC using the conditions described in Example 1. The desired title compound elutes after 11 minutes. Example 3 Cvclo-S-acetvl-Gly-Lvs-Glv-Asp-Cvs-OH Bromoacetyl-Gly-Lys(t-butyloxycarbonyl)-Gly-Asp(beta-t-butyl)-Cys(S- triphenylmethyl)-O-(polymer resin) is prepared using standard solid phase peptide synthesis utilizing fiuorenylmethoxycarbonyl (FMOC) protecting group chemistry on a p-alkoxybenzyl alcohol resin. Repeated treatment of the resin bound peptide with a 1% solution of trifluoroacetic acid in dichtoromethane results in cleavage of the S-triphenylmethyl group as evidenced by the bright' yellow of the solution. Treatment is continued until dissipation of the yellow color (ca. 1.5 L of the cleavage solution is required per gram of resin bound peptide.) After complete cleavage of the S-triphenylmethyl group, the resin bound peptide is washed several times with a 5% solution of N-methylmorphoiine in N,N-dimethylacetamide and then shaken in pure N,N-dimethylacetamide for 12 hrto complete the cyclization. Treatment of the cyclized resin bound peptide with trifluoroacetic acid containing (v/v) 1% phenol, 1% anisole and 1% ethanedithiol effects concomitant cleavage of the remaining protective groups and cleavage of the desired product from the resin. Purification of the crude product as described in Example 2 affords the title compound identical to that described above. Example 4 Synthesis of Other Compounds of Formula I Using the methods described in Examples 1 and 2, the compounds listed in Table I may be prepared. The compounds are depicted by Formula I wherein m = 1 and n = 3, Ri and Rg are OH, R14 is hydrogen and X is S. Crude products are purified using HPLC as described in Example 2. The following amino acid derivatives may be used in place of Boc-Gly for coupling to the alpha-amine group of Lys to obtain the substituents R2 and R3 shown below. When Boc- Glycine is used, R2 and R3 are both hydrogen (H). Amino Acid Derivative R 2 R3 benzyloxymethyl) His Boc-L-(N .i"m 4-imidazolylmethyl H be nzyloxy methyl) His Boc-D-Trp H 3-indolylmethyl Boc-L-Tφ 3-indolylmethyl H Boc-D-(N ε -2- H 4-amino-1 -butyl chlorocarbobenzy l-oxy) Lys Boc-L-(N ε -2- 4-amino-1 -butyl H chlorocarbobenzyl-oxy)Lys Boc-D-(N δ - H 3-amino-1-propyl carbobenzyoxy) Orn Boc-L-(N δ - 3-amino-1-propyl H carbobenzyoxy)Orn Boc-D-(0-benzyi)Thr H 1-hydoxy-1 -ethyl Boc-L-(O-benzyl)Thr 1-hydoxy-1 -ethyl H Boc-L-Pro gives R2 + R4 = CH2CH2CH2 Boc-D-Pro gives R3 + R4 = CH2CH2CH2 The substituted bromoacetic acids listed below may be used in place of bromoacetic acid in Example 1 and in combination with the amino acid derivatives listed above to provide the compounds shown in Table 1 with variable substituents at R5, R&. When bromoacetic acid is used in combination with the amino acid derivatives listed above R5 and Q are hydrogen (H). 1-naphthyl-α-bromoacetic acid 2-naphthyl-α-bromoacetic acid phenyl-α-bromoacetic acid 2-trifluromethylphenyl-α-bromoacetic acid 3-trifluromethylphenyl-α-bromoacetic acid 4-trifluromethylphenyl-α-bromoacetic acid 4-biphenyl-α-bromoacetic acid 2-bromopropionic acid 2-bromobutyric acid 2-bromopentanoic acid L-Pennicillamine may be substituted for L-cysteine in Example 1 to produce compounds in Table 1 where R7 and Re are methyl. TABLE I Selected compounds of Formula I R2 R3 R4 R5. 6 R7 δ H (4) (4) H, H H H H 1-hydroxy-1- H H,H H H ethyl H H H H, 1 -naphthyl H H H 1-hydroxy-1- H H, phenyl H H ethyl H H H H,4-biphenyl H H H 4-hydroxy- H H,H H H benzyl H H H H, phenyl H H H H H H,H CH3 CH3 H H H H H H H H H 4-imidazolyl- H H,H H H methyl H 2-methyl- H H,H H H 1-propyl H 4-hydroxy- H H, phenyl H H benzyl H H H H, 4-biphenyl H H H H H H, 1-naphthyl H H H 2-methyl- H H,H H H thioethyl H hydroxy¬ H H,H H H methyl H 3-indolyl- H H,H H H methyl H carboxamidoe H H,H H H thyl H H H H, 4-tri- H H fluoromethylp henyl H H H H, 2-naphthyl H H H H H H, propyl H H H H H H H benzyl H H H,H H H H 1-hydroxy-1- H H, phenyl H H ethyl H 4-hydroxy- H H,H CH3 CH3 benzyl H 4-amino-1- H H,H H H butyl CH3 H H H,H H H (5) H (5) H,H H H H H H H, 2-naphthyl H H 1-hydroxy-1- H H H,H H H ethyl H H H H, phenyl H H H 2-carboxy-1- H H,H H H ethyl 1 -butyl H H H,H H H 2-propyl H H H,H H H 2-butyl H H H,H H H 4-hydroxy- H H H,H H H benzyl H H H H, ethyl H H 4-amino-1- H H H,H H H butyl H H H H, 1-propyl H H benzyl H H H,H H H fluoromethylp henyl 2-methyl- H H H.H H H 1-propyl H H H H,CH 3 H H H H H H, H H pentafluoro- phenyl 2-carbox- H H H,H H H amidoethyl H H H H, 4-tri- H H fluoromethylp henyl 2-carboxy- H H H. H H H ethyl carboxy- H H H, H H H methyl Example 5 Cvcto-S-ace.vl-.D-Tvrt-Lvs-Glv-AsD-Cvs-NH2 Synthesis of the title compound is accomplished using standard Boc-synthetic protocols on a 4-methylbenzylhydrylamine resin to obtain first a linear peptide as described in Example 1. Cleavage of the linear peptide from the resin with hydrogen fluoride foltowed by cyclization as described in Example 2 affords the title compound after HPLC purification. Using this procedure the following compounds are analogously obtained. Cycto-S-acetyi-(D-Ala)-Lys-Gly-Asp-Cys-NH2 Cycto-S-acetyl- (D-Val)-Lys-Gly-Asp-Cys-NH2 Cyclo-S-acetyl-(D-Leu)-Lys-Gly-Asp-Cys-NH2 Cyclo-S-acetyl-(D-lle)-Lys-Gly-Asp-Cys-NH2 Cycto-S-acetyl-(D-Phe)-Lys-Gly-Asp-Cys-NH2 Cycto-S-acetyl-(D-Pro)-Lys-Gly-Asp-Cys-NH2 Cyclo-S-acetyl-Gly-Lys-Gly-Asp-Cys-NH2 Example 6 Cvclo-S-acetv-Glv-Lvs-Glv-AsD-Cvs-OH sulfoxide The purified product from Example 2 is dissolved in water at a concentration of 10 mg per mL. The pH of the solution is adjusted to 7. A 50% solution of hydrogen peroxide is added to make a final concentration of 3% hydrogen peroxide and the resulting reaction mixture is stirred overnight at room temperature. The solutton is toaded directly onto an octadecylsilyl reverse phase chromatography column. The sulfoxide isomers formed in the reactton are eluted with a linear gradient of acetonitrile in 1% trifluoroacetic acid in water. Example 7 Inhibition of fibrinogen binding to GP Ifc lll a Microtiter plates are coated with fibrinogen (10 μg/ml) and then blocked with TACTS buffer containing 0.5% BSA. (TACTS buffer contains 20mM Tris.HCI, pH 7.5, 0.02% sodium azkJe, 2 mM calcium chloride, 0.05% Tween 20, 150 mM sodium chtoride.) The plate is washed with phosphate buffered saline containing 0.01% Tween 20 and a dilution of the sample to be determined added, foltowed by addition of solubilized HbHIa receptor (40 μg/ml) in TACTS, 0.5% BSA. After incubation, the plate is washed and murine monoclonal anti-platelet antibody AP3 (1 μg/ml) added. After another wash goat and anti-mouse IgG conjugated to horseradish peroxidase is added. A final wash is performed and developing reagent buffer (10 mg o- phenylenediamine dihydrochloride, 0.0212% hydrogen peroxide, 0.22 mM citrate, 50 mM phosphate, pH 5.0) is added and then incubated until color developed. The reaction is stopped with 1 N suifuric acid and the absorbance at 492 nm is recorded and the IC50 values determined. Example 8 Inhibition of Vitronectin Binding to Vitronectin Receptor a. Coat 96 -well microliter plates (Nunc Maxisorp) with human vitronectin (Telios) made up at 15 μg/ml in PBS. Use 50 μl/well. Incubate overnight 4 * C. b. Remove coat solutton. Wash plates one time with 200 μl of assay buffer (50 mM Tris, 100 mM NaCI, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.4) with the addition of 3.5% BSA in assay buffer. Add additional 150μl assay buffer/well. Block plate for 1 hour at room temperature. c. Prepare test compounds in assay buffer. Prepare human vitronectin receptor, purified at 50 μg/ml concentration. d. Add 25 μl of test compounds or buffer control into the plates. Add 25 μl of the receptor solution into the plates. Incubate at room temperature with shaking for 1 hour, e . Prepare antibody solution during incubation. Combine 4B12 mab (a β3 specific antibody) at 1 :1650 with a rabbit F(ab')2 anti-murine Fc-HRP conjugate (Pel-Freez)l :7500 in assay buffer. f. Decant plates. Wash 4 times (150 μl/well) with PBS, .05% Tween 20. Add in 50 μl of the antibody solution per well. Incubate 1 hour at RT with shaking, g. Prepare OPD (orthophenylenediamine) substrate, 10mg OPD (Sigma) into 15 ml of phosphate-citrate. Then add 6 μl of 30% H2O2 to the solutton. Do this 5 minutes before use. h. Wash plates with PBS/Tween 20 4 times. Add 75 μl of the OPD solution to each well. Let reaction proceed 20-30 minutes. Add 75 μl of 1 M H2SO4 to stop reaction. Read absorbance at 492nm. In view of the efficacy of these cyclic polypeptides as inhibitors of fibrinogen binding to GP lib Hla > and the feasibility as demonstrated herein of producing these cyclic polypeptides, the present invention may have application in the treatment of a large group of disorders associated with, or characterized by, a hyperthrombotto state. Representative of such disorders are genetic or acquired deficiencies of factors which normally prevent a hyperthrombotto state; medical procedures such as angioplasty and thrombolytic therapy; mechanical obstructtons to blood flow, such as tumor masses, prosthetic synthetic cardiac valves, and extracorporeal pertuston devices; atherosclerosis; and coronary artery disease. The present invention has of necessity been discussed herein by reference to certain specific methods and materials. It is to be understood that the discussion of these specific methods and materials in no way constitutes any limitation on the scope of the present invention, which extends to any and all alternative materials and methods suitable for accomplishing the objectives of the present invention.

Claims: What is claimed is: 1. A polypeptide containing the tripeptide sequence Lys-Gly-Asp capable of inhibiting ADP-induced human platelet aggregation, and having fewer than about 345 amino acid residues. 2. The polypeptide of claim 1 having an IC50 of less than about 100nM in a Fg/GP HbHIa solid phase ELISA. 3. The polypeptide of claim 2 having an IC50 of less than about 10nM. 4. The polypeptide of claim 2 that is a cyclic polypeptide wherein the cycle contains the tripeptide sequence. 5. The polypeptide of claim 4 comprising about 20 α-amino acid residues. 6. The polypeptide of claim 5 comprising fewer than 10 α-amino acid residues. 7. The polypeptide of claim 6 where in the cycle extends through the sidechain of at least one α-amino acid. 8. The polypeptide of claim 2 that is a synthetic polypeptide. 9. The polypeptide of claim 3 further having an IC50 of greater than 10OnM in a Vitronectin/Vitronectin receptor (Vn/VnR) solid phase ELISA. 10. The polypeptide of claim 1 that is not a member of the lipocortin or calpactin family of proteins. 11. The polypeptide of claim 10 wherein the family members are selected from the group placental anticoagulant protein (PAP), Lipocortin I and Lipocortin II. 12. The polypeptide of claim 7 having the structure Xaa ! -Xaa 2 -Gly- Asp-Xaa 3 -R I — z — I wherein Xaa is a D or L α-amino acid linked to Z through the α amino group; Xaa2 is Om or Lys; Xaa3 is a D or L α-amino acid linked through the side chain to Z; Z is an amide bond, disulfide, COCH2S, COCH2SO, or COCH(C6H5)S; and R is OH or NH2. 13. The polypeptide of claim 12 wherein Xaai is a Gly or a D α-amino acid, Xaa2 is Lys, and Xaa3 is Cys. 14. The polypeptide of claim 13 wherein Xaa, is selected from the group D-tyr D-Val D-Phe D-leu D-Ala D-his Gly D-lle D-Lys D-Asn D-met D-GIn D-Pro D-Ser D-Arg D-Thrand D-Trp Xaa2 is Lys;and Xaa3 is selected from the group Cys and amino adip ' ic acid. 15. The polypeptide of claim 14 wherein Z is COCH2S or COCH (CβHsJS. 16. A polypeptide containing the tripeptide sequence Xaa-Gly-Asp, where Xaa is omithine (Om) or Lysine (Lys), having higher inhibition potency in a Fg/GPIIbIHa solid phase ELISA, than the polypeptide has in a Vn/Vn R solid phase ELISA, the polypeptide being other than placental anticoagulant Protein (PAP) ,[Orn 2 ]Echistatin, Om-Gly-Asp-Phe, and Orn-Gly -Asp- Asp. 17. The polypeptide of claim 16 having an IC50 of less than about 10OnM in the Fg/GPII¬ bIHa ELISA . 18. The polypeptide of claim 17 having an IC50 of less than about 10nM in the Fg/GPIIbIHa ELISA. 19. The polypeptide of claim 18 wherein the inhibition potency is more than 10-fold higher in the Fg. GPIIbHIa ELISA than in the Vn/VnR ELISA. 20. The polypeptide of claim 19 wherein the inhibition potency is more than 100-fold higher in the Fg/ GP llblll a ELISA than in the Vn/VnR ELISA. 21. The polypeptide of claim 19 that is a cyclic polypeptide wherein the tripeptide sequence is in the cycle. 22. The polypeptide of claim 21 comprising from 4 to 10 amino acids. 23. The polypeptide of claim 22 where in the cycle extends through the sidechain of at least one α-amino acid sidechain. 24. The polypeptide of claim 23 wherein the α-amino acid is Cys. 25. The polypeptide of claim 16 that is a synthetic polypeptide. 26. The polypeptide of claim 22 represented by Formula 1. Formula 1 wherein Ri and Rg are the same or different and are selected from hydroxy, C-|-Cβ alkoxy, C3-C12 alkenoxy, C6-Ci2 aryloxy, di-Ci -Cβ alkylamino-Cι -Cβ-alkoxy, acylamino-Ci -Cβ-alkoxy selected from the group acetylaminoethoxy, nicotinoylaminoethoxy, and succinamidoethoxy, pivaloyloxyethoxy, C6-C12 aryl-Ci -Cβ-alkoxy where the aryl group is unsubstituted or substituted with one or more of the groups nitro, halo (F, Cl, Br, I), C-|-C4-alkoxy, and amino, hydroxy-C2-Cβ-alkoxy, dihydroxy-C3-Cβ-alkoxy, and NR10R11 wherein Rio and Rn are the same or different and are hydrogen, C1 -Cβ-alkyl, C3- Cβ-alkenyl, Cβ-Ci 2-aryl where the aryl group is unsubstituted or substituted with one or more of the groups nitro, halo (F, Cl, Br, I), Cι-C4-alkoxy, and amino, C6-Ci2-aryl- C1 -Cβ-alkyl where the aryl group is unsubstituted or substituted by one or more of the groups nitro, halo (F, Cl, Br,l), Cι-C4-alkoxy, and amino; R2, R3, R5, Re. R7. Rδ are the same or different and are selected from hydrogen, C6- -12 aryl where the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), Ci-Cβ alkyl, hato-Ci- Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, phenyl, acetamido, benzamido, di-Ci-Cβ alkylamino, Ci-Cβ alkylamino, C6-C12 aroyl, Ci-Cβ alkanoyl, and hydroxy-Ci-Cβ alkyl, C1 -Cι 2 alkyl either substituted or unsubstituted, branched or straight chain where the substituents are selected from halo (F, Cl, Br, I), Ci-Cβ alkoxy, C6-C12 aryloxy where the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), Ci-Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di-Ci-Cβ alkylamino, C -Cβ alkylamino, C6-C12 aroyl, and Ci-Cβ alkanoyl, isothioureido, C3-C7 cyctoalkyl, ureido, amino, Ci-Cβ alkylamino, di-Ci-Cβ alkylamino, hydroxy, amino-C2-Cβ alkylthio, amino-C2-Cβ alkoxy, acetamido, benzamido wherein the phenyl ring is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), Ci-Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di-Ci-Cβ alkylamino, Ci-Cβ alkylamino, C6-C 2 aroyl, and Ci-Cβ alkanoyl, C6- 12 arylamino wherein the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo, Ci-Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di- C -Cβ alkylamino, Ci-Cβ alkylamino, C6-C12 aroyl, and C1- Cβ alkanoyl, guanidino, phthalimido, mercapto, Ci-Cβ alkylthio, C6-C12 arylthio, carboxy, carboxamide, carbo-Ci-Cβ alkoxy, C6-C12 aryl wherein the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo, Ci-Cβ alkyl, C1 -Cβ-alkoxy, amino, phenyloxy, acetamido, benzamido, di- Ci-Cβ alkylamino, Ci-Cβ alkylamino, hydroxy-Ci-Cβ alkyl, C6-C12 aroyl, and C -Cβ alkanoyl, and aromatic heterocycle wherein the heterocyclic groups have 5-10 ring atoms and contain up to two O, N, or S heteroatoms; R2 and R3, R5 and Re, or R7 and Re may optionally and independently be joined together to form a carbocyclic or heterocyclic ring of from four to seven atoms where the heteroatoms are selected from O, S or NR12 where R12 is selected from hydrogen, C -Cβ-alkyl, C3-Cβ-alkenyl, C6-C12-aryl, C6-Ci2-aryl-Cι- Cβ-alkyl, Ci-Cβ alkanoyl, and C6-C12 aroyl, R4 is selected from hydrogen, Ci-Cβ alkyl, C3-Cιo cycloalkyl, C6-C12 aryl, and C6-C12 aryl-Ci -Cβ-alkyl; R2 or R3 may be optionally joined with R4 to form a piperidine, pyrrolidine or thiazolidine ring; R 4 is selected from hydrogen, C1 -Cβ-alkyl, C3-Cβ-alkenyl, Cβ-Ci 2-aryl, and Ce-Ci 2 aryl-Ci -Cβ- alkyl; X is selected from an O or S atom, an S atom bearing one or two O atoms, NR13 wherein R13 is hydrogen, C -Cβ-alkyl, C3-Cβ-alkenyl, Cβ-Ci 2-aryl, C6-Ci2-aryl-Cι -Cβ-alkyl, Ci-Cβ alkanoyl, and C6-C12 aroyl, and C6-Ci2 aryl, Ci-Cβ alkanoyl, (CH2)k where k is an integer from 0 to 5; n is an integer from 1 to 6; m is an integer from 0 to 4; and pharmaceutically acceptable salts thereof. 27. A pharmaceutical compositton comprising a pharmaceutically acceptable excipient and the compound of Claim 26. 28. A method for inhibiting platelet aggregation which method comprises administering a platelet aggregation inhibiting amount of the compound of Claim 26. 29. A method for reducing platelet aggregation in a mammal, comprising administering a pharmaceutically effective amount of the composition of matter as defined by claim 26 to the mammal. 30. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the compositton of matter as defined by claim 26 to the mammal. 31. A composition of matter for reducing platelet aggregation in a mammal, comprising the compositton of matter as defined by claim 12. 32. A composition of matter for treating a mammal who has an increased propensity for thrombus formation, comprising the composition of matter as defined by claim 12. 33. A composition of matter for inhibiting fibrinogen binding to platelets in a mammal, comprising the composition of matter as defined by claim 12. 34. A method for treating a mammal who has an Increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the composition of matter as defined by claim 26 in combination with a thrombolytic agent. 35. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the composition of matter as defined by claim 26 in combination with an anticoagulant. 36. A method for treating a mammal who has an increased propensity for thrombus formation, comprising administering a pharmaceutically effective amount of the compositton of matter as defined by claim 26 following angioplasty. 37. The cyclic peptide of claim 26 wherein the cycle contains 17 or 1 δ atoms in a ring. 38. The cyclic peptide of claim 37 wherein the cyclic peptide contains at least one D-α- amino acid. 39. The cyclic peptide of claim 38 wherein the D-amino acid is in any position in the cycle except the Lys-Gly-Asp sequence.

Inventors: BURNIER JOHN P, BURNIER, JOHN, P.
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1991
Main classification: A61K37
IPC codes: A61K38, A61P7, C07K7, C07K11, C07K14