WO1995017421A1 - PEPTIDE ANALOGS OF THE FACTOR IXa PLATELET BINDING SITE - Google Patents

Abstract

Synthetic peptide analogs of human factor IXa are provided which are conformationally restricted by means of intramolecular bonding. The peptides compete with native factor IXa for binding to a platelet surface, and thereby inhibit the factor IXa-induced activation of factor X on the surface of platelet. The peptides are designed by means of an equilibrium conformational model of the factor IXa Gla domain.

Description

PEPTIDE ANALOGS OF THE FACTOR IXa

PLATELET BINDING SITE

Field of the Invention

The invention relates to synthetic peptide analogs of the factor IXa platelet binding site.

Background of the Invention

Factor IXa.

Human factor IXa is the activated form of the zymogen, factor IX. The non-activated form of factor IX is a single-chain glycoprotein of 415 residues and has considerable amino acid sequence similarities with other vitamin K-dependent proteins, such as factor VII, factor X, prothrombin, and protein C. Kurachi, et al. Proc. Natl. Acad. Sci. USA 79 6461-6464 (1982) (incorporated herein by reference) disclose the amino acid sequence of factor IX, deduced from the sequence of a cDNA insert coding for factor IX. The most easily definable functional domains of the factor IX sequence (as described from the amino to the carboxyl terminus, respectively) are: the vitamin K-dependent domain containing post-translationally modified glutamic acid residues (γ-carboxyglutamic acid or "Gla"; two epidermal growth factor (EGF) - like domain residues (which include an endothelial cell binding site ) ; an activation peptide region ; and a catalytic domain, which confers the protease function.

Factor IX activation to factor IXa involves a two-step mechanism. The Arg¹⁴⁵ - Ala¹⁴⁶ bond is cleaved, giving rise to a two-chain intermediate, which is then cleaved at the Arg¹⁸⁰ - Val¹⁸¹ bond. The second cleavage yields factor IXa as a disulfide-linked serine protease and a 35-residue activation peptide having a molecular weight of 11 kDa. During hemostasis, factor IX may be activated by factor XIa in the presence of Ca²⁺ ions or by factor Vila in a reaction requiring Ca²⁺ ions and tissue factor. Both enzymes cleave the Arg¹⁴⁵ - Ala¹⁴⁶ and Arg¹⁸⁰ - Val¹⁸¹ bonds in factor IX.

The activated form of factor IX, factor IXa, is a disulfide-linked serine protease which forms a complex with its cofactor, factor Villa. Factor IXa and factor Villa bind in close proximity to one another on the platelet surface, which facilitates the formation of the complex. Factor IXa catalyzes the activation of factor X to factor Xa by hydrolyzing internal arginine-isoleucine and arginine-glycine bonds in the factor X heavy chain.

The hydrolysis of factor X by factor IXa provides two active molecules of factor Xa. Each factor Xa molecule can associate with factor Va and calcium ions to form a prothrombinase complex on a negatively charged phospholipid surface (e.g., a platelet surface). The resulting prothrombinase complex then converts prothrombin to thrombin, which in turn catalyzes the conversion of fibrinogen to fibrin and results in clot formation. Thus, platelet binding of factor IXa and conversion of factor X to factor Xa by the platelet-bound factor IXa/factor VIII complex are essential steps for efficient clot formation in the intrinsic blood-clotting cascade.

Both human factor IX and factor IXa compete with one another to bind reversibly to 250-300 shared binding sites on the surface membrane of an activated platelet. Additionally, factor IXa binds to 250-300 more sites to which factor IX cannot bind. Hence, factor IXa can bind to twice the number of platelet sites (500-600 per platelet) as factor IX (250-300 sites per platelet). Furthermore, the presence of saturating concentrations of factor Villa and factor X can increase the affinity of factor IXa binding five fold (from a K_d (dissociation constant) of approximately 2.5 nmol\L to about 0.5 nmol\L). But, the presence of factor Villa and factor X does not affect the affinity of factor IX binding (K_d of approximately 2.5 nmol\L). Thus, the presence of factors Villa and X favor the binding of factor IXa over factor IX.

Moreover, platelet binding site occupancy with factor IXa is closely coupled with rate enhancements of factor X activation to factor Xa. These rate enhancements are achieved as a consequence of a decrease in K_m (Michaelis constant) by activated platelets to a value near the plasma concentration of factor X, combined with an increase in K_cat (turnover number, i.e. amount of inactive factor X enzymatically converted to its active form) in the presence of activated platelets and factor Villa.

The roles of Ca²⁺ concentration, the catalytic active site residues, and the Gla domain residues inbinding factor IXa on the activated platelet surface and in the assembly of the factor X activating complex have been studied. Only full-length factor IX or modified full-length factor IX was utilized for the above studies. In Factor IX, either an average of 1-3 γ-carboxyglutamic acid residues were chemically modified (Mod-Gla) or all γ-carboxyglutamic acid residues were enzymatically removed (Des-Gla). See, Rawala-Sheikh et al., Blood 79 398-405 (1992).

The enzymatic active site of factor IXa, which is specific for factor X, may be blocked by reaction with a peptide such as glutamyl-glycyl-arginyl-chloromethyl-ketone. Such full-length active site-inhibited factor IXa molecules are competitive inhibitors of both factor IXa binding to platelets and factor X activation. The K_A (inhibition constant) for factor IXa with its enzymatic active site-blocked is identical to the K_d for factor IXa binding to platelets. See, Ahmad et al. J. Biol. Chem. 264 20012-20016 (1989). Thus, the catalytic active site of factor IXa is not involved in factor IXa binding to platelet receptors. However, the modification of as little as one mole of Gla per mole of factor IX results in a complete loss of factor IXa-related coagulant activity even though the factor IXa Gla domain is not the active enzymatic site. See Rawala-Sheikh, et al. Blood, 79 398-405 (1992) at page 402. This loss of activity apparently occurs when any one of eleven of the twelve Gla residues of factor IX is modified.

The factor IX Gla domain has been studied because of its relationship to the binding of factor IXa to endothelial cells. Native peptide fragments comprising the intact Gla domain of factor IX were prepared by partial digestion of factor IX with enzymes. The partial digestion provided native peptide fragments predominantly comprising at least factor IX amino acid residues 1-42 which were purified by eluting through a Sephadex column. These native fragments of factor IX comprising the intact Gla domain inhibited the binding of factor IX to endothelium, see Ryan et al. J. Biol. Chem 264 20283-20287 (1989).

Further, a computer-generated model approximating the human factor IX Gla sequence which contains a binding site for endothelial cells was provided by Cheung, et al. J. Biol. Chem. 267 20529-20531 (1992). The computer-generated model was produced as a hypothetical approximation based on coordinates from the bovine calcium prothrombin crystalline structure. A model was produced which theoretically implicated amino acid residues 3-11 as a binding site to endothelial cells. The title of the Cheung, et al . article states that the endothelial binding site is made up of amino acid residues 3-11. However, at page 20531, col. 1, in vitro testing is described which showed that atom 11 was not part of the endothelial cell binding site. Bovine and human factor IX were found to bind equally well to bovine endothelial cells even though human factor IX has glutamine and bovine factor IX has arginine at position 11. Thus, in contrast with the hypothetical model of Cheung, et al. the in vitro tests of Cheung, et al. indicate that amino acid position number 11 of the Gla domain is not part of the factor IX endothelial cell binding site.

The accuracy of the computer model of Cheung, et al., supra, is questionable, since it does not explain the in vitro test results. Perhaps, the inconsistencies between the in vitro test data and the proposed theoretical model of the endothelial cell binding site of factor IX results from the model being an approximation. The Cheung model is hypothetical and unverified since it is based on prothrombin crystalline data and approximations from a computer program to provide a theoretical three-dimensional model of the factor IX Gla domain. Moreover, the coordinates of the Cheung, et. al , computer model are not listed. Thus, the model cannot be generated. Cheung et al. synthesized DNA constructs for producing mutant full-length factor IX by recombinant DNA techniques. Only full-length factor IX molecules were produced. The synthetic DNA constructs coded for mutant factor IX molecules containing Gla domain modifications in amino acids 1-11. All of the mutant factor IX molecules produced, preserved the two adjacent γ-carboxyglutamic acid (Gla) amino acids at positions 7 and 8 in the Gla domain. Only amino acids other than Gla amino acids were varied. Preservation of the Gla-7 and Gla-8 was necessary, since the Gla amino acids located in the Gla domain of factor IX associate with calcium ions which are critical to the three-dimensional constraint of the Gla domain in the intact factor IX. There were no attempts by Cheung, et al., supra, to synthesize short peptides of Gla domain amino acid residues as candidates for inhibiting the binding of human factor IX to endothelium cells .

The Gla-domain of factor IXa has also been implicated as being involved in the binding of factor IX to a platelet receptor. See Rawala-Sheikh, et al . Blood 79398- 405 (1992). Mutant full-length factor IXa produced by recombinant DNA techniques has been studied to investigate the role of Gla residues in the binding of factor IXa to the platelet surface. Tests with these mutant factor IXa molecules indicated that other determinants outside of the Gla domain were also responsible for the binding of factor IXa. See Ahmad et al. J. Biol. Chem. 268 8571-8576 (1992). Two of the factor IX mutants studied are chimeric proteins of factor IX wherein the EGF-1 and EGF-2 amino acid sequence segments of factor X have been substituted for the N-terminal EGF-1 of factor IX (IX_Xegf1) or the C-terminal EGF-2 domain of factor IX (IX_Xegf2), respectively. The studies with intact chimeric activated factor IX_Xegf1 (factor IXa_Xegf1) suggest either (1) that the EGF-1 domain of factor IX/IXa is not involved in factor IX/IXa binding to platelets, or (2) that the EGF-1 domain from factor X, when inserted into factor IX, suffices to promote normal factor IX/IXa binding.

In nature the Gla amino acid residues of the factor IX Gla domain are synthesized as glutamic acid residues which are post-translationally modified to the dicarboxylic Gla form by a vitamin-K dependent carboxylase. Thus, it is difficult to synthesize peptides comprising the critical γ-carboxyglutamic acid residues. Hence, recombinant DNA techniques have been used previously, which provide mutant Gla domains located in full-length factor IXa. These techniques take advantage of post-translational modification of glutamic acid to Gla by host enzymes.

Synthetic peptide analogs of the factor IXa binding site, which do not contain the Gla amino acid residues at amino acid positions 7 and 8 of the Gla domain, have not been produced prior to the present invention. Moreover, it was not known that short synthetically conformationally restricted peptides free of Gla amino acid residues that are adjacent to one another could compete with factor IXa for binding sites on the activated platelet surface.

Antithrombotic Therapy.

Existing methods for preventing or treating arterial and venous thrombosis involve inhibiting both the intrinsic and extrinsic blood coagulation cascades with oral anticoagulants, heparin or other anticoagulants, or alternatively by pharmacologically inhibiting platelets. For example, oral anticoagulants such as coumarin-like drugs are used to inhibit the syntheses of vitamin K-dependent proteins. They block many coagulation reactions, involving proteins such as prothrombin, factor VII, factor IX and factor X. Heparin, by potentiating the action of antithrombin III, accelerates inactivation of thrombin, factor Xa and a variety of other plasma serine proteases.

These therapeutic approaches are nonselective and inhibit coagulation reactions involved in the development of venous and arterial thrombosis while at the same time inhibiting reactions which are essential for the maintenance of normal hemostasis. Similarly, most platelet inhibitor drugs block a wide variety of platelet responses. Thus, while some drugs may be effective in preventing thrombotic processes, they can enhance the risk of bleeding. What is needed is a therapeutic agent which specifically interferes with intrinsic coagulation reactions leading to the activation of factor X, while leaving extrinsic coagulation reactions intact. This will permit normal hemostatic plug formation at sites of vascular injury (extrinsic coagulation), thereby minimizing the risk of bleeding during the antithrombotic therapy.

Prevention of factor IXa binding to activated platelets would limit the biologically important platelet contribution to intrinsic coagulation reactions. Accordingly, effective anti-thrombotic agents which inhibit the binding of factor IXa to surfaces of activated platelets are needed.

Summary of the Invention

A synthetic peptide is provided having an amino acid sequence length of from at least 5 to about 75 aminoacids, which sequence comprises an amino acid sequence segment corresponding to a portion of the sequence of the binding site on factor IXa for activated platelets, or a pharmaceutically acceptable salt of the peptide;

wherein the peptide has an artificially restricted conformation and is free of adjacent γ-carboxyglutamic acid residues; and

wherein the peptide has the ability to inhibit the binding of factor IXa to an activated platelet surface. Preferably, the synthetic peptide is free of γ-carboxyglutamic acid residues.

In another embodiment, a synthetic peptide is provided having an amino acid sequence length of from at least 5 to about 75 amino acids, which sequence comprises an amino acid sequence segment corresponding to a portion of the sequence of the binding site on factor IXa for activated platelets, or a pharmaceutically acceptable salt of the peptide;

wherein the peptide has an artificially restricted conformation provided at least in part by a covalent bond other than a cysteine-cysteine disulfide bond, or by a cysteine-cysteine disulfide bond which comprises at least one cysteine residue not present in the corresponding native factor IXa amino acid sequence; and wherein the peptide has the ability to inhibit the binding of factor IXa to an activated platelet surface.

In a further embodiment the invention is directed to a method of designing and synthesizing a synthetic peptide analog to the site on the factor IXa chain for binding to activated platelets. The distance between two parts of a molecular model of the factor IXa platelet binding site is determined at conformational equilibrium. The primary structure of the binding site is then modified to restrict that distance to the determined distance. The designed peptide analog having from at least five to about seventy-five amino acids and comprising the modified primary structure is then synthesized.

The invention further provides pharmaceutical compositions comprising one or more of the peptides according to the invention corresponding to the portion of the sequence of the binding site for activated platelets on the factor IXa chain, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. Preferred pharmaceutical compositions comprise a peptide according to the invention having an amino acid sequence from five to about forty-five amino acids in length, and more preferred compositions comprise a peptide having an amino acid sequence from five to about twenty amino acids in length.

The invention also provides a method of inhibiting the binding of factor IXa to the surface of an activated platelet. The activated platelets are contacted with one or more peptides of the invention, corresponding to a portion of the sequence of the binding site for activated platelets on the factor IXa chain, which compete with factor IXa in binding to the activated platelet. Inhibition of factor IXa attachment to the platelet surface, and factor IXa enzymatic activity on the platelet surface, inhibits factor IXa coagulant activity. Thus, the peptides of the invention are potent anticoagulants, having antithrombotic utility.

By "platelet binding sites" or "activated platelet binding site" on factor IX or factor IXa is meant the region of the intact IX mature polypeptide chain comprising from amino acid 1 (Tyr) to about amino acid 14 (Leu) of the mature polypeptide, corresponding to amino acids 1-14 of SEQ IDNO.1. By an amino acid sequence which "corresponds to a portion of the platelet binding site" on the factor IXa chain is meant a sequence which comprises a sequence segment identical to a portion of the platelet binding site sequence or a sequence segment derived from a three-dimensional model of a portion of the platelet binding site sequence.

By "sequence segment" it is meant a continuous portion of an amino acid sequence comprising two or more amino acid residues.

Detailed Description of the Invention

We prepared synthetic peptides based upon a testable computer model to approximating the platelet binding site of factor IX. The synthetic peptides, artificially constrained to the three-dimensional structure of our computer model (see Appendix 1 below), are potent inhibitors of factor IXa binding to platelets. The fact that the constrained, synthetic peptides are potent inhibitors of factor IXa binding to platelets establishes that our computer model is an accurate three-dimensional representation of the factor IXa platelet binding site.

The synthetic peptides compete with factor IXa for binding sites on the platelet surface. Activation of factor IX to factor IXa and the expression of factor IXa enzymatic activity on the platelet surface are key biological events in hemostasis. The binding of factor IXa to platelets is essential for efficient factor X activation. Prevention of factor IXa binding to activated platelets inhibits the biologically important platelet contribution to coagulation reactions involving factor IXa. Thus, the synthetic peptides provide a potent therapeutic effect by inhibiting the binding of factor IXa to platelets. Importantly, peptides which have the same amino acid sequence as the constrained peptides, but not constrained to the three-dimensional structure of the computer model, are inactive or substantially less active than the constrained peptides.

Computer modeling has provided a testable three-dimensional representation of the factor IXa platelet binding site. The space-filling model was calculated by utilizing the calculated structure's primary amino acid sequence. Hypothetical disulfide linkages were located within the Gla domain model equal to the distances between molecules.

The computer model of the Gla domain three-dimensional structure was constructed by starting with the coordinates for the crystalline structure of the prothrombin fragment 1 Gla domain (Soriano-Garcia, et al. Biochem. 31 2554-2566, (1992). The initial coordinates were developed further by changing the model's prothrombin Gla domain amino acid sequence to the amino acid sequence of residues 1 to 47 of the factor IX Gla domain. The three-dimensional mathematical modeling was performed using the biopolymer module provided within the SYBYL computational chemistry package. The Amber force field, as implemented in SYBYL, was utilized in all the subsequent calculations. Atomic parameters describing calcium and the γ-carboxylated glutamic acid residues were added to the force field tables to increase the accuracy of the computer model.

After the changes to the model due to the amino acid replacements were completed, the structure was energy minimized to convergence using a conjugate-gradient approach. The newly minimized structure was then solvated with water using the Silverware algorithm as implemented in SYBYL. The water/protein complex was again energy minimized prior to an energy-dependent simulation of molecular motion.

Our resulting three-dimensional model of the Gla domain structure of factor IXa was used as a design template for synthesizing constrained peptides according to the present invention. These peptides are expected to adopt a conformational repertoire overlapping that of the native protein. The modified, constrained peptides identified herein are free of adjacent γ-carboxyglutamic acid residues except when they are constrained by a covalent bond other than a cysteinecysteine disulfide bond. The synthetic peptides inhibit factor IXa binding to platelets. Thus the peptides according to the present invention are potent anticoagulates, which are believed useful as antithrombotic agents. Ideally, an antithrombotic agent should interfere with intrinsic coagulation reactions leading to the activation of factors XI and IX while leaving extrinsic coagulation reactions intact. Normal hemostatic plug formation can occur at sites of vascular injury via intact extrinsic coagulation reactions. The synthetic peptides of the invention are specific for the platelet binding site on factor IXa and have the ability to inhibit the binding of factor IXa to platelets. Thus, the synthetic peptides will inhibit factor IXa enzymatic activity on the surface of platelets, without affecting the extrinsic pathway of blood coagulation involving factor VII, X and V, and prothrombin.

The inventive peptides are specific in their inhibitory effect on the intrinsic coagulation pathway. Hence, the peptides will inhibit or minimize intravascular thrombus formation without sacrificing normal hemostatic plug formation.

Traditional synthesis of the linear amino acid sequence of biologically interesting proteins may result in peptides that are either biologically inactive or, at best, marginally active. Previously the three-dimensional structure of the factor IX has been preserved by modifying the binding site portion of the molecule and leaving the rest of the molecule intact . For example, activity of the modified binding site of factor IX with endothelial cells depended upon the intact molecule to provide a three-dimensional conformation.

Our molecular model of the factor IXa Gla domain provides a template for designing conformationally-restricted synthetic analogs to the factor IXa site which binds to the platelet surface. The synthetic conformationally-restricted analogs have the ability to compete with factor IX and factor IXa for binding sites on platelet surface. Thus, the synthetic analogs inhibit the binding of factor IX or factor IXa to sites on the platelet surface. This in turn prevents the enzymatic activity of factor IXa from being expressed on the platelet surface.

Factor IXa binds to 500-600 binding sites per platelet. Factor IX will only bind to half of those sites. Thus, the constrained peptides mimic factor IXa, since they have the ability to bind to all 500-600 platelet sites. Using both distance and geometric constraints imparted through measurement of the subdomains within the calculated factor IXa binding site structure, constraints are artificially introduced, e.g., disulfide bonds to limit the conformational freedom of a synthetic peptide that incorporates the relevant amino acids. Certain conformationally restricted synthetic analogs having the ability to inhibit the binding of factor IX and factor IXa to platelets correspond to factor IXa chain residues 3-11, according to the numbering of the amino acids of the mature polypeptide. The model disclosed may be utilized to prepare additional conformationally-restricted synthetic peptides having similar activity.

Appendix 1 included herein contains the set of

Brookhaven coordinates and connect statements specifying our equilibrium conformation model of the major portion of factor IXa Gla chain domain comprising the 48 amino acids spanning positions Tyr 1 To Gly 48, inclusive. (SEQ ID NO:1, amino acids 1-48). The remaining amino acids of the factor IXa sequence were truncated.

A corresponding graphic molecular model satisfying these coordinates may be generated by inputting the coordinates and connect statements into any of the many commercially available molecular modeling programs which are capable of reading files in the Brookhaven format. Such programs include, for example, those of BioDesign, Inc., Pasadena, CA; Biosym Technologies, San Diego, CA; Tripos, St. Louis, MO; Polygen, Waltham, MA; and Chemical Design Ltd., Oxford, UK. The data may be entered as an ASCII file.

According to the Brookhaven format shown in Appendix 1, each of the atoms of factor IXa chain Gla domain, residues 1-48 is assigned a number and respective X, Y and Z coordinates. The coordinate portion of the listing begins with the Tyrosine residue (Tyr 1) at position one of the mature factor IXa heavy chain. The atom types are identified as "N" for nitrogen, "HN" for hydrogen which is connected to a nitrogen atom, "C" for carbon, "CA" for a carbon, "CB" for β carbon, "CG" for γ carbon, and so forth. Identical atoms of branched side chains are indicated by numbers. Thus, the two γ carbons of VAL 10 are designated "CG 1" and "CG 2" respectively.

The data file further comprises a connect statement which begins immediately after the coordinates for atom 539. The connect statement identifies the covalent bonding pattern of each of the 525 atoms of the 48 amino acids residue chain and atoms 526-539 which are the unnamed carboxylic terminating sequence. The complete data file of 525 coordinates, together with the connect statement for these entries, specifies the equilibrium conformation of the factor IXa Gla domain.

The amino acid sequence in the computer model can be modified as follows to eliminate Gla amino acid residues in the three-dimensional structure and maintain the binding surfaces represented by amino acid Gly 4 - Lys 5 - Leu 6 and by Phe 9 - Val 10 - Gln 11. The Gla residues at position 7 and 8 are changed to asparginine and glutamine residues, respectively. Also proline amino acid residues are substituted for serine at position 3 and glycine at position 11. Surprisingly, these changes which eliminate the two Gla amino acid residues introduce a folding pattern similar to that present in the native structure. The predicted folding pattern for this putative structure was tested for its ability to mimic the structure observed in our model of the Gla domain of factor IXa. Satisfactory agreement was found between our proposed model according to Appendix 1 and the modified putative structure.

The analogs of the invention generally have an amino acid sequence similar to the native Gla domain sequence in the vicinity of the platelet binding site. However, a covalent modification is artificially introduced to restrict each analog to the conformation (or one close to it) displayed by the above model. Preferably, the analogs consist essentially of a peptide having from at least five (5) to about seventy-five (75) amino acid residues. Preferably the analog has at least five (5) to about forty-five (45) amino acid residues, most preferably from about five (5) to about twenty (20) amino acid residues. Generally, the covalent modification is accomplished by determining a distance between two noncontiguous parts of the amino acid chain according to the model. Then a chemical moiety is introduced to fix that determined distance in the analog. For example, a 5-6A distance can be fixed using a disulfide bond. Cysteine residues can be introduced at the appropriate positions in the model followed by testing the new cysteine-containing model for its ability to mimic the structure observed in the model. Alternatively, the disulfide bond can be artificially introduced by generating a disulfide bond between native cysteine residues in the synthetic polypeptide when this will produce a polypeptide with a restricted conformation corresponding to the above model.

In constraining the peptide analogs it is sometimes necessary to compensate for the orientation of amino acid side chains such that torsional stress does not misalign the peptide structure. Thus, in some instances, it is desirable to employ D-Cys analogs or appropriate combinations of D-L cysteine to mimic the correct stereochemistry. In general, these peptides are then synthesized according to the standard chemistry described below.

The use of native or artificially introduced cysteine residues to create the artificially introduced disulfide bridge is one way to conformationally restrict the peptides. Disulfide bonds, however, can be intrinsically unstable and it is sometimes difficult to obtain a homogeneous solution of intradisulfide-bonded species without concomitant mixed disulfides. If a biologically active conformationally restricted peptide having a cysteine-cysteine disulfide bond tends to unfold, it may be more effective to constrain the peptide in a folded conformation via a covalent bond which is more stable than a disulfide bridge. There are several strategies which can be utilized in the covalent closure of the peptides. Two of these strategies are described below.

The peptide can be internally cross-linked via the side chains of a lysine e -amino group and the carboxylic acid function of a glutamic or aspartic acid side chain, thus creating an amide bond. The peptide is synthesized according to standard procedures on a low substitution (0.2 mmol/gm or less) paramethylbenzhydrylamine resin. The first residue added to the resin is an N-α-tBOC,∈-fMOC lysine. The rest of the peptide synthesis is continued normally using tBOC chemistry until the final residue is added. The last residue to be added is a Z-protected glutamic acid, where the carboxylic acid moiety is protected with a tertbutyl group. Treatment of the peptide resin with piperidine/DMF removes the fMOC group from the ∈-amino group of the initial lysine without affecting any other protection groups. Subsequent treatment with trifluoroacetic acid removes the protection of the carboxylic acid group of the glutamic acid. Following neutralization, the peptide is covalently closed using a standard diimide-mediated coupling reaction. It should be emphasized that this is only one of the ways in which the synthetic peptide can be covalently closed.

Other fMOC/tBOC strategies include covalent closure of the peptide between two free amino groups utilizing toluene-2,4-diisocyanate (TDI), a heterobifunctional cross-linker. The methyl group of the aromatic ring of TDI prevents the isocyanate group in the 2 position from reacting at a pH 7.5 or below, whereas the isocyanate group in the para position is highly reactive. A shift in pH to greater than 9.0 will initiate a reaction with the isocyanate group in the 2-position, thus enabling highly specific and controlled conditions for covalent closure of the peptide.

By utilizing a variety of different strategies for restricting the conformation of peptides, distance geometries and orientation of the folded peptide can be controlled. Any such strategies employing chemical reactions known in the art may be used.

Using the above described techniques, synthetic peptide analogs can be made and tested for their ability to inhibit factor IXa binding to the platelet surface and to inhibit factor IXa enzymatic activity on the platelet surface.

Particularly useful peptide analogs which were derived using the techniques described herein comprise amino acids corresponding to segments of the factor IXa Gla domain sequence residues 1-14. Conformationally restricted peptides corresponding to factor IXa residues 4-11 (SEQ ID NO: 2) and residues 4-8 and 9-11 (SEQ ID NOS: 3-4), respectively. The predicted folding pattern of the peptides were tested for their ability to mimic the structure observed in our model of the Gla domain of factor IXa. Finding satisfactory agreement, the peptides were synthesized according to conventional solid phase procedures on an Applied Biosystems 43 OA Peptide Synthesizer by a modification of the procedure described by Kent et al. in Synthetic Peptides in Biology and Medicine eds. Alitalo et al. (Elsevier Science Publishers, Amsterdam, pp. 29-58 (1985)), in which dimethylformamide replaced methylene chloride in the routine wash cycles. The synthesis was carried out using a paramethylbenzhydrylamine resin (United States Biochemical Corp., Cleveland, OH). The solvents and protected amino acids were synthesis grade biotechnology products purchased from Fischer Scientific Co., Pittsburgh, PA. The resulting peptide was folded into a three-dimensional constrained conformation in a separate chemical reaction step after the peptide was purified.

The folded peptides were examined by both reverse phase and gel filtration high performance liquid chromatography (HPLC). Each of the three folded peptides demonstrated a single homogenous peak with a retention time identical to the corresponding unfolded peptide. This indicated the presence of a single homogeneous mixture for each folded peptide, and not a mixed population of diverse polymers.

The hexapeptide, SEQ ID NO: 3, has an amino acid sequence identical to Factor IXa amino acids 2-7, except that the first and last amino acids have been replaced with cysteine residues. Similarly the peptide, SEQ ID NO: 4, is identical to factor IXa amino acids 8-12, except for the replacement of the first and last amino acids with cysteine residues.

The peptide, SEQ ID NO: 2, is identical to amino acids 2-13 of native factor IXa except (i) the first and last amino acids (Asn 2 and Asn 13) are replaced with cysteine residues; (ii) the serine corresponding to position 3 and the glycine corresponding to position 12 of the native peptide are replaced with proline residues; and (iii) the two Gla residues at position 7 and 8 are replaced with Asp and Glu residues, respectively. Hence, the SEQ ID NO : 2 peptide consists of the amino acid sequence Cys - Pro - Gly - Lys - Leu - Asp - Glu - Phe - Val - Gln - Pro - Cys .

Each of the three peptides, SEQ ID NOS: 2-4, were conformationally restricted using cysteine-cysteine disulfide bonds. Other restricting means may be advantageously used. Each peptide inhibits the binding of factor IXa to the platelet surface. As a consequence, each peptide may be used to inhibit the procoagulant function of factor IXa by limiting platelet involvement in intrinsic coagulation. Methods of assaying factor IXa binding to the platelet surface are known in the art. One such method is described hereinafter in Example 5(d).

The present peptides are relatively short in length and therefore they are easily synthesized by chemical means. Moreover, the peptides are preferably free of the traditionally difficult-to-synthesize adjacent (consecutively occurring) γ-carboxyglutamic acid residues. More preferably, the peptides are free of γ-carboxyglutamic acid residues. Such synthetic peptides have many advantages over the use of native amino acids 1-48 of the Gla domain of factor IXa or the entire factor IXa chain.

Historically, portions of the factor IXa chain comprising a γ-carboxyglutamic acid residue have not been readily produced by synthetic techniques. Such peptides are usually made by recombinant DNA techniques, which are expensive and time consuming. Further, the native factor IXa Gla domain requires calcium ions to interact with its Gla residues to assume the proper three-dimensional shape. Synthetic peptides which are conformationally constrained without Gla residues do not require the presence of calcium ions for proper three-dimensional shape. Also, shorter synthetic peptides may be more soluble and less immunogenic than larger proteins.

As used herein, "peptide" refers to a linear series of no more than about seventy-five (75) amino acid residues connected to one another by peptide bonds between the alpha-amino groups and carboxy groups of adjacent amino acid residues. Additional covalent bonds between portions of the peptide are also present to restrain the conformation of the molecule, such as amide and disulfide bonds. The term "synthetic peptide" means a chemically derived chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.

The term "homology" as describing the relationship between two amino acid sequences means the extent to which the sequences, viewed from the N-terminal to the C-terminal direction, have segments of their sequences which are identical and which occur in the same N-terminal to C-terminal order in the overall sequence. The synthetic peptides according to the invention have an amino acid sequence which is the same as that of the native amino acid sequence, but for inserted, deleted, or interchanged (one or more amino acids is substituted for the same number of other amino acids) portions.

The degree of amino acid sequence homology between the amino acid sequence of a synthetic peptide according to the invention and that of the native peptide is expressed as a percentage. This percentage is obtained by determining the number of amino acids in the sequence of the synthetic peptide which occur in segments that are identical to segments of the native amino acid sequence and which occur in the same N-terminal to C-terminal order as the native segments, divided by the total number of amino acids in the native sequence.

A "substantial amino acid sequence homology" is any amino acid sequence homology greater that 30 percent. Preferably the homology is greater than 80 percent, most preferably greater than 90 percent.

Peptides of the present invention include any analog, fragment or chemical derivative of the peptides capable of inhibiting the binding of factor IXa binding to platelets. The term "analog" includes any peptide having substantial amino acid sequence homology to the peptides of the invention in which one or more amino acids have been substituted with other amino acids, and the substituted amino acids allow or require the peptide to assume the equilibrium conformation of the domain of the parent protein. Often, cysteine, lysine and glutamic acid will be used for their side chains which can form covalent linkages to restrict the conformation of a peptide. In addition, conservative amino acid changes may be made which do not alter the biological function of the peptide. For instance, one polar amino acid, such as glycine, may be substituted for another polar amino acid; or one acidic amino acid, such as aspartic acid may be substituted for another acidic amino acid, such as glutamic acid; or a basic amino acid, such as lysine, arginine or histidine may be substituted for another basic amino acid; or a non-polar amino acid, such as alanine, leucine or isoleucine may be substituted for another non-polar amino acid.

The term "analog" shall also include any peptide which has one or more amino acids deleted from or added to an amino acid sequence identical to that of the native fragment of the amino acid sequence in the Gla-domain in the factor IX chain, but which still retains a substantial amino acid sequence homology to the platelet binding site on factor IXa, as well as the ability to inhibit the binding of factor IXa to platelets. Further, the preferred peptides do not contain adjacent γ-carboxy-glutamic acid amino acid residues, and more preferably have no γ-carboxyglutamic acid amino acid residues .

The term "fragment" shall refer to any shorter version of the peptides identified herein having at least five amino acid residues, wherein the fragment is a synthetic peptide which is capable of inhibiting the binding of factor IXa to platelets .

The three-letter symbols used to represent the amino acid residues in the peptides of the present invention are those symbols commonly used in the art. The amino acid residues are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid, as long as the desired functional property of inhibition of factor IXa-induced factor IX activation is retained by the peptide. The three-letter symbols used herein refer to the following amino acids: Ser is serine; lie is isoleucine; Gln is glutamine; Phe is phenylalanine; His is histidine; Trp is tryptophan; Lys is lysine; Asn is asparagine; Leu is leucine; Gly is glycine; Thr is threonine; Asp is aspartic acid; Arg is arginine; Gla is γ-carboxyglutamic acid; and Ala is alanine.

The peptides of the present invention may be prepared by any of the following known techniques. Conveniently, the peptides may be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc. 15, 2149-2154 (1963). Other peptide synthesis techniques may be found, for example, in M. Bodanszky et. al., Peptide Synthesis, John Wiley & Sons, 2d Ed. (1976); Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, eds. Alitalo, K., Partanen, P. and Vakeri, A., (Elsevier Science Publishers, Amsterdam, 1985) p. 295-58; as well as other reference works known to those skilled in the art. A summary of peptide synthesis techniques may be found in J. Stuart and J.D. Young, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, IL (1984). The synthesis of peptides by solution methods may also be used, as described in The Proteins, vol II, 3d Ed., Neurath, H. et al., Eds., p. 105-237, Academic Press, New York, NY (1976). Appropriate protective groups for use in such syntheses will be found in the above texts as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, NY (1973). Of course, the present peptides may also be prepared by recombinant DNA techniques. But, such methods are not preferred because of the need for purification and subsequent chemical modifications to conformationally restrain the peptides.

In general, these synthetic methods involve the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively-removable protecting group. A different, selectively-removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.

Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide. The peptides of the invention are devoid of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.

The peptides of the present invention generally contain at least five (5) amino acid residues and up to seventy-five (75) amino acid residues, preferably from about five (5) to about forty-five (45) amino acid residues, and as small as five (5) to about twenty (20) amino acids. The peptides may be linked to an additional sequence of amino acids either or both at the N-terminus and at the C-terminus, wherein the additional sequences are from 1-100 amino acids in length. Such additional amino acid sequences, or linker sequences, can be conveniently affixed to a detectable label or solid matrix, or carrier. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid and aspartic acid, or the like.

Any peptide of the present invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids which are capable of forming salts with the peptides of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.

Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such a mono-, di- and trialkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine and the like) .

The present peptides are useful in a pharmaceutical composition for treatment to prevent intrinsic blood clotting. Such a pharmaceutical composition may be used to inhibit the binding of a platelet to factor IXa, or to inhibit the coagulant activity of factor IXa on the platelet surface. Thus, one or more of the synthetic peptides of the present invention may be present in a pharmaceutical composition in admixture with a pharmaceutically-acceptable carrier. The pharmaceutical composition may be compounded according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral or parenteral.

Compositions for oral dosage form may include any of the usual pharmaceutical media, such as, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (e.g., suspensions, elixirs and solutions) or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (e.g., powders, capsules and tablets). Controlled release forms may also be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.

For compositions comprising the peptide according to the invention to be administered parenterally, the carrier will usually comprise sterile water, although other ingredients to aid solubility or for preservation purposes may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The parenteral routes of administration may be intravenous injection, intramuscular injection or subcutaneous injection.

For intravenous administration, the peptides may be dissolved in an appropriate intravenous delivery vehicle containing physiologically compatible substances such as sodium chloride, glycine and the like, having a buffered pH compatible with physiologic conditions. Such intravenous delivery vehicles are known to those skilled in the art.

It is contemplated that the peptides of the present invention have utility as anticoagulant and/or antithrombotic agents. It is contemplated that the peptides may be administered to patients either at risk for developing arterial or venous thrombosis, or to patients with established thromboembolism to prevent extension of the thrombi. For example, it is contemplated that the peptides may find utility in the prevention and treatment of deep venous thrombosis and pulmonary embolism, treatment and prevention of cerebral vascular thromboembolism, the treatment and prevention of systemic arterial thrombosis and embolism, and the treatment and possibly the prophylaxis of established disseminated intravascular coagulation. Patients suffering from transient ischemic attacks are, in particular, at increased risk of brain damage through thrombus formation.

In particular, it is contemplated that the peptides of the present invention will find utility in the prevention of rethrombosis following lytic therapy. While lytic agents such as tissue plasminogen activator, urokinase and strep-tokinase have been utilized to dissolve vascular thrombi, their use is associated with a significant rate of rethrombosis, about 20-30%. This is because lytic therapy results in the exposure of a thrombogenic site, at the location of the prior thrombus. While lytic agents are effective in dissolving vascular thrombi, they offer no protection from clot reformation. The peptides of the present invention, by virtue of their inhibition of the binding of factor IXa to the platelet surface and thus inhibition of factor IXa-induced activation of factor X on the platelet surface, are expected to possess substantial rethrombosis inhibiting activity. They may thus be administered as an adjuvant to lytic therapy to prevent reformation of dissolved vascular thrombi.

The peptides may be administered by any convenient means which will result in the delivery to the bloodstream of an amount effective to inhibit the binding of factor IXa to the platelet surface. Intravenous administration is presently contemplated as the preferred administration route. The amount administered will depend on the activity of the particular compound administered, which may be readily determined by those of ordinary skill in the art. The amount may also vary depending on the nature and extent of the lesion which is to be protected from rethrombosis; the size and weight of the patient; the route of administration, the age, sex and health of the patient; and other factors. Generally, the peptides may be administered in an amount sufficient to provide a plasma concentration in the range of from about 10^-9 to about 10^-5 M, more preferably in the range of from about 1 × 10^-8 to about 5 × 10^-6 M. Plasma concentrations higher or lower than these may be utilized, depending upon the activity of the particular compound being administered, and the nature of treatment.

It may be appreciated that a single bolus injection of 1 mg peptide per kilogram of treated subject body weight would achieve a maximum in vivo plasma concentration of 100 nM, assuming 100% recovery of drug. It is therefore contemplated that bolus administration will comprise a dosage of from about 0.1 mg to about 1 gram, per kilogram subject body weight. The bolus administration is most advantageously followed by a continuous infusion of peptide, as needed. The amount of peptide continuously infused depends on the approximate half- life of the peptide in the circulation. Those skilled in the art would, for any factor IXa platelet-binding-inhibiting peptide, be readily able to determine the half-life from routine experimentation. The peptides of the invention are expected to inhibit intrinsic coagulation without affecting extrinsic coagulation. According to one exemplary treatment protocol, an amount of peptide shown effective by the in vivo assay described elsewhere herein, is administered to a patient by bolus administration and/or continuous infusion. The potency of the peptide and its clearance from the circulation is then monitored by drawing and assaying blood samples at timed intervals. The samples are assayed in parallel with control samples to compare clotting times. At the end of the evaluation period, the dosage is adjusted to provide the desired in vivo effect.

The following non-limiting examples serve to illustrate the practice of the invention.

Example 1

Computer Model

A structural model approximating the Factor IXa Gla domain (residues Tyr 1-Gly 48) was constructed using the computational chemistry package supplied by Molecular Simulations, Inc., Pasadena CA and a Silicon Graphics 4D 280 Parallel Processing Supercomputer. A description of the modeling package and methods has been previously published (Jameson, Nature 349, 465-466 (1989)). The coordinates from the bovine prothrombin a Gla crystal structure (Soriano-Garcia, et al. Biochem. 31 2554-2566 (1992) were used as a guideline. The amino acids of factor IXa Gla domain, residues 1-48, were substituted for the amino acids in the bovine prothrombin 1 Gla crystal structure based on sequence alignment of the prothrombin Gla domain and the factor IXa Gla domain. Thus, exchanges of the amino acids and resulting changes to coordinates were performed using the biopolymer module provided within the SYBYL computational chemistry package (Tripos Associates INC, St. Louis MO.). The Amber forcefield, as implemented in the SYBYL package, was utilized in all the subsequent calculations (Weiner , et al . J . Am . Chem . Soc . 106765-784 (1984). Atomic parameters describing calcium and γ-carboxylated glutamic acid residues were added to the force field table. The atomic properties of calcium (a transition state metal) are inadequately described within the force field tables to account for the coordination complexes formed between calcium atoms and the negative charges of the γ-carboxylated glutamic acids. Therefore, the distance-geometry measurements related to Ca²⁺ atoms and Gla residues in the prothrombin Gla domain coordinated complexes were held as restraints (not allowed to vary) during the modeling process of creating the factor IXa structure.

After all amino acid replacements were completed, and the additional atomic parameters and coordinates restraints added, the structure was energy minimized to convergence using a conjugate-gradient approach. Several ten picosecond high energy (900°K) dynamic runs (energy-dependent simulations of molecular motion) were used to dislodge inappropriate amino acid contacts. The structure was allowed to cool to 300°K over a 100 picosecond dynamics calculation, followed by minimization of the resulting structure. The newly energy minimized structure was then solvated with water (2 solvent shells were added to insure that all portions of the surface were adequately solvated) using the Silverware algorithm as implemented by SYBYL. The water-protein complex was again energy minimized prior to an energy-dependant simulation of molecular motion (t=100 picoseconds). A trajectory file, recorded during this entire dynamic run, indicated that after ~12 picoseconds of dynamics, the calculated backbone structure had stabilized, i.e., reached a low energy well. Thus, a stable low energy structure was obtained. Since a disulfide-bonded cysteine has an ideal bond length from α-carbon to a-carbon of ~5-6A, we searched the factor IXa Gla domain structure model for ideal disulfide distances as well as for locations where a disulfide bond would not be expected to induce torsional stress. The calculated structure coordinates for the stable structure are set forth in Appendix 1.

Examples 2-4

Particularly useful peptide analogs which were derived using the techniques described herein comprise amino acids corresponding to segments of the factor IXa Gla domain sequence residues 1-14. Conformationally restricted peptides corresponding to factor IXa amino acid residues 4-11 (SEQ ID NO: 2) and residues 4-8 (SEQ ID NO: 3) and residues 9-11 (SEQ ID NO: 4) were produced. To maintain the binding surfaces represented by amino acids Gly 4-Lys 5-Leu 6 and by Phe 9-Val 10-Gln 11 and eliminate γ-carboxyglutamic acid residues, the Gla residues at position 7 and 8 were changed to introduce a folding pattern similar to that present in the native structure. The predicted folding pattern of the putative structure was tested for its ability to mimic the structure observed in our model of the Gla domain of factor IXa. Finding satisfactory agreement, the peptides were synthesized according to conventional solid phase procedures on an Applied Biosystems 430A Peptide Synthesizer by a modification of the procedure described by Kent et al. in Synthetic Peptides in Biology and Medicine (Elsevier Science Publishers, Amsterdam, pp. 29-58 (1985)), in which dimethylformamide replaced methylene chloride in the routine wash cycles. The synthesis was carried out using a paramethylbenzhydrylamine resin (United States Biochemical Corp., Cleveland, OH). The solvents and protected amino acids were synthesis grade biotechnology products purchased from Fischer Scientific Co., Pittsburgh, PA. The resulting peptides were folded into a three-dimensional constrained conformation in a separate chemical reaction step after the peptide was purified as follows.

The peptides were each dissolved in deionized water as a 0.1 mg/ml solution in a flask containing a stir bar. The pH was adjusted to 8.5 with NH₄OH and each of the three solutions were allowed to stir at 5°C for at least three days. Each of the resulting solutions was lyophilized.

The folded peptides were examined by both reverse phase and gel filtration high performance liquid chromatography (HPLC). The HPLC system was the Waters 600 Gradient Module, Model 740 Data Module, Model 46K Universal Injector and Lambda-Max Model 481 Detector. Reverse phase chromatography was performed using a Waters C8 μBondapak Column equilibrated with 0.1% (V/V) trifluoroacetic acid. The column was eluted with a linear gradient of aqueous acetonitrile containing 0.1% trifluoroacetic acid with a detector set at a wavelength of 206 nm. Gel filtration of the peptides was also carried out using a Waters Protein-Pak 60 column which was run isocratically with 0.1% (V/V) trifluoroacetic in 20% acetonitrile. Each of the three folded peptides demonstrated a single homogenous peak with a retention time identical to the corresponding unfolded peptide. This indicates the presence of a single homogeneous mixture for each folded peptide, and not a mixed population of diverse polymers.

Example 5

Effect of Gla-Domain Derived Peptide on the Binding of Factor IXa to Platelets

A. Purif ication of Human Coagulation Factors.

Human coagulation proteins, including factor IX, factor IXa, factor VIII, factor X and α-thrombin, were purified, assayed and characterized as previously published (Ahmad, et al. J. Biol. Chem. 264 3244-3251, (1989). The conditions used for activation of factor VIII with human α-thrombin were identical to those previously published. All proteins were >98% pure as determined by polyacrylamide slab gel electrophoresis. Factor IX molecule was radiolabeled with ¹²⁵I by the iodogen method as previously described (Ahmad, et al. supra). Specific radioactivities of all proteins were in the range of 2.0-2.5 × 10⁶ cpm/μg. Activation of factor IX by purified factor XIa was carried out as described by Ahmad, et al. supra. The p-aminobenzamidine fluorescence assay was employed to quantitatively examine the activation of factor IX as previously reported by Lin, et al. J. Biol. Chem. 265 144 - 150 ( 1990 ) .

B. Purified IXa

Purified IXa was labeled with ^12SI by a minor modification (Sinha et. al., J. Biol. Chem. 260 10714-10719

(1985)) of the iodogen method to a specific activity of 5 x

10⁶ cpm/mg. The radiolabeled protein retained >90% of its biological activity compared with unlabeled factor IX. C. Assay of Factor IXa Binding to Platelets

All incubations were performed at 37°C without stirring the reaction mixture. Gel-filtered platelets (3-4 × 10⁸/ml) in calcium-free 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) Tyrode's buffer, pH 7.4, were incubated at 37°C in a 1.5 ml Eppendorf plastic centrifuge tube with mixtures of unlabeled and radiolabeled factor IXa (0.1-20 nM), calcium chloride (5 mM), and human α-thrombin(0.1 U/ml) in the presence or absence of factor X (1.5 μM) and thrombin activated factor VIII (2 U/ml) as detailed previously (Ahmad, et al., supra). At various times after the addition of the platelet stimulus, aliquots were removed and centrifuged through a mixture of silicone oils as described in Greengard et al .. Biochem.. 25, 3884-3890 (1986). The data were analyzed and the number of binding sites and dissociation constants (K_d) were calculated from the means of three independent determinations, each done in duplicate, as previously described (Ahmad, et al.. supra) using a Mac Plus Computer and the LIGAND Program as modified by G.A. McPherson (Elsevier Science Publishers BV, The Netherlands, 1985). Total binding was not corrected for any nonsaturable component. More than 86% of the platelets were sedimented under these conditions. D. Effect of Peptides on Factor

IXa-Platelet Binding.

Platelets were incubated as described in Example 5C, above, with various concentrations of synthetic peptides, factor IXa or buffer, followed by incubation with radiolabeled factor IXa. After 20 minutes, samples were centrifuged. Binding of ¹²⁵I-factor IXa was compared to control binding in the absence of competing synthetic peptides or unlabelled factor IXa.

The IC₅₀ method of Cha, Biochem. Pharmacol. 242177-2185 (1975) was used to determine the inhibitor constants K_i as previously described ( Sinha et. a ll . , Biochem . 26 3768 - 3775 (1987)). In the case of classical competitive inhibition, IC₅₀ (total inhibitor concentration at which the enzyme reaction velocity is 50% of the uninhibited reaction) is related to the substrate concentration as follows,

I₅₀ = 1/2 Et + K_i + K_iS/K_m where Et it the total enzyme concentration and S is the substrate concentration. K_i was thus determined from the plot of IC₅₀ vs S. The results are set forth in Table 1:

TABLE 1

Competing K_i of Peptide

Factor IXa or Inhibition of Factor

Gla Domain Peptide of IXa Binding to Platelet Factor IXa 0.5 × 10^-9

SEQ ID NO:2 3.5 × 10^-8

SEQ ID NO: 3 1.0 × 10^-6

SEQ ID NO:4 1.0 × 10^-5

Synergism Between SEQ ID NO: 3 and SEQ ID NO: 4

The above binding assay was repeated with a mixture comprising equimolar amounts of the two peptides SEQ ID NO: 3 and SEQ ID NO: 4. These peptides, as shown in Table 1, separately displayed inhibitory activities in the binding assay with K_i's of 10^-6 and 10^-5 M respectively. When tested together in equimolar concentrations, the two peptides demonstrated striking synergism with a K. of 1 × 10^-7 see table 2, below. The concentrations of the combined peptides required to inhibit factor IXa binding to platelets 50% were 50-fold lower than expected on the basis of their inhibitory activities when used alone.

TABLE 2

Competing K_i of Peptide

Factor IXa or Inhibition of Factor IXa Gla

Domain Peptide Binding to Platelet

SEQ ID NO: 3 + SEQ ID NO: 4^* 1.0 × 10^{-7 *}Two peptides added together at equimolar concentration. Example 6

Anti -Coagulant Effect of Gla Domain-Derived Peptides

The peptides derived from the factor IXa Gla domain are assayed for possible inhibitory effects on blood coagulation as follows. Phospholipids can substitute for platelets in most coagulation reactions. Thus, parallel assays are run with the peptides to determine whether their inhibitory effects were specific for their interaction with platelets.

The experimental protocol involves the assay of factor IXa activity by minor modifications of the method according to Scott et al., Blood 63 42-50 (1984). This assay determines the kaolin-activated partial thromboplastin time

(Proctor et al., Am. J. Clin. Pathol. 36 212-219 (1961)).

APPENDIX 1

Factor IX GLA-Domain Brookhaven Format

ATOM 1 N TYR 1 -10.697 -8.531 14.159 1.00 0.00

ATOM 2 CA TYR 1 -11.866 -8.660 13.303 1.00 0.00

ATOM 3 C TYR 1 -13.118 -9.144 14.040 1.00 0.00

ATOM 4 O TYR 1 -13.534 -10.290 13.864 1.00 0.00

ATOM 5 CB TYR 1 -11.549 -9.458 12.028 1.00 0.00

ATOM 6 CG TYR 1 -11.242 -10.940 12.147 1.00 0.00

ATOM 7 CD1 TYR 1 -10.541 -11.442 13.258 1.00 0.00

ATOM 8 CD2 TYR 1 -11.605 -11.812 11.102 1.00 0.00

ATOM 9 CE1 TYR 1 -10.178 -12.797 13.309 1.00 o.oo

ATOM 10 CE2 TYR 1 -11.266 -13.177 11.164 1.00 0.00

ATOM 11 CZ TYR 1 -10.548 -13.662 12.271 1.00 0.00

ATOM 12 OH TYR 1 -10.179 -14.973 12.336 1.00 0.00

ATOM 13 H TYR 1 -10.473 -7.617 14.557 1.00 0.00

ATOM 14 HH TYR 1 -9.713 -15.124 13.163 1.00 0.00

ATOM 15 N ASN 2 -13.741 -8.252 14.816 1.00 0.00

ATOM 16 CA ASN 2 -15.060 -8.399 15.428 1.00 0.00

ATOM 17 C ASN 2 -15.418 -9.827 15.829 1.00 0.00

ATOM 18 O ASN 2 -16.231 -10.473 15.163 1.00 0.00

ATOM 19 CB ASN 2 -16.098 -7.826 14.467 1.00 0.00

ATOM 20 CG ASN 2 -16.930 -6.753 15.141 1.00 0.00

ATOM 21 OD1 ASN 2 -18.131 -6.939 15.340 1.00 0.00

ATOM 22 ND2 ASN 2 -16.299 -5.638 15.480 1.00 0.00

ATOM 23 H ASN 2 -13.376 -7.295 14.830 1.00 0.00

ATOM 24 HD21ASN 2 -15.299 -5.531 15.251 1.00 0.00

ATOM 25 HD22ASN 2 -16.794 -4.854 15.921 1.00 0.00

ATOM 26 N SER 3 -14.811 -10.348 16.895 1.00 0.00

ATOM 27 CA SER 3 -14.828 -11.785 17.098 1.00 0.00

ATOM 28 C SER 3 -15.369 -12.259 18.443 1.00 0.00

ATOM 29 O SER 3 -15.958 -13.340 18.501 1.00 0.00

ATOM 30 CB SER 3 -13.481 -12.406 16.738 1.00 0.00

ATOM 31 OG SER 3 -13.431 -12.634 15.345 1.00 0.00

ATOM 32 H SER 3 -14.286 -9.752 17.544 1.00 0.00

ATOM 33 HG SER 3 -13.369 -11.776 14.902 1.00 0.00

ATOM 34 N GLY 4 -15.182 -11.498 19.521 1.00 0.00

ATOM 35 CA GLY 4 -15.652 -11.915 20.830 1.00 0.00

ATOM 36 C GLY 4 -17.142 -11.640 20.980 1.00 0.00

ATOM 37 O GLy 4 -17.981 -12.460 20.606 1.00 0.00

ATOM 38 H GLY 4 -14.707 -10.595 19.430 1.00 0.00

ATOM 39 N LYS 5 -17.459 -10.483 21.551 1.00 0.00

ATOM 40 CA LYS 5 -18.791 -9.933 21.668 1.00 0.00

ATOM 41 C LYS S -18.618 -8.443 21.901 1.00 0.00

ATOM 42 O LYS 5 -18.469 -8.016 23.044 1.00 0.00

ATOM 43 CB LYS 5 -19.567 -10.573 22.827 1.00 0.00

ATOM 44 CG LYS 5 -20.956 -9.935 22.966 1.00 0.00

ATOM 45 CD LYS 5 -21.027 -9.029 24.199 1.00 0.00

ATOM 46 CE LYS 5 -21.537 -7.633 23.830 1.00 0.00

ATOM 47 NZ LYS 5 -20.491 -6.611 24.023 1.00 0.00

ATOM 48 H LYS 5 -16.688 -9.885 21.863 1.00 0.00

ATOM 49 HZ3 LYS 5 -19.620 -6.937 23.606 1.00 0.00

ATOM 50 HZ2 LYS 5 -20.315 -6.443 25.003 1.00 0.00

ATOM 51 HZ1 LYS 5 -20.743 -5.747 23.559 1.00 0.00

ATOM 52 N LEU 6 -18.696 -7.655 20.831 1.00 0.00

ATOM 53 CA LEU 6 -18.738 -6.203 20.902 1.00 0.00

ATOM 54 C LEU 6 -17.598 -5.627 21.753 1.00 0.00

ATOM 55 O LEU 6 -17.817 -5.228 22.904 1.00 0.00

ATOM 56 CB LEU 6 -20.128 -5.724 21.335 1.00 0.00

ATOM 57 CG LEU 6 -21.211 -6.052 20.310 1.00 0.00

ATOM 58 CD1 LEU 6 -21.768 -7.449 20.560 1.00 0.00 ATOM 59 CD2 LEU 6 -22.354 -5.054 20.465 1.00 0.00

ATOM 60 H LEU 6 -18.720 -8.074 19.913 1.00 0.00

ATOM 61 N GLA 7 -16.395 -5.658 21.175 1.00 0.00

ATOM 62 CA GLA 7 -15.117 -5.304 21.775 1.00 0.00

ATOM. 63 C GLA 7 -14.847 -3.796 21.765 1.00 0.00

ATOM 64 O GLA 7 -14.492 -3.235 22.805 1.00 0.00

ATOM 65 CB GLA 7 -14.009 -6.085 21.062 1.00 0.00

ATOM 66 CG GLA 7 -14.404 -7.560 21.009 1.00 0.00

ATOM 67 CD GLA 7 -14.578 -8.149 22.418 1.00 0.00

ATOM 68 OE2 GLA 7 -15.392 -9.092 22.568 1.00 0.00

ATOM 69 OE1 GLA 7 -13.930 -7.654 23.371 1.00 0.00

ATOM 70 C12 GLA 7 -13.527 -8.425 20.104 1.00 0.00

ATOM 71 O13 GLA 7 -12.473 -8.861 20.615 1.00 0.00

ATOM 72 O14 GLA 7 -14.111 -9.012 19.157 1.00 0.00

ATOM 73 N GLA 8 -15.024 -3.168 20.597 1.00 0.00

ATOM 74 CA GLA 8 -14.962 -1.745 20.266 1.00 0.00

ATOM 75 C GLA 8 -15.037 -0.823 21.501 1.00 0.00

ATOM 76 O GLA 8 -14.054 -0.183 21.891 1.00 0.00

ATOM 77 CB GLA 8 -15-976 -1.433 19.135 1.00 0.00

ATOM 78 CG GLA 8 -16.847 -2.658 18.750 1.00 0.00

ATOM 79 CD GLA 8 -17.650 -2.577 17.450 1.00 0.00

ATOM 80 OE2 GLA 8 -17.498 -3.505 16.619 1.00 0.00

ATOM 81 OE1 GLA 8 -18.417 -1.600 17.274 1.00 0.00

ATOM 82 C12 GLA 8 -17.872 -2.974 19.853 1.00 0.00

ATOM 83 O13 GLA 8 -18.052 -2.102 20.732 1.00 0.00

ATOM 84 O14 GLA 8 -18.758 -3.824 19.610 1.00 0.00

ATOM 85 N PHE 9 -16.201 -0.829 22.159 1.00 0.00

ATOM 86 CA PHE 9 -16.568 -0.169 23.409 1.00 0.00

ATOM 87 C PHE 9 -15.422 -0.007 24.413 1.00 0.00

ATOM 88 O PHE 9 -15.319 1.033 25.065 1.00 0.00

ATOM 89 CB PHE 9 -17.720 -0.962 24.038 1.00 0.00

ATOM 90 CG PHE 9 -18.653 -0.212 24.967 1.00 0.00

ATOM 91 CD1 PHE 9 -19.800 0.408 24.440 1.00 0.00

ATOM 92 CD2 PHE 9 -18.550 -0.407 26.357 1.00 0.00

ATOM 93 CE1 PHE 9 -20.803 0.891 25.300 1.00 0.00

ATOM 94 CE2 PHE 9 -19.552 0.074 27.219 1.00 0.00

ATOM 95 CZ PHE 9 -20.678 0.729 26.691 1.00 0.00

ATOM 96 H PHE 9 -16.926 -1.385 21.708 1.00 0.00

ATOM 97 N VAL 10 -14.586 -1.035 24.581 1.00 0.00

ATOM 98 CA VAL 10 -13.539 -1.062 25.591 1.00 0.00

ATOM 99 C VAL 10 -12.507 0.055 25.443 1.00 0.00

ATOM 100 O VAL 10 -12.127 0.667 26.444 1.00 0.00

ATOM 101 CB VAL 10 -12.875 -2.442 25.622 1.00 0.00

ATOM 102 CGI VAL 10 -11.606 -2.460 26.474 1.00 0.00

ATOM 103 CG2 VAL 10 -13.851 -3.471 26.185 1.00 0.00

ATOM 104 H VAL 10 -14.691 -1.839 23.965 1.00 0.00

ATOM 105 N GLN 11 -11.986 0.270 24.233 1.00 0.00

ATOM 106 CA GLN 11 -10.729 0.986 24.090 1.00 0.00

ATOM 107 C GLN 11 -9.892 0.446 22.933 1.00 0.00

ATOM 108 O GLN 11 -9.274 -0.610 23.067 1.00 0.00

ATOM 109 CB GLN 11 -10.937 2.504 24.086 1.00 0.00

ATOM 110 CG GLN 11 -9.610 3.273 24.139 1.00 0.00

ATOM 111 CD GLN 11 -8.606 2.697 25.133 1.00 0.00

ATOM 112 OE1 GLN 11 -8.701 2.923 26.336 1.00 0.00

ATOM 113 NE2 GLN 11 -7.608 1-977 24.629 1.00 0.00

ATOM 114 HE2 1GLN 11 -7.525 1.844 23.620 1.00 0.00

ATOM 115 HE22GLN 11 -6.939 549 25.245 1.00 0.00

ATOM 116 H GLN 11 -12.390 200 23.427 1.00 0.00

ATOM 117 N GLY 12 -9.853 184 21.823 1.00 0.00

ATOM 118 CA GLY 12 -9.056 830 20.662 1.00 0.00

ATOM 119 C GLY 12 -7 558 862 20.953 1.00 0.00

ATOM 120 O GLY 12 -7 112 557 21.875 1.00 0.00

ATOM 121 H GLY 12 -10.399 030 21.771 1.00 0.00

ATOM 122 N ASN 13 -6.800 0.117 20.150 1.00 0.00

ATOM 123 CA ASN 13 -5.353 -0.055 20 184 1.00 0.00

ATOM 124 C ASN 13 -5 005 -1.227 19.272 1.00 0.00

ATOM 125 O ASN 13 -5.491 -2.331 19 498 1.00 0.00

ATOM 126 CB ASN 13 -4.860 -0.249 21.628 1.00 0.00

ATOM 127 CG ASN 13 -3.572 -1.054 21.818 1.00 0.00

ATOM 128 OD1 ASN 13 -3.447 -1.772 22.807 1.00 0.00

ATOM 129 ND2 ASN 13 -2.577 -0 894 20 947 1.00 0.00

ATOM 130 H ASN 13 -7.316 -0 461 19 475 1.00 0.00

ATOM 131 HD21ASN 13 -2 670 -0 316 20 131 1.00 0.00 ATOM 132 HD22ASN 13 -1.755 -1.512 21.010 1.00 0.00

ATOM 133 N LEU 14 -4.171 -0.955 18.255 1.00 0.00

ATOM 134 CA LEU 14 -3.688 -1.915 17.261 1.00 0.00

ATOM 13S C LEU 14 -3.657 -3.367 17.754 1.00 0.00

ATOM 136 O LEU 14 -4.352 -4.216 17.195 1.00 0.00

ATOM 137 CB LEU 14 -2.311 -1.485 16.749 1.00 0.00

ATOM 138 CG LEU 14 -2.356 -0.154 16.002 1.00 0.00

ATOM 139 CD1 LEU 14 -0.968 0.159 15.450 1.00 0.00

ATOM 140 CD2 LEU 14 -3.337 -0.239 14.840 1.00 0.00

ATOM 141 H LEU 14 -3.884 0.002 18.124 1.00 0.00

ATOM 142 N GLA 15 -2.867 -3.632 18.801 1.00 0.00

ATOM 143 CA GLA 15 -2.834 -4.905 19.509 1.00 0.00

ATOM 144 C GLA 15 -4.177 -5.153 20.230 1.00 0.00

ATOM 145 O GLA 15 -4.275 -4.903 21.434 1.00 0.00

ATOM 146 CB GLA IS -1.639 -4.939 20.488 1.00 0.00

ATOM 147 CG GLA 15 -0.238 -4.613 19.917 1.00 0.00

ATOM 148 CD GLA 15 0.906 -5.357 20.626 1.00 0.00

ATOM 149 OE2 GLA 15 1.867 -5.753 19.922 1.00 0.00

ATOM 150 OE1 GLA 15 0.836 -5.528 21.868 1.00 0.00

ATOM 151 C12 GLA 15 0.033 -3.102 19.838 1.00 0.00

ATOM 152 013 GLA 15 -0.336 -2.374 20.794 1.00 0.00

ATOM 153 OK 1 GLA 15 0.577 -2.657 18.799 1.00 0.00

ATOM 154 N ARG 16 -5.191 -5.649 19.502 1.00 0.00

ATOM 155 CA ARG 16 -6.532 -5.992 19.987 1.00 0.00

ATOM 156 C ARG 16 -7.558 -6.110 18.854 1.00 0.00

ATOM 157 O ARG 16 -7.759 -7.204 18.334 1.00 0.00

ATOM 158 CB ARG 16 -7.034 -5.092 21.122 1.00 0.00

ATOM 159 CG ARG 16 -7.018 -5.851 22.453 1.00 0.00

ATOM 160 CD ARG 16 -8.285 -5.633 23.286 1.00 0.00

ATOM 161 NE ARG 16 -8.870 -4.309 23.060 1.00 0.00

ATOM 162 CZ ARG 16 -10.047 -4.118 22.449 1.00 0.00

ATOM 163 NH1 ARG 16 -10.530 -2.881 22.320 1.00 0.00

ATOM 164 NH2 ARG 16 -10.714 -5-180 21.992 1.00 0.00

ATOM 165 H ARG 16 -4.994 -5.865 18.525 1.00 0.00

ATOM 166 HE ARG 16 -8.284 -3.505 23.237 1.00 0.00

ATOM 167 HH11ARG 16 -9.839 -2.126 22.252 1.00 0.00

ATOM 168 HH12ARG 16 -11.331 -2.740 21.692 1.00 0.00

ATOM 169 HH21ARG 16 -10.302 -6.097 22.063 1.00 0.00

ATOM 170 HH22ARG 16 -11.343 -5.026 21.191 1.00 0.00

ATOM 171 N GLA 17 -8.220 -5.005 18.481 1.00 0.00

ATOM 172 CA GLA 17 -9.289 -5.039 17.484 1.00 0.00

ATOM 173 C GLA 17 -8.727 -5.468 16.127 1.00 0.00

ATOM 174 O GLA 17 -9.222 -6.378 15.461 1.00 0.00

ATOM 175 CB GLA 17 -9.929 -3.654 17.303 1.00 0.00

ATOM 176 CG GLA 17 -10.214 -2.800 18.545 1.00 0.00

ATOM 177 CD GLA 17 -11.312 -3.289 19.480 1.00 0.00

ATOM 178 OE2 GLA 17 -11.924 -2.434 20.161 1.00 0.00

ATOM 179 OE1 GLA 17 -11.487 -4.519 19.618 1.00 0.00

ATOM 180 C12 GLA 17 -8.978 -2.141 19.178 1.00 0.00

ATOM 181 O13 GLA 17 -8.854 -2.142 20.427 1.00 0.00

ATOM 182 O14 GLA 17 -8.409 -1.276 18.471 1.00 0.00

ATOM 183 N CVS 18 -7.702 -4.728 15.705 1.00 0.00

ATOM 184 CA CYS 18 -7.161 -4.804 14.366 1.00 0.00

ATOM 185 C CYS 18 -6.151 -5.934 14.243 1.00 0.00

ATOM 186 O CYS 18 -6.316 -6.822 13.402 1.00 0.00

ATOM 187 CB CYS 18 -6.544 -3.459 13.980 1.00 0.00

ATOM 188 SG CYS 18 -7.601 -2.436 12.927 1.00 0.00

ATOM 189 H CYS 18 -7.377 -3.998 16.330 1.00 0.00

ATOM 190 LPG2 CYS 18 -7.097 -2.089 12.623 1.00 0.00

ATOM 191 LPG1 CYS 18 -8.174 -2.522 13.279 1.00 0.00

ATOM 192 N MET 19 -5.092 -5.872 15.053 1.00 0.00

ATOM 193 CA MET 19 -3.981 -6.808 15.042 1.00 0.00

ATOM 194 C MET 19 -4.035 -7.725 16.268 1.00 0.00

ATOM 195 O MET 19 -4.662 -7.385 17.274 1.00 0.00

ATOM 196 CB MET 19 -2.663 -6.032 14.954 1.00 0.00

ATOM 197 CG MET 19 -2.652 -5.163 13.695 1.00 0.00

ATOM 198 SD MET 19 -1.960 -3.497 13.874 1.00 0.00

ATOM 199 CE MET 19 -0.221 -3.921 14.146 1.00 0.00

ATOM 200 LPD1 MET 19 -1 -905 -3.361 13.210 1.00 0.00

ATOM 201 LPD2 MET 19 -2.126 -3.410 14.525 1.00 0.00

ATOM 202 H MET 19 -S.060 -5.159 15.772 1.00 0.00

ATOM 203 N GLA 20 -3.382 -8.888 16.136 1.00 0.00

ATOM 204 CA GLA 20 -3.758 - 10.161 16.743 1.00 0.00 ATOM 205 C GLA 20 -5.042 -10.721 16.097 1.00 0.00

ATOM 206 O GLA 20 -4.982 -11.760 15.433 1.00 0.00

ATOM 207 CB GLA 20 -3-664 -10.168 18.281 1.00 0.00

ATOM 208 CG GLA 20 -2.450 -10.969 18.810 1.00 0.00

ATOM 209 CD GLA 20 -2.763 -12.065 19.841 1.00 0.00

ATOM 210 OE2 GLA 20 -2.315 -13.214 19.611 1.00 0.00

ATOM 211 OE1 GLA 20 -3.451 -11.782 20.850 1.00 0.00

ATOM 212 C12 GLA 20 -1.168 -10.174 19.072 1.00 0.00

ATOM 213 013 GLA 20 -1.175 -9.327 19.992 1.00 0.00

ATOM 214 014 GLA 20 -0.129 -10.585 18.502 1.00 0.00

ATOM 215 N GLA 21 -6.162 -9.983 16.168 1.00 0.00

ATOM 216 CA GLA 21 -7.341 -10.046 15.297 1.00 0.00

ATOM 217 C GLA 21 -6.999 -9.867 13.802 1.00 0.00

ATOM 218 O GLA 21 -5.848 -10.041 13.400 1.00 0.00

ATOM 219 CB GLA 21 -8.253 -11.259 15.569 1.00 0.00

ATOM 220 CG GIA 21 -8.184 -11.880 16.974 1.00 0.00

ATOM 221 CD GLA 21 -7.487 -13.248 16.947 1.00 0.00

ATOM 222 OE2 GLA 21 -7.493 -13.897 15.869 1.00 0.00

ATOM 223 OE1 GLA 21 -6.920 -13.662 17.982 1.00 0.00

ATOM 224 C12 GLA 21 -9.531 -11.948 17.694 1.00 0.00

ATOM 225 O13 GLA 21 -10.332 -12.840 17.334 1.00 0.00

ATOM 226 O14 GLA 21 -9.589 -11.397 18.814 1.00 0.00

ATOM 227 N LYS 22 -7.997 -9.618 12.950 1.00 0.00

ATOM 228 CA LYS 22 -7.795 -9.380 11.521 1.00 0.00

ATOM 229 C LYS 22 -8.766 -8.335 10.972 1.00 0.00

ATOM 230 O LYS 22 -9.564 -8.626 10.080 1.00 0.00

ATOM 231 CB LYS 22 -7.764 -10.654 10.659 1.00 0.00

ATOM 232 CG LYS 22 -7.817 -11.974 11.419 1.00 0.00

ATOM 233 CD LYS 22 -6.520 -12.759 11.232 1.00 0.00

ATOM 234 CE LYS 22 -6.267 -13.601 12.478 1.00 0.00

ATOM 235 NZ LYS 22 -6.595 -12.843 13.695 1.00 0.00

ATOM 236 H LYS 22 -8.928 -9.489 13.326 1.00 0.00

ATOM 237 HZ3 LYS 22 -6.694 -13.437 14.515 1.00 0.00

ATOM 238 HZ2 LYS 22 -7.484 -12.376 13.599 1.00 0.00

ATOM 239 HZ1 LYS 22 -5.890 -12.148 13.943 1.00 0.00

ATOM 240 N CYS 23 -8.694 -7.118 11.513 1.00 0.00

ATOM 241 CA CYS 23 -9.616 -6.030 11.190 1.00 0.00

ATOM 242 C CYS 23 -9.612 -5.617 9.712 1.00 0.00

ATOM 243 O CYS 23 -8.739 -6.021 8.944 1.00 0.00

ATOM 244 CB CYS 23 -9.285 -4.825 12.060 1.00 0.00

ATOM 245 SG CYS 23 -8.054 -3.704 11.349 1.00 0.00

ATOM 246 H CYS 23 -7.993 -6.956 12.228 1.00 0.00

ATOM 247 LPG2 CYS 23 -8.493 -3.298 11.026 1.00 0.00

ATOM 248 LPG1 CYS 23 -7.520 -4.124 11.414 1.00 0.00

ATOM 249 N SER 24 -10.572 -4.771 9.320 1.00 0.00

ATOM 250 CA SER 24 -10.554 -4.199 7.987 1.00 0.00

ATOM 251 C SER 24 -10.661 -2.671 7.973 1.00 0.00

ATOM 252 O SER 24 -9.663 -1.977 8.242 1.00 0.00

ATOM 253 CB SER 24 -11.527 -4.927 7.056 1.00 0.00

ATOM 254 OG SER 24 -12.843 -4.894 7.570 1.00 0.00

ATOM 255 H SER 24 -11.251 -4.437 9.995 1.00 0.00

ATOM 256 HG SER 24 -12.824 -5.171 8.494 1.00 0.00

ATOM 257 N PHE 25 -11.840 -2-151 7.627 1.00 0.00

ATOM 258 CA PHE 25 -12.046 -0.719 7.466 1.00 0.00

ATOM 259 C PHE 25 -12.551 -0.075 8.763 1.00 0.00

ATOM 260 O PHE 25 -11.746 0.457 9.530 1.00 0.00

ATOM 261 CB PHE 25 -12.958 -0.438 6.267 1.00 0.00

ATOM 262 CG PHE 25 -12.261 -0.191 4.945 1.00 0.00

ATOM 263 CD1 PHE 25 -12.342 -1.153 3.920 1.00 0.00

ATOM 264 CD2 PHE 25 -11.675 1.061 4.684 1.00 0.00

ATOM 265 CE1 PHE 25 -11.801 -0.882 2.650 1.00 0.00

ATOM 266 CE2 PHE 25 -11.131 1.331 3.415 1.00 0.00

ATOM 267 CZ PHE 25 -11.196 0.362 2.399 1.00 0.00

ATOM 268 H PHE 25 -12.615 -2.785 7.465 1.00 0.00

ATOM 269 N GLA 26 -13.874 -0.118 8.967 1.00 0.00

ATOM 270 CA GLA 26 -14.560 0.472 10.129 1.00 0.00

ATOM 271 C GLA 26 -13.804 0.352 11.451 1.00 0.00

ATOM 272 O GLA 26 -13.464 1.374 12.047 1.00 0.00

ATOM 273 CB GLA 26 -16.020 -0.065 10.183 1.00 0.00

ATOM 274 CG GLA 26 -17.125 0.934 10.580 1.00 0.00

ATOM 275 CD GLA 26 -16.852 2.364 10.080 1.00 0.00

ATOM 276 OE2 GLA 26 -16.884 2.592 8.848 1.00 0.00

ATOM 277 OE1 GLA 26 -16.591 3.246 10.930 1.00 0.00 ATOM 2761 C12 GLA 26 -18.534 0.421 10.213 1.00 0.00

ATOM 279 O13 GLA 26 -19.528 0.859 10.843 1.00 0.00

ATOM 280 O14 GLA 26 -18.645 -0.349 9.230 1.00 0.00

ATOM 281 N GLA 27 -13.439 -0.872 11.864 1.00 0.00

ATOM 282 CA GLA 27 -12.679 -1.173 13.081 1.00 0.00

ATOM 283 C GIA 27 -11.442 -0.266 13.259 1.00 0.00

ATOM 284 O GLA 27 -11.085 0.123 14.373 1.00 0.00

ATOM 285 CB GLA 27 -12.305 -2.670 13.129 1.00 0.00

ATOM 286 CG GLA 27 -13.477 -3.637 13.409 1.00 0.00

ATOM 287 CD GLA 27 -13.027 -5.030 13.859 1.00 0.00

ATOM 288 OE2 GLA 27 -13-677 -5.S80 14.778 1.00 0.00

ATOM 289 OE1 GLA 27 -12.065 -5.577 13.274 1.00 0.00

ATOM 290 C12 GLA 27 -14.419 -3.750 12.195 1.00 0.00

ATOM 291 O13 GIA 27 -13.927 -4.095 11.089 1.00 0.00

ATOM 292 O14 GLA 27 -15.653 -3.726 12.417 1.00 0.00

ATOM 293 N ALA 28 -10.776 0.092 12.157 1.00 0.00

ATOM 294 CA ALA 28 -9.564 0.896 12.186 1.00 0.00

ATOM 295 C ALA 28 -9.806 2.383 12.481 1.00 0.00

ATOM 296 O ALA 28 -8.865 3.094 12.844 1.00 0.00

ATOM 297 CB ALA 28 -8.772 0.680 10.898 1.00 0.00

ATOM 298 H ALA 28 -11.171 -0.143 11.254 1.00 0.00

ATOM 299 N ARG 29 -11.041 2.872 12.330 1.00 0.00

ATOM 300 CA ARG 29 -11.359 4.241 12.697 1.00 0.00

ATOM 301 C ARG 29 -11.349 4.382 14.216 1.00 0.00

ATOM 302 O ARG 29 -10.652 5.246 14.749 1.00 0.00

ATOM 303 CB ARG 29 -12.696 4.673 12.090 1.00 0.00

ATOM 304 CG ARG 29 -12.749 6.192 11.915 1.00 0.00

ATOM 305 CD ARG 29 -13.939 6.778 12.678 1.00 0.00

ATOM 306 NE ARG 29 -13.788 6.575 14.123 1.00 0.00

ATOM 307 CZ ARG 29 -12.827 7.203 14.812 1.00 0.00

ATOM 308 NH1 ARG 29 -12.093 8.104 14.156 1.00 0.00

ATOM 309 NH2 ARG 29 -12.596 6.958 16.103 1.00 0.00

ATOM 310 H ARG 29 -11.810 2.246 12.100 1.00 0.00

ATOM 311 HE ARG 29 -14.366 5.848 14.567 1.00 0.00

ATOM 312 HH21ARG 29 -13.193 6.275 16.588 1.00 0.00

ATOM 313 HH22ARG 29 -11.737 7.251 16.540 1.00 0.00

ATOM 314 HH11ARG 29 -12.244 8.188 13.160 1.00 0.00

ATOM 315 HH12ARG 29 -11.294 8.569 14.580 1.00 0.00

ATOM 316 N GLA 30 -12.122 3.537 14.907 1.00 0.00

ATOM 317 CA GLA 30 -12.218 3.504 16.360 1.00 0.00

ATOM 318 C GLA 30 -10.838 3.335 17.007 1.00 0.00

ATOM 319 O GLA 30 -10.510 4.062 17.945 1.00 0.00

ATOM 320 CB GLA 30 -13.253 2.460 16.826 1.00 0.00

ATOM 321 CG GLA 30 -14.724 2.951 16.851 1.00 0.00

ATOM 322 CD GLA 30 -14.856 4.429 16.438 1.00 0.00

ATOM 323 OE2 GLA 30 -15.359 4.703 15.325 1.00 0.00

ATOM 324 OE1 GLA 30 -14.387 5.334 17.174 1.00 0.00

ATOM 325 C12 GLA 30 -15.436 2.701 18.189 1.00 0.00

ATOM 326 013 GLA 30 -16.668 2.926 16.191 1.00 0.00

ATOM 327 014 GLA 30 -14.742 2.772 19.231 1.00 0.00

ATOM 328 N VAL 31 -10.013 2.416 16.486 1.00 0.00

ATOM 329 CA VAL 31 -8.689 2.124 17.030 1.00 0.00

ATOM 330 C VAL 31 -7.759 3.335 17.164 1.00 0.00

ATOM 331 O VAL 31 -7.436 3.735 18.282 1.00 0.00

ATOM 332 CB VAL 31 -8.033 0.926 16.334 1.00 0.00

ATOM 333 CGI VAL 31 -7.934 1.110 14.824 1.00 0.00

ATOM 334 CG2 VAL 31 -6.629 0.691 16.884 1.00 0.00

ATOM 335 H VAL 31 -10.369 1.821 15.744 1.00 0.00

ATOM 336 N PHE 32 -7.266 3.882 16.047 1.00 0.00

ATOM 337 CA PHE 32 -6.215 4.894 16.127 1.00 0.00

ATOM 338 C PHE 32 -6.627 6.276 15.615 1.00 0.00

ATOM 339 O PHE 32 -5.957 6.846 14.746 1.00 0.00

ATOM 340 CB PHE 32 -4.869 4.388 15.562 1.00 0.00

ATOM 341 CG PHE 32 -3.677 4.777 16.387 1.00 0.00

ATOM 342 CD1 PHE 32 -3.626 4.461 17.757 1.00 0.00

ATOM 343 CD2 PHE 32 -2.601 5.453 15.787 1.00 0.00

ATOM 344 CE1 PHE 32 -2.507 4.827 18.527 1.00 0.00

ATOM 345 CE2 PHE 32 -1.480 5.820 16.557 1.00 0.00

ATOM 346 CZ PHE 32 -1.434 5.506 17.926 1.00 0.00

ATOM 347 H PHE 32 -7.575 3.551 15.142 1.00 0.00

ATOM 348 N GLA 33 -7.676 6.852 16.210 1.00 0.00

ATOM 349 CA GLA 33 -8.010 8.270 16.148 1.00 0.00

ATOM 350 C GLA 33 -8.625 8.732 14.826 1.00 0.00 ATOM 351 O GLA 33 -9.769 9.184 14.811 1.00 0.00 ATOM 352 CB GLA 33 -6.800 9.130 16.497 1.00 0.00

ATOM 353 CG GLA 33 -6.696 9.571 17.962 1.00 0.00

ATOM 354 CD GLA 33 -7.894 10.411 18.437 1.00 0.00

ATOM 355 OE2 GLA 33 -7.990 10.683 19.657 1.00 0.00

ATOM 356 OE1 GLA 33 -8.733 10.774 17.581 1.00 0.00

ATOM 357 C12 GLA 33 -5.347 10.254 18.208 1.00 0.00

ATOM 358 O13 GLA 33 -4.829 10.809 17.205 1.00 0.00

ATOM 359 O14 GLA 33 -4.664 9.805 19.159 1.00 0.00

ATOM 360 N ASN 34 -7.879 8.697 13.720 1.00 0.00

ATOM 361 CA ASN 34 -8.290 9.433 12.532 1.00 0.00

ATOM 362 C ASN 34 -8.120 8.690 11.211 1.00 0.00

ATOM 363 O ASN 34 -7.011 8.309 10.841 1.00 0.00

ATOM 364 CB ASN 34 -7.762 10.874 12.535 1.00 0.00

ATOM 365 CG ASN 34 -6.464 11.076 11.759 1.00 0.00

ATOM 366 OD1 ASN 34 -5.372 11.001 12.320 1.00 0.00

ATOM 367 ND2 ASN 34 -6.563 11.385 10.472 1.00 0.00

ATOM 368 H ASN 34 -6.982 8.226 13.746 1.00 0.00

ATOM 369 HD21ASN 34 -7.480 11.502 10.017 1.00 0.00

ATOM 370 HD22ASN 34 -5.728 11.509 9.925 1.00 0.00

ATOM 371 N THR 35 -9.244 8.524 10.511 1.00 0.00

ATOM 372 CA THR 35 -9.430 7.841 9.238 1.00 0.00

ATOM 373 C THR 35 -8.316 8.113 8.226 1.00 0.00

ATOM 374 O THR 35 -7.825 7.197 7.562 1.00 0.00

ATOM 375 CB THR 3S -10.781 8.296 8.677 1.00 0.00

ATOM 376 OG1 THR 35 -11.675 8.608 9.734 1.00 0.00

ATOM 377 CG2 THR 35 -11.594 7.236 7.766 1.00 0.00

ATOM 378 H THR 35 -10.109 8.895 10.884 1.00 0.00

ATOM 379 HG1 THR 35 -12.563 8.373 9.455 1.00 0.00

ATOM 380 V GLU 36 -7.928 9.380 8.099 1.00 0.00

ATOM 381 CA GLU 36 -6.874 9.836 7.216 1.00 0.00

ATOM 382 C GLU 36 -5.564 9.079 7.459 1.00 0.00

ATOM 383 O GLU 36 -4.876 8.726 6.504 1.00 0.00

ATOM 384 CB GLU 36 -6.711 11.347 7.395 1.00 0.00

ATOM 38S CG GLU 36 -7.971 12.106 6.950 1.00 0.00

ATOM 386 CD GLU 36 -9.059 12.252 8.016 1.00 0.00

ATOM 387 OE1 GLU 36 -8.878 11.681 9.119 1.00 0.00

ATOM 388 OE2 GLU 36 -10.077 12.901 7.694 1.00 0.00

ATOM 389 H GLU 36 -8.395 10.098 8.653 1.00 0.00

ATOM 390 N ARG 37 -5.237 8.818 8.729 1.00 0.00

ATOM 391 CA ARG 37 -4.113 7.974 9.106 1.00 0.00

ATOM 392 C ARG 37 -4.525 6.505 9.007 1.00 0.00

ATOM 393 O ARG 37 -3.865 5.708 8.340 1.00 0.00

ATOM 394 CB ARG 37 -3.716 8.284 10.546 1.00 0.00

ATOM 39S CG ARG 37 -2.522 9.229 10.620 1.00 0.00

ATOM 396 CD ARG 37 -2.006 9.244 12.057 1.00 0.00

ATOM 397 NE ARG 37 -3.072 9.609 12.993 1.00 0.00

ATOM 398 CZ ARG 37 -3.761 8.722 13.721 1.00 0.00

ATOM 399 NH1 ARG 37 -3.589 7.414 13.518 1.00 0.00

ATOM 400 NH2 ARG 37 -4.625 9.128 14.651 1.00 0.00

ATOM 401 H ARG 37 -5.897 9.064 9.459 1.00 0.00

ATOM 402 HE ARG 37 -3.480 10.532 12.895 1.00 0.00

ATOM 403 HH21ARG 37 -4.682 10.077 15.010 1.00 0.00

ATOM 404 HH22ARG 37 -5.176 8.410 15.121 1.00 0.00

ATOM 405 HH11ARG 37 -2.907 7.082 12.848 1.00 0.00

ATOM 406 HH12ARG 37 -4.179 6.755 14.023 1.00 0.00

ATOM 407 N THR 38 -5.634 6.165 9.673 1.00 0.00

ATOM 408 CA THR 38 -6.247 4.848 9.722 1.00 0.00

ATOM 409 C THR 38 -6.116 4.081 8.408 1.00 0.00

ATOM 410 O THR 38 -5.618 2.961 8.398 1.00 0.00

ATOM 411 CB THR 38 -7.702 5.003 10.166 1.00 0.00

ATOM 412 OG1 THR 38 -7.758 5.325 11.538 1.00 0.00

ATOM 413 CG2 THR 38 -8.530 3.752 9.895 1.00 0.00

ATOM 414 H THR 38 -6.101 6.883 10.213 1.00 0.00

ATOM 415 HG1 THR 38 -8.087 4.551 12.020 1.00 0.00

ATOM 416 N THR 39 -6.528 4.685 7.293 1.00 0.00

ATOM 417 CA THR 39 -6.468 4.053 5.983 1.00 0.00

ATOM 418 C THR 39 -5.055 3.634 5.566 1.00 0.00

ATOM 419 O THR 39 -4.893 2.586 4.934 1.00 0.00

ATOM 420 CB THR 39 -7.175 4.904 4.924 1.00 0.00

ATOM 421 OG1 THR 39 -6.886 6.278 5.075 1.00 0.00

ATOM 422 CG2 THR 39 -8.684 4.715 5.039 1.00 0.00

ATOM 423 H THR 39 -6.909 5.620 7.365 1.00 0.00 ATOM 424 HG1 THR 39 -7.357 6.609 5.852 1.00 0.00 ATOM 425 N GLU 40 -4.044 4.439 5.911 1.00 0.00 ATOM 426 CA GLU 40 -2.640 4.135 5.680 1.00 0.00 ATOM 427 C GLU 40 -2.185 2.968 6.557 1.00 0.00 ATOM 428 O GLU 40 -1.577 2.019 6.062 1.00 0.00 ATOM 429 CB GLU 40 -1.770 5.382 5.885 1.00 0.00 ATOM 430 CG GLU 40 -2.355 6.642 5.236 1.00 0.00 ATOM 431 CD GLU 40 -1.355 7.378 4.344 1.00 0.00 ATOM 432 OE1 GLU 40 -0.636 8.259 4.860 1.00 0.00 ATOM 433 OE2 GLU 40 -1.360 7.037 3.137 1.00 0.00 ATOM 434 H GLU 40 -4.234 5.230 6.520 1.00 0.00 ATOM 435 N PHE 41 -2.519 3.016 7.850 1.00 0.00 ATOM 436 CA PHE 41 -2.310 1.888 8.752 1.00 0.00 ATOM 437 C PHE 41 -2.931 0.612 8.171 1.00 0.00 ATOM 438 O PHE 41 -2.304 -0.445 8.140 1.00 0.00 ATOM 439 CB PHE 41 -2.903 2.208 10.124 1.00 0.00 ATOM 440 CG PHE 41 -2.081 3.159 10.963 1.00 0.00 ATOM 441 CD1 PHE 41 -1.131 2.655 11.870 1.00 0.00 ATOM 442 CD2 PHE 41 -2.321 4.544 10.897 1.00 0.00 ATOM 443 CE1 PHE 41 -0.417 3.537 12.701 1.00 0.00 ATOM 444 CE2 PHE 41 -1.597 5.426 11.717 1.00 0.00 ATOM 445 CZ PHE 41 -0.647 4.923 12.623 1.00 0.00 ATOM 446 H PHE 41 -2.995 3.845 8.201 1.00 0.00 ATOM 447 N TRP 42 -4.160 0.739 7.673 1.00 0.00 ATOM 448 CA TRP 42 -4.919 -0.266 6.956 1.00 0.00 ATOM 449 C TRP 42 -4.134 -0.795 5.755 1.00 0.00 ATOM 450 O TRP 42 -4.042 -2.006 5.565 1.00 0.00 ATOM 451 CB TRP 42 -6.230 0.390 6.532 1.00 0.00 ATOM 452 CG TRP 42 -7.285 -0.454 5.895 1.00 0.00 ATOM 453 CD1 TRP 42 -8.311 0.049 5.178 1.00 0.00 ATOM 454 CD2 TRP 42 -7.484 -1.901 5.946 1.00 0.00 ATOM 455 NE1 TRP 42 -9.094 -0.995 4.732 1.00 0.00 ATOM 456 CE2 TRP 42 -8.636 -2.216 5.170 1.00 0.00 ATOM 457 CE3 TRP 42 -6.820 -2.981 6.569 1.00 0.00 ATOM 458 CZ2 TRP 42 -9.100 -3.530 5.001 1.00 0.00 ATOM 459 CZ3 TRP 42 -7.290 -4.299 6.430 1.00 0.00 ATOM 460 CH2 TRP 42 -8.415 -4.579 5.632 1.00 0.00 ATOM 461 H TRP 42 -4.603 1.640 7.791 1.00 0.00 ATOM 462 HE1 TRP 42 -9.938 -0 838 4.197 1.00 0.00 ATOM 463 N LYS 43 -3.551 0.103 4.950 1.00 0.00 ATOM 464 CA LYS 43 -2 611 -0 284 3 904 1.00 0.00 ATOM 46S C LYS 43 -1.527 -1.197 4 479 1 00 0.00 ATOM 466 O LYS 43 -1.429 -2.359 4.087 1.00 0.00 ATOM 467 CB LYS 43 -1.975 0.952 3.273 1.00 0.00 ATOM 468 CG LYS 43 -3.030 1 856 2.652 1.00 0.00 ATOM 469 CD LYS 43 -2.662 3.303 2 953 1.00 0.00 ATOM 470 CE LYS 43 -3.699 4 247 2 362 1.00 0.00 ATOM 471 NZ LYS 43 -3.509 5.607 2.873 1.00 0.00 ATOM 472 H LYS 43 -3.668 1.093 5.149 1.00 0.00 ATOM 473 HZ3 LYS 43 -2.539 5.913 2.775 1.00 0.00

ATOM 474 HZ2 LYS 43 -3.699 5.642 3.871 1.00 0.00 ATOM 475 HZ1 LYS 43 -4 .097 6.274 2.392 1.00 0.00 ATOM 476 N GLN 44 -0.757 0.673 5.442 1.00 0.00 ATOM 477 CA GLN 14 0.254 -1.411 6.192 1.00 0.00 ATOM 478 C GLN 44 -0.226 -2.823 6.531 1.00 0.00 ATOM 479 O GLN 44 0.468 -3.806 6.286 1.00 0.00 ATOM 460 CB GLN 44 0.544 -0.656 7.467 1.00 0.00 ATOM 481 CG GLN 44 1.801 0.211 7.426 1.00 0.00 ATOM 482 CD GLN 4 4 1.919 1.117 8.640 1.00 0.00 ATOM 463 OE1 GLN 4 4 2.706 2.056 8.648 1.00 0.00

ATOM 484 NE2 GLN 4 4 1.13S 0.865 9..684 1.00 0.00 ATOM 465 H GLN 44 -0.936 0.292 5.705 1.00 0.00 ATOM 486 HE21GLN 44 0.423 0.148 9.629 1.00 0.00 ATOM 487 HE22GLN 44 1.119 1.554 10.429 1.00 0.00 ATOM 488 N TYR 45 -1.428 -2.897 7.099 1.00 0.00 ATOM 489 CA TYR 45 -2.111 -4.124 7.445 1.00 0.00 ATOM 490 C TYR 45 -2.309 -5.041 6.233 1.00 0.00 ATOM 491 O TYR 45 -1.640 -6.06S 6.109 1.00 0.00 ATOM 492 CB TYR 45 -3.446 -3.753 8.091 1.00 0.00 ATOM 493 CC TYR 45 -4.056 -4.643 8.930 1.00 0.00 ATOM 494 CD1 TYR 45 -3 .975 -4.773 10.330 1.00 0.00 ATOM 495 CD2 TYR 45 -4.631 -5.966 8.310 1.00 0.00 ATOM 498 CE1 TYR 45 -4.446 -5.839 11.112 1.00 0.00 ATOM 497 CE2 TYR 45 -5.075 -7.044 9.092 1.00 0.00

ATOM 498 CZ TYR 45 -4.980 -6.980 10.491 1.00 0.00

ATOM 499 OH TYR 45 -5.305 -8.071 11.232 1.00 0.00

ATOM 500 H TYR 45 -1.912 -2.027 7.281 1.00 0.00

ATOM 501 HH TYR 45 -5.445 -7.857 12.167 1.00 0.00

ATOM 502 N VAL 46 -3.268 -4.708 5.369 1.00 0.00

ATOM 503 CA VAL 46 -3.804 -5.684 4.436 1.00 0.00

ATOM 504 C VAL 46 -3.153 -5.684 3.053 1.00 0.00

ATOM 505 O VAL 46 -3.278 -6.680 2.340 1.00 0.00

ATOM 506 CB VAL 46 -5.330 -5.588 4.377 1.00 0.00

ATOM 507 CG1 VAL 46 -5.798 -4.499 3.411 1.00 0.00

ATOM 508 CG2 VAL 46 -5.945 -6.933 4.002 1.00 0.00

ATOM 509 H VAL 46 -3.745 -3.818 5.484 1.00 0.00

ATOM 510 N ASP 47 -2.488 -4.587 2.661 1.00 0.00

ATOM 511 CA ASP 47 -1.986 -4.347 1.307 1.00 0.00

ATOM 512 C ASP 47 -1.397 -5.608 0.677 1.00 0.00

ATOM 513 O ASP 47 -1.870 -6.090 -0.351 1.00 0.00

ATOM 514 CB ASP 47 -0.958 -3.218 1.355 1.00 0.00

ATOM 515 CG ASP 47 -1.085 -2.236 0.201 1.00 0.00

ATOM 516 OD1 ASP 47 -2.174 -1.639 0.055 1.00 0.00

ATOM 517 OD2 ASP 47 -0.062 -2.076 -0.501 1.00 0.00

ATOM 518 H ASP 47 -2.348 -3.836 3.330 1.00 0.00

ATOM 519 N GLY 48 -0.408 -6.181 1.362 1.00 0.00

ATOM 520 CA GLY 48 0.081 -7.512 1.064 1.00 0.00

ATOM 521 C GLY 48 0.053 -8.373 2.325 1.00 0.00

ATOM 522 O GLY 48 1.087 -8.923 2.697 1.00 0.00

ATOM 523 OXT GLY 48 -1.113 -8.465 2.968 1.00 0.00

ATOM 524 H GLY 48 -0.082 -5.708 2.194 1.00 0.00

TER 525 GLY 48

ATOM 526 CO UNN -17.483 2.887 14.115 1.00 0.00

TER 527 UNN

ATOM 528 CO UNN -16.224 -1.256 15.982 1.00 0.00

TER 529 UNN

ATOM 530 CO UNN -12.372 -1.241 17.496 1.00 0.00

TER 531 UNN

ATOM 532 CO UNN -12.763 -4.581 17.359 1.00 0.00

TER 533 UNN

ATOM 534 CO UNN -10.810 -8.201 18.729 1.00 0.00

TER 535 UNN

ATOM 536 CO UNN -6.910 -11.401 20.105 1.00 0.00

TER 537 UNN

ATOM 538 CO UNN 1.480 -8.643 19.524 1.00 0.00

TER 539 UNN

CONECT 188 187 190 191 245

CONECT 245 186 244 247 248

MASTER 0 0 0 0 0 0 0 0 531 8 2 1 1

END

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Temple University - Of The Commonwealth System of Higher Education (ii) INVENTORS: Walsh, Peter N., Ahmad,

Syed S. and Jameson, Bradford A.

(iii) TITLE OF INVENTION: PEPTIDE ANALOGS OF THE FACTOR IXa PLATELET BINDING SITE

(iv) NUMBER OF SEQUENCES: 4

(v) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Seidel, Gonda, Lavorgna

& Monaco, P.C.

(B) STREET: Two Penn Center Plaza,

Suite 1800

(C) CITY: Philadelphia

(D) STATE: Pennsylvania

(E) COUNTRY: U.S.A.

(F) ZIP: 19103

(vi) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch,

720 Kb

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect 5.1

(vii) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) PRIORIRY APPLICATION DATA:

(A) APPLICATION NUMBER: 08/172,330

(B) FILING DATE: 12/22/93

(ix) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A.

(B) REGISTRATION NUMBER: 30,480

(C) REFERENCE/DOCKET NUMBER: 6056-187PC

(x) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 568-8383

(B) TELEFAX: (215) 568-5549 (2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa

5 10 15 Arg Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa

20 25 30

Val Phe Xaa Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr

35 40 45

Val Asp Gln

48 (3) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Cys Pro Gly Lys Leu Asp Glu Phe Val Gln Pro Cys

5 10 (4) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Cys Ser Gly Lys Leu Cys

5

(5) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Cys Phe Val Gln Cys

5

Claims

Claims

1. A synthetic peptide, or a pharmaceutically acceptable salt thereof, said peptide consisting essentially of an amino acid sequence from at least 5 to about 75 amino acids in length, which sequence corresponds to a portion of the amino acid sequence of the platelet binding site on factor IXa, said peptide having an artificially introduced restricted conformation free of adjacent γ-carboxyglutamic acid residues and the ability to inhibit the binding of factor IXa to a platelet surface.

2. A peptide according to claim 1 wherein the peptide is free of γ-carboxyglutamic acid residues.

3. A peptide according to claim 2 wherein the peptide is from 5 to about 45 amino acids in length.

4. A peptide according to claim 3 wherein the peptide is from about 5 to about 20 amino acids in length.

5. A peptide according to claim 2 wherein the conformation is restricted by means of at least one cysteinecysteine disulfide bond.

6. A peptide according to claim 2 wherein the restricted conformation is determined from the equilibrium conformation model comprising the set of coordinates and connect statements of Appendix 1.

7. A synthetic peptide, or a pharmaceutically acceptable salt thereof, said peptide consisting essentially of an amino acid sequence from at least 5 to about 75 amino acids in length, which amino acid sequence corresponds to a portion of the amino acid sequence of the platelet binding site on factor IXa,

wherein said restricted conformation is provided at least in part by: (i) at least one cysteine-cysteine disulfide bond which is not present in the native amino acid sequence of factor XIa, or

(ii) at least one artificially introduced covalent bond other than a disulfide bond.

8. A peptide according to claim 7 wherein the peptide is from about 5 to about 45 amino acids in length.

9. A peptide according to claim 8 wherein the peptide is from about 5 to about 20 amino acids in length.

10. A peptide according to claim 7, wherein the conformation is restricted by means of at least one cysteine-cysteine disulfide bond.

11. A peptide according to claim 7 wherein the restricted conformation is determined from the equilibrium conformation model comprising the set of coordinates and connect statements of Appendix 1.

12. A peptide according to claim 7 wherein the conformation is restricted at least in part by at least one amide bond.

13. A peptide according to claim 7 wherein the conformation is restricted at least in part by at least one toluene-2 , 4-diisocyanate cross-link between two free amino groups of the peptide.

14. A peptide according to claim 13 wherein the conformation is restricted at least in part by at least one amide bond formed between side chains of a lysine residue and a glutamic or aspartic acid residue of the peptide.

15. A peptide according to claim 7 wherein the amino acid sequence of said peptide comprises amino acids 4-6 or 9-11 of SEQ ID NO:l, or combinations thereof.

16. A peptide according to claim 15 having an amino acid sequence selected from the group of sequences consisting of:

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4 and

combinations thereof.

17. A peptide according to claim 16 having an amino acid sequence of SEQ ID NO: 2.

18. A method of designing a synthetic peptide or a pharmaceutically acceptable salt thereof, said peptide consisting essentially of an amino acid sequence from at least 5 to about 75 amino acids in length, which sequence corresponds to a portion of the amino acid sequence of the platelet binding site on factor IXa, said peptide having an artificially introduced restricted conformation and the ability to inhibit the binding of factor IXa to a platelet surface, comprising:

determining the distance between two parts of a molecular model including the factor IXa platelet binding site at conformational equilibrium;

modifying the primary structure of the platelet binding site to restrict the distance between said two parts to the predetermined distance; and

synthesizing a peptide comprising said modified primary structure.

19. A method according to claim 18, wherein said modified primary structure is free of adjacent γ-carboxyglutamic acid residues.

20. A method according to claim 19, wherein said modified primary structure is free of γ-carboxyglutamic acid residues .

21. The method of claim 20 wherein the step of modifying the primary structure comprises introducing one or more cysteine residues to form an intramolecular disulfide bond, or forming an amide bond linking two parts of the primary structure of the platelet binding site.

22. The method according to claim 21 wherein the step of modifying the primary structure comprises introducing an amino acid selected from the group consisting of lysine, glutamic acid and aspartic acid and reacting side chains of a lysine with a glutamic or aspartic acid residue to form an amide bond internally cross-linking two parts of the platelet binding site.

23. The method according to claim 18 wherein the step of modifying the primary structure comprises introducing a toluene-2, 4-diisocyanate structure to internally cross-link two free amino groups of the peptide.

24. The method according to claim 18 wherein the molecular model comprises the set of coordinates and connect statements of Appendix 1.

25. A method of producing a synthetic peptide, or a pharmaceutically acceptable salt thereof, said peptide consisting essentially of an amino acid sequence from at least 5 to about 75 amino acids in length, which sequence corresponds to a portion of the amino acid sequence of the platelet binding site on factor IXa, said peptide having an artificially introduced restricted conformation and the ability to inhibit the binding of factor IXa to a platelet surface, comprising:

providing a peptide having an amino acid sequence corresponding to a portion of the sequence of the platelet binding site on the factor IXa chain;

determining the conformational equilibrium of that portion of the factor IXa chain; and

introducing a covalent modification into the peptide to restrict a distance determined to be between two parts of the peptide to a distance between two corresponding parts of the peptide in the equilibrium conformation.

26. A method according to claim 25 whereirintroducing a covalent modification comprises restricting a distance determined to be between two parts of the peptide to a distance between two corresponding parts of the peptide in the equilibrium conformation by introducing a cysteine residue not present in the native amino acid sequence to form a cysteine-cysteine disulfide bond with another cysteine residue or by introducing a covalent bond other than a cysteine-cysteine disulfide bond.

27. A method according to claim 26 wherein said covalent modification comprises an amide bond cross-linking two parts of the peptide.

28. A method according to claim 26 wherein said synthetic peptide is free of γ-carboxyglutamic acid residues.

29. A pharmaceutical composition comprising one or more peptides of claim 1, or a pharmaceutically acceptable salt of said peptide, and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising a peptide of claim 2, or a pharmaceutically acceptable salt of said peptide, and a pharmaceutically acceptable carrier.

31. A pharmaceutical composition comprising a peptide of claim 7, or a pharmaceutically acceptable salt of said peptide, and a pharmaceutically acceptable carrier.

32. A method of inhibiting factor IXa-induced activation of factor X on the platelet surface comprising contacting a platelet with one or more synthetic peptides according to claim 1, or a pharmaceutically acceptable salt thereof.

33. A method according to claim 32 wherein an amino acid sequence segment of said peptide is selected from the group of consisting of: SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO:4; and

combinations thereof.

34. A method according to claim 32 wherein said peptide is free of γ-carboxyglutamic acid residues.

35. A method of inhibiting the binding of factor IXa to a platelet surface comprising contacting a platelet surface with one or more synthetic peptides comprising an amino acid sequence corresponding to a portion of the sequence of the platelet binding site on the factor IXa chain, said peptide having an artificially introduced restricted conformation and the ability to inhibit the binding of factor IXa to a platelet surface.

36. A method according to claim 35 wherein said peptide is free of γ-carboxyglutamic acid residues.

37. A method according to claim 36 wherein said peptide comprises an amino acid sequence selected from the group consisting of:

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4; and

combinations thereof.

38. A method for inhibiting thrombosis comprising administering to a mammal in need of such treatment an effective amount of one or more synthetic peptides according to claim 1, or a pharmaceutically acceptable salt of said peptide.

39. A method for inhibiting thrombosis comprising administering to a mammal in need of such treatment an effective amount of one or more synthetic peptides according to claim 2, or a pharmaceutically acceptable salt of said peptide .