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Vorapaxar is a novel protease-activated receptor-1 (PAR-1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Patients who received vorapaxar in addition to standard of care antiplatelet therapy had an increased incidence of major bleeding events compared with placebo. To assess whether platelet transfusion can restore hemostasis in primates on triple antiplatelet therapy, template bleeding times were assessed concurrently in the buccal mucosa, finger pad, and distolateral tail of anesthetized cynomolgus macaques to evaluate bleeding with vorapaxar as either monotherapy or in combination with aspirin or aspirin and clopidogrel. Aspirin (5mg/kg, IV) or vorapaxar (1mg/kg, PO) alone had no significant effect on bleeding times in the three vascular beds examined. A modest (

Antagonism of the bradykinin B1 receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating α-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B1 receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of L-734,217, a potent fibrinogen receptor antagonist, were studied in male dogs. L-734,217 was given intravenously at 0.01 mg kg-1, in a cross-over fashion, to conscious dogs or to dogs anesthetized with pentobarbital. Plasma concentrations of L-734,217 were measured using a radioimmunoassay and inhibitory effects on ex vivo platelet aggregation induced by ADP or collagen were determined. In pentobarbital-treated dogs, L-734,217 plasma concentrations during the first 3 h collection period were significantly higher than those in the control animals. Corresponding to the increased plasma levels, the mean ex vivo inhibitory effects on ADP- or collagen-induced platelet aggregation in dogs under anesthesia appeared greater than in those without the anesthetic treatment. Pharmacokinetic analysis revealed a modest, but significant (up to 40%) elevation in the area under the plasma concentration-time curve during 6 h of the drug administration, and a reduction in L-734,217 plasma clearance and volumes of distribution, in the anesthetized dogs. Analysis of pharmacodynamic data indicated that the EC50 and the Hill coefficient of the platelet aggregation response-plasma concentration curve were not altered by pentobarbital treatment. The results are in agreement with the findings that the administration of pentobarbital alone (in the absence of L-734,217) did not affect appreciably the ex vivo platelet aggregatory responses. In a separate group of dogs, L-734,217 was found to be metabolically stable, and was eliminated unchanged renally (64 +/- 4%) and hepatically (32 +/- 6%). In addition, L-734,217 did not bind substantially to canine plasma proteins or blood cellular components. It is possible that alterations of regional hemodynamics, reportedly mediated by pentobarbital, contributed to changes observed in the present study. That is, alterations occurred in L-734,217 elimination and distribution processes which resulted in an increase in drug plasma levels. Since pentobarbital anesthesia influenced only the pharmacokinetics, and not the pharmacodynamics, of L-734,217, the apparent increases in the inhibition of platelet aggregation responses observed following L-734,217 administration to the anesthetized dogs were probably sequential effects of the pharmacokinetic interactions.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Centrally constrained thieno[2,3- b ]thiophene sulfonamides have provided a potent, selective, orally active series of platelet aggregation inhibitors. Compound 21 showed excellent activity in the dog after a single oral dose of 200 μg/kg.

Potency enhancing features of two series of fibrinogen receptor antagonists were combined to give analogs with improved potency and oral activity. Antagonists containing either alkyl or aryl sulfonamides and a central isoindolinone structural constraint demonstrate high affinity for both activated and unactivated platelet receptors.

The structure-activity relationship of a series of orally active glycoprotein IIb/IIIa antagonists containing a nitrogen heterocycle grafted onto a 3,4-dihydro-1 (1H)-isoquinolinone core is described. These compounds are structurally novel analogs of the progenitor compound 1 (L-734,217,[[3(R)-[2-(piperidin-4-yl)ethyl]-2-oxopiperidinyl ]acetyl]-3(R)- methyl-beta-alanine) in which the lactam chiral center has been removed. The 4-piperazinyl- and 4-piperidinyl-substituted 3,4-dihydro-1(1H)-isoquinolinones were found to be optimal for in vitro potency. In addition, substitution at the 3-position of the beta-amino acid enhanced potency with the 3-pyridyl and 3-ethynyl analogs being the most potent prepared. Attempts to improve the in vivo profile of these compounds focused on modification of the physical properties. Ester prodrugs were prepared to increase the lipophilicity and remove the zwitterionic nature of the antagonists. The prodrug approach, coupled with the arylpiperazine terminus (pKa = approximately 9.0), afforded moderately basic and relatively nonpolar compounds. The acid N-[[7-(piperazin-1-yl)-3,4-dihydro-1(1H)-oxoisoquinolin-2-yl ]acetyl]-3(S)- ethynyl-beta-alanine, 6d (L-767,679), is a potent fibrinogen receptor antagonist able to inhibit the ADP-induced aggregation of human gel-filtered platelets with an IC50 of 12 nM. Although 6d is orally active based on the results of an ex vivo dog assay at 0.3 mg/kg, the ethyl ester prodrug of this compound, 19 (L-767,685), is better absorbed at this dose than 6d. Upon oral dosing, the ester 19 is converted to 6d in vivo in dog with an estimated oral systemic availability of > 17% (0-8 h, AUC19po/AUC6div). In addition, studies in monkey at an oral dose of 1 mg/kg show that 19 affects the complete inhibition of the ex vivo platelet aggregation in response to ADP between 2 and 8 h postdose with the level of inhibition remaining at 40% at 12 h postdose. This level of activity was superior to that observed for 6d and 1 at the same dose. Using ex vivo ADP-induced aggregation data from rhesus monkey (n = 2, 0-8 h using the AUC19po/AUC6div), the estimated systemic oral availability of 6d when dosed as 19 is 32%.

SummaryThe antithrombotic efficacies of the coagulation factor Xa inhibitor recombinant tick anticoagulant peptide (rTAP) and heparin were compared in a canine model of left circumflex (LCX) coronary artery electrolytic lesion. Intravenous infusions of saline (controls), rTAP (50 μg/kg/min continuous infusion) or heparin (200 U/kg bolus followed by 2 U/kg/min continuous infusion) were started 60 min prior to the initiation of LCX coronary artery electrolytic injury (150 μA continuous anodal current). All 6/6 saline-treated control animals developed occlusive thrombi at 49.8 ± 13.6 min after the initiation of vessel injury, and possessed a residual thrombus mass of 20.7 ± 3.3 mg. In the rTAP treatment group, 4/6 preparations developed occlusive thrombi, but with times to thrombosis delayed significantly compared to both the saline control as well as to the heparin treatment group (202.7 ± 28.9 min; p

Tick anticoagulant peptide (TAP) is a 60 amino acid protein originally isolated from the soft tick, Ornithodoros moubata, which exhibits potent anticoagulant properties due to its selective inhibition of blood coagulation factor Xa. We evaluated a recombinant version of TAP (rTAP) for its ability to accelerate recombinant tissue plasminogen activator (rt-PA) -mediated lysis of an occlusive thrombus and prevent acute reocclusion in a canine model of femoral artery thrombosis. An occlusive thrombus was formed by insertion of a thrombogenic copper coil into the femoral artery of anesthetized dogs. Blood flow velocity was monitored directly and continuously by Doppler flowmetry. 60 min after occlusion, dogs received an i.v. infusion of either saline or rTAP (0.5, 2.5 or 8.0 μg/kg/min), followed 45 min later by rt-PA (0.8 mg/kg, i.v. over 90 min; n=8/group). The saline and rTAP infusions were discontinued 1 h after stopping the rt-PA. All dogs achieved reperfusion, with a time to reperfusion in the saline-treated (vehicle) group (administered rt-PA alone) of 61 ±7 min. The time to reperfusion was slightly decreased in the 0.5ug/kg/min rTAP group (47±4min, p=NS). In the groups administered rTAP at 2.5 and 8.0 ug/kg/min, significant reductions in the time to reperfusion were observed (28±4 and 32±5mins, respectively, p

The effect of tissue-type plasminogen activator (t-PA) alone or in combination with heparin, the Arg-Gly-Asp-containing peptide bitistatin, or both heparin and bitistatin was evaluated on thrombolysis time and acute reocclusion in a canine model of coronary thrombosis. Thrombus formation was elicited by electrolytic injury with a needle electrode to the endothelial surface of the circumflex coronary artery in the open-chest, anesthetized dog in the presence of a flow-limiting critical stenosis. Thirty minutes after spontaneous coronary artery occlusion, t-PA (1 mg/kg i.v. over 90 minutes) was administered. Group 1 was given t-PA alone; reperfusion occurred at 78.2 +/- 5.6 minutes with a reperfusion incidence of 60% (6/10). Group 2 received t-PA plus heparin (100 units/kg plus 50 units/kg/hr); reperfusion occurred at 61.9 +/- 9.1 minutes with a reperfusion incidence of 90% (9/10). Group 3 received t-PA plus heparin plus bitistatin (30 micrograms/kg plus 3 micrograms/kg/min); reperfusion occurred at 47.3 +/- 7.6 minutes (p less than 0.05 versus group 1) with a reperfusion incidence of 90% (9/10). Group 4 received t-PA plus bitistatin, and reperfusion occurred at 51.8 +/- 8.5 minutes; however, the reperfusion incidence was only 60% (6/10). In groups 1, 2, and 4, acute reocclusion occurred in more than 80% of the reperfused dogs, whereas in group 3 reocclusion occurred in 22% (2/9) of the reperfused dogs (p less than 0.05 versus group 1). The dose of heparin used in this study increased activated partial thromboplastin times 1.5-2.0-fold over control.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5--221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.

The small molecule direct thrombin inhibitor L-374,087 was characterized across species in an in vitro activated partial thromboplastin clotting time (aPTT) assay and in vivo in rhesus monkey and dog thrombosis models. In vitro in rhesus, dog, and human plasma, L-374,087 concentrations eliciting 2-fold increases in aPTT were 0.25, 1.9, and 0.28 microM, respectively. In anesthetized rhesus monkeys, 300 microgram/kg bolus plus 12 microgram/kg/min and 300 microgram/kg bolus plus 30 microgram/kg/min L-374,087 i.v. infusions significantly reduced jugular vein thrombus extension, with both regimens limiting venous thrombus extension to 2-fold that of baseline thrombus mass compared with a 5-fold extension observed in the vehicle control group. Antithrombotic efficacy in the rhesus with the lower-dose regimen was achieved with 2.3- to 2.4-fold increases in aPTT and prothrombin time. In a conscious instrumented dog model of electrolytic vessel injury, the oral administration of two 10 mg/kg L-374,087 doses 12 h apart significantly reduced jugular vein thrombus mass, reduced the incidence of and delayed time to occlusive coronary artery thrombosis, and significantly reduced coronary artery thrombus mass and ensuing posterolateral myocardial infarct size. Antithrombotic efficacy in the dog was achieved with 1.6- to 2.0-fold increases in aPTT at 1 to 6 h after oral dosing with L-374,087. These results indicate significant antithrombotic efficacy against both venous and coronary arterial thrombosis with L-374,087 with only moderate elevations in aPTT or prothrombin time. The oral efficacy of L-374,087 characterizes this compound as a prototype for the further development of orally active direct thrombin inhibitors.

Effects of pentobarbital on pharmacokinetics and pharmacodynamics of L-734,217, a potent fibrinogen receptor antagonist, were studied in male dogs. L-734,217 was given intravenously at 0.01 mg kg-1, in a cross-over fashion, to conscious dogs or to dogs anesthetized with pentobarbital. Plasma concentrations of L-734,217 were measured using a radioimmunoassay and inhibitory effects on ex vivo platelet aggregation induced by ADP or collagen were determined. In pentobarbital-treated dogs, L-734,217 plasma concentrations during the first 3 h collection period were significantly higher than those in the control animals. Corresponding to the increased plasma levels, the mean ex vivo inhibitory effects on ADP- or collagen-induced platelet aggregation in dogs under anesthesia appeared greater than in those without the anesthetic treatment. Pharmacokinetic analysis revealed a modest, but significant (up to 40%) elevation in the area under the plasma concentration-time curve during 6 h of the drug administration, and a reduction in L-734,217 plasma clearance and volumes of distribution, in the anesthetized dogs. Analysis of pharmacodynamic data indicated that the EC50 and the Hill coefficient of the platelet aggregation response-plasma concentration curve were not altered by pentobarbital treatment. The results are in agreement with the findings that the administration of pentobarbital alone (in the absence of L-734,217) did not affect appreciably the ex vivo platelet aggregatory responses. In a separate group of dogs, L-734,217 was found to be metabolically stable, and was eliminated unchanged renally (64 +/- 4%) and hepatically (32 +/- 6%). In addition, L-734,217 did not bind substantially to canine plasma proteins or blood cellular components. It is possible that alterations of regional hemodynamics, reportedly mediated by pentobarbital, contributed to changes observed in the present study. That is, alterations occurred in L-734,217 elimination and distribution processes which resulted in an increase in drug plasma levels. Since pentobarbital anesthesia influenced only the pharmacokinetics, and not the pharmacodynamics, of L-734,217, the apparent increases in the inhibition of platelet aggregation responses observed following L-734,217 administration to the anesthetized dogs were probably sequential effects of the pharmacokinetic interactions.

A conscious dog model of left circumflex coronary artery electrolytic injury was used to assess the oral antithrombotic efficacy of L-738,167, a potent nonpeptide antagonist of platelet GP IIb/IIIa. L-738,167 was administered either as a single oral pretreatment dose 2 hours before initiation of vessel injury or as two oral doses administered 24 hours apart, 12 hours before and after initiation of vessel injury.In untreated controls, electrolytic coronary injury (50 microA, 3 hours) resulted in thrombotic occlusion and myocardial ischemia in 15 of 16 dogs, with 4 developing lethal arrhythmias. Significant reductions in thrombus mass and complete prevention of myocardial ischemia and infarction were achieved with a single 100- to 300-microg/kg dose of L-738,167 pretreatment and with two 100-microg/kg doses administered 12 hours before and after initiation of vessel injury. Delays and/or reductions in incidence of ischemia, thrombus mass, and infarct sizes also were achieved with 10- to 30-microg/kg pretreatment and with two 30-microg/kg doses administered 12 hours before and after initiation of vessel injury. None of the L-738,167-treated animals developed lethal arrhythmias. A single oral 100-microg/kg dose of L-738,167 achieved90% inhibitions of ADP (extent)- and collagen (rate)-induced ex vivo platelet aggregation and fivefold to sixfold or greater elevations in bleeding time; a single oral 30-microg/kg dose of L-738,167 achieved sustained 40% to 70% inhibitions of ADP- and collagen-induced ex vivo platelet aggregation and modest twofold to threefold elevations in bleeding time. At 12 to 24 hours after single oral 30- and 100-microg/kg doses of L-738,167, a substantially greater L-738,167 concentration was associated with platelets than free in plasma.These findings are indicative of potent and sustained oral antithrombotic efficacy and suggest that L-738,167 possesses potential for the oral management of chronic thrombotic occlusive disorders.

The nonpeptide platelet glycoprotein IIb/IIIa antagonist, L-738, 167, was characterized in dog and nonhuman primate. In an anesthetized canine model of coronary artery electrolytic lesion, L-738,167 elicited dose-dependent (3, 4, 4.5 and 5 micrograms/kg i.v.) decreases in incidence of occlusion, reductions in thrombus mass and elevations in bleeding time. Antithrombotic efficacy correlated with inhibition of adenosine diphosphate-induced platelet aggregation but was dissociated from marked bleeding time elevation. Similarly, suppression of platelet-dependent cyclic flow reductions with L-738,167 in the canine coronary artery (5 micrograms/kg i.v.) and African green monkey carotid artery (10 micrograms/kg i.v.) correlated with inhibition of adenosine diphosphate-induced platelet aggregation but not with inhibition of thrombin-induced platelet aggregation or significant prolongation of bleeding time. In conscious dogs and sedated chimpanzees, single dose intravenous bolus (5-20 micrograms/kg) and oral (25-200 micrograms/kg) administration of L-738,167 exhibited long duration (or = 8 hr) inhibition of ex vivo platelet aggregation. Once daily oral administration to conscious dogs (10-30 micrograms/kg/day for 15 days) and rhesus monkeys (200-250 micrograms/kg/day for 11 days) maintained significant but submaximal (50-90% inhibition) trough levels of inhibition of adenosine diphosphate-induced ex vivo platelet aggregation. Platelet sensitivity to adenosine diphosphate after multiple days of oral dosing in dogs was similar to pretreatment sensitivity. L-738,167 showed characteristics suitable for chronic oral therapy with a glycoprotein IIb/IIIa inhibitor.

The antiplatelet activity of L-734,217, a nonpeptide platelet GPIIb/IIIa antagonist, was evaluated in the rat, guinea pig and dog. IC50 for inhibition of in vitro platelet aggregation for these species (agonists: adenosine diphosphate, collagen) were rat, 838,000 and1,100,000 nM; guinea pig, 124 and 156 nM; dog, 42 and 50 nM. In an in vivo rat/in vitro dog platelet aggregation assay, effective antiaggregatory plasma concentrations of L-734,217 were achieved after 8.0 to 16.0 mg/kg p.o. vs. 0.3 to 1.0 mg/kg i.v. to rats. Delays in platelet-dependent hemostatic plug formation in severed mesenteric arteries were observed after 2.0 to 5.0 mg/kg p.o. vs. 0.1 to 0.2 mg/kg i.v. to guinea pigs. Dose-dependent inhibitions of ex vivo platelet aggregation after 0.3 to 3.0 mg/kg p.o. and 0.03 to 0.3 mg/kg i.v. L-734,217 to conscious dogs yielded estimates of 8 to 16% oral bioavailability. The antiplatelet activity of 3.0 mg/kg p.o. L-734,217 in dogs was unaffected by dosage form or food. In a conscious dog model of left circumflex coronary artery electrolytic lesion, 3.0 mg/kg p.o. L-734,217 q4 to 8 hr reduced thrombus mass, prevented occlusive coronary artery thrombosis and reduced or prevented myocardial infarction and ventricular ectopy. In anesthetized dogs, a dissociation between inhibition of ex vivo platelet aggregation and template bleeding time prolongation was observed with i.v. L-734,217. The results of the coadministration of heparin, aspirin and L-734,217 to anesthetized dogs suggested a synergistic effect on template bleeding time with no effect on plasma L-734,217 concentrations. These findings indicate L-734,217 to be an important lead structure for the development of therapeutically useful oral antiplatelet agents.

The antithrombotic efficacies of the coagulation factor Xa inhibitor recombinant tick anticoagulant peptide (rTAP) and heparin were compared in a canine model of left circumflex (LCX) coronary artery electrolytic lesion. Intravenous infusions of saline (controls), rTAP (50 micrograms/kg/min continuous infusion) or heparin (200 U/kg bolus followed by 2 U/kg/min continuous infusion) were started 60 min prior to the initiation of LCX coronary artery electrolytic injury (150 microA continuous anodal current). All 6/6 saline-treated control animals developed occlusive thrombi at 49.8 +/- 13.6 min after the initiation of vessel injury, and possessed a residual thrombus mass of 20.7 +/- 3.3 mg. In the rTAP treatment group, 4/6 preparations developed occlusive thrombi, but with times to thrombosis delayed significantly compared to both the saline control as well as to the heparin treatment group (202.7 +/- 28.9 min; p0.01 to both saline and heparin groups). The remaining 2 rTAP-treated preparations remained patent despite the continued electrical stimulation of the coronary vessel for 5 h. Residual thrombus mass in the rTAP treatment group was reduced markedly compared to the saline control group (4.4 +/- 1.0 mg; p0.01). Heparin infusion resulted in a modest but statistically insignificant delay in occlusive LCX coronary artery thrombosis compared to saline controls, with all 6/6 heparin-treated preparations occluding at 79.7 +/- 16.5 min after the initiation of vessel injury. Residual thrombus mass in heparin-treated animals, however, was reduced compared to saline controls (9.4 +/- 1.4 mg; p0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Background Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. Methods and Results The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor–derived peptide SFLLR-NH 2 . IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of ≈64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. Conclusions These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.

The antiaggregatory and antithrombotic actions of MK-0383, a low molecular weight, nonpeptide antagonist of the platelet glycoprotein IIb/IIIa, were evaluated in a variety of canine models. Inhibition of ex vivo platelet aggregation responses to ADP and collagen were observed after the acute sequential i.v. administrations of 10 to 500 micrograms/kg or 360-min continuous i.v. infusions of 1 to 10 micrograms/kg/min of MK-0383. Hemostatic function normalized within 30 (platelet response to collagen, template bleeding times) to 90 min (platelet response and sensitivity to ADP) after the termination of 360-min i.v. MK-0383 infusions, suggesting no protracted, direct effects on platelet function. With acute sequential i.v. administrations of MK-0383, platelet response to ADP was abolished without significant extension of bleeding time. In a model of platelet-dependent cyclic flow reductions in injured, stenosed left circumflex coronary artery, the bolus i.v. administrations of 300 and 1000 micrograms/kg of MK-0383 totally abolished cyclic flow reductions for periods of 18 +/- 1 and 37 +/- 5 min, respectively. In a model of electrically induced left circumflex coronary artery occlusive thrombosis, 10 micrograms/kg/min i.v. of MK-0383 initiated 15 min before electrical injury prevented occlusive thrombosis in three of six preparations despite continued electrical stimulation of the vessel for 300 min, delayed occlusion in three of six preparations (160.3 +/- 5.5 min) and reduced thrombus mass (5.1 +/- 1.3 mg), compared to the development of occlusive thrombosis in six of six saline-treated preparations (50.5 +/- 8.7 min; 19.1 +/- 3.0 mg). When administered as an adjunct to thrombolytic agents in the presence of background heparin for lysis of electrically induced left circumflex coronary artery occlusive thrombus, 10 micrograms/kg/min i.v. of MK-0383 initiated 15 min before tissue-type plasminogen activator or streptokinase increased the incidence of (tissue-type plasminogen activator: eight of nine MK-0383 vs. three of eight saline; streptokinase: eight of eight MK-0383 vs. two of eight saline) and accelerated reperfusion, and reduced the incidence of acute thrombotic reocclusion during continued MK-0383 infusion. These findings indicate significant antithrombotic potential for MK-0383 alone or as an adjunct to thrombolytic therapy in the treatment of coronary artery ischemic syndromes.

The antiaggregatory and antithrombotic actions of MK-0852, a cyclic heptapeptide antagonist of the platelet GP IIb/IIIa, were evaluated in a variety of canine models. In vitro, MK-0852 inhibited the aggregation of canine platelet-rich plasma induced by 10 microM ADP in the presence of 1 microM epinephrine with an IC50 value of 0.10 microM. The i.v. infusion of 1.0 and 3.0 micrograms/kg/min MK-0852 to anesthetized dogs significantly inhibited ex vivo platelet aggregation responses to ADP and collagen, with the 3.0 micrograms/kg/min infusion completely inhibiting ex vivo aggregation responses to both agonists. The i.v. administrations of 100 and 300 micrograms/kg MK-0852 suppressed platelet-dependent cyclic flow reductions in stenosed canine left circumflex (LCX) coronary artery for periods of 24 +/- 3 and 64 +/- 4 min, respectively. In a canine model of copper coil-induced femoral arterial thrombosis, i.v. MK-0852 (100 micrograms/kg + 1 microgram/kg/min), initiated 15 min before coil placement, reduced the incidence of occlusive thrombosis during the 45-min post-coil time period of continued therapy (1/5 MK-0852 vs. 7/7 saline; P.01). MK-0852 was administered as an adjunctive therapy with tPA to evaluate its effects on thrombolysis after copper coil-induced femoral arterial thrombus formation. MK-0852 (i.v.; 100 micrograms/kg + 1 microgram/kg/min), initiated 15 min before tPA, reduced the incidence of post-thrombolysis reocclusion. During the 60-min period of continued drug infusion after the termination of tPA, 0 of 5 animals receiving MK-0852 reoccluded vs. 7/8 saline (P.01). In a canine model of electrically induced LCX coronary artery thrombosis, i.v. MK-0852 (100 micrograms/kg + 3 micrograms/kg/min), initiated 15 min before the initiation of electrical injury, prevented occlusive thrombosis in 4 of 6 preparations despite the continued electrical stimulation of the vessel for 180 min. Thrombotic occlusion was delayed in the remaining two preparations (99 and 100 min), compared with occlusion in 4 of 4 saline-treated preparations (69.3 +/- 6.3 min). When administered as an adjunct to thrombolytic agents for lysis of electrically induced LCX coronary artery thrombi, i.v. MK-0852 (300 micrograms/kg + 3 micrograms/kg/min), initiated 15 min before tPA or streptokinase, both increased the incidence of reperfusion (tPA: 8/8 MK-0852 vs. 3/8 saline; streptokinase: 5/8 MK-0852 vs. 2/8 saline) and accelerated reperfusion. The incidence of reocclusion during continued adjunctive therapy was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND Effective thrombolytic recanalization of an occluded coronary vessel is often limited by acute thrombotic reocclusion, which has galvanized the search for effective adjunctive or conjunctive antithrombotic agents. METHODS AND RESULTS Recombinant versions of tick anticoagulant peptide (rTAP) and hirudin (rHIR) are highly selective and potent polypeptide inhibitors of factor Xa and thrombin, respectively. The comparative antithrombotic efficacies of rTAP, rHIR, and heparin, administered conjunctively with recombinant tissue-type plasminogen activator (rt-PA), on thrombolytic reperfusion and reocclusion, were determined in a canine model of occlusive coronary artery thrombosis with a superimposed critical stenosis. In this model, a platelet-rich occlusive thrombus was formed after damage to the intimal surface of the left circumflex coronary artery induced by electrolytic injury. Fifteen minutes after occlusion, the dogs received a systemic intravenous administration of either saline (control), heparin (200 units/kg bolus + 2 units/kg/min, heparin (HEP) 200 or 100 units/kg bolus + 1 unit/kg/min, HEP 100), rHIR (50 or 100 micrograms/kg/min, rHIR 50 or 100, respectively), or rTAP (100 micrograms/kg/min, rTAP 100) followed 15 minutes later by rt-PA (100 micrograms/kg bolus + 10 micrograms/kg/min over 90 minutes). Infusions of the conjunctive agents were discontinued 60 minutes after termination of rt-PA. The incidence and time (mean +/- SEM) to thrombolytic reperfusion were determined for control (five of 12; 68.0 +/- 7.8 minutes), HEP 100 (six of eight; 40.1 +/- 8.3 minutes), HEP 200 (six of eight; 39.8 +/- 9.5 minutes), rHIR 50 (six of eight; 51.7 +/- 14.6 minutes), rHIR 100 (eight of eight; 19.5 +/- 4.2 minutes), and rTAP 100 (eight of eight; 22.8 +/- 10.0 minutes). The incidence and time to reocclusion after rt-PA were determined for control (four of five; 45.7 +/- 12.5 minutes), HEP 100 (four of six; 18.2 +/- 10.7 minutes), HEP 200 (five of six; 26.2 +/- 20.7 minutes), rHIR 50 (four of six; 47.3 +/- 21.6 minutes), rHIR 100 (six of eight; 89.8 +/- 5.9 minutes), and rTAP 100 (three of eight; 54.0 +/- 16.3 minutes). All of the dogs that reoccluded in the rHIR 100 group did so after termination of the inhibitor infusion, whereas two of the three dogs in the rTAP 100 group that reoccluded did so during the inhibitor infusion. Coronary artery blood flow was characterized by intermittent periods of reocclusion and recanalization in all groups except rTAP 100. CONCLUSIONS The potent antithrombotic effects of rTAP in this model directly implicate de novo thrombin formation as a major source of thrombin activity within the highly thrombogenic residual thrombus. These findings suggest that direct inhibition of prothrombinase activity may be an effective strategy in the development of a new class of conjunctive agents.

A model of acute, platelet-dependent canine coronary artery thrombosis was utilized to assess the antithrombotic effect of a synthetic, RGD-containing 49-residue protein termed echistatin. This protein is derived from the venom of the viper, Echis carinatus. In vitro, echistatin inhibited ADP (10 microM)-induced platelet aggregation with IC50 values in human and canine platelet-rich plasma of 101 +/- 4 and 127 +/- 32 nM, respectively. In vivo, in the dog, infusion of echistatin for 30 min at 20 micrograms kg-1 min-1 or 2.6 nM kg-1 min-1 resulted in total abolition of acute platelet-dependent coronary thrombus formation in all dogs tested (n = 5). Infusion of a lower dose (10 micrograms kg-1 min-1) was not effective in prevention of thrombus formation. Blood samples were taken before and after infusion of echistatin in order to determine ex vivo platelet aggregatory responses. Echistatin (20 micrograms kg-1 min-1, i.v.) attenuated ex vivo platelet aggregation elicited by ADP, U-46619 and collagen and increased bleeding time by 2.9 +/- 0.5-fold over control. Thus, in the dog, echistatin is an effective antithrombotic agent inhibiting both platelet aggregation in vivo in the coronary artery as well as ex vivo with a concomitant increase in bleeding time. Furthermore, the effects of echistatin on platelet aggregation and bleeding time are reversible with restoration to control levels occurring 30-60 min after termination of the infusion.

The effect of inhibition of factor XIIIa with 2-(l-acetonylthio)-5-methylthiazolo[2,3-b]1,3,4-thiadiazo lium perchlorate (L-722,151) on coronary thrombolysis and reocclusion was studied in an acute dog model of electrically induced coronary thrombosis. L-722,151 (0.1 mg/kg/min intravenously [IV] or placebo was administered 15 minutes before current initiation (150 microA) and for the duration of the experiment (270 minutes). Fifteen minutes after thrombus formation, heparin (300 U/kg, IV) was administered, followed 45 minutes later by recombinant tissue-type plasminogen activator (tPA) (10 micrograms/kg/min, IV for 90 minutes). Placebo-treated animals thrombosed at 48.9 +/- 8.1 minutes (mean +/- SEM) and reperfused in response to tPA at 49.1 +/- 9.3 minutes. L-722,151 pretreated animals thrombosed at 44.4 +/- 9.7 minutes and reperfused in response to tPA at 16.4 +/- 2.8 minutes (P less than .05 v vehicle). Furthermore, residual thrombus mass was reduced by L-722,151 from 6.9 +/- 1.9 mg in placebo-treated animals to 1.7 +/- 0.6 mg (P less than .05 v vehicle). Acute reocclusion occurred in 86% of placebo and in 75% of L-722,151-treated animals. The incidence of tPA-induced reperfusion in L-722,151-treated dogs was 100% (8 of 8), whereas only 70% (7 of 10) of placebo-treated dogs reperfused. These results demonstrate that pretreatment with L-722,151 hastens reperfusion time threefold and reduces residual thrombus mass. These effects occurred with no change in systemic blood pressure in response to L-722,151. When L-722,151 was administered 15 minutes after thrombus formation in a separate group of dogs (n = 5), no beneficial effect on thrombolysis time or thrombus mass was observed. Thus, the specific factor XIIIa catalyzed crosslinking reaction(s), which may determine(s) resistance to plasmin-mediated fibrin degradation, occur(s) rapidly. Inhibition of this crosslinking by pretreatment with L-722,151 promotes tPA-induced thrombolysis.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.