Pharmacological Characterization of a Novel Factor Xa Inhibitor, FXV673

Thrombosis Research 103 (2001) 309 – 324

REGULAR ARTICLE

Pharmacological Characterization of a Novel Factor Xa Inhibitor, FXV673$

Valeria Chu1, Karen Brown1, Dennis Colussi2, Jingbo Gao1, Jeffery Bostwick1, Charles Kasiewski1, Ross Bentley3, Susan Morgan1, Kevin Guertin4, Henry W. Pauls1, Yong Gong1, Alison Zulli5, Mark H. Perrone6, Christopher T. Dunwiddie7 and Robert J. Leadley8 1 Aventis Pharmaceuticals, Mail Stop: EM-A1B, Route 202 and 206, P.O. Box 6800, Bridgewater, NJ 08807, USA; 2Merck Sharp and Dome, West Point, PA, USA; 3SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA; 4Roche Pharmaceuticals, Nutley, NJ, USA; 5Cephalon, King of Prussia, PA, USA; 6Bristol-Myers Squibb Pharmaceuticals, Princeton, NJ, USA; 7Eli Lilly & Company, Indianapolis, IN, USA and 8Pfizer Global R&D, Ann Arbor, MI, USA (Received 10 May 2001 by Editor L. Medved; revised/accepted 15 June 2001)

Abstract FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a Ki of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (mM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (mM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was

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This work was completed at Aventis Pharmaceuticals by the departments of Cardiovascular Drug Discovery and Medicinal Chemistry, Collegeville, PA, USA.

Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time. Corresponding author: Valeria Chu, PhD, Aventis Pharmaceuticals, Mail Stop: EM-A1B, Route 202 and 206, P.O. Box 6800, Bridgewater, NJ 08807, USA. Tel: +1 (908) 231 4607; Fax: 1 (908) 231 4760; E-mail: .

performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC50s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (  2.5fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent. D 2001 Elsevier Science Ltd. All rights reserved. Key Words: Antithrombotic; Anticoagulant; Canine arterial venous thrombosis models; Factor Xa inhibitor; FXV673

F

actor Xa (FXa) is the penultimate enzyme in the coagulation cascade. It directly converts prothrombin to thrombin through the prothrombinase complex, which consists of FXa,

0049-3848/01/$ – see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII S0049-3848(01)00328-0

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factor Va (FVa), phospholipid surface, and Ca2 + , and it leads to platelet activation and fibrin clot formation. FXa can be formed via the intrinsic tenase (FIXa/FVIIIa) or the extrinsic tenase (FVIIa/tissue factor) complexes [1–4]. It is due to the amplification nature of the coagulation cascade that one molecule of FXa is able to generate more than 1000 molecules of thrombin [5]. In addition, the reaction rate of prothrombinasebound FXa increases 300,000-fold compared to the rate catalyzed by free FXa and causes an explosive burst of thrombin generation [6,7]. Unfractionated heparin and low molecular weight heparin are antithrombin III-dependent antithrombotic agents. Their inhibitory effects are mainly on fluid phase thrombin and FXa and are resistant to clot-bound thrombin and clot-bound prothrombinase-associated FXa [8– 10]. Attempts to target the inhibition of thrombin generation as an antithrombotic strategy have been validated through the use of naturally occurring, highly selective FXa inhibitors [11,12]. In addition, recent studies demonstrated that clot-bound FXa could activate prothrombin thus exerting procoagulant activity on thrombi. Small direct and indirect FXa inhibitors could inhibit further thrombus propagation [13,14]. Both peptides and the small molecule Xa inhibitors are currently available. Among the best-studied natural FXa inhibitors are tick anticoagulant peptide (TAP) [15,16] and antistasin (ATS) [17,18]. Both recombinant TAP and recombinant ATS have been studied in various experimental models of arterial and venous thrombosis as well as disseminated intravascular coagulation. The results have demonstrated that specific inhibition of FXa provides equal or superior efficacy to that achieved by heparin or the inhibition of thrombin directly [8,11,13]. Recent intensive research focused in this area has led to the discovery of several potent and selective small molecule FXa inhibitors, such as DX-9065a (Ki = 41 nM) [19], YM-60828 (Ki = 1.3 nM) [20], YM-75466 (Ki = 1.3 nM) [21], SEL-2711 (Ki = 3 nM) [22], BX-807834 (Ki = 0.11 nM) [23], SK549 (K i = 0.52 nM) [24], and RPR 130737 (Ki = 2.4 nM) [25]. In addition, these compounds have shown efficacy in several animal models of thrombosis including venous thrombosis in rats,

arterio-venous shunt in rats, venous thrombosis in rabbits, and carotid electrical vascular injury in rabbits [13,26–30]. In an intensive research effort, 2-(R)-(3-carbamimidoylbenzyl)-3-(R)-[4-(1-oxypyridin-4yl)benzoylamino]-butyric acid methyl ester (FXV673) was found to be a potent and selective FXa inhibitor and could be developed into a therapeutic agent. FXV673 shows selectivity greater than 1000-fold for enzymes such as plasmin and tissue-plasminogen activator (tPA); therefore, there is no concern about compromising the physiological or pharmacological fibrinolytic pathway. FXV673 could inhibit FXa, which has been assembled into the prothrombinase complex, consequently inhibits further thrombin generation. FXV673 does not interfere with the existing thrombin activity. Furthermore, FXV673 does not inhibit activated protein C (aPC), thus allowing for proper functioning of this endogenous thrombin-mediated antithrombotic pathway. This report describes the in vitro biochemical characterization of FXV673 and its in vivo antithrombotic efficacy in a canine model of acute arterial and venous thrombosis.

1. Material and Methods 1.1. Enzyme Assays Using Chromogenic Substrates FXV673 was synthesized by the Medicinal Chemistry Section in the Department of Cardiovascular Drug Discovery at Rhoˆne-Poulenc Rorer (now Aventis Pharmaceuticals). FXa of human, dog, and rabbit origins, thrombin and aPC were obtained from Enzyme Research Laboratories (South Bend, IN). Bovine trypsin was obtained from Sigma (St. Louis, MO). Human plasmin was purchased from Pharmacia Hepar (Franklin, OH). Human recombinant t-PA (Activase) was obtained from Genentech (San Francisco, CA). The chromogenic substrates used were: Spectrozyme FXa (Km = 90.5 mM, American Diagnostica, Greenwich, CT) for FXa; Pefachrome TH (Km = 50 mM, Centerchem, Stamford, CT) for thrombin; S-2765 (Km = 250 mM, Diapharma Group, Franklin, OH) for trypsin; Pefachrome-tPA (Km = 192 mM, Centerchem, Stanford,

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CT) for t-PA. The chromogenic substrate S-2366 (Km = 91 mM, Diapharma Group) was used for assay of both plasmin and aPC. All enzyme assays were carried out at room temperature in 96-well microtiter plates following the methods described previously [25]. The enzyme reactions were initiated by the addition of substrate, and the color developed from the release of p-nitroanilide from each chromogenic substrate was monitored continuously for 5 min at 405 nm on a Thermomax microtiter plate reader (Molecular Devices, Sunnyvale, CA). The human FXa reactions were initiated either by addition of FXa (1 nM) to the mixture-containing substrate and increasing concentrations of FXV673 (0–30.5 nM), or by the addition of substrate to the preincubated FXa–FXV673 mixture. The progress curves were recorded. Under the experimental conditions, less than 10% of the substrate was consumed in all assays. The IC50 of the inhibitor was determined by measuring the amount of inhibitor required to diminish 50% of the initial velocity of the control [31]. Assuming a competitive mechanism of inhibition, the apparent Ki values were then calculated according to the Cheng–Prusoff equation, Ki = IC50/(1+[S]/Km) [32]. 1.2. Prothrombinase Assay 1.2.1. Liposomes as the Source of Phospholipids Phosphatidylcholine (PC; Bovine brain, Sigma) and phosphatidylserine (PS; Bovine brain, Sigma) were used to prepare PS(20%)/PC(80%) lipid vesicles following the procedure of Bloom [33] in 0.05 M Tris–HCl, 0.15 M NaCl, pH 7.5 buffer. The prothrombinase complex was assembled with the addition of PS/PC, CaCl2, FXa, and FVa to the PEG-buffer in a total volume of 4.5 ml. Forty-five microliters of the aliquot was combined with 25 ml of the inhibitor dilutions and preincubated for 5 min followed by the addition of 30 ml of prothrombin. At this stage, the final concentrations of PS/PC, CaCl2, FXa, FVa, and prothrombin were 50 mM, 5 mM, 0.01 nM, 5 nM, and 1.2 mM, respectively. The reaction mixture was incubated for 2 min and stopped by the addition of EDTA to a final concentration of 50 mM. Prothrombin was also added to the reaction mixture without FXV673 to initiate the thrombin

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generation. And then followed after 1 min by additions of FXV673, ranging from 0 to 12.5 nM. Aliquots of the reaction mixture were removed at different time intervals for a period of 30 min. The thrombin generation reaction of each aliquot was stopped by the addition of 50 mM EDTA. A coupled-enzyme assay was used to measure the prothrombinase complex activity by measuring the subsequent thrombin generation. Twenty-five microliters of the reaction mixture was removed, and thrombin activity was monitored at 405 nm for 5 min on a Thermomax microtiter plate reader using 110 mM of S-2238 as a substrate [34]. The initial velocities of thrombin generation were recorded for the control and the FXV673-treated samples to determine the IC50, which is the concentration of FXV673 required to inhibit 50% of thrombin generation. 1.2.2. Gel-Filtered Platelets as the Source of Phospholipid Blood was obtained from human volunteers; the subjects had taken no medications for at least 14 days prior to the blood donation. Blood samples were mixed with 1/10 volume of 3.8% sodium citrate and then centrifuged at 120  g for 15 min, after which the platelet-rich plasma (PRP) was collected. Platelets were pelleted from the platelet concentrates by centrifugation at 650  g for 15 min. The platelet pellets were washed once with a buffer containing 137 mM NaCl (Fisher), 20 mM HEPES (Sigma), 5.5 mM Glucose (Sigma), and 1.4 mM PGE1 (Sigma), pH 6.6. The platelets were resuspended in modified calcium-free Tyrode’s buffer–HSA (human serum albumin, Albuminar-25, Armour Pharmaceutical, Kankakee, IL), composed of 0.35% HSA in 137 mM NaCl, 2 mM MgCl2, 0.42 mM Na2HPO4, 11.9 mM NaHCO3, 2.9 mM KCl, 5.5 mM glucose, and 10 mM HEPES, pH 7.35. The platelets were further processed by passing them through a Sepharose2B gel-filtration column (2.5  25 cm) equilibrated with the same buffer [35,36]. The isolated platelet suspensions were adjusted to a cell count of 2  108 cells/ml in the modified Tyrode’s buffer containing 2 mM of CaCl2. To 250 ml of platelets, 10 ml of FVa was added to a final concentration of 5 nM. The platelets were then incubated for 15 min followed by the addition of 4.5 ml of FXa to achieve a final concentration of 10

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pM. The mixture was incubated at room temperature for 5 min followed by the addition of serial dilutions of FXV673. Five minutes later, 5.5 ml of prothrombin (final concentration 1.2 mM) was added to initiate the reaction. Two minutes later, the reaction was stopped by the addition of 0.1 ml of 0.1 M EDTA, and the thrombin generation assays were performed as before. 1.3. Coagulation Assays 1.3.1. Activated partial thromboplastin time (APTT) and prothrombin time (PT) APTT and PT were measured with an MLA Electra 800 automatic coagulation timer (Orthodiagnostics, Raritan, NJ). Citrated human (George King Biomedical, Overland Park, KS), monkey (rhesus, Covance Research Product, Alice, TX), dog (mongrel, Covance Research Product), rabbit (New Zealand White, Cocalico Biologics, Roamestown, PA), and rat (Sprague – Dawley, Charles River, Wilmington, MA) plasmas were used in the assays. For the APTT measurement, 100 ml of freshly thawed plasma was mixed with 100 ml of compound dilutions followed by the automatic addition of 100 ml of actin-activated cephaloplastin reagent (Dade, Miami, FL) and 100 ml of 0.035 M calcium chloride to start clot formation. For the PT assay, plasma and serial dilutions of compound were mixed as in the APTT assay followed by the automatic addition of 200 ml of Innovin (Dade) to start clot formation. Anticoagulant activity of the compound was evaluated in determining the concentration required for doubling the plasma clotting time. 1.3.2. FX-Deficient Plasma Clotting Time Correction Assay Various concentrations of FXa in 100 ml aliquots were incubated with 100 ml FXa assay buffer followed by the automatic addition of 100 ml of 0.035 M calcium chloride and 100 ml of the FXdeficient plasma to initiate clot formation. The concentration of FXa added to achieve the APTT for pooled normal human plasma (American Diagnostica) was determined to be 18 nM. Serial dilutions of the FXa inhibitor in 100 ml aliquots were incubated with 100 ml of 18 nM FXa followed by the automatic addition of 100 ml of 0.035 M calcium chloride and 100 ml of FX-deficient

plasma (American Diagnostica). Anticoagulant activity of the compound was evaluated by determining the concentration required for doubling the FX-deficient plasma clotting time in the presence of a predetermined concentration of FXa. 1.4. Platelet Aggregation Blood was obtained from human volunteers; subjects had taken no medications for at least 14 days prior to the blood donation. Blood samples were mixed with 1/10 volume of 3.8% sodium citrate and then centrifuged at 120  g for 15 min, after which the PRP was collected. The platelet count was adjusted to 2  108 cells/ ml [35,36]. Platelet aggregation was performed according to the turbidometric method of Born [36]. FXV673 at concentrations ranging from 1 to 100 mM were incubated with 0.25 ml of PRP for 1 min followed by activation of the platelet with various agonists. Aggregation was induced by ADP (Sigma), collagen (equine tendon, Helena Lab., Beaumont, TX), and TRAP1 – 6 (thrombin receptor activation peptide, Peninsula Lab., Belmont, CA) at final concentrations of 10 mM, 5 mg/ml, and 25 mM, respectively. A platelet aggregation profiler Model PAP-4 (Bio Data, Hatboro, PA) was used to record platelet aggregation. The inhibition of aggregation was expressed as the percentage of the maximal rate of aggregation observed in the absence of antagonists [36]. 1.5. Kinetic Studies of Prothrombinase-Bound FXa Inhibition A coupled-enzyme assay was used to study the inhibition mechanism of prothrombinase-bound FXa. The concentrations of FXV673 used were 0– 2.5 nM, and the concentrations of the natural substrate for prothrombinase, prothrombin, ranged from 0.01 to 1.2 mM. The subsequent thrombin generation was measured by 110 mM of S-2238. The results were analyzed with a Cornish-Bowden plot (S/v = Km/V + S/V + Km/VI/Ki) and a Dixon plot (1/v = K m /VS + 1/V + K m /VSI/K i ) [37] using the Leonora Steady-state Enzyme Kinetics Program by Dr. A. Cornish-Bowden (distributed with Analysis of Enzyme Kinetic Data, Oxford Univ. Press, 1995).

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1.6. Acute Arterial and Venous Electrolytic Injury Canine Model All procedures in this study were performed in compliance with the Animal Welfare Act Regulations and with the Guide for the Care and Use of Laboratory Animals (DHEW Publication No. NIH 85-23, 1996). A previously published procedure [12,38] was followed with slight modifications. Briefly, mongrel dogs of either sex (10–20 kg) were anesthetized with sodium pentobarbital (30 mg/kg iv, with supplements given as needed), intubated, and ventilated with room air using a Harvard respirator (Harvard Apparatus, S. Natick, MA). A tri-lumen catheter (SAFEDWELL plus, Becton Dickinson, Sandy, UT) was placed in the right femoral vein for the administration of test agents and supplemental anesthesia. The right femoral artery was cannulated for measurement of arterial blood pressure and for obtaining blood samples. Heart rate was monitored using standard limb lead II of the electrocardiograph. The left common carotid artery and the right jugular vein were exposed and isolated using blunt dissection. A calibrated ultrasonic flow probe was placed around the carotid artery and jugular vein for continuous monitoring of blood flow. In these studies, an electric current and a stenosis to restrict blood flow (to mimic a stenotic vessel and create shear stress on the neighboring endothelial cells) were used to provide the vascular damage necessary for thrombus formation. Stimulating electrodes were constructed from a 26-gauge, stainless steel hypodermic needle, bent at a 90 angle, and soldered to a 32-gauge insulated wire. The electrodes were inserted through the vessel wall and positioned so that the intraluminal portion of the electrode was in firm contact with the intimal surface of the vessel. A stainless steel clamp with a screw adjuster was used to create the arterial stenosis. After recording the baseline blood flow, the screw clamp was adjusted to reduce blood flow to 60% of baseline. The jugular vein stenosis was created by placing a short segment of PE-205 tubing parallel to the vessel and tying a ligature around the vessel and tubing, after which the tubing was removed. Electrolytic injury to the intimal surface of each vessel was

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accomplished by delivery of 150 mA of anodal current through the intravascular electrodes. Fifteen minutes after starting the electric current, FXV673 was administered as an intravenous bolus (5 ml) plus an intravenous infusion (0.25 ml/min for 225 min). FXV673 was prepared in isotonic saline containing 5% dimethyl sulfoxide (DMSO) and the doses administered were: 10 mg/ kg + 1 mg/kg/min (n = 5), 25 mg/kg + 2.5 mg/kg/ min (n = 5), 83 mg/kg + 8.3 mg/kg/min (n = 4), and 300 mg/kg + 30 mg/kg/min (n = 4). Seven animals received vehicle alone. The time to zero blood flow (i.e., vessel occlusion) in each vessel was measured. The time to vessel occlusion was defined as the time from the initiation of current to when blood flow was equal to zero for  5 min. Four hours after the start of the electrolytic injury (225 min of drug infusion), each vessel was ligated and removed. The vessel segment was opened along its longitudinal axis and the thrombus was removed and weighed on an analytical balance. 1.7. Ex Vivo Measurement of APTT, PT, and FXV673 Plasma Concentration in Dogs Blood samples for determination of APTT, PT, and plasma drug levels were obtained prior to initiation of electrical current and drug administration, and then 15, 45, 105, 165, and 225 min after beginning the drug infusion. Blood was drawn into chilled syringes containing 1/10 volume 3.8% trisodium citrate and aliquoted into 1.7 ml Eppendorf centrifuge tubes. Blood samples used for determining plasma drug levels were kept on ice and were spun at 10,000 RPM to separate plasma from the cellular components of

Fig. 1. Chemical structure of FXV673, a 2-(R)-(3carbamimidoylbenzyl)-3-(R)-[4-(1-oxypyridin-4-yl)benzoylamino]-butyric acid methyl ester.

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Table 1. Inhibitory effect and selectivity of FXV673 for FXa and other serine proteases (n = 3) Enzymes

Ki (nM)

FXa Thrombin Trypsin aPC Plasmin t-PA

0.5 3956 301 >18,491 656 8681

Selectivity Ki,enzyme/Ki,FXa 1 > 7900 600 >36,000 1300 >17,000

the blood. These were stored at 70C until assayed. Plasma used for determining the APTT and PT was kept at room temperature and assayed the same day. The concentration of FXV673 in plasma was determined by using chromogenic assays for ex vivo anti-Xa activity. Factor Xa activity was determined by chromogenic assay kit (American Diagnostics) utilizing bovine FXa and Spectrozyme FXa, prepared according to manufacturer’s instructions, and assay buffer (0.05 M Tris, 0.15 M NaCl, 0.1% PEG-8000, pH 7.5). The enzyme reactions were initiated by the addition of substrate and were monitored continuously for 5 min at 405 nm on a Spectra Max 250 plate reader (Molecular Devices) at 37C. Plasma samples collected from the electrolytic injury experiment were thawed and added to a 96-well plate (20 ml in a final volume of 200 ml) with the assay

reagents (60 ml assay buffer, 60 ml FXa, and 60 ml Spectrozyme FXa). Initial velocities were used for calculation of FXa activity. The inhibition of FXa activity was determined as follows: % inhibition of FXa activity = 1 (Vi with FXV673/Vi of the predrug control)  100. Plasma drug concentrations were estimated from a standard curve that was prepared by adding varying concentrations of FXV673 to control dog plasma and then plotting the concentration of FXV673 against the % inhibition of FXa activity. Unknown concentrations were interpolated electronically from the curve, then multiplied by the dilution factor to estimate the drug concentration in the plasma (including any active metabolites). 1.8. Statistics Data were expressed as mean ± S.E.M. The effect of FXV673 on platelet aggregation was analyzed by using the ANOVA one-way Dunnett’s test. The GraphPad Prism (GraphPad Software, San Diego, CA) computer program was used for analysis. A P value of less than .05 was considered to be statistically significant. Statistical significance between treatment groups in animal studies was tested using two-way analysis of variance. The least significant difference test was used to perform multiple comparisons of means. Differences were considered significant when the P value was < .05.

Fig. 2. Progress curve depicting the inhibition of FXa by FXV673. The tracings were monitored for 40 min for reactions initiated by the addition of 1 nM FXa to 200 mM of Spectrozyme Xa containing increasing concentrations of FXV673. The concentrations of FXV673 were (nM): (a) 0, (b) 1.91, (c) 3.8, (d) 7.6, (e) 15.3, and (f) 30.5.

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2. Results 2.1. Selective Inhibitory Effect of FXV673 on FXa and Other Serine Proteases Chromogenic substrates were used to assess the inhibitory effect of FXV673 on coagulation enzymes FXa and thrombin as well as other trypsin-like serine proteases. FXV673 (chemical structure shown in Fig. 1) was a potent FXa inhibitor with a Ki of 0.52 nM. FXV673 also inhibited other serine proteases, albeit at much higher concentrations (Ki’s ranged from  300 nM to greater than 36,000 nM). The results in Table 1 show that FXV673 is a potent and very selective FXa inhibitor (selectivity against other proteases >1000-fold, except selectivity against trypsin which is approximately 600-fold). The progress curves of FXa–FXV673 interactions were followed when reactions were initiated by the addition of enzyme to solutions containing substrate and increasing concentrations of FXV673. The progress tracings demonstrated that FXV673 is a fast-binding and reversible inhibitor (Fig. 2). Also FXV673 exhibits significant inhibition of FXa at a concentration similar to that of FXa, indicating that FXV673 is a tight-binding inhibitor of FXa [39,40]. FXV673 exhibits species-specificity, it inhibits human, dog, and rabbit FXa with a Ki of 0.52, 1.41, and 0.27 nM, respectively. When ranking the sensitivity of FXV673, it is found to be the most sensitive inhibitor of rabbit FXa among the three species tested. FXV673 is an approximately 2.5-fold more sensitive inhibitor of human FXa than dog FXa.

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the cleavage of prothrombin and the generation of thrombin as subsequently measured in the amidolytic chromogenic assay is dependent on FXa activity. The results in Fig. 3A and B show that FXV673 inhibited thrombin generation dosedependently, with IC50s of 1.38 ± 0.09 nM (n = 3) and 2.55 ± 0.11 nM (n = 3) for the prothrombinase-bound FXa on liposomes and platelets, respectively. To study if FXV673 is efficacious after the onset of a thrombotic event, the timecourse of thrombin generation was studied in the

2.2. Studies of Inhibitory Effect of FXV673 on Prothrombinase-Bound FXa Using Liposomes or Fresh Platelets as Phospholipid Sources To determine if FXV673 is an effective inhibitor of FXa when it is complexed with FVa, and Ca2 + on a phospholipid membrane, we reconstituted the prothrombinase complex on synthetic phospholipid membranes and platelets. FXa was the only component that exhibited proteolytic activity in the prothrombinase complex, regardless of whether the source of the phospholipid was from liposomes or the platelet membranes. Therefore,

Fig. 3. Inhibitory effect of FXV673 on prothrombinasebound FXa. The phospholipid source was PS/PC (panel A) and the gel-filtered platelet surface (panel B). Attenuation of prothrombinase activity was expressed as the inhibition of thrombin generation measured in a coupled-enzyme assay using chromogenic substrate, S2238, for thrombin. The initial velocities of thrombin generation were recorded for the control and the FXV673-treated samples to determine the IC50, which was the concentration of FXV673 required to inhibit 50% of thrombin generation.

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The concentration of FXV673 required to double the APTT time of FX-deficient plasma to which FXa had been added was 0.20 ± 0.003 mM (n = 2), indicating that FXV673 could inhibit prothrombinase-bound FXa in the physiological condition. 2.4. Platelet Aggregation Studies The effect of FXV673 on platelet aggregation was studied in PRP. Platelet aggregation was induced by ADP, TRAP1 – 6, and collagen. At the concentrations from 1 to 100 mM, FXV673 showed no effect on platelet aggregation in PRP as shown in Table 3. Fig. 4. Time-course of inhibitory effect of FXV673 on prothrombinase-bound FXa after initiation of thrombin generation. FXV673 at concentrations ranging from 0 to 12.5 nM was added to a thrombin generation mixture that had been proceeded for 1 min. Aliquots of the reaction mixture were removed at different time intervals and the initial velocity of thrombin generation at each time point was monitored by a coupled enzyme assay. The % residual thrombin activity as compared to the control was calculated.

presence of various concentrations of FXV673 after the initiation of thrombin generation. The results indicated that FXV673 dose-dependently inhibited further generation of thrombin via the inhibition of prothrombinase-bound FXa (Fig. 4). 2.3. Anticoagulant Activity Reflected in Plasma Clotting Time Assays The clotting time prolongation assays (APTT and PT) were evaluated with pooled plasma derived from humans, monkeys, dogs, rabbits, and rats. The concentrations of FXV673 required to double the clotting times are listed in Table 2. A species-specific anticoagulant effect of FXV673 was observed. There were approximately fourfold and twofold differences in concentrations required for doubling the clotting time in APTT and PT assays, between the rabbit and the least active species, the dog. Humans are approximately 2.5-fold more sensitive than dogs in both assays. The rank order of the anticoagulant potency of FXV673 in plasma was rabbit>human>monkey>rat>dog.

2.5. Monitoring Thrombin Generation to Determine the Mechanism of Prothrombinase-Bound FXa Inhibition by FXV673 It was demonstrated that the cofactors of the prothrombinase assembly (FVa, FXa, Ca2 + , and phospholipid) interact with each other simultaneously. The resulting modulation of conformation and orientation of both the substrate (prothrombin) and the enzyme (FXa) enhances the rate of thrombin conversion dramatically [6,41,42]. However, the small molecule substrates or inhibitors are restricted to binding to the active site of FXa without the extended macromolecular interactions. Therefore, we studied the mechanism of prothrombinase complex inhibition by FXV673 using the natural substrate and monitored the generation of thrombin. The results from the Cornish-Bowden plots in Fig. 5A show lines that intersect at the X-axis,

Table 2. Effect of FXV673 on prolongation of PT and APTT in human, monkey, dog, rabbit, and rat plasma in vitro (mean ± S.E.M., n = 2) Species

PT doubling concentration (MM)

APTT doubling concentration (MM)

Human Monkey Dog Rabbit Rat

1.1 ± 0.07 1.32 ± 0.065 2.31 ± 0.085 0.92 ± 0.035 1.69 ± 0.045

0.41 ± 0.02 0.65 ± 0.02 1.12 ± 0.06 0.25 ± 0.17 0.80 ± 0.13

PT doubling concentration and APTT doubling concentration indicate concentration of FXV673 required to double the clotting times in PT and APTT, respectively.

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Table 3. Effect of FXV673 on platelet aggregation in the PRP system (n = 2) Concentration of FXV673 (MM) 100 10 1 0

Rate of platelet aggregation induced with various agonists ADP (10 MM) 43 ± 1 43 ± 1 43 ± 5 41 ± 0.5

TRAP1 – 6 (25 MM) 63 ± 4 61 ± 1 52 ± 1 59 ± 4

Collagen (5 Mg/ml) 42 ± 3 46 ± 7 43 ± 4 45 ± 5

P > .05, there is no significant difference in rate of platelet aggregation in the presence/absence of FXV673 from 1 – 100 mM.

and the Dixon plots, in Fig. 5B, also display lines which intersect at the X-axis. The kinetic analyses indicate that in the presence of prothrombin, the natural substrate of FXa, the small molecule FXV673 is a noncompetitive inhibitor

Fig. 5. Kinetic analyses of the inhibitory effect of FXV673 on prothrombinase-bound FXa when its natural substrate, prothrombin, was used. Cornish-Bowden plots for FXV673 (panel A): [S]/v (concentration of prothrombin/initial velocity, mM/mOD/min) versus [I] (concentration of FXV673, nM) at different concentrations of [S], prothrombin, ranged from 0.01 to 1.2 mM. Dixon plots for FXV673 (panel B): 1/v (1/initial velocity, 1/mOD/min) versus [I] (concentration of FXV673, nM) in the presence of different concentrations of [S] prothrombin, ranged from 0.01 to 1.2 mM.

for the prothrombinase-bound FXa-mediated thrombin generation.

Fig. 6. Dose-dependent increases in the time to vessel occlusion (panel A) and decreases in thrombus mass (panel B) by intravenous bolus plus infusion of FXV673. Doses shown are the constant infusion portion of FXV673 administration. Values are the mean ± S.E.M. n = 7 for the vehicle-treated group, n = 5 and n = 4 for the first two lower dosing groups and the next two higher dosing groups, respectively. * P < .05 as compared to vehicletreated animals.

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2.6. Studies in the Electrolytic Injured Canine Arterial Venous Thrombosis Models 2.6.1. Time to Vessel Occlusion and Thrombus Mass A treatment with an antithrombotic agent tends to increase the time to vessel occlusion and to decrease the thrombus mass formed in the vessels of the experimental animals. FXV673 produced significant, dose-dependent prolongation of the time to vessel occlusion and reduction of thrombus formation in the carotid artery and jugular vein as well. The results following the bolus plus intravenous infusion regimens of FXV673 are presented in Fig. 6A and B. 2.6.2. Correlations of FXV673 Plasma Concentrations (Measured at Termination of FXV673 Infusions) and Time to Carotid Artery Occlusion Administration of FXV673 resulted in dosedependent increases in the plasma concentration of the compound. The measurement of plasma concentrations of FXV673 was based on ex vivo Factor Xa inhibition chromogenic assays. Blood samples were collected at preselected time points throughout the studies. The plasma-concentration profile of FXV673 is presented in Fig. 7.

Fig. 7. Time-course of plasma FXV673 concentrations, measured in response to the administration of different doses of FXV673. Plasma concentrations were determined by ex vivo anti-FXa activity using chromogenic assays. Doses are those shown on the graph. At the end of the infusion period the plasma concentrations were approximately 100 ng/ml (0.21 mM), 250 ng/ml (0.52 mM), and 850 ng/ml (1.76 mM), respectively, for the first three doses administered.

Fig. 8. Correlation of FXV673 plasma concentrations (measured at the termination of infusions) with the prolongation of the time to carotid artery occlusion or with the reduction of carotid artery thrombus mass. Dose-dependent prolongation of the time to carotid artery occlusion (panel A) and reduction of thrombus mass (panel B) are illustrated. The effective doses achieved a 50 – 90% prolongation of the time to occlusion resulting in approximate plasma concentrations from 100 ng/ml (0.21 mM) to 850 ng/ml (1.76 mM) as shown on the graph. These plasma concentrations also correspond to the effective doses that achieved a 50 – 90% reduction of thrombus mass.

At the termination of FXV673 infusions, the plasma concentrations of FXV673 were approximately 100 ng/ml (0.21 mM), 250 ng/ml (0.52

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mM), 850 ng/ml (1.76 mM), and 2.9 mg/ml (6.01 mM) for the administration of 10 mg/kg + 1 mg/kg/min, 25 mg/kg + 2.5 mg/kg/min, 83 mg/ kg + 8.3mg/kg/min, and 30 mg/kg + 300 mg/kg/ min, respectively. The mean carotid artery time-to-occlusion in the vehicle-treated group (n = 6) is 82 ± 12 min. Treatment of FXV673 dose-dependently prolonged the time-to-occlusion to 164 ± 44, 208 ± 32, and 240 ± 0 min,

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respectively. At the conclusion of the experiment the thrombi were removed from the injured vessels and weighed. A dose-dependent reduction in the carotid artery thrombus mass was observed in the FXV673-treated animals as compared to the controls. The thrombus mass reduced from 108 ± 9 mg (control) to 54 ± 21, 29 ± 19, and 6 ± 2 mg, respectively, with the treatment of aforementioned increasing concentrations of FXV673. The effective doses (ED) that achieved approximately 50–90% of prolongation of the time to carotid artery occlusion resulted in plasma concentrations (measured at the termination of infusion) between 100 ng/ml (0.21 mM) and 850 ng/ ml (1.76 mM) as shown in Fig. 8A. Likewise these plasma concentrations achieved a carotid artery thrombus mass reduction of approximately 50% to 90% as shown in Fig. 8B. 2.6.3. APTT and PT The time-course of APTT fold-change and PT fold-change in anesthetized dogs in response to the administration of different doses of FXV673 are shown in Fig. 9. The results are expressed as fold-change as compared to the pretreatment control values. Pretreatment APTT and PT values ranged from a mean of 11.8 to 13.5 s, and 8.5 to 9.4 s, respectively. Values are the mean ± S.E.M. n = 7 for the vehicle-treated group, n = 5 for the first two lower FXV673 dosing groups, and n = 4 for the next two higher FXV673 dosing groups.

3. Discussion

Fig. 9. Time-course of APTT fold-change (panel A) and PT fold-change (panel B) in anesthetized dogs in response to the administration of different doses of FXV673. Doses (bolus + intravenous infusion) are shown on the graph. Blood samples were obtained at the indicated time points. The results are expressed as fold-change as compared to the pretreatment control values. Values are the mean ± S.E.M. n = 7 for the vehicle-treated group, n = 5 for the first two lower FXV673 dosing groups, and n = 4 for the next two higher FXV673 dosing groups.

FXV673 is a selective and potent FXa inhibitor. It can block further thrombin generation by interference with the prothrombinase-bound FXa. The inhibitory effect of FXV673 is specific for coagulation without impairing platelet function, potentially resulting in an improved efficacy-to-risk factor. The antithrombotic effect of FXV673 has been demonstrated in well-established canine arterial venous thrombosis model at doses that did not significantly alter systemic coagulation as measured by PT and APTT [19,27]. FXV673 significantly attenuates prothrombinase activity, as do natural FXa inhibitors rTAP [43] and ATS [44]. In inhibition studies of pro-

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thrombinase complex, assembled with liposomes or gel-filtered platelets, the IC50 for FXV673 was 1.38 and 2.55 nM, respectively. A slightly higher IC50 value obtained with the gel-filtered platelets could be due to nonspecific drug–protein interactions. Under the pathological condition in which thrombus is being formed, FXa could be in the fluid phase or locally concentrated by forming a prothrombinase complex or by binding to the clot formed. In the studies of inhibition of further thrombin generation, we found that FXV673, a small molecule inhibitor, was able to inhibit prothrombinase-bound FXa, and thus to inhibit further generation of thrombin. By extrapolation FXV673 could be expected to inhibit clotbound FXa, which has been targeted as a major culprit of further thrombus propagation [7,8]. This phenomenon cannot be achieved by heparin or low molecular weight heparin, probably due to steric hindrance [7,8,45]. It was reported previously that FXV673 is a competitive, thus, active site-directed, reversible and fast binding inhibitor for fluid phase FXa [45]. However, kinetic analyses indicate that in the presence of prothrombin, the natural substrate, FXV673 is a noncompetitive inhibitor for the prothrombinase-bound FXa. In addition to binding to the FXa active site, prothrombin, a macromolecular substrate, could interact with FXa at the exosite [46]. In fact this process can modify prothrombin specificity and thus, enhances the affinity to FXa [40–42]. Since FXV673 is a synthetic, small molecular inhibitor targeted for the active site, it cannot mimic the simultaneous binding of prothrombin to the exosite of FXa. Therefore, FXV673 or any other small molecule FXa inhibitor is expected to be a noncompetitive inhibitor [47]. The anticoagulant activity of FXV673 was evidenced by the prolongation of the clotting time in the plasma-based PT and APTT assays. The clotting time assays measure the lag time of thrombin generation. The PT assay reflects a faster generation of thrombin activity than the APTT assay. Some small molecule FXa inhibitors, DX9065a [19] and YM-75466 [21], were more potent in prolonging PT than APTT. However, other inhibitors, SEL-2711 [22] and RPR 130737 [25], were more sensitive in APTT assays. YM-60828 exhibited similar potency in both the PT and the

APTT assays [20]. We found that FXV673 belonged to the group of FXa inhibitors that were more efficient in prolonging APTT than PT. These results indicate that the kinetics of FXa inactivation in these assays is dependent on the compound class. In the fluid phase FXa assays, PT and APTT assays, the species-specific phenomena described with DX-9065a [19], YM-60828 [20], ZK-807834 [23], and RPR 130737 [25] were also observed with FXV673. However, the rank order of anticoagulant effect of these inhibitors in different species varied, depending on the class of compound. Among all species studied, the anticoagulant effect of FXV673 was most potent in rabbit plasma. It is known that FXa has no effect on platelet activation; however, once it is assembled into the prothrombinase complex, it triggers enormous thrombin generation. Thrombin is the major enzyme responsible for clot formation and for platelet activation. The kinetic analysis demonstrates that thrombin has an approximately 10,000-fold higher affinity for platelets than for fibrinogen [48,49]. The platelet aggregation studies in PRP demonstrated that up to 100 mM of FXV673 had no effect on platelet aggregation either with or without the addition of agonists. Thus, treatment with FXV673 would not compromise primary hemostasis, allowing platelets to aggregate normally in response to several agonists and potentially yielding a clinical profile benefited by a reduced bleeding risk. In this report, we demonstrated the efficacy of FXV673, a noncompetitive inhibitor for prothrombinase-bound FXa-mediated thrombin generation in the canine arterial and venous thrombosis models as illustrated by its ability to reduce the thrombus mass and to prolong the time to occlusion. The results also indicated that the maximal antithrombotic effect was achieved at 83 mg/kg iv bolus plus 8.3 mg/kg/min infusion for 225 min. At this dose, APTT and PT results were, respectively, 2.5- and 1.6-fold higher than in controls, indicating that systemic hypocoagulability is not required to achieve antithrombotic efficacy with this agent. However, at a dose 3– 10-fold higher than the maximally effective antithrombotic dose, APTT and PT clotting times were markedly prolonged to approximately nine- and threefold greater than control times.

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Further analyses of the results from the canine arterial thrombosis model indicate that a clinical effect in terms of thrombus mass reduction and prolongation of the time to occlusion is expected to occur at doses between ED50 and ED90 for these two parameters. Thus, FXV673 plasma concentrations ranging between 100 ng/ml (0.21 mM) and 850 ng/ml (1.76 mM) should be achieved for efficacy. The ED70 –ED80 of these two parameters corresponding to a plasma concentration of approximately 250 ng/ml (0.52 mM) in dogs increases APTT less than 1.5-fold and would not cause bleeding risk. FXV673 is an approximately 2.5-fold more potent inhibitor of human FXa than dog FXa as determined by FXa assay and APTT assay. Using interspecies extrapolation of pharmacokinetic parameters from dogs to humans, it was found that a 100 ng/ml (0.21 mM) concentration in human plasma might be targeted for clinical efficacy. Several in vitro studies have demonstrated that clot-bound FXa and thrombin provide sustained procoagulant activity, which probably plays an important role in the progression of arterial thrombosis [7,8]. In addition, clinical studies of thrombin inhibitors have indicated that thrombin generation continues during treatment. For example, both antithrombin–thrombin complex and fibrinopeptide A were detected in the hirudin studies. These results support the conclusion that thrombin is generated persistently even during treatment with thrombin inhibitors [50,51]. Clinical studies of Argatroban revealed that there is a rebound coagulation phenomenon in patients with unstable angina pectoris after discontinuation of treatment [52]. In a preclinical canine model of coronary artery thrombosis, it was shown that treatment by the FXa inhibitor, TAP, provided more effective attenuation of thrombus procoagulant activity than did direct thrombin inhibitor treatment [11]. Therefore, in several clinical trials [51,52] clot-bound FXa may account for the recurrent thrombin activity observed after cessation of thrombin inhibitor treatment in patients after thrombolysis or mechanical interventions. These observations suggest that treatment with a small molecule inhibitor such as FXV673 that can access the clot-bound or pro-

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thrombinase-bound FXa could have a superior clinical outcome.

References 1. Davie EW. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemostasis 1995;74:1–6. 2. Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance and regulation. Biochemistry 1991;30:10363–70. 3. Hoffman M, Monroe DM, Oliver JA, Roberts HR. Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation. Blood 1995;86:1794–801. 4. Kirchofer D, Tschopp TB, Baumgartner HR. Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model. Arterioscler Thromb Vasc Biol 1995;15:1098–106. 5. Barrowcliffe TW. LMW: relationship between antithrombotic and anticoagulant effects. In: Land DA, editor. Heparin and related polysaccharides. New York: Plenum, 1992. pp. 205–19. 6. Mann KG. The assembly of blood clotting complexes on membranes. TIBS 1987;12: 229–33. 7. Hemker HC, Beguin S. Mode of action of heparin and related drugs. Semin Thromb Haemostasis 1991;17(Suppl 1):29–34. 8. Prager NA, Abendschein DR, McKenzie CR, Eisenberg PR. Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots. Circulation 1995;92: 962–7. 9. Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Importance of factor Xa in determining the procoagulant activity of whole blood clots. J Clin Invest 1993;91:1877–83. 10. Mckenzie CR, Abendschein DR, Eisenberg PR. Sustained inhibition of whole-blood clot procoagulant activity by inhibition of thrombus-associated factor Xa. Arterioscler Thromb Vasc Biol 1996;16:1285–91. 11. Vlasuk GP. Structural and functional characterization of tick anticoagulant peptide

322

12.

13.

14.

15.

16.

17.

18.

19.

20.

V. Chu et al./Thrombosis Research 103 (2001) 309–324

(TAP): a potent and selective inhibitor of blood coagulation factor Xa. Thromb Haemostasis 1993;70:212–6. Lynch JJ, Sitko GR, Mellott MJ, Nutt EM, Lehman ED, Friedman PA, Dunwiddie CT, Vlasuk GP. Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy. A comparison of factor Xa versus thrombin inhibition. Cardiovasc Res 1994;28:78–85. Hauptmann J, Stu¨rzebecher J. Synthetic inhibitors of thrombin and factor Xa: from bench to bedside. Thromb Res 1999;94:203–41. He´rault JP, Bernat A, Pflieger AM, Lormeau JC, Herbert JM. Comparative effects of two direct and indirect factor Xa inhibitors on free and clot-bound prothrombinase. J Pharmacol Exp Ther 1997;283:16–22. Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa. Science 1990;48:593–6. Neeper MP, Waxman L, Smith DE, Schulman CA, Sardana M, Ellis RW, Schaffer LW, Siegel KS, Vlasuk GP. Characterization of recombinant tick anticoagulant peptide. A highly selective inhibitor of blood coagulation factor Xa. J Biol Chem 1990;265:17746–52. Tuszynski GP, Gasic TB, Gasic GJ. Isolation and characterization of antistasin. J Biol Chem 1987;262:9718–23. Dunwiddie C, Thornberry NA, Bull HG, Sardana M, Friedman PA, Jacobs JW, Simpson E. Antistasin, a leech-derived inhibitor of factor Xa. J Biol Chem 1989;264:16694–9. Hara T, Yokoyama A, Ishihara H, Yokoyama Y, Nagahara T, Iwamoto M. DX-9065a, a new synthetic, potent anticoagulant and selective inhibitor for factor Xa. Thromb Haemostasis 1994;71:314–9. Taniuchi Y, Sakai Y, Hisamichi N, Kayama M, Mano Y, Sato K, Hirayama F, Koshio H, Matsumoto Y, Kawasaki T. Biochemical and pharmacological characterization of YM-60828, a newly synthesized and orally active inhibitor of human Factor Xa. Thromb Haemostasis 1998;79:543–8.

21. Sato K, Taniuchi Y, Kawaski T, Hirayama F, Koshio H, Matsumoto Y. Relationship between the antithrombotic effect of YM-75466, a novel factor Xa inhibitor, and coagulation parameters in rats. Eur J Pharmacol 1998; 347:231–6. 22. Zeneca Ltd.: W09610022. Novel aminoheterocyclic derivatives as inhibitors of factor Xa. Expert Opin Ther Pat 1996;6:795–9. 23. Abendschein DR, Baum PK, Verhallen P, Eisenberg PR, Sullivan M, Light DR. A novel synthetic inhibitor of Factor Xa decreases early reocclusion and improves 24-h patency after coronary fibrinolysis in dogs. J Pharmacol Exp Ther 2001;296:567–72. 24. Wong CP, Crain EJ, Knabb RM, Meade RP, Quan ML, Watson CA, Wexler RR, Wright MR, Slee AM. Nonpeptide factor Xa inhibitors: II. Antithrombotic evaluation in a rabbit model of electrically induced carotid artery thrombosis. J Pharmacol Exp Ther 2000;295:212–8. 25. Chu V, Brown K, Colussi D, Choi YM, Green D, Pauls HW, Spada AP, Perrone MH, Leadley RJ, Dunwiddie CT. In vitro characterization of a novel Factor Xa inhibitor, RPR 130737. Thromb Res 2000;99:71–82. 26. Hara T, Yokohama A, Tanabe K, Ishihara H, Iwamoto M. DX-9065a, an orally active, specific inhibitor of Factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb Haemostasis 1995;74:635–9. 27. Sato K, Kawasaki T, Hisamichi N, Taniuchi Y, Hirayama F, Koshio H, Matsumoto Y. Antithrombotic effects of YM-60828, a newly synthesized factor Xa inhibitor, in rat thrombosis models and its effects on bleeding time. Br J Pharmacol 1998;123:92–6. 28. Bostwick J, Bentley R, Morgan S, Brown K, Chu V, Ewing WR, Spada AP, Pauls H, Perrone MH, Dunwiddie CT, Leadley RJ. RPR 120844, a novel, specific inhibitor of coagulation factor Xa inhibits venous thrombosis in the rabbit. Thromb Haemostasis 1999;81: 157–60. 29. Walenga JM, Jeske WP, Hoppensteadt D, Kaiser B. Factor Xa inhibitors: today and be-

V. Chu et al./Thrombosis Research 103 (2001) 309–324

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

yond. Curr Opin Cardiovasc Pulm Res Invest Drugs 1999;1:13–27. Dunwiddie CT, Waxman L, Vlasuk GP, Friedman PA. Purification and characterization of inhibitors of blood coagulation Factor Xa from hematophagous organisms. Lorand L, Mann K, editors. Methods Enzymol, vol. 223. New York: Academic Press, 1993. pp. 291–3. Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973;22: 3099–108. Bloom J, Nesheim ME, Mann KG. Phospholipid binding properties of bovine factor V and Va. Biochemistry 1979;18:4419–25. Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980;255:274–83. Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979;254:5357–63. Plow EF, Pierschbacher MD, Rusolahti E, Marguerie GA, Ginsberg MH. The effect of arg–gly–asp-containing peptides on fibrinogen and von Willebrand binding to platelets. Proc Natl Acad Sci USA 1985;82: 8057–61. Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962;194:927–9. Cornish-Bowden A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J 1974;137: 143–4. Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine: a model for in vivo evaluation of anti-thrombotic agents. Thromb Res 1980;17:841–53. Williams JW, Morrison JF. The kinetics of reversible tight-binding inhibition. In: Purich L, editor. Methods Enzymol vol. 63. New York: Academic Press, 1979. pp. 437–67.

323

40. Betz A, Vlasuk GP, Bergum PW, Krishnaswamy S. Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by Factor Xa. Biochemistry 1997;36:181–91. 41. Krishnaswamy S, Betz A. Exosites determine macromolecular substrate recognition by prothrombinase. Biochemistry 1997;36: 12080–6. 42. Krishnaswamy S, Vlasuk GP, Bergum P. Assembly of the prothrombinase complex enhances the inhibition of bovine factor Xa by tick anticoagulant peptide. Biochemistry 1994;33:7897–907. 43. Dunwiddie CT, Smith DE, Nutt EM, Vlasuk GP. Anticoagulant effects of the selective factor Xa inhibitors tick anticoagulant peptide and antistasin in the APTT assay are determined by the relative rate of prothrombinase inhibition. Thromb Res 1994;64:787–94. 44. Weitz JI. Low-molecular-weight heparins. N Engl J Med 1997;337:688–98. 45. Chu V, Brown K, Colussi D, Gao J, Guertin K, Zulli A, Pauls H, Spada A, Perone MH, Dunwiddie CT, Leadley R. In vitro characterization of a novel FXa inhibitor, RPR 130673. Blood 2000;96:57a. 46. Krishnaswamy S, Betz A. Macromolecular substrate recognition by prothrombinase is mediated by interactions at exosites in the enzyme complex. Blood 1996;88(Suppl 1):518a. 47. Berndt MC, Gregore C, Dowden G, Castaldi PA. Thrombin interactions with platelet membrane proteins. Ann NY Acad Sci 1986; 485:374–86. 48. Higgins DL, Lewis SD, Shafer JA. Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin. J Biol Chem 1983;258:9276–82. 49. Teitel JM, Rosenberg RD. Protection of factor Xa from neutralization by the heparin– antithrombin complex. J Clin Invest 1983;71: 1383–91. 50. Ginsberg JS, Nurmohamed MT, Gent M, MacKinnon B, Stevens P, Weitz J, Maraganore J, Hirsh J. Effects on thrombin generation of single injections of hirulog in patients

324

V. Chu et al./Thrombosis Research 103 (2001) 309–324

with calf vein thrombosis. Thromb Haemostasis 1994;72:523–5. 51. Zoldhelyi P, Bichler J, Owen WG, Grill DE, Fuster V, Mruk JS, Chesebro JH. Persistent thrombin generation in humans during specific thrombin inhibition with hirudin. Circulation 1994;90:2671–8. 52. Gold HK, Torres FW, Garabedian HD, Wer-

ner W, Jang I, Khan A, Hagstrom JN, Yasu da T, Leinbach RC, Newell JB, Bovill EG, Stump DC, Collen D. Evidence for a rebound coagulation phenomenon after cessation of a 4-hour infusion of a specific thrombin inhibitor in patients with unstable angina pectoris. J Am Coll Cardiol 1993;21: 1039–47.