WO1998055130A1 - Exosite assay for anticoagulants - Google Patents

Abstract

Formation of blood clots requires the catalytic formation of the protein thrombin, and applicant has developed a biochemical assay to measure certain exosite interactions of the prothrombinase complex involved in such catalytic formation.

Description

TITLE OF THE INVENTION

EXOSITE ASSAY FOR ANTICOAGULANTS

BACKGROUND OF THE INVENTION

In the coagulation cascade that forms insoluble blood clots, the enzyme thrombin catalyzes the formation of fibrin from its soluble precursor fibrinogen. Fibrin is an insoluble protein that is a fundamental component in the molecular network of blood clots. Thrombin formation is catalysed by a membrane-assembled macromolecular enzyme complex referred to as prothrombinase. The basis for the substrate specificity of prothrombinase is poorly understood. Inhibition kinetics of the cleavage of prethrombin 2 (an analog of prothrombin) by prothrombinase using active site directed inhibitors of factor Xa, suggest that productive recognition of the macromolecular substrate results from an initial interaction at enzymic sites distinct from the active site (or exosite) of prothrombinase followed by interactions at the active site prior to cleavage, to give thrombin. Substrate affinity is largely determined by exosite interactions while the second step contributes to the maximum catalytic rate. These findings indicate limitations to the common approach to targeting the active site of factor Xa for therapeutic purposes. Present applicant has discovered that exosite interactions underlie the macromolecular substrate specificity in the catalytic formation of thrombin, an essential step in the coagulation or clotting cascade.

Applicant has discovered an assay to measure exosite interactions. Several of the highly specific proteolytic activation steps of the clotting cascade are catalysed by enzyme complexes assembled through interactions between an arginine-specific serine proteinase and a cofactor protein on membranes or surfaces. The proteolytic conversion of prothrombin to thrombin is a key step of the coagulation cascade. The specific recognition and cleavage of two peptide bonds in prothrombin is catalysed by the prothrombinase complex, which assembles through reversible interactions between factor Xa (a serine protease) and factor Va, in the presence of calcium ions and negatively charged membranes. See, e.g., Mann, K.G., et al. (1988) Annu. Rev. Biochem. 57, 915-956. The assembly of factor Xa into prothrombinase leads to a profound increase of 100, 000-fold in the catalytic efficiency of prothrombinase for prothrombin activation. Alterations in factor Xa which result from its interaction with factor Na on the membrane surface probably play an important role in these changes. However, the increased catalytic efficiency for prothrombin activation is not paralleled by increases in the cleavage of synthetic peptidyl substrates, binding of ligands to the active site of factor Xa or in the reaction with macromolecular serine proteinase inhibitors such as antithrombin III. Thus, the accelerating effects of the cofactor appear related in some way to the macromolecular substrate specificity of factor Xa. The macromolecular substrate specificity of prothrombinase provides an enzymatic assay for inhibitors of prothrombinase having a variety of therapeutic utilities.

Recent studies with tick anticoagulant peptide have implicated factor Na-induced changes at extended macromolecular recognition sites in factor Xa. The contribution of such effects at exosites, defined as enzymic sites removed from the catalytic residues or the traditional PI -P3 determinants of protease specificity, to the enhanced rate of prothrombin activation by prothrombinase is unknown. Applicant has used classical reversible inhibitors or alternate substrates directed towards the active sites of factor Xa to probe the role of interactions between prothrombinase and the macromolecular substrate at extended macromolecular recognition sites. There is considerable interest in the therapeutic modulation of coagulation by specific factor Xa inhibitors. The recent availability of X-ray structures of factor Xa will likely direct structure aided design of specific active site directed inhibitors of factor Xa. While these inhibitors may display appropriate properties when assessed with synthetic peptidyl substrates for factor Xa in vitro, applicant's observations indicate that targeting the active site of factor Xa will fail to yield competitive inhibitors of macromolecular substrate cleavage by the prothrombinase complex.

Applicant has constructed an enzymatic assay for the purpose of measuring interactions at such exosites in prothrombinase, particularly factor Xa. The exosite assay is suitable for screening inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote for the catalytic site of prothrombinase. Such inhibitors constitute a new class of anticoagulants suitable in the clinical management of cardiovascular disease, stroke and a variety of hematological disorders.

BRIEF DESCRIPTION OF THE INVENTION

An essential step in the formation of clots in the coagulation cascade is the formation of thrombin by the catalytic cleavage of its precursor prothrombin by the enzyme complex prothrombinase. Applicant has discovered that interactions at exosites remote from the enzymatically active site of prothrombinase substantially affect the formation rate of thrombin, and therefore provide an assay suitable for identifying therapeutics useful in the therapy and management of diseases involved with abnormalities in coagulation or clotting, e.g., stroke.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Linear Competitive Inhibition of Peptidyl Substrate

Hydrolysis by PAB. Initial velocities were measured using 0.5 nM prothrombinase (0.5 nM Xa, 20 nM Va, 50 μM PCPS), increasing concentrations of Spectrozyme Xa with 0 (V), 15 μM (Δ), 30 μM(Δ), 60 μM(#) and 120 μM (O) PAB. These lines are drawn according to linear competitive inhibition with the fitted constants: Km=85±5 μM, kcat = 234±1.2 μM. Inset: Double reciprocal plot showing effect of PAB on Km and not on the Vmax for the reaction.

Figure 2: Noncompetitive Inhibition of Prethrombin 2 plus

Fragment 1.2 by PAB. The initial velocity for thrombin formation was determined at increasing concentrations of prethrombin 2 plus fragment 1.2 using prothrombinase assembled with 0.25 nM factor Xa, 54 μM PCPS and 24 nM Va. Rates normalized/nM prothrombinase were measured using 0 (O), 189 μM (•) or 409 μM PAB (Δ). The lines are drawn following analysis according to classical noncompetitive inhibition, with the constants Kmo _s = 38±0.02 μM, VmaX_obJE_T = 23±4 s^"1 and Ki= 57.3±4.7 μM. Inset: Double reciprocal plot showing that PAB alters Vmax but not Km. Figure 3 : Noncompetitive Inhibition of PAB on the cleavage of

Prethrombin 2. The initial velocity for thrombin formation was determined at increasing concentrations of prethrombin 2 using 5 nM prothrombinase (5 nM Xa, 54 μM PCPS and 24 nM Va). Initial rates normalized/nM prothrombinase were measured using 0 (O), 60 μM (•) and 160 μM PAB (Δ). The lines are drawn following analysis according to classical noncompetitive inhibition, with the constants: Km_obs= 3.39±0.1 μM, VmaXob/E_T = 1.46---0.02 s^"1 and Ki= 31.8±9.64 μM. Inset: Double reciprocal plot showing that PAB alters Vmax but has no effect on Km. Figure 4: Competitive Inhibition of Inactivated Thrombin on Prethrombin 2 Cleavage by Prothrombinase. Initial velocities were determined at increasing concentrations of prethrombin 2, 5 nM prothrombinase (5 nM Xa, 25 nM factor Va, 44 μM PCPS) in the presence of 0 μM (V), 3 μM (A), 6 μM (Δ) and 12 μM (•) thrombin inactivated with APMSF. The lines are drawn following analysis according to classical noncompetitive inhibition, with the constants: Kmobs = 3.17±0.13 μM, VmaXobJE_T = 1.28±0.02 s^"1 and Kp= 2.2±0.08 μM.

Figure 5: Effect of Inactivated Thrombin on Peptidyl Substrate

Cleavage. Initial velocities were determined at increasing concentrations of Spectrozyme Xa and 0.5 nM prothrombinase (0.5 nM Xa, 25 nM factor Va, 44 μM PCPS) in the presence of 0 μM (O), 3 μM (•), 6 μM (Δ) and 12 μM (A) inactivated thrombin. Fitting of all data to the Michaelis Menten equation yielded Km = 82.3±2 μM and kcat = 7 s-¹.

Figure 6: A schematic diagram of the prothrombinase complex in various conformations. Prethrombin 2 (the substrate S) is cleaved to the two chain product, thrombin (P) by prothrombinase (E) in the presence of active site-directed reversible inhibitor (I). K_s, Kj, and K_P refer to the equilibrium dissociation constants for the binding of S, I and P to E, respectively. K_s* is the equilibrium dissociation constant for substrate binding to the active site. Initial velocity measurements indicate that multipliers α and β are indistinguishable from 1. The rate constant for the catalytic step is denoted kcat- Figure 7: A schematic illustration of cleavage sites in prothrombin and derivatives. Figure 8: A schematic illustration of other Prethrombin 2 derivatives.

ABBREVIATIONS AND DEFINITIONS

APMSF amidino-phenylmethylsulfonylfluoride

Fragment 1.2 N-terminal fragment of prothrombin

PCPS phosphatidylcholine and phosphatidylserine

Prethrombin 2 An analog of prothrombin.

Ha, Prethrombin 2 inhibited with APMSF

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to an exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of: (a) providing a substrate solution comprising a substrate at a concentration between about 0.05 μM and about 20 μM, factor Va at a concentration between about 0.05 nM and about 200 nM, and phospholipids at a concentration of between about 30 μM and about 500 μM, and a test inhibitor, in buffer containing between about 1 mM to about 10 mM Ca²⁺ at pH between about 7.0 and about 9.0, said buffer lacking a chelator of Ca²⁺, said substrate comprising a protease cleavage site and an exosite binding determinant;

(b) initiating catalytic cleavage of the substrate by adding to the substrate solution an aliquot of factor Xa, to give a final concentration of Xa of between about 0.05 nM and about 200 nM, such that there is excess Va over Xa, to form a substrate-prothrombinase complex;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin. In one embodiment of the exosite assay of the present invention, the substrate comprises prethrombin 2.

In another embodiment of the exosite assay of the present invention, the substrate is selected from the group consisting of prethrombin 2, ζ-prethrombin 2, a mixture of fragments ζl -prethrombin 2 and ζ2-prethrombin 2, and a fusion polypeptide comprising ζl -prethrombin 2 and ζ2-prethrombin 2, any of which polypeptides has bovine origin, human origin, or both.

This invention also relates to an exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of:

(a) providing a prothrombinase solution comprising factor Xa at a concentration between about 0.05 nM and about 200 nM, factor Na at a concentration between about 0.05 nM and about 200 nM, such that there is excess of factor Na over Xa, and phospholipids at a concentration of between about 30 μM and about 500 μM, and a test inhibitor, in buffer containing between about 1 mM to about 10 mM Ca²⁺ at pH between about 7.0 and about 9.0, said buffer lacking a chelator of Ca²⁺;

(b) initiating catalytic cleavage of a substrate by adding to the prothrombinase solution an aliquot of substrate, to give a final concentration of substrate of between about 0.05 μM and about 20 μM, to form a substrate- prothrombinase complex, said substrate comprising a protease cleavage site and an exosite binding determinant;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

In one embodiment of the exosite assay of the present invention, the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200 nm, said phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, and phosphatidylglycerol, and mixtures thereof. In another embodiment of the exosite assay of the present invention, the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200nm, said phospholipids comprising about 75% (w/w) L-alpha- phosphatidylcholine and about 25% (w/w) L-alpha-phosphatidylserine. In another embodiment of the exosite assay of the present invention, the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200 nm, said phospholipids comprising at least about 5% to about 50% phosphatidylserine, the remainder selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and phosphatidylglycerol, and mixtures thereof.

In another embodiment of the exosite assay of the present invention, the phospholipids are provided from rabbit brain cephalin or membranes prepared from activated platelets.

In another embodiment of the exosite assay of the present invention, the quenching of aliquots in step (c) is carried out by adding buffer containing an inhibitor of factor Xa, and a chelator of Ca²⁺.

In another embodiment of the exosite assay of the present invention, the inhibitor of factor Xa is Tick Anticoagulant Peptide.

In another embodiment of the exosite assay of the present invention, the inhibitor of factor Xa is recombinant Tick Anticoagulant Peptide.

In another embodiment of the exosite assay of the present invention, the concentration of thrombin in step (d) is carried out with a calorimetric assay.

In another embodiment of the exosite assay of the present invention, the concentration of thrombin is calculated from the initial velocity in the hydrolysis of thrombin substrate H-D-phenylalanylpipecoylarginine pnitroaniline.

The present invention also relates to an exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of:

(a) providing a substrate solution comprising prethrombin 2 at a concentration between about 1 μM and about 2 μM, factor Na at a concentration of about 20 nM, and phospholipids at a concentration of about 50 μM, and a test inhibitor, in buffer containing about 2 mM Ca²⁺ at pH of about 7.4, said buffer lacking a chelator of Ca²⁺, said phospholipids being small unilamellar vesicles of diameter between about 20 nm and about 200nm, said vesicles comprising about 75% (w/w) L- alpha-phosphatidylcholine and about 25% (w/w) L-alpha-phosphatidylserine; (b) initiating catalytic cleavage of prethrombin 2 by adding to the substrate solution an aliquot of factor Xa, to give a final concentration of Xa of between about 2 nM and about 5 nM, such that there is excess Na over Xa, to form a substrate-prothrombinase complex;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

The present invention also relates to an exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of:

(a) providing a prothrombinase solution comprising factor Xa at a concentration between about 2 nM and about 5 nM, factor Na at a concentration of about 20 nM, such that there is excess of factor Na over Xa, and phospholipids at a concentration of about 50 μM, and a test inhibitor, in buffer containing about 2 mM Ca ⁺ at pH of about 7.4, said buffer lacking a chelator of Ca²⁺, said phospholipids being small unilamellar vesicles of diameter between about 20 nm and about 200nm, comprising about 75% (w/w) L-alpha-phosphatidylcholine and about 25% (w/w) L- alpha-phosphatidylserine;

(b) initiating catalytic cleavage of prethrombin 2 by adding to the prothrombinase solution an aliquot of prethrombin 2, to give a final concentration of prethrombin 2 of between about 1 μM and about 22 μM, to form a substrate- prothrombinase complex;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots; (d) assaying the quenched cleaved aliquots for the concentration of thrombin. The present invention also relates to inhibitors giving an IC₅₀ in the exosite assay of the present invention of less than about 1.0 μM.

Unless otherwise specifically noted, the enzymes, protein factors, and protein substrates of the exosite assay and counterscreen of the present invention, e.g., prothrombin, prethrombin 2, factor Na, factor Xa, are intended to originate from any mammalian species, preferably human or bovine, most preferably human. Bovine prothrombin (SEQ. ID NO. 1), bovine prethrombin 2 (SEQ. ID NO. 2), Human prothrombin (SEQ. ID NO. 3), and human prethrombin 2 (SEQ. ID NO. 4) are illustrative substrates of the exosite assay. Fragments of prothrombin mentioned in the present invention have the following characteristics in the bovine and human sequences:

Fragment Amino Acid Residues of Prothrombin

Bovine Fragment 1 1-156 of bovine prothrombin

Bovine Fragment 2 157-274 of bovine prothrombin Bovine Fragment 1.2 1-274 of bovine prothrombin

Human Fragment 1 1 - 155 of human prothrombin

Human Fragment 2 156-271 of human prothrombin

Human Fragment 1.2 1 -271 of human prothrombin

Factor Xa and other arginine-specific serine proteinases are inhibited by 4-aminobenzamidine (PAB) which binds reversibly to the S 1 or the primary specificity pocket in the protease. This is consistent with the kinetic behavior of PAB as a linear competitive inhibitor of synthetic peptide hydrolysis by the prothrombinase complex (Fig. 1). Linear competitive inhibition implies that PAB binding to the primary specificity pocket of factor Xa within prothrombinase precludes synthetic peptidyl substrate binding to the active site. Thus, apparent affinity for the peptidyl substrate is systematically decreased to undetectable levels by increasing concentrations of inhibitor, leading to an increase in Km while the Vmax remains unchanged.

The conversion of prothrombin to thrombin results from cleavage at R³²³-I²⁴ followed by cleavage at R²⁷⁴.χ²⁷⁵ by prothrombinase. The first cleavage is the one most significantly affected by factor Va or membranes. The kinetics of recognition and cleavage at this site in prethrombin 2 is indistinguishable from the cleavage at the same site in intact prothrombin.

Initial velocity studies of prethrombin 2 plus fragment 1.2 cleavage by prothrombinase indicate that PAB acts as a classical noncompetitive inhibitor of this reaction (Fig. 2). Classical noncompetitive inhibition, evident as a change in the apparent Nmax with no affect on the Km, implies that PAB bound to the active site of the enzyme does not alter the affinity for prethrombin 2 plus fragment 1.2 but changes the rate constant for catalysis.

The fragment 1.2 activation peptide associates tightly but reversibly with prethrombin 2 and imparts membrane binding and factor Na binding properties to the substrate. Classical noncompetitive inhibition by PAB was also observed for prethrombin 2 cleavage by prothrombinase in the absence of fragment 1.2 (Fig. 3).

Occupation of the primary specificity pocket of factor Xa within the prothrombinase complex by PAB has no obvious effect on the Km for macromolecular substrate cleavage even in the absence of activation peptide domains responsible for membrane and factor Na binding by the substrate.

This discrepancy between the kinetics of cleavage of tripeptidyl substrates and prethrombin 2 by prothrombinase was sustained with other inhibitors and alternate peptidyl substrates. See Table I. Table I. Inhibition kinetics of synthetic peptidyl or macromolecular substrate cleavage by prothrombinase. Kinetic constants were determined from data as illustrated in Figure 2 and are reported ±95% confidence limits following analysis according to the indicated rate expression.

Substrate Inhibitor* Inhibition Type Km kcat Ki

(μM)±S.D. (s-1) ±S.D. (μlvQ±S.D.

Spectrozyme Xa¹ PAB Competitive 85 ± 5 234 ± 4 24.5 ± 1.2

TAPA Competitive 82.7 ± 3.5 233 ± 3 1.87 ± 0.3

S2238 Competitive 114 ± 3.2 238 ± 3 51.3 ± 1.3 Prethrombin 2 PAB Noncompetitive 3.39 ± 0.1 1.46 ± 0.02 31.8 ± 0.64

TAPA Noncompetitive 2.97 ± 0.14 1.49 ± 0.02 4.37 ± 0.15

S2238 Noncompetitive 2.35 ± 0.1 1.37 ± 0.02 28.9 ± 1.6

S2222 Noncompetitive 2.51 ± 0.22 1.02 ± 0.03 361.8 ± 22.3

PAB is 4-aminobenzamidine; TAPA is Nα-tosylglycyl-3-DL- amidinophenylalanyl methyl ester; S2238 is H-D-phenylalanyl-L-pipecoyl-L-arginyl p- nitroanilide; S2222 is isoleucylglutamyl-glycinyl-arginyl p-nitroanilide.

A peptidyl p-nitroanilide substrate which is methoxycarbonyl cyclohexyglycyl glycyl arginyl p-nitroanilide.

In each case, reagents directed towards the active site of factor Xa within the prothrombinase complex, acted as linear competitive inhibitors of Spectrozyme Xa cleavage but acted as classical noncompetitive inhibitors of prethrombin 2 cleavage. Noncompetitive inhibition of prethrombin 2 hydrolysis by alternate peptide substrates of prothrombinase indicates that the Km for prethrombin 2 is not changed even when a peptidyl substrate is productively bound and cleaved at the active site of the protease. The results with S2222 are particularly significant because the peptidyl sequence of this substrate is identical to the P4-P1 sequence preceding the scissile bond in prethrombin 2. Hence the primary determinants of macromolecular substrate affinity for the prothrombinase complex result from interactions on sites on the enzyme complex, termed exosites, that are distinct from those involved in the binding of active site directed ligands or oligopeptidyl substrate analogs. Since cleavage of prethrombin 2 by factor Xa within the prothrombinase complex must involve binding interactions between the substrate and the active site of factor Xa, at least two binding steps precede cleavage of the scissile bond in prethrombin 2 (Figure 6). The first step involves interactions between the macromolecular substrate and exosites in prothrombinase, which is the step with equilibrium constant K_s. This bimolecular step is not influenced by binding of small active site directed inhibitors or synthetic peptidyl substrates to the active site of factor Xa. Equivalently, formation of the ES complex has no detectable effect on the binding of small molecules to the active site of factor Xa within prothrombinase. The formation of the initial complex is then followed by interactions between structures surrounding the scissile bond in the protein substrate and the active site of factor Xa in a unimolecular step before catalysis, requiring prior dissociation of alternate substrates or inhibitors from the active site of the enzyme. Inhibition of macromolecular substrate cleavage by these reagents is not achieved by interfering with the bimolecular combination of enzyme and substrate but rather by influencing the formation of ES*. Since the unimolecular transformation of ES to ES* precedes catalysis, it is expected to contribute to the maximum catalytic rate. As a result, inhibitors that interfere with the interactions between the macromolecular substrate and the active site of the enzyme will be expected to reduce ES* formation and decrease the Nmax for the reaction. This interpretation is consistent with the observed kinetics of prethrombin 2 inhibition by active site directed reagents.

Additional insights into the multi-step mechanism for macromolecular substrate binding and cleavage are provided by the composite nature of the observed kinetic constants readily illustrated using the rapid equilibrium assumption:

Nmax_obs

The equilibrium constant (Ks*) for the interaction between the macromolecular substrate and the active site of factor Xa influences both the observed Km and Nmax. Yet, the presence of inhibitor, I, leads to a decrease in velocity without affecting Kmob_s, even though I and Ki terms modify both composite kinetic constants. Such findings are only expected when Ks* is much greater than 1, implying that the ES to ES* transition is unfavorable. This conclusion can also be drawn from the relevant steady state rate expressions.

The kinetic measurements support three major conclusions. Affinity of prothrombinase for the macromolecular substrate is determined entirely by interactions at exosites. Cleavage at R³²³_I³²⁴ i_n prethrombin 2 (the step with equilibrium constant K_P ) results from subsequent binding interactions between the active site and structures surrounding the scissile bond in a unimolecular step that is unfavorable. The Nmax contains significant contributions from the equilibrium dissociation constant for the active site interactions between prethrombin 2 and the active site of factor Xa within the prothrombinase complex.

It follows that reagents competing with prethrombin 2 for its initial interaction with the enzyme should behave as linear competitive inhibitors. Additionally, if inhibition of exosite binding is indeed achieved at site(s) distinct from the active site, this type of inhibitor should not restrict access to the active site and therefore not affect synthetic tripeptidyl substrate cleavage by prothrombinase.

Thrombin, inactivated with APMSF (Ha,), was found to be an effective product inhibitor of prethrombin 2 cleavage of prothrombinase. Initial velocity studies established that Ilai was a linear competitive inhibitor of prethrombin 2 activation with Kp = 2.2 μM (figure 4). The same concentrations of Ilai had a very small effect on the rate of Spectrozyme Xa hydrolysis by prothrombinase (Figure 5). These properties are predicted for an inhibitor that competes with the macromolecular substrate for interactions at the exosite of prothrombinase. Thus, the product release steps following cleavage of the R¹²¹-l¹²⁴ peptide bond in prethrombin 2 also involve interactions between newly-formed thrombin and some exosite on prothrombinase that precludes prethrombin 2 binding but does not obscure access of small ligands, such as Spectrozyme Xa, to the active site of Xa.

Binding specificity for the macromolecular substrate is determined by two resolvable steps (having equilibrium constants Ks and Ks in Figure 6). Interactions at an exosite predominate the perceived affinity of enzyme for substrate in activity measurements. Consequently, inhibitors directed to this site may help resolve the difficulties in preparing reversible inhibitors (I) of the active site that can specifically react only with a particular protease belonging to a family of arginine- specific coagulation and digestive serine proteases, all with homologous active sites. Exosite-directed inhibitors may also offer the added advantage of permitting the further regulation of the already exosite-inhibited enzyme by circulating serine proteinase inhibitors that is otherwise abrogated upon binding of reversible inhibitors to the active site of factor Xa.

The known structure of prethrombin 2 indicates that structures surrounding the R³²³_I³²⁴ scissile bond in the zymogen require significant rearrangement before they can be docked with the active site of factor Xa. This provides a structural correlate for our conclusion that the active site interaction is governed by an unfavorable step. Consequently, substrate binding specificity for cleavage at R³²³-I³²⁴ may be largely determined by the exosite rather than the active site interactions.

The Exosite Assay

The exosite assay measures enzymatic hydrolysis of prothrombin or derivatives thereof by the prothrombinase complex. This complex comprises factor Na, phospholipids, Ca²⁺ and factor Xa, in buffer lacking a chelator of Ca²⁺. It is preferred that there is excess Na over Xa. Reaction is initiated by adding substrate, such as prothrombin, prethrombin 2 or related analogs. Alternatively, instead of mixing substrate and prothrombinase complex to initiate, reaction protocol allows the addition of factor Xa as the last component to the mixture of factor Na, phospholipids, Ca²⁺, buffer backing a chelator of Ca²⁺, and substrate. In either case, reaction is initiated when substrate, e.g., prothrombin or related analogs, is mixed a complex containing factor Xa.

There is a range of final concentrations of components. The substrate concentration varies from between about 0.05 μM and about 200 μM, preferably between about 1 μM and about 2 μM, most preferably about 1 μM. Factor Na varies between about 0.05 nM and about 200 nM, preferably about 20 nM. Phospholipids range from between about 30 μM and about 500 μM, preferably about 50 μM. The concentration of Ca²⁺ may vary between about lmM and about 10 mM, preferably about 2 mM. The pH range is between about 7.0 and about 9.0, preferably about pH 7.4. The buffer employed may not have a Ca²⁺ chelating function, e.g., phosphate buffer will not work for this reason. Factor Xa, which contains the active site for hydrolysis, may range in concentration between about 0.05 nM and about 200 nM, preferably between about 2 nM and about 5 nM.

The phospholipids provide a molecular template for the reaction as a negatively-charged surface. They are typically small unilamellar vesicles of diameter between above 20 nm and about 200 nm, of natural or synthetic origin or of mixed origin. Suitable phospholipids of natural origin include rabbit brain cephalin or membranes prepared from activated platelets or other blood cells. Synthetic phospholipids comprise at least 5% (w/w) of phosphotidylserine, the remainder selected from synthetic phospholipids including, but not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, or phosphatidic acid, or mixtures thereof. A preferred composition of synthetic phospholipids is unilamellar vesicles of diameter between about 20 nm and about 200 nm, said phospholipids comprising about 75% (w/w) phosphahdylcholine and about 25% (w/w) phosphatidylserine.

The exosite assay measures inhibition of exosite interaction in the presence of a putative inhibitor, when the substrate is a macromolecular substance, such as prothrombin or prethrombin 2. Detectable inhibition in this exosite assay is then further evaluated in a counterscreen that detects competitive inhibition of small molecular weight substances of factor Xa, e.g.. Spectrozyme. Components that inhibit the exosite assay but fail to competitively inhibit hydrolysis in the counterscreen are the compounds of choice, i.e., they interfere at sites remote from the active site on factor Xa. Compounds that inhibit both the exosite assay and the counterscreen are likely interfering with the reaction at the active site of factor Xa.

Prothrombin and related Analogs as Macromolecular Substrates for Prothrombinase Thrombin formation requires cleavage of prothrombin at Arg -lie (R³²³) followed by Arg²⁷⁴-Thr²⁷⁵ (R²⁷⁴) by prothrombinase. See Figure 7. Prior cleavage at R²⁷⁴ yields prethrombin 2 plus fragment 1.2. The fragment 1 domain mediates the high-affinity interaction of prothrombin with membranes, and fragment 2 is responsible for binding factor Na. The site denoted by R¹⁵⁵ is subject to cleavage by thrombin which liberates fragment 1 from either prothrombin or fragment 1.2. The shaded area of Figure 7 denote the ability of prethrombin 2 to interact tightly with the fragment 2 domain. The prethrombin 2 derivatives require further cleavage at R³²³ to form thrombin and represent substrates which cannot interact with membranes or factor Na (Prethrombin 2), which can bind to factor Na but not membranes (Prethrombin 2 plus Fragment 2) and which can bind both factor Na and membranes (Prethrombin 2 plus Fragment 1.2). The substrate for the exosite assay must contain two functional sites, which are a protease cleavage site and an exosite binding determinant. The protease cleavage site is the sequence

He Glu Gly Arg He Nal (SEQ ID 9 ) , or He Asp Gly Arg He Nal (SEQ ID 10 ) or any other homologous amino acid sequence cleavable, whether or not contained within a larger amino acid sequence, by the prothrombinase complex in the exosite assays of the present invention. It is preferred that the protease cleavage site and the exosite binding determinant are situated on the same polypeptide chain, e.g. in human or bovine prethrombin 2. These two funtional sites may be linked in a fusion polypeptide, e.g. with a polyglycine linker.

The exosite binding determinant is contained within the following C- terminal regions of prethrombin 2:

PROTEIN OR PEPTIDE RESIDUES OF EXOSITE

Bovine prethrombin 2 198-308

Bovine Prothrombin 472-582

Human Prethrombin 2 198-308

It will be understood that homologous sequences, or fragments thereof, are readily testable as exosite binding determinants, in the exosite assays of the present invention. Accordingly, the substrates of the exosites assays of the present invention include exosite binding determinants having the amino acide sequences as listed above, as well as homologous sequences, or fragments thereof.

Suitable substrates include, but are not limited to, prethrombin 2, ζ- prethrombin 2, a mixture of fragments ζl -prethrombin 2 and ζ2 -prethrombin 2, and a fusion polypeptide comprising ζl -prethrombin 2 and ζ2-prethrombin 2, any of which polypeptides may have bovine origin, human origin, or both.

Small Substrates for Prothrombinase

A variety of small substrates are suitable for measuring thrombin formation. These include, but are not limited to:

COMPOUND SYNONYM

Arg-OMe

Tos-Arg-OMe TAME

Bz-Arg-OMe BAME

Ac- Arg-OMe

Tos-Ornithine-OMe TOME

Lys-OMe LME

Tos-Lys-OMe TLME

Ac-Lys-OMe ALME

Boc-Lys-OMe

Boc-Ala-Lys-OMe

Boc-Gly-Lys-OMe

Boc-D-Ala-Lys-OMe

Boc-Phe-Lys-OMe

Boc-Asn-Lys-OMe

Boc-Gly-Gly-Lys-OMe

Bz-Ala-OMe

Ac-Ala-OMe

His-OMe HME COMPOUND SYNONYM

Phe-OMe PME

Tos-Gph-OMe -

R-Arg-Gly-OMe -

Bz-Arg-OEt BAEE

Lys-OEt LEe

Bz-Tyr-OEt -

Ac-Tyr-OEt -

Tos-Tyr-OBe -

Z-Lys-ONp -

Z-Ala-ONp -

Z-Tyr-ONp - p-nitrophenyl acetate NPhAc p-nitrophenyl-p -guanidino benzoate NPGB p-nitrophenyl-amino-p-toulate NPMT p-nitrobenzy p- toluensulfonyl-L-arginine NBTA

Many of the substrates listed immediately above are also suitable in the small substrate counterscreen, an assay to detect competitive inhibition of factor Xa hydrolysis of prothrombin. Other suitable small substrates for the small substrate counterscreen include, but are not limited to:

COMPOUND SYNONYM

Bz-Arg-/?NA BAPNA

Bz-Lys-_jPNA

Ac-Tyr-/?NA

Z-Trp-Arg- ?NA

H-D-CAT-Ala-Arg- NA Spectrozyme-TH

H-D-Phe-Aze- Arg-/?NA S-2388

H-D-Chg-But-Arg-^NA CBS 34.47 COMPOUND SYNONYM

PyrGlu-Gly-Arg-/?NA S-2244

Bz-PyrGlu-Gly-Arg-/?NA S-2405

Z-Ala-Gly-Arg-^NA

Z-Asn-Gly-Arg- )NA

Z-Glu-Gly-Arg-/?NA

Z-Lys-Gly-Arg-y^NA

Z-Phe-Gly-Arg-/?NA

Z-Pro-Gly-Arg-pNA

Z-Ser-Gly-Arg-/?NA

CBz-Val-Gly-Arg-/?NA Chromozym-TRY

H-D-Val-Leu-Arg- >NA S-2266

Z-Ala-Phe-Arg-/?NA

Z-Asn-Phe-Arg-/?NA

Z-Glu-Phe-Arg-/>NA

Z-Lys-Phe-Arg-/?NA

Z-Phe-Phe-Arg-/?NA

Z-Ser-Phe-Arg-/?NA

H-D-Pro-Phe-Arg-/?NA S2302

Bz-Pro-Phe-Arg-^NA Chromozym-PK

H-D-Val-Phe-Arg-^NA S-2325

H-D-Phe-Pip-Arg- NA S-2238

H-D-Nal-Pip-Arg-pNA

H-D-Arg-Pro-Arg-/?NA

Z-Arg-Pro-Arg- ?NA

CBz-Gly-Pro-Arg-/?NA Chromozym-TH

Tos-Gly-Pro-Arg-^NA Chromozym-TH

H-D-Ile-Pro-Arg- NA S-2288

H-D-Lys-Pro-Arg-^NA

H-D-Phe-Pro-Arg-pNA COMPOUND SYNONYM

Bz-D-Phe-Pro-Arg- ?NA

Bz-Phe-Pro-Arg-/>NA

Z-D-Phe-Pro-Arg-/?NA

Z-Phe-Pro-Arg-^NA

PyrGlu-Pro-Arg-^NA S-2366

Sarc-Pro-Arg- ?NA

H-D-Val-Pro-Arg-pNA S-2234

Z-D-Val-Pro-Arg-/?NA H-D-Phe-Val-Arg-/?NA Bz-Phe-Val-Arg-^NA S-2160

Bz-Phe-Pro-GPA- ?NA H-D-Val-Leu-Lys-pNA S-2251

PyrGlu-Phe-Lys-pNA S-2403

H-D-Val-Phe-Lys-/.NA S-2390

Tos-Gly-Pro-Lys-pNA Chromozym-PL

Bz-He-Glu-Gly-Arg- NA S-2222

Bz-Ile-Glu(pip)-Gly-Arg-/?NA S2337

Tos-Gly-Pro-Arg-ANBA-R H-D-Phe-Pro-Arg-ANBA-R

CBo-Gly-Pro-Arg-MBNA

Boc-Val-Pro-Arg-MCA

H-D-Phe-Pro-Arg-AIE

CBz-Gly-Pro-Arg-βNA

CBz-Gly-Gly-Arg-MCA

Z-Trp-Arg-MCA COMPOUND SYNONYM

Abz ...Arg-Gly...Nba

H-D-Phe-Pip-Arg- ?ADA S-2497

Boc-Arg-SBu-i

Boc-Arg-SPe-i

Boc-Arg-SPe-s

Boc-Arg-SBzl

Boc-Met-Arg-SBu-i

Boc-Pro-Arg-SBu-i

Boc-Phe-Ser-Arg-SBzl

Boc-Leu-Ser-Arg-SBzl

Boc-Leu-Thr-Arg-SBzl

Boc-Val-Val-Arg-SBzl

Boc-Val-Phe-Arg-SBzl

Boc-Val-Trp-Arg-SBzl

Boc-Phe-Phe-Arg-SBzl

Boc-Phe-Trp-Arg-SBzl

Boc-Trp-Phe-Arg-SBzl

Boc-Trp-Trp-Arg-SBzl

Z-Arg-SBu-i

Z-Arg-SBzl

Z-Arg-SBzl(NO₂)

Z-Arg-SBzl(Cl)

Z-Arg-SBzl(OCH₃)

Z-Pro-Arg-SBu-i

Z-Val-Arg-SBu-i COMPOUND SYNONYM

Z-Gly-Arg-SBu-i

Z-Ala-Arg-SBu-i

Z-Phe-Arg-SBu-i

Z-Trp-Arg-SBu-i

Z-Ser-Arg-SBu-i

Z-Thr-Arg-SBu-i

Z-Asn-Arg-SBu-i

Z-Glu-Arg-SBu-i

Z-Lys-Arg-SBu-i

Z-Arg-SGly-Pro-NH₂

D-Phe-Pro-Arg-SBzl

CBz-Lys-SBzl

Small Substrate Counterscreen

The exosite assay measures inhibition of exosite interaction in the presence of a putative inhibitor, when the substrate is a macromolecular substrate, such as prothrombin or prethrombin 2. Detectable inhibition in this exosite assay is then further evaluated in a counterscreen that detects competitive inhibition of small molecular weight substrates of factor Xa, e.g., Spectrozyme Xa. Compounds that inhibit the exosite assay but fail to competitively inhibit hydrolysis in the counterscreen are the compounds of choice, i.e. they interfere at sites remote from the active site on factor Xa. Compounds that inhibit both the exosite assay and the counterscreen are likely interfering with the reaction at the active site of factor Xa, not at the exosite. Reaction conditions for the counterscreen are substantially similar to the exosite assay, except that the substrate is a small molecular weight oligopeptide instead of a macromolecular weight substrate such as prothrombin. Typical small molecular weight oligopeptides include natural or synthetic tripeptides or tetrapeptides or analogs thereof. One preferred small molecular weight substrate for the counterscreen is Spectrozyme Xa. Alternative substrates are listed in Tables given above. For the counterscreen, there is a range of final concentrations of components. The small molecular weight substrate concentration varies from between about 10 μM and about 800 μM, preferably about 100 μM. Factor Va varies from between about 15 nM and about 100 nM, and is preferably about 20 nM. Phospholipids range from between about 30 μM and about 500 μM, preferably about 50 μM. The concentration of Ca²⁺ may vary from between about lmM and about lOmM, preferably at about 2 mM. The pH range is between about 7.0 and about 9.0, preferably about 7.4. The buffer employed may not have a Ca²⁺ chelating function, e.g. phosphate buffer will not work for this reason. Factor Xa, which contains the active site for hydrolysis of substrate, may range in concentration from between about 0.05 nM and about 200 nM, preferably 0.5nM. Phospholipid composition for the counterscreen is essentially identical with phospholipid composition of the exosite assay. (See supra).

EXAMPLE 1

Preparation of Enzymes and Protein Factors

Prothrombin and factor X was purified from bovine plasma as previously described [Krishnaswamy, S., et al.(1986), J. Biol. Chem. 261 : 8977; Krishnaswamy, S. (1990) J. Biol. Chem. 265, 3708-3718]. Factor X was activated using the purified activator from Russell's viper venom and the resultant factor Xa was purified using benzamidine sepharose (Jesty, J. et al. (1976) Methods Enzymol. 45, 95; Krishnaswamy, S., et al., (1987) J. Biol. Chem. 262, 3291-3299). Bovine factor Va was purified using established procedures (Krishnaswamy, S., et al., (1988) J. Biol. Chem.. 263, 5714-5723; Kalafatis, M, et al. (1993) Methods Enzymol 222, 224-236) The recombinant protein wt TAP was expressed in Pichia Pastoris and purified as described (Laroche, Y., et al. (1994) Biotechnology (NY) 12, 1119-1124). The purified monoclonal IgG, αHII-5, directed against a peptidyl sequence present in the kringle 2 domain of prothrombin was obtained according to Church, W.R., et al., (1991) J. Biol. Chem. 266, 8384-8391. Prothrombin fragment 1.2, prethrombin 1, and thrombin were prepared as previously described (Mann, K.G., et al. (1981)

Methods Enzymol. 80, 286-302; Lundblad, R.L., et al., (1976) Methods Enzymol. 45, 156-176). The isolation of prethrombin 2 and fragment 2 was according to modifications of existing methods (Mann, K.G et al. (1981) Methods Enzymol. 80,286-302; Carlisle, T.L.,et al., (1990), J. Biol. Chem. 265, 22044-22055).

Prethrombin 1 (180 μM, 150 mg) in 0.85 M Na₃ Citrate was treated with 220 nM factor Xa. Following cleavage for 30 min at room temperature, the reaction mixture was directly applied to benzamidine sepharose (1.5 x 8 cm), equilibrated in the same buffer. Development of the resin with 20 mM Hepes, 0.15 M NaCl, pH 7.4, resulted in the elution of a sharp peak containing prethrombin 2 and fragment 2. Thrombin and factor Xa bound to the resin could be eluted with the same buffer containing 4 mM benzamidine. Fractions containing a prethrombin 2 and fragment 2 were pooled, applied to a column to trypsin inhibitor sepharose (1.6 x 5 cm) to remove trace amounts of factor Xa, dialyzed against 25 mM sodium phosphate, pH 6.5., and applied to a column (2.5 x 30 cm) of S-sepharose equilibrated in the same buffer. Fragment 2 was not retained by the resin. Bound prethrombin 2 was eluted with a linear gradient of increasing NaCl (0 to 1.0 M, 4mL/min, 150 min) prepared in the same buffer. Fractions containing prethrombin 2 and fragment 2 were pooled separately and precipitated with ammonium sulfate (80% saturation). Precipitated protein was collected by centrifugation (53000g, 30 min) and dissolved in 50% glycerol. The individual protein preparations were then subject to gel filtration chromatography using Sephadex G-75 (2.5 x 100 cm) equilibrated in 20 mM Hepes, 2.5 M NaCl, pH 7.4., to remove possible traces of cross-contaminating fragments (Carlisle, T.L., et al., (1990), J. Biol. Chem. 265, 22044-22055.) In each case, the pooled fractions were dialyzed against 20 mM Hepes, pH 7.4, concentrated by precipitation with ammonium sulfate and centrifugation and stored at -20°C as concentrated solutions (>200 μM) in 50% glycerol. Typical yields were 40 mg of prethrombin 2 and 10 mg of fragment 2. Prethrombin 2, fragment 2, and fragment 1.2 were exchanged into Assay Buffer (20 mM Hepes, 0.15 NaCl, 2.0 mM CaCl₂, 0.1% (w/v) PEG, Ph 7.4), either by dialysis or by desalting using centrifuge columns (Sephadex G-25, 5 mL) before use. The purity of protein preparations was established by SDS-PAGE followed by staining with Coomassie Brilliant Blue (Laemmli, U.K. (1970) Nature 227, 680-685). SDS-PAGE analysis revealed three closely spaced bands in all fragment 2 preparations. N-terminal sequencing yielded a single sequence expected for bovine fragment 2 (Mann, K.G., et al. (1981) Methods Enzmol. 80, 286-302). Laser desorption mass spectrometry of fragment 2 (peptide formula weight 12 791) yielded a major species at 12 825 with a minor peak at 13 456. The basis for this heterogeneity was not further investigated but may be related to partial glycosylation (Carlisle, T.L., et al., (1990), J. Biol. Chem. 265, 22044-22055)) or heterogeneity in some other modification (Owen, W. G, et al. (1974) J. Biol. Chem. 249, 594-605). Titration of factor Xa or thrombin with p-nitrophenyl p'-guanidinobenzoate (Chase, T. Jr., et al. (1967) Methods Enzymol. 19, 20-27) gave 1.12 and 0.96 mol active sites/mol protein, respectively. Protein concentrations were determined using the following molecular weights and extinction coefficients, at a concentration of 0.1% at 280nm: factor Xa, 45 300, 1.24; factor Va, 168 000, 1.74;. prethrombin 1, 50 200, 1.64; prethrombin 2, 37 400, 1.95; fragment 1.2, 34 800, 1.12; fragment 2, 12 800, 1.25; wt-TAP, 6 980, 2.54.

EXAMPLE 2

Preparation of Prethrombin 2 Denvatives

The fragment -thrombin is a prothrombinase digest of prothrombin, prepared according to Krishnaswamy, S. et al., Biochemistry 36, 12080 (1997); Lundblad, R. L. et al., Methods Enzymol. 45, 156 (1976).

For the preparation of bovine γτ-thrombin, α-thrombin in Assay Buffer (22 μM, 45 ml) was incubated with 0.7 μM trypsin for 3 hours at room temperature. The reaction was quenched with 1 OμM soybean trypsin inhibitor, dialysed against 20 mM Tris-PO₄ pH 5.8, 40 mM NaCl, 0.1% (w/v) PEG for 4h at 4°C and applied to a column (1.5x12.5 cm) of S-sepharose equilibrated in the same buffer. Bound protein was eluted (4 ml/min, 120 min) with a linear gradient of increasing NaCl (40 mM-700 mM) in 20 mM Tris-PO₄ 0.1% (w/v) PEG, pH 5.8. All fractions containing protein exhibited equivalent specific activities to α- thrombin in the cleavage of S2238. Fractions from the leading peak, containing γ-r- thrombin with an estimated contamination of 5% undigested material determined by SDS-PAGE were pooled, dialyzed against 20 mM Hepes, 40 mM NaCl, 0.1% (w/v) PEG, pH 7.4 and subject to affinity chromatography using a 4.5x17 cm column of fibrin sepharose equilibrated in the same buffer. Bound protein was eluted with a linear gradient of increasing NaCl (40-600 mM) in 20 mM Hepes, 0.1 % (w/v) PEG, pH 7.4. The γχ-thrombin containing fractions were pooled and reapplied to a second fibrin sepharose column with isocratic elution to remove traces of remaining α-thrombin. The flowthrough fractions were characterized by the same specific activity as α-thrombin towards S2238 with ~1% of the specific activity of α-thrombin in a fibrinogen clotting assay . Pooled material was concentrated by ultrafiltration in a stirred cell to a concentration of ~ 100 μM, inactivated by the addition of 1 mM APMSF followed by brief incubation at room temperature and dialysed against Assay Buffer. The resulting preparation of inactivated γτ-thrombin (γτ-IIai) possessed <0.01% catalytic activity when compared to α-thrombin. Protein sequencing of the fragments resolved by SDS- PAGE yielded the expected sequence for bovine γτ-thrombin. In addition, the mass of γτ-thrombin was determined by MALDI mass spectrometry and found to be consistent with removal of the undecapeptide (He¹¹² - Arg ¹²²) using the numbering system for prethrombin 2.

Bovine ς-thrombin was prepared by treatment of α-thrombin (27 μM, 15 ml) in 0.25 M sodium phosphate buffer pH 6.5 with 4.2 nM chymotrypsin for 4 hrs at room temperature. The digest, terminated by addition of 10 μM TPCK, was dialysed against 20 mM MES, 80 mM NaCl, pH 6.5 and applied to a column (1.5x8cm) of S-sepharose equilibrated in the same buffer. Elution with a linear gradient (4 ml/min, 130 min) of increasing NaCl (80-800 mM) in 20 mM MES, pH 6.5 resulted in the separation of chymotrypsin, undigested thrombin and two peaks of cleaved material. Protein in the two resolved peaks of cleaved material appeared identical by SDS-PAGE, protein sequencing and specific activity measured with S2238. The cleaved material was pooled, concentrated, inactivated with AMPSF and dialysed into Assay Buffer as described above. Protein sequence analysis indicated a single cleavage at Tip 197 (of bovine prethrombin 2) consistent with the expected result for the action of chymotrypsin on human thrombin to yield ζ- thrombin. On this basis, the resulting inactivated preparation was designated ζ-IIa;. The two chymotryptic fragments of thrombin were isolated by HPLC separation of the products generated following chymotryptic cleavage of α- thrombin. Thrombin (30 mg) treated with chymotrypsin (as above) was dialysed against 20 mM Tris, 30% (v/v) CH₃CN, pH 9.0 for 4h at room temperature. Following clarification by centrifugation (50,000xg, 20 min), aliquots (3 ml) were fractionated by cation exchange HPLC (Aquapore Cation 7 μm, 0.46 x 22.2 cm, ABI). Elution (1 ml/min,30 min) with a linear gradient of increasing NaCl (0-350 mM) in 20 mM Tris, 30% (v/v) CH₃CN, pH 9.0 resolved two peaks corresponding to the two thrombin fragments. Material from each of the peaks accumulated from successive runs was pooled, dialysed against 0.1 % (v/v) TFA, concentrated by Iyophilization and further purified by reversed phase HPLC. Each of the pools was dialysed against Buffer A (20 mM Net₃-PO₄, pH 2.5) and fractionated in ~1 mg aliquots using an Aquapore Phenyl column (0.46 x 22.2 cm, ABI). Bound protein was eluted (1 ml/min) with a biphasic gradient of increasing Buffer B (20 mM Net₃-PO₄, 80% (v/v) CH₃CN, pH 2.5) of 0-24% Buffer B in 25 min followed by 30-37% Buffer B in 80 min. Analysis by SDS-PAGE confirmed quantitative separation of the two fragments which were identified as ζl-thrombin and ζ2- thrombin based on N-terminal sequence analysis and mass spectrometry. Pools were prepared for each of fragments, lyophilized, dialysed against Assay Buffer and clarified by centrifugation (50,000xg, 20 min). Dialysis resulted in variable amounts of protein loss due to precipitation. The final concentrations were in the range of 15 μM for ζ-1 thrombin and 40 μM ζ-2 thrombin.

The two chymotryptic fragments of prethrombin 2 were prepared by treating prethrombin 2 (27 μM, 5 ml) in 0.25 M sodium phosphate pH 6.5 with 1 nM chymotryspin for 3 h at room temperature. The reaction mixture was dialysed against Buffer A (above) and fragments were separated by reversed phase HPLC as described above for the thrombin fragments. The resulting peptides were lyophilized, dialysed against Assay Buffer and clarified by centrifugation (50,000xg, 20 min). The fragments were designated ζl-prethrombin 2 and ζ2- prethrombin 2 on the basis of SDS-PAGE, N-terminal sequence analysis and mass spectrometry.

The purity of all protein preparations was judged by SDS-PAGE. The concentrations of the thrombin and prethrombin 2 derivatives were determined using molecular weights determined from the primary structure and extinction coefficients: γτ-thrombin, 34, 100, 1.89; ζ-thrombin 37, 400, 1.85; ζl-thrombin or ζl- prethrombin 2, 23, 000, 1.83; ζ2-thrombin or ζ2-prethrombin 2, 12, 450, 1.90.

EXAMPLE 3

Exosite Assay A. Measurement for Thrombin Formation

All kinetic measurements were performed in 20 mM Hepes, 0. 15 M NaCl, 0. 1% (w/v) PEG, 2 mM CaCl₂, pH 7.4. Final concentrations of 1.4 μM prethrombin 2, 1.4 μM prethrombin 2 plus 2.8 μM fragment 2, or 1.4 μM prethrombin 2 plus 2.8 μM fragment 1.2, or 1.4 μM prothrombin, were used for these measurements. Reaction mixtures (290 μL) were prepared containing 1.4 μM substrate with no addition, with 50 μM PCPS, 30 nM Va or 30 nM Va plus 50 μM PCPS and were incubated for 10 min at 25°C. Thrombin formation was initiated by the addition of a factor Xa solution (10 μL) to achieve the indicated concentration and the reactions were maintained at 25°C. Samples (10 μL) were withdrawn at various times and quenched by mixing with 90 μL of 20 mM Hepes, 0.15 M NaCl, 50 mM EDTA, 0.1% (w/v) PEG, 2 μM wt- TAP, pH 7.4. Quenched samples were then further diluted in the same buffer lacking wt-TAP and initial velocities of S2238 hydrolysis were determined in 96-well plates at ambient temperature in a Vmax kinetic plate reader, using 200 μM S2238 in 20 mM Hepes, 0.15 M NaCl, 50 mM EDTA, 0.1% (w/v) PEG, pH 7.4. Measured rates were related to the concentration of thrombin from the linear dependence of initial velocity on known concentrations of thrombin determined in each experiment. The addition of fragment 2 or fragment 1.2 at concentrations exceeding those carried over into the assay had no detectable effect on the rate of S2238 hydrolysis by thrombin. For certain substrate combinations, the apparent lack of quantitative conversion of substrate to thrombin in complete progress curves was established to arise from significant product inhibition by thrombin.

Control experiments established that the quenched samples were sufficiently diluted so that the added inhibitors had a negligible effect on thrombin activity during initial velocity measurements. Initial velocity data were analyzed by nonlinear least squares regression analysis using established steady state rate expressions for linear competitive or classical noncompetitive inhibition. See Segel, I.H., (1975) Enzyme Kinetics. Behaviour and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. (John Wiley & Sons, New York). Alternate inhibition mechanisms were excluded on the basis of significantly poorer fits as judged by the root mean squared deviation and errors of the fitted parameters. The fitted constants are listed ±95% confidence limits. Data are presented in double reciprocal form to provide a convenient visual confirmation of the conclusions obtained by direct analysis according to the stated rate expressions.

EXAMPLE 4

Small Substrate Counterscreen for Exosite Assay

The kinetics of Spectrozyme Xa hydrolysis by prothrombinase was measured using increasing concentrations of inhibitor at one fixed concentration of substrate. Prothrombinase (0.5 nM, 175 μl) ) was assembled by mixing 0.5 nM Xa, 20 nM factor Va, 44 μM PCPS in 20 mM Hepes, 0.15 M NaCl, 0.1 %(w/v) PEG-8000 and 2 mM CaCl₂ , pH 7.4 and incubated with the stated concentrations of inhibitor. Peptidyl substrate hydrolysis was initiated by adding 25 μl of Spectrozyme Xa prepared in the same buffer to achieve a final concentration of 50 μM. Following mixing by brief vibration, the initial steady state rate of Spectrozyme hydrolysis was determined by continuously monitoring the change in absorbance at 405 nm at room temperature in a Vmax kinetic plate reader.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, or modifications, as come within the scope of the following claims and its equivalents.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Emory University and Krishnaswamy, Sriram

(ii) TITLE OF INVENTION: EXOSITE ASSAY FOR ANTICOAGULA-SfTS

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Kilpatrick Stockton LLP

(B) STREET: Suite 2800, 1100 Peachtree Street

(C) CITY: Atlanta

(D) STATE: Georgia

(E) COUNTRY: USA

(F) ZIP: 30309-4530

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US98/

(B) FILING DATE: 27 May 1998

(C) CLASSIFICATION:

(vii) PRIOR APPLICATON DATA:

(A) APPLICATION NUMBER: US60/081030

(B) FILING DATE: 08 April 1998

(C) CLASSIFICATION:

(vii) (A) APPLICATION NUMBER: US60/048864

(B) FILING DATE: 06 June 1997

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Meredith, Roy. D.

(B) REGISTRATION NUMBER: 30,777

(C) REFERENCE/DOCKET NUMBER: EMU141PCT

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (404) 815-6500 (B) TELEFAX: (404) 815-6555

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 582 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Bovine Prothrombin

(iii) HYPOTHETICAL: NO

(iv) AN I-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Cow

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

Ala Asn Lys Gly Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg 1 5 10 15

Glu Cys Leu Glu Glu Pro Cys Ser Arg Glu Glu Ala Phe Glu Ala Leu 20 25 30

Glu Ser Leu Ser Ala Thr Asp Ala Phe Trp Ala Lys Tyr Thr Ala Cys 35 40 45

Glu Ser Ala Arg Asn Pro Arg Glu Lys Leu Asn Glu Cys Leu Glu Gly 50 55 60

Asn Cys Ala Glu Gly Val Gly Met Asn Tyr Arg Gly Asn Val Ser Val 65 70 75 80

Thr Arg Ser Gly lie Glu Cys Gin Leu Trp Arg Ser Arg Tyr Pro His

85 90 95

Lys Pro Glu lie Asn Ser Thr Thr His Pro Gly Ala Asp Leu Arg Glu 100 105 110

Asn Phe Cys Arg Asn Pro Asp Gly Ser lie Thr Gly Pro Trp Cys Tyr 115 120 125 Thr Thr Ser Pro Thr Leu Arg Arg Glu Glu Cys Ser Val Pro Val Cys 130 135 140

Gly Gin Asp Arg Val Thr Val Glu Val lie Pro Arg Ser Gly Gly Ser 145 150 155 160

Thr Thr Ser Gin Ser Pro Leu Leu Glu Thr Cys Val Pro Asp Arg Gly

165 170 175

Arg Glu Tyr Arg Gly Arg Leu Ala Val Thr Thr Ser Gly Ser Arg Cys 180 185 190

Leu Ala Trp Ser Ser Glu Gin Ala Lys Ala Leu Ser Lys Asp Gin Asp 195 200 205

Phe Asn Pro Ala Val Pro Leu Ala Glu Asn Phe Cys Arg Asn Pro Asp 210 215 220

Gly Asp Glu Glu Gly Ala Trp Cys Tyr Val Ala Asp Gin Pro Gly Asp 225 230 235 240

Phe Glu Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Pro Val Asp Gly Asp

245 250 255

Leu Gly Asp Arg Leu Gly Glu Asp Pro Asp Pro Asp Ala Ala lie Glu 260 265 270

Gly Arg Thr Ser Glu Asp His Phe Gin Pro Phe Phe Asn Glu Lys Thr 275 280 285

Phe Gly Ala Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys 290 295 300

Lys Gin Val Gin Asp Gin Thr Glu Lys Glu Leu Phe Glu Ser Tyr lie 305 310 315 320

Glu Gly Arg lie Val Glu Gly Gin Asp Ala Glu Val Gly Leu Ser Pro

325 330 335

Trp Gin Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly 340 345 350

Ala Ser Leu lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu 355 360 365 Leu Tyr Pro Pro Trp Asp Lys Asn Phe Thr Val Asp Asp Leu Leu Val 370 375 380

Arg lie Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Lys Val Glu Lys 385 390 395 400

lie Ser Met Leu Asp Lys lie Tyr lie His Pro Arg Tyr Asn Trp Lys

405 410 415

Glu Asn Leu Asp Arg Asp lie Ala Leu Leu Lys Leu Lys Arg Pro lie 420 425 430

Glu Leu Ser Asp Tyr lie His Pro Val Cys Leu Pro Asp Lys Gin Thr 435 440 445

Ala Ala Lys Leu Leu His Ala Gly Phe Lys Gly Arg Val Thr Gly Trp 450 455 460

Gly Asn Arg Arg Glu Thr Trp Thr Thr Ser Val Ala Glu Val Gin Pro 465 470 475 480

Ser Val Leu Gin Val Val Asn Leu Pro Leu Val Glu Arg Pro Val Cys

485 490 495

Lys Ala Ser Thr Arg lie Arg lie Thr Asp Asn Met Phe Cys Ala Gly 500 505 510

Tyr Lys Pro Gly Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser 515 520 525

Gly Gly Pro Phe Val Met Lys Ser Pro Tyr Asn Asn Arg Trp Tyr Gin 530 535 540

Met Gly lie Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr 545 550 555 560

Gly Phe Tyr Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val

565 570 575

lie Asp Arg Leu Gly Ser 580 (2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 308 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Bovine Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Cow

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

Thr Ser Glu Asp His Phe Gin Pro Phe Phe Asn Glu Lys Thr Phe Gly 1 5 10 15

Ala Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Gin 20 25 30

Val Gin Asp Gin Thr Glu Lys Glu Leu Phe Glu Ser Tyr lie Glu Gly 35 40 45

Arg lie Val Glu Gly Gin Asp Ala Glu Val Gly Leu Ser Pro Trp Gin 50 55 60

Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser 65 70 75 80

Leu lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr

85 90 95

Pro Pro Trp Asp Lys Asn Phe Thr Val Asp Asp Leu Leu Val Arg lie 100 105 110

Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Lys Val Glu Lys lie Ser 115 120 125

Met Leu Asp Lys lie Tyr lie His Pro Arg Tyr Asn Trp Lys Glu Asn 130 135 140 Leu Asp Arg Asp lie Ala Leu Leu Lys Leu Lys Arg Pro lie Glu Leu 145 150 155 160

Ser Asp Tyr lie His Pro Val Cys Leu Pro Asp Lys Gin Thr Ala Ala

165 170 175

Lys Leu Leu His Ala Gly Phe Lys Gly Arg Val Thr Gly Trp Gly Asn 180 185 190

Arg Arg Glu Thr Trp Thr Thr Ser Val Ala Glu Val Gin Pro Ser Val 195 200 205

Leu Gin Val Val Asn Leu Pro Leu Val Glu Arg Pro Val Cys Lys Ala 210 215 220

Ser Thr Arg lie Arg lie Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys 225 230 235 240

Pro Gly Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly

245 250 255

Pro Phe Val Met Lys Ser Pro Tyr Asn Asn Arg Trp Tyr Gin Met Gly 260 265 270

lie Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe 275 280 285

Tyr Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val lie Asp 290 295 300

Arg Leu Gly Ser 305

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 579 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Human Prothrombin

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

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

Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu 1 5 10 15

Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu 20 25 30

Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu 35 40 45

Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn 50 55 60

Cys Ala Glu Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn lie Thr 65 70 75 80

Arg Ser Gly lie Glu Cys Gin Leu Trp Arg Ser Arg Tyr Pro His Lys

85 90 95

Pro Glu lie Asn Ser Thr Thr His Pro Gly Ala Asp Leu Gin Glu Asn 100 105 110

Phe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr 115 120 125

Thr Asp Pro Thr Val Arg Arg Gin Glu Cys Ser lie Pro Val Cys Gly 130 135 140

Gin Asp Gin Val Thr Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser 145 150 155 160

Val Asn Leu Ser Pro Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin

165 170 175

Gin Tyr Gin Gly Arg Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu 180 185 190

Ala Trp Ala Ser Ala Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe 195 200 205

Asn Ser Ala Val Gin Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly 210 215 220 Asp Glu Glu Gly Val Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe 225 230 235 240

Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr

245 250 255

Gly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala lie Glu Gly Arg Thr 260 265 270

Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser 275 280 285

Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu 290 295 300

Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr lie Asp Gly Arg 305 310 315 320

lie Val Glu Gly Ser Asp Ala Glu lie Gly Met Ser Pro Trp Gin Val

325 330 335

Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser Leu 340 345 350

lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro 355 360 365

Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg lie Gly 370 375 380

Lys His Ser Arg Thr Arg Tyr Glu Arg Asn lie Glu Lys lie Ser Met 385 390 395 400

Leu Glu Lys lie Tyr lie His Pro Arg Tyr Asn Trp Arg Glu Asn Leu

405 410 415

Asp Arg Asp lie Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser 420 425 430

Asp Tyr lie His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser 435 440 445

Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu 450 455 460 Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu 465 470 475 480

Gin Val Val Asn Leu Pro lie Val Glu Arg Pro Val Cys Lys Asp Ser

485 490 495

Thr Arg lie Arg lie Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 500 505 510

Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 515 520 525

Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly lie 530 535 540

Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr 545 550 555 560

Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val lie Asp Gin

565 570 575

Phe Gly Glu

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 308 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Human Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Thr Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly 1 5 10 15 Ser Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser 20 25 30

Leu Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr lie Asp Gly 35 40 45

Arg lie Val Glu Gly Ser Asp Ala Glu lie Gly Met Ser Pro Trp Gin 50 55 60

Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser 65 70 75 80

Leu lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr

85 90 95

Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg lie 100 105 110

Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn lie Glu Lys lie Ser 115 120 125

Met Leu Glu Lys lie Tyr lie His Pro Arg Tyr Asn Trp Arg Glu Asn 130 135 140

Leu Asp Arg Asp lie Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe 145 150 155 160

Ser Asp Tyr lie His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala

165 170 175

Ser Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn 180 185 190

Leu Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val 195 200 205

Leu Gin Val Val Asn Leu Pro lie Val Glu Arg Pro Val Cys Lys Asp 210 215 220

Ser Thr Arg lie Arg lie Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys 225 230 235 240

Pro Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly

245 250 255 Pro Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly 260 265 270

lie Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe 275 280 285

Tyr Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val lie Asp 290 295 300

Gin Phe Gly Glu 305

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 197 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Bovine Zeta 1 Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Cow

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

Thr Ser Glu Asp His Phe Gin Pro Phe Phe Asn Glu Lys Thr Phe Gly 1 5 10 15

Ala Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Gin 20 25 30

Val Gin Asp Gin Thr Glu Lys Glu Leu Phe Glu Ser Tyr lie Glu Gly 35 40 45

Arg lie Val Glu Gly Gin Asp Ala Glu Val Gly Leu Ser Pro Trp Gin 50 55 60

Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser 65 70 75 80 Leu lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr

85 90 95

Pro Pro Trp Asp Lys Asn Phe Thr Val Asp Asp Leu Leu Val Arg lie 100 105 110

Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Lys Val Glu Lys lie Ser 115 120 125

Met Leu Asp Lys lie Tyr lie His Pro Arg Tyr Asn Trp Lys Glu Asn 130 135 140

Leu Asp Arg Asp lie Ala Leu Leu Lys Leu Lys Arg Pro lie Glu Leu 145 150 155 160

Ser Asp Tyr lie His Pro Val Cys Leu Pro Asp Lys Gin Thr Ala Ala

165 170 175

Lys Leu Leu His Ala Gly Phe Lys Gly Arg Val Thr Gly Trp Gly Asn 180 185 190

Arg Arg Glu Thr Trp 195

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 111 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Bovine Zeta 2 Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Cow

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Thr Thr Ser Val Ala Glu Val Gin Pro Ser Val Leu Gin Val Val Asn 1 5 10 15

Leu Pro Leu Val Glu Arg Pro Val Cys Lys Ala Ser Thr Arg lie Arg 20 25 30 lie Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Gly Glu Gly Lys 35 40 45

Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys 50 55 60

Ser Pro Tyr Asn Asn Arg Trp Tyr Gin Met Gly lie Val Ser Trp Gly 65 70 75 80

Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe

85 90 95

Arg Leu Lys Lys Trp lie Gin Lys Val lie Asp Arg Leu Gly Ser 100 105 110

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 192 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Human Zeta 1 Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Thr Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly 1 5 10 15

Ser Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser 20 25 30

Leu Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr lie Asp Gly 35 40 45

Arg lie Val Glu Gly Ser Asp Ala Glu lie Gly Met Ser Pro Trp Gin 50 55 60 Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser 65 70 75 80

Leu lie Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr

85 90 95

Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg lie 100 105 110

Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn lie Glu Lys lie Ser 115 120 125

Met Leu Glu Lys lie Tyr lie His Pro Arg Tyr Asn Trp Arg Glu Asn 130 135 140

Leu Asp Arg Asp lie Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe 145 150 155 160

Ser Asp Tyr lie His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala

165 170 175

Ser Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn 180 185 190

(2 ) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 116 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Human Zeta 2 Prethrombin 2

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Leu Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val 1 5 10 15 Leu Gin Val Val Asn Leu Pro lie Val Glu Arg Pro Val Cys Lys Asp 20 25 30

Ser Thr Arg lie Arg lie Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys 35 40 45

Pro Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly 50 55 60

Pro Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly 65 70 75 80

lie Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe

85 90 95

Tyr Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val lie Asp 100 105 110

Gin Phe Gly Glu 115

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Protease Cleavage Site

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Cow

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

He Glu Gly Arg He Val 1 5 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(A) DESCRIPTION: Protease Cleavage Site

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

He Asp Gly Arg He Val 1 5

Claims

What is Claimed is:

1. An exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of: (a) providing a substrate solution comprising a substrate at a concentration between about 0.05 ╬╝M and about 20 ╬╝M, factor Va at a concentration between about 0.05 nM and about 200 nM, and phospholipids at a concentration of between about 30 ╬╝M and about 500 ╬╝M, and a test inhibitor, in buffer containing between about 1 mM to about 10 mM Ca²⁺ at pH between about 7.0 and about 9.0, said buffer lacking a chelator of Ca²⁺, said substrate comprising a protease cleavage site and an exosite binding determinant;

(b) initiating catalytic cleavage of the substrate by adding to the substrate solution an aliquot of factor Xa, to give a final concentration of Xa of between about 0.05 nM and about 200 nM, such that there is excess Va over Xa, to form a substrate- prothrombinase complex;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

2. The exosite assay of claim 1, wherein the substrate comprises prethrombin 2.

3. The exosite assay of claim 1, wherein the substrate is selected from the group consisting of prethrombin 2, ╬╢-prethrombin 2, a mixture of fragments ╬╢l-prethrombin 2 and ╬╢2-prethrombin 2, and a fusion polypeptide comprising ╬╢l-prethrombin 2 and ╬╢2- prethrombin 2, any of which polypeptides has bovine origin, human origin, or both.

4. An exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of:

(a) providing a prothrombinase solution comprising factor Xa at a concentration between about 0.05 nM and about 200 nM, factor Va at a concentration between about 0.05 nM and about 200 nM, such that there is excess of factor Va over Xa, and phospholipids at a concentration of between about 30 ╬╝M and about 500 ╬╝M, and a test inhibitor, in buffer containing between about 1 mM to about 10 mM Ca²⁺ at pH between about 7.0 and about 9.0, said buffer lacking a chelator of Ca²⁺;

(b) initiating catalytic cleavage of a substate by adding to the prothrombinase solution an aliquot of substrate, to give a final concentration of substrate of between about 0.05 ╬╝M and about 20 ╬╝M, to form a substrate- prothrombinase complex, said substrate comprising protease cleavage site and an exosite binding determinant;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

5. The exosite assay of claim 4, wherein the substrate comprises prethrombin 2.

6. The exosite assay of claim 4, wherein the substrate is selected from the group consisting of prethrombin 2, ╬╢-prethrombin 2, and a mixture of fragments ╬╢l- prethrombin 2 and ╬╢2-prethrombin 2, and a fusion polypeptide comprising ╬╢l- prethrombin 2 and ╬╢2 -prethrombin 2, any of which polypeptides may have bovine origin, human origin, or both.

7. The exosite assay of any of claims 1-6, wherein the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200 nm, said phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, and phosphatidylglycerol, and mixtures thereof.

8. The exosite assay of any of claims 1-6, wherein the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200nm, said phospholipids comprising about 75% (w/w) L-alpha-phosphatidylcholine and about 25%o (w/w) L-alpha-phosphatidylserine.

9. The exosite assay of any of claims 1-6, wherein the phospholipids are small unilamellar vesicles of diameter between about 20 nm and about 200 nm, said phospholipids comprising at least about 5% to about 50% phosphatidylserine, the remainder selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and phosphatidylglycerol, and mixtures thereof.

10. The exosite assay of any of claims 1-6, wherein the phospholipids are provided from rabbit brain cephalin or membranes prepared from activated platelets.

11. The exosite assay of any of claims 1 -6, wherein the quenching of aliquots in step (c) is carried out by adding buffer containing an inhibitor of factor Xa, and a chelator of Ca²⁺.

12. The exosite assay of claim 11, wherein the inhibitor of factor Xa is Tick Anticoagulant Peptide.

13. The exosite assay of claim 11 , wherein the inhibitor of factor Xa is recombinant Tick Anticoagulant Peptide.

14. The exosite assay of any of claims 1-6, wherein the concentration of thrombin in step (d) is carried out with a calorimetric assay.

15. The exosite assay of claim 14, wherein the concentration of thrombin is calculated from the initial velocity in the hydrolysis of thrombin substrate H-D- phenylalanylpipecoylarginine pnitroaniline.

16. An exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of: (a) providing a substrate solution comprising prethrombin 2 at a concentration between about 1 ╬╝M and about 2 ╬╝M, factor Va at a concentration of about 20 nM, and phospholipids at a concentration of about 50 ╬╝M, and a test inhibitor, in buffer containing about 2 mM Ca²⁺ at pH of about 7.4, said buffer lacking a chelator of Ca²⁺, said phospholipids being small unilamellar vesicles of diameter between about 20 nm and about 200nm, said vesicles comprising about 75% (w/w) L- alpha-phosphatidylcholine and about 25% (w/w) L-alpha-phosphatidylserine;

(b) initiating catalytic cleavage of prethrombin 2 by adding to the substrate solution ah aliquot of factor Xa, to give a final concentration of Xa of between about 2 nM and about 5 nM, such that there is excess Va over Xa, to form a substrate- prothrombinase complex; (c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

17. The exosite assay of claim 16, wherein the substrate comprises prethrombin 2.

18. The exosite assay of claim 16, wherein the substrate is selected from the group consisting of prethrombin 2, ╬╢-prethrombin 2, and a mixture of fragments ╬╢l- prethrombin 2 and ╬╢2-prethrombin 2, and a fusion polypeptide comprising ╬╢l- prethrombin 2 and ╬╢2-prethrombin 2, any of which polypeptides may have bovine origin, human origin, or both.

19. An exosite assay for inhibitors of the catalytic cleavage of prothrombin to thrombin by prothrombinase, at sites remote from the catalytic site of prothrombinase, comprising the steps of:

(a) providing a prothrombinase solution comprising factor Xa at a concentration between about 2 nM and about 5 nM, factor Va at a concentration of about 20 nM, such that there is excess of factor Va over Xa, and phospholipids at a concentration of about 50 ╬╝M, and a test inhibitor, in buffer containing about 2 mM Ca²⁺ at pH of about 7.4, said buffer lacking a chelator of Ca²⁺, said phospholipids being small unilamellar vesicles of diameter between about 20 nm and about 200nm, comprising about 75%) (w/w) L-alpha-phosphatidylcholine and about 25% (w/w) L- alpha-phosphatidylserine;

(b) initiating catalytic cleavage of prethrombin 2 by adding to the prothrombinase solution an aliquot of prethrombin 2, to give a final concentration of prethrombin 2 of between about 1 ╬╝M and about 22 ╬╝M, to form a substrate- prothrombinase complex;

(c) withdrawing aliquots and quenching them, to give quenched cleaved aliquots;

(d) assaying the quenched cleaved aliquots for the concentration of thrombin.

20. The exosite assay of claim 19, wherein the substrate comprises prethrombin 2.

21. The exosite assay of claim 19, wherein the substrate is selected from the group consisting of prethrombin 2, ╬╢-prethrombin 2, and a mixture of fragments ╬╢l- prethrombin 2 and ╬╢2-prethrombin 2, and a fusion polypeptide comprising ╬╢l- prethrombin 2 and ╬╢2 -prethrombin 2, any of which polypeptides may have bovine origin, human origin, or both.

22. Inhibitors giving an IC₅₀ of less than about 1.0 ╬╝M, in the assay of any of claims 1-6, or 16ΓÇö21.

23. The exosite assay of any of claims 1, 3,16, or 19, wherein prothrombin, prethrombin 2, factor Va , factor Xa are of bovine origin.

24. The exosite assay of any of claims 1, 3,16, or 19, wherein prothrombin, prethrombin 2, factor Va , factor Xa are of human origin.

25. The exosite assay of any of claims 1, 3, 16 or 19, wherein each of prothrombin, prethrombin 2, factor Va and factor Xa is of either human or bovine origin.