Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer.

BACKGROUND: Exposure of the plasma protein factor XII (FXII) to an anionic surface generates activated FXII that not only triggers the intrinsic pathway of blood coagulation through the activation of FXI but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of FXII are not associated with excessive bleeding, thrombosis models in factor-deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, FXII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage. OBJECTIVES: Using an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease-resistant RNA aptamer that binds specifically to FXII and directly inhibits FXII coagulant function. METHODS AND RESULTS: We describe the isolation and characterization of a high-affinity RNA aptamer targeting FXII/activated FXII (FXIIa) that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of FXII, as well as inhibiting intrinsic pathway activation (FXI activation). However, the aptamer does not affect the FXIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation). CONCLUSIONS: We have generated a specific and potent FXII/FXIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation.

Effects of factor XI deficiency on ferric chloride-induced vena cava thrombosis in mice.

BACKGROUND: Increased plasma levels of coagulation factor (F) XI are a risk factor for venous thrombosis. OBJECTIVE: To further explore the relationship between FXI and venous thrombosis, we evaluated FXI-deficient and wild-type mice in a ferric chloride (FeCl(3))-induced vena cava thrombosis model. METHODS AND RESULTS: Thrombosis was induced by 3-min topical application of filter papers containing increasing concentrations of FeCl(3) and the thrombus was measured at 30 min. In contrast to wild-type mice, FXI-deficient mice failed to form a thrombus with 5% FeCl(3,) and were partially protected against 7.5% and 10% FeCl(3,) respectively. The protective effect was substantially stronger than a high dose of heparin (1,000 units kg(-1), i.v.), clopidogrel (30 mg kg(-1), p.o.) or argatroban (30 mg kg(-1), i.p.). These antithrombotic agents resulted in off-scale bleeding in a tail bleeding time assay, whereas the bleeding time of FXI-deficient mice was unchanged compared to wild-type mice. In addition to its known effect on the coagulation cascade, enhanced clot lysis was demonstrated in FXI-deficient mouse and human plasma compared to those supplemented with FXIa. CONCLUSION: Given the strong antithrombotic efficacy (possibly contributed by strong anticoagulant activity associated with increased fibrinolytic activity) and mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable therapeutic strategy to treat or prevent venous thrombosis.

Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor.

BACKGROUND/OBJECTIVE: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. In the present study, we describe the development of a murine model of vena cava thrombosis and its use to characterize the antithrombotic activity of potato carboxypeptidase inhibitor (PCI) of TAFIa (activated TAFI) in mice. METHODS/RESULTS: Vena cava thrombosis was induced by various concentrations of FeCl(3) in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl(3)) induced thrombosis that was consistent and sensitive to reference antithrombotic agents such as clopidogrel and heparin. Dose-response studies identified a PCI dose (5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1), i.v.) that produced a maximum 45% decrease in vena cava thrombus mass as assessed by protein content (n = 8, P < 0.01 compared to vehicle) in the 3.5% FeCl(3)-induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl(3)-induced carotid artery thrombosis in mice. In a tail transection bleeding model, the 5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1) dose of PCI increased tail-bleeding time up to 3.5 times control (n = 8, P < 0.05). The ex vivo activity of antithrombotic doses of PCI was also demonstrated by the enhanced lysis of whole blood clots formed in a thrombelastograph with the addition of a sub-threshold concentration of tPA. CONCLUSION: These studies provide evidence for a role of TAFIa in venous thrombosis in mice, and describe an optimized vena cava injury model appropriate for the evaluation of antithrombotic drugs and the characterization of novel therapeutic targets.

Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice.

Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.

Characterization of thromboxane A2/prostaglandin endoperoxide receptors in aorta.

Thromboxane A2/prostaglandin endoperoxide receptor antagonists were studied in rat and guinea-pig aortas contracted with U-46619 (9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F2 alpha) or 8-epi-prostaglandin F2 alpha. In rat aorta, the antagonists competitively inhibited contractions evoked by either agonist with a rank order of potency as follows: BMS-180291 ([1s-(exo,exo)]-2-[[3-[4-[(pentylamino) carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benz enepropanoic acid) > or = SQ 29,548 ([1s-[1 alpha,2 beta-(5z), 3 beta,4 alpha)]-7-[3-[[2-[(phenylamino)carbonyl]hydrozino] methyl]-7-oxobicyclo-[2.2.1]hept-2-yl]-5-heptanoic acid) > daltroban (4-[2-(4-chlorobenzenesulfonylamino) methyl]-benzene acetic acid) > or = SQ 30,741 ([1s-[1 beta,2 alpha(5z),3 alpha,4 beta]]-7-[3-[[[[(oxa)amino]acetyl] amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl-5-heptanoic acid) = AA-2414 (2,4,5-trimethyl-3,6-dioxo-zeta-phenyl-1,4-cyclohexadien-1-heptano ic acid). In guinea-pig aorta, the antagonists competitively antagonized contractions elicited by either agonist with the following rank order of potency: SQ 29,548 = AA-2414 > or = SQ 30,741 > daltroban. Antagonism by BMS-180291 in guinea-pig aorta was not strictly competitive. These findings indicate that thromboxane A2/prostaglandin endoperoxide receptors in rat aortas are different from those in guinea pigs. Because the actions of both agonists were equivalently antagonized by each of the antagonists in both rat and guinea-pig aortas, the results do not support the hypothesis that U-46619 and 8-epi-prostaglandin F2 alpha elicit contractions via different receptor subtypes in the aorta.

Myocardial calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts.

OBJECTIVES: To study calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts by measuring the hydrolysis of the endogenous choline phospholipids. METHODS: Langendorff perfused rabbit hearts were exposed to global ischemia for 15 or 60 min, or control perfusion for the same length of time. The hearts were then rapidly frozen in liquid nitrogen and lyophilized. Calcium-independent phospholipase A2 activity in the lyophilized tissue was studied by measuring accumulation of lysophospholipids resulting from hydrolysis of both the choline diacylphospholipid and the choline plasmalogen pool. RESULTS: The calcium-independent phospholipase A2 activity showed the same pH, temperature and calcium sensitivity in control and ischemic (15 min of ischemia) lyophilized myocardial tissue. Incubation of control and ischemic tissue showed no difference in the rate of accumulation of lysophospholipids when the ischemic tissue was obtained from hearts exposed to 15 min of ischemia (107 +/- 4 vs 111 +/- 7 nmol/g dry wt x min, ischemia versus control, mean +/- s.e.m., n = 8), but a significant decrease was noticed in tissue from hearts that had been exposed to 60 min of ischemia (31 +/- 9 vs 86 +/- 18 nmol/g dry wt x min, P < 0.05, n = 4). The decreased phospholipase A2 activity in tissue exposed to 60 min of ischemia was not due to enhanced metabolism of the lysophospholipids (84 +/- 15 vs 79 +/- 8 nmol/g dry wt x min, n = 4). The calcium-independent phospholipase A2 activity was considerably lower in fresh myocardial tissue compared with lyophilized tissue, but comparison of control and ischemic fresh tissue gave results comparable to those found using lyophilized tissue. The myocardial calcium-independent phospholipase A2 activity showed no plasmalogen selectivity in either control or ischemic myocardium. CONCLUSIONS: In isolated perfused rabbit hearts we found no evidence for activation of calcium-independent phospholipase A2 activity during global ischemia. With prolonged time of ischemia there was a significant decrease in calcium-independent phospholipase A2 activity.

Dissociation of endothelial cell dysfunction and blood pressure in SHR.

This study was designed to examine the impairment of endothelium-dependent relaxation in spontaneously hypertensive rats (SHR), to determine whether endothelial cell function is normalized by in vivo treatment with a thromboxane A2-prostaglandin endoperoxide (TP)-receptor blocker, and to establish whether endothelial dysfunction contributes to the elevated blood pressure. In isolated aortic rings from SHR, endothelium-dependent relaxations caused by acetylcholine, adenosine diphosphate, and alpha-thrombin were markedly impaired compared with those from Wistar-Kyoto (WKY) normotensive rats. Arachidonic acid-induced contractions were significantly enhanced in aorta from SHR. In contrast, relaxations caused by direct smooth muscle vasodilators, nitroprusside and cromakalim, and contractions caused by U-46619 were not different between SHR and WKY rats. Treatment of SHR with the oral TP-receptor antagonist, ifetroban, at 20 and 50 mg.kg-1.day-1 fully restored endothelium-dependent relaxation toward normal. However, ifetroban produced no effect on blood pressure in SHR. In vitro incubation of aortic rings from SHR with ifetroban also normalized relaxations to acetylcholine but had no effect in aorta from WKY. In contrast, the thromboxane A synthase inhibitor, dazoxiben, only partially improved abnormal acetylcholine-induced relaxations in aorta from SHR. The results demonstrate that endothelial cell dysfunction in hypertension can be restored to normal by selective TP-receptor blockade. Furthermore, endothelial cell dysfunction and TP-receptor activation may not significantly contribute to elevated systemic blood pressure in SHR.

Protective effect of the serotonin receptor antagonist cinanserin in two canine models of pacing-induced myocardial ischemia.

Previous studies have indicated that structurally dissimilar serotonin2 (5HT2) antagonists can protect globally ischemic/reperfused reperfused rat hearts. We therefore determined if the 5HT2 antagonist cinanserin can protect the ischemic myocardium in two models of pacing-induced ischemia in anesthetized dogs. In one model, dogs were subjected to repeated 5-min episodes of pacing superimposed upon left anterior descending coronary artery stenosis such that an ST segment elevation of 10-11 mV was obtained and upon cessation of pacing, a return of ST segment elevation to baseline. In vehicle-treated animals, this ST segment elevation to baseline. In vehicle-treated animals, this ST segment elevation was constant for all pacing-induced ischemic episodes. After infusion of 20 micrograms/kg/min cinanserin (intracoronary, i.c.), ST segment elevation was significantly reduced during pacing-induced ischemia at all times measured postdrug. In the second model, animals were subjected to pacing-induced ischemia for 15 min such that segmental shortening was reduced to zero. Upon cessation of pacing-induced ischemia, recovery of shortening was assessed. In vehicle-treated dogs, function recovered to only 40% of baseline at 3 h into recovery. Pretreatment with 5 micrograms/kg/min cinanserin i.c. significantly improved recovery of postischemic function. In both protocols, cinanserin had no effect on ischemic regional blood flow or peripheral hemodynamics. Thus, cinanserin appears to exert profound protective effects in two models of effort-induced ischemia.

Dose-related cardioprotection by ifetroban in relation to inhibition of thrombosis and ex vivo platelet function.

The dose-related cardioprotective efficacy of the thromboxane A2/prostaglandin endoperoxide (TP) receptor antagonist, ifetroban (BMS-180291), was investigated in an anesthetized ferret model of myocardial ischemia (90 min) followed by reperfusion (5 h). Treatment was begun at either the 75th minute of ischemia or 5 min after initiating reperfusion. The magnitude of TP receptor blockade was evaluated by ex vivo platelet function. Additional experiments in ferrets tested the antithrombotic potency of ifetroban as an inhibitor of thrombotic cyclic flow reduction (CFR) in the stenosed abdominal aorta (Folts model). Continuous ifetroban infusions of 0.03, 0.1 and 0.3 mg/kg/h reduced myocardial infarct size from 22% of the left ventricle in vehicle-control ferrets to 20, 12 and 9%, respectively. These represented reductions in infarct size of 8, 43 and 56%. Delaying initiation of treatment with high-dose ifetroban until 5 min into reperfusion also significantly reduced infarct size by 45%. High-dose ifetroban treatment did not prevent neutrophil (PMNL) accumulation measured as tissue myeloperoxidase activity in infarcted tissue. At the end of the 5-hour reperfusion period, the low, medium and high doses produced 90, 98 and 98% blockade of platelet TP receptors, respectively, measured as inhibition of ex vivo platelet shape change responses to U-46,619. Ifetroban inhibited thrombotic CFR at a threshold dose of 0.03 +/- 0.004 mg/kg, which antagonized 92% of ferret platelet TP receptors. Thus, ifetroban exhibited cardioprotective and antithrombotic activities and was effective at doses producing > 90% TP receptor blockade. Cardioprotective activity was not associated with any reductions of PMNL accumulation in infarcted tissue and was demonstrable even when treatment was delayed until 5 min after initiation of reperfusion.

Myocardial salvage efficacy of the thromboxane receptor antagonist ifetroban in ferrets and dogs.

The effects of the thromboxane A2 (TXA2)/prostaglandin endoperoxide (TP) receptor antagonist ifetroban (BMS-180291) on infarct size (IS) resulting from coronary occlusion/reperfusion was determined in anesthetized dogs and ferrets. In dogs, ifetroban (1 + 1 mg/kg/h, intravenously, i.v.) or vehicle administration was initiated 10 min before left circumflex coronary artery (LCX) occlusion. In ferrets, the left anterior descending coronary artery (LAD) was occluded; after 75 min, a continuous infusion of ifetroban (0.3 + 0.3 mg/kg/h i.v.) or vehicle was started. After 90-min ischemia in both species, the LCX or LAD occlusion was released; reperfusion was continued for 5 h, at which time IS was determined. Regional myocardial blood flow (RMBF) before and during occlusion and during reperfusion were measured with radioactive microspheres. Ifetroban significantly decreased the extent of infarction to 39 +/- 5% of the area at risk (AAR) from 64 +/- 5% in dogs and to 15 +/- 2% of the left ventricle as compared with a control of 22 +/- 2% in ferrets. The protective effect of ifetroban in both species occurred with no increase in collateral BF or treatment-related alterations in peripheral hemodynamic status. Ifetroban, at the doses that reduced IS in ferrets, inhibited 99% of platelet TP receptors throughout the experiment, as measured by inhibition of the ex vivo platelet shape change response to U-46,619, a TP receptor agonist. Thus, doses of ifetroban causing profound TP receptor blockade also salvaged jeopardized myocardium in dogs and ferrets without changing collateral BF or peripheral hemodynamics.

Failure of aspirin to interfere with the cardioprotective effects of ifetroban.

The thromboxane receptor antagonist ifetroban ([1S-(1 alpha,2 alpha,3 alpha, 4 alpha)]-2-[[3-[4-[(pentylamino)carbonyl]-2-oxazolyl]- 7-oxabicyclo[2.2.1]hept-2-yl]methyl]benzenepropanoic acid) and aspirin were evaluated for direct and combined effects on myocardial infarct size in anesthetized ferrets subjected to coronary artery occlusion (90 min) and reperfusion (5 h). Aspirin (10 mg/kg) or vehicle was administered as an i.v. bolus dose at the 45th min of occlusion in an initial assessment of its cardioprotective potential in this species. In interaction studies, aspirin was injected i.v. 10 min prior to occlusion (10 mg/kg) and at the 45th min of ischemia (5 mg/kg) both with and without subsequent administration of ifetroban (0.3 mg/kg + 0.3 mg/kg per h) beginning at the 75th min of occlusion. Aspirin administration alone caused non-significant (P > 0.05) 5-7% reductions in tissue damage (19.8-21.8% of left ventricle) from that observed in vehicle-controls (20.4-22.9% of left ventricle). Ifetroban alone significantly (P < 0.05) reduced infarct size compared to vehicle treatment (13 +/- 1% vs. 23 +/- 2% of left ventricle), and this was not prevented by combination with aspirin (12 +/- 2% vs. 22 +/- 3% of left ventricle). In the absence and presence of aspirin, ifetroban reduced infarct size by 42% and 43%, respectively. Concurrently, thromboxane A2-generating capacity in blood (measured as thromboxane B2 in clotted serum) was decreased ca. 99% by aspirin treatment. Thus, virtually complete platelet cyclooxygenase inhibition by aspirin afforded no cardioprotective action in the ferret and, more importantly, this inhibition did not interfere with the myocardial salvage efficacy of ifetroban.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboxane receptor antagonist BMS-180291, but not aspirin, reduces the severity of pacing-induced ischemia in dogs.

We determined the effect of thromboxane A2 (TXA2) prostaglandin endoperoxide (TP) receptor antagonism, using BMS-180291 or aspirin, on the severity of pacing-induced ischemia in anesthetized dogs. Thromboxane receptor antagonists may not only have antithrombotic activity, but may also have direct cardioprotective effects, unlike aspirin. Left anterior descending coronary artery (LAD) stenosis was adjusted so that a significant (10-12 mV) ST segment elevation was observed only when superimposed on atrial pacing. Each heart was subjected to 5-min episodes of pacing-induced ischemia 10, 40, and 70 min after initiation of BMS-180291 (1 mg/kg + 1 mg/kg/h) or vehicle. In the vehicle group, ST segment elevation was reproducible at all pacing-induced ischemia episodes, whereas BMS-180291 significantly reduced it by 30% at the later ischemia episodes. This reduction in ST segment increase was not accompanied by alterations in regional myocardial blood flow (RMBF) nor in hemodynamic status. Aspirin in the same model [10 mg/kg intravenously (i.v.) given 10 min before pacing-induced ischemia] did not significantly reduce ST segment elevation, indicating a lack of protective effect in this model. Thromboxane receptor blockade appears to protect myocardium subjected to pacing-induced ischemia, an effect not produced by aspirin.

Characterization of rabbit myocardial phospholipase A2 activity using endogenous phospholipid substrates.

We have developed an assay for studying myocardial phospholipase A2 activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This assay was used to characterize phospholipase A2 activity in rabbit myocardium. Lyophilized rabbit myocardium was incubated at 37 degrees C in Tris-HCl buffer containing either ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl-lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-lysoplasmenylcholine were measured using a recently developed HPLC method. The identity of the individual species was confirmed by ion-spray LC-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min caused a linear increase in all lysophospholipids. The main increases were found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 +/- 3 (mean +/- SE, N = 8) and 48 +/- 3 nmol/g dry wt x min, respectively. The apparent phospholipase A2 activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0, and incubation at room temperature and 45 degrees C decreased the activity by 80 and 40%, respectively. Studies of the metabolism of the formed lysophospholipids showed a substantial metabolism of the lysophospholipids that accounted for about 40% of the total phospholipase A2 activity. This method offers a novel approach to study phospholipase A2 activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools.

Comparison of thrombin active site and exosite inhibitors and heparin in experimental models of arterial and venous thrombosis and bleeding.

Different pharmacological approaches to thrombin inhibition were compared for their effects on thrombosis and bleeding time in anesthetized rats. Thrombosis was induced in the carotid artery by transmural vessel injury and in the vena cava by partial blood flow stasis combined with mild endothelial disruption. Small mesenteric arteries were punctured with a hypodermic needle to measure the bleeding time. Dose-response relationships were determined with a thrombin active site inhibitor, N-methyl (GYKI 14,766); a thrombin exosite inhibitor, succinyl-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-Gln (BMS 180,742); and heparin. BMS 180,742 interferes with fibrinogen binding to the thrombin exosite but, unlike GYKI 14,766, it does not block thrombin's catalytic site. The effects on thrombosis and bleeding time were correlated with ex vivo clotting times using the activated partial thromboplastin time for heparin and the thrombin time for GYKI 14,766 and BMS 180,742. Venous thrombosis was inhibited more than 90% by all three inhibitors at doses that either produced threshold increases or had no effect on bleeding and clotting times. Arterial thrombosis was inhibited 82% by GYKI 14,766 and 63% by heparin but it was not inhibited by BMS 180,742. These antithrombotic activities were accompanied by a maximal activated partial thromboplastin time increase and doubling of the bleeding time with heparin and a maximal thrombin time prolongation and 35% increase in bleeding time with GYKI 14,766. These results suggest that thrombin inhibitors, which act at the active site or exosite or through antithrombin III, are equally efficacious against venous thrombosis but active site inhibitors are the most effective against arterial thrombosis.

Superior activity of a thromboxane receptor antagonist as compared with aspirin in rat models of arterial and venous thrombosis.

We determined the effects of aspirin and a novel thromboxane A2/prostaglandin endoperoxide (TP)-receptor antagonist, BMS-180291, on thrombosis and bleeding times in skin and mesenteric arteries. In anesthetized rats, occlusive thrombosis was induced in the carotid artery by topical application of ferrous chloride and in the vena cava by blood flow stasis combined with either infusion of thromboplastin or hypotonic saline. Aspirin (1, 10, and 50 mg/kg) did not reduce arterial or venous thrombus weight significantly. BMS 180,291 (150 micrograms/kg/min) decreased arterial thrombus weight and hypotonic saline-induced caval thrombus weight by 58 and 57%, respectively. BMS-180291 lacked antithrombotic activity at a lower dose (50 micrograms/kg/min) and failed to inhibit thromboplastin-induced caval thrombosis. BMS-180291 (150 micrograms/kg/min) significantly reduced arterial thrombus weight by 40% when plasma epinephrine concentration was increased to 5 ng/ml. BMS-180291 and aspirin produced increases of only < or = 30% in bleeding times. These results demonstrate that BMS-180291 has antithrombotic activity in experimental aspirin-resistant arterial and venous thrombosis. Both aspirin and BMS-180291 have only modest effects on small artery hemostasis in rats.

Myocardial salvage efficacy of a thromboxane receptor antagonist, SQ, 30,741, in relation to inhibition ex vivo of platelet function in the ferret.

The myocardial salvage efficacy of a thromboxane A2/prostaglandin endoperoxide (TP) receptor antagonist has not been previously determined in a ferret model of ischemia and reperfusion. Assessments of the reproducibility of infarct size resulting from a 90 min period of occlusion followed by 5 hr of reperfusion of the left anterior descending coronary artery in saline-treated control ferrets revealed a consistent mean level of tissue damage representing 23.1 +/- 1.4% of the left ventricle. In subsequent studies, ferrets were given the thromboxane receptor antagonist SQ 30,741 (1 mg/kg bolus and 1 mg/kg/hr infusion, i.v.) or vehicle. At this dose, SQ 30,741 significantly reduced infarct size from that measured in control ferrets by 44%. Concurrently, the drug produced a 97% inhibition of platelet TP receptors as measured by inhibition of the ex vivo platelet shape change response to U-46,619. Drug administration was not associated with measurable alterations in mean blood pressure, heart rate or the rate-pressure-product. The importance of this finding to clinical utility and the mechanism of the observed cardioprotective action, however, remain unclear. These data indicate that the ferret represents a useful model for the assessment of the myocardial salvage efficacy of TP receptor antagonists and are consistent with attenuation of ischemic myocardial damage by doses of these agents which produce > 96% TP receptor blockade.

Effects of endogenous and exogenous lysophosphatidylcholine in isolated perfused rat hearts.

In isolated Langendorff perfused rat hearts, treatment with exogenous palmitoyl-lysophosphatidylcholine (P-LPC; 3-50 microM) under normoxic conditions, resulted in reduced heart rate (HR), coronary flow (CF) and contractile function. After 30 min Krebs perfusion, following P-LPC infusion, HR and CF remained reduced and contractile function continued to deteriorate. End diastolic pressure (EDP) and lactate dehydrogenase (LDH) release in LPC treated hearts were significantly increased from controls. Myocardial lysophosphatidylcholine (LPC) levels after 25 min global ischemia were significantly higher than controls (463 +/- 10 for control vs 550 +/- 15 nmol/g dry wt for ischemia). Following 30 min reperfusion an increase from control was still observed (475 +/- 11 for control vs. 594 +/- 17 for ischemia+reperfusion). Analysis of molecular species of LPC demonstrated that palmitoyl, oleoyl and stearoyl were increased after 25 min ischemia. After 30 min of reperfusion only palmitoyl and stearoyl were significantly increased. After 25 min treatment with 3 microM P-LPC and 30 min normoxic perfusion, myocardial LPC was three-fold higher than after 25 min ischemia. Treatment with 0.2 microM exogenous P-LPC resulted in myocardial tissue LPC levels (562 +/- 23) equivalent to those seen after 25 min ischemia (550 +/- 15). Compared to time matched controls hearts perfused with 0.2 microM P-LPC displayed no significant reductions in contractile function nor increase in LDH release. Thus, in isolated rat hearts, the increase in LPC seen after 25 min of global ischemia may not solely mediate the contractile dysfunction and LDH release observed.

Isolation and identification of lysophosphatidylcholines as endogenous modulators of thromboxane receptors.

Inhibition of thromboxane receptor radioligand binding to human platelet membranes has been employed as the basis for a radioreceptor assay designed to measure thromboxane receptor binding activity in samples of biological fluids. This method was used during phase 1 clinical evaluation of the thromboxane receptor antagonist SQ 30,741. Frequently, baseline plasma samples as well as plasma samples from placebo-treated subjects showed significant inhibition of radioligand binding in the radioreceptor assay, suggesting the presence of endogenous thromboxane receptor ligands. This receptor binding activity was stable and could be monitored in blood from normal volunteers using a modification of the radioreceptor assay. In order to identify the substance responsible for the observed activity, the activity present in pooled bovine blood was isolated and evaluated by a combination of FAB/MS, 1H-NMR, 13C-NMR and co-injection with reference standards on HPLC. Several endogenous thromboxane receptor ligands were identified as L-alpha-lysophosphatidylcholine (LPC) species. One major species, palmitoyl-LPC, contracted isolated rat aortic spirals, and these contractions could be delayed or prevented, but not reversed by the thromboxane receptor antagonist SQ 29,548. Palmitoyl-LPC slightly potentiated aortic contractions induced by the thromboxane receptor agonist, U-46,619, and diminished in a concentration-dependent manner the antagonism by SQ 29,548 of contractile responses to U-46,619. These findings are consistent with a potential for LPC species to bind and activate thromboxane receptors.

Pharmacological profile of BMS 180,291: a potent, long-acting, orally active thromboxane A2/prostaglandin endoperoxide receptor antagonist.

180,291 1S-(1 alpha, 2 alpha, 3 alpha, 4 alpha)-2-[[3-[4- [(pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept- 2- yl]methyl]benzenepropanoic acid (BMS) is a potent and highly selective antagonist of thromboxane A2/prostaglandin endoperoxide (TP) receptors. In human platelet-rich plasma, BMS 180,291 inhibited platelet aggregation induced by arachidonate (800 microM) and U-46,619 (10 microM) with respective IC50 values of 7 +/- 1 (S.E.M.) and 21 +/- 2 nM. Inhibition of both the rate and full extent of 11,9-epoxymethano-prostaglandin H2 (U-46,619)-induced platelet aggregation were insurmountable at antagonist concentrations > 10 nM, but BMS 180,291 antagonized U-46,619-induced platelet shape change competitively with a KB of 11 +/- 2 nM. BMS 180,291 concentrations < or = 1 mM did not inhibit platelet aggregation induced by high concentrations of ADP (20 microM) or human alpha-thrombin (1 U/ml). BMS 180,291 inhibited binding of [3H]1S-[1 alpha,2 alpha(5Z),3 alpha,4 alpha]-7-[3-[[2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]-hept-2- yl]-5-heptenoic acid to human platelet membranes with a kd of 4.0 +/- 1.0 nM and slope factor of 1.06 +/- 0.13. U-46,619-induced concentrations of rat aortae were competitively antagonized by BMS 180,291 with a KB of 0.6 +/- 0.1 nM. Aortic responses to norepinephrine, serotonin and angiotensin II were not inhibited by BMS 180,291 at 1 microM. U-46,619-induced contractions of guinea pig tracheal rings were antagonized in an almost all-or-none manner, with maximal blockade at > or = 1 nM BMS 180,291, but little effect at lower concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiplatelet activity of the long-acting thromboxane receptor antagonist BMS 180,291 in monkeys.

The effects of the novel TxA2/prostaglandin endoperoxide (TP) receptor antagonist BMS 180,291 on platelet reactivity was determined ex vivo in conscious African green monkeys. Platelet aggregation responses to U-46,619 were decreased 50% and 100% at 23 to 24 hrs after BMS 180,291 oral doses of 1 and 3 mg/kg, respectively. In addition to inhibiting aggregation, a 3 mg/kg oral dose of BMS 180,291 also produced an 11 +/- 3-fold shift to the right in the U-46,619 concentration-response relationship for platelet shape change at 24 hrs after dosing. When the 3 mg/kg oral dose was continued for 11 days, the shift in this concentration-response relationship increased to 26 +/- 10- and 93 +/- 30-fold at 24 hrs after the 8th and 11th doses, respectively. This progressive inhibition corresponds to 93 +/- 3 and 99 +/- 1% blockade of platelet TP-receptors responsible for shape change, respectively. Comparable levels of TP-receptor blockade have been previously correlated with antithrombotic and antiischemic activities of TP-receptor antagonists in vivo. Platelet reactivity to U-46,619 had completely recovered on the 7th day after the final dose of BMS 180,291, indicating effective elimination from the circulation over this interval. In separate experiments, a 3-mg/kg i.v. dose of BMS 180,291 produced only marginal and transient hemodynamic effects in anesthetized African green monkeys. Overall, these data demonstrate that BMS 180,291 given orally once a day produces a sustained and therapeutically-relevant level of TP-receptor antagonism.