Effects of selective cyclooxygenase-2 inhibition on vascular responses and thrombosis in canine coronary arteries.

BACKGROUND: Prostanoid synthesis via the action of cyclooxygenase-2 (COX-2) is a component of the inflammatory response. Prostacyclin, a product of COX-2 in vascular endothelium, has important physiological roles, such as increasing blood flow to injured tissues, reducing leukocyte adherence, and inhibiting platelet aggregation. We examined the possibility that selective COX-2 inhibition could suppress the protective effects of prostacyclin, resulting in an alteration of the hemostatic balance and vascular tone. METHODS AND RESULTS: Circumflex coronary artery thrombosis was induced in dogs by vascular electrolytic injury. Orally administered celecoxib (COX-2 inhibition) or high-dose aspirin (HDA) (COX-1 and COX-2 inhibition) did not alter time to occlusive thrombus formation compared with controls (celecoxib 77.7+/-7.2 minutes, HDA 72.0+/-18.5 minutes, control 93.0+/-21.8 minutes). Oral HDA with an endothelial recovery period (HDA-ER) (COX-1 inhibition) produced a significant increase in time to vessel occlusion (257.0+/-41.6 minutes). The observed increase in time to occlusion was abolished when celecoxib was administered to animals dosed with HDA-ER (80.7+/-20.6 minutes). The vasomotor effect of endothelium-derived prostacyclin was examined by monitoring coronary flow during intracoronary administration of arachidonic acid or acetylcholine. In celecoxib-treated animals, vasodilation in response to arachidonic acid was reduced significantly compared with controls. CONCLUSIONS: The results indicate important physiological roles for COX-2-derived prostacyclin and raise concerns regarding an increased risk of acute vascular events in patients receiving COX-2 inhibitors. The risk may be increased in individuals with underlying inflammatory disorders, including coronary artery disease.

Reduction of myocardial infarct size after ischemia and reperfusion by the glycosaminoglycan pentosan polysulfate.

Activation of the complement system contributes to the tissue destruction associated with myocardial ischemia/reperfusion. Pentosan polysulfate (PPS), a negatively charged sulfated glycosaminoglycan (GAG) and an effective inhibitor of complement activation, was studied for its potential to decrease infarct size in an experimental model of myocardial ischemia/reperfusion injury. Open-chest rabbits were subjected to 30-min occlusion of the left coronary artery followed by 5 h of reperfusion. Vehicle (saline) or PPS (30 mg/kg/h) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Treatment with PPS significantly (p < 0.05) reduced infarct size as compared with vehicle-treated animals (27.5+/-2.9% vs. 13.34+/-2.6%). Analysis of tissue demonstrated decreased deposition of membrane-attack complex and neutrophil accumulation in the area at risk. The results indicate that, like heparin and related GAGs, PPS possesses the ability to decrease infarct size after an acute period of myocardial ischemia and reperfusion. The observations are consistent with the suggestion that PPS may mediate its cytoprotective effect through modulation of the complement cascade.

Correlation between the in vivo efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) and ex vivo platelet inhibition.

In vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) was studied with respect to their ex vivo platelet inhibition in heparinized platelet-rich plasma (hPRP) and citrated platelet-rich plasma (cPRP) using a canine model of carotid artery thrombosis. For each drug group (n = 6), the right carotid artery was used as control vessel and resulting occlusive thrombus was kept in situ to examine the direct thrombolytic efficacy of the antagonists. Thirty minutes after occlusion of control vessel, a low or high dose of each antagonist was administered and the left carotid artery was used as test vessel. All control vessels occluded within 86-96 min in response to electrolytic injury. The incidence of occlusion with lower doses of m7E3, DMP-728, and MK-383 was 100, 33 and 100%, respectively; corresponding times to occlusion were 174, 220 and 118 min. Lower doses inhibited ADP- or AA-induced platelet aggregation in cPRP (>80%). Incidence of occlusion with high doses of m7E3, DMP-728 and MK-383 was 33, 0 and 0%, respectively; corresponding times to occlusion were 209, >240 and >240 min. Higher doses inhibited aggregation in cPRP (>80%), but only partially in hPRP (45-66%). Dose-dependent prolongation of bleeding time occurred with all antagonists. None of the antagonists lyzed preformed thrombi in control vessels. The results indicate that ex vivo platelet aggregation conducted in hPRP, as opposed to conventional cPRP, provides a better assessment of the in vivo efficacy of GPIIb/IIIa receptor antagonists.

TP-9201, a glycoprotein IIb/IIIa platelet receptor antagonist, prevents rethrombosis after successful arterial thrombolysis in the dog.

BACKGROUND AND PURPOSE: We examined the ability of TP-9201, a platelet glycoprotein IIb/IIIa receptor antagonist, to prevent carotid artery rethrombosis in the anesthetized dog. METHODS: Occlusive thrombosis was induced by electrolytic injury of the left carotid artery. Thirty minutes later, 0.05 U/kg of anisoylated plasminogen streptokinase activator complex (APSAC) was infused locally to achieve clot lysis. Carotid artery recanalization was followed immediately by the infusion of either saline (10 mL/h, 240 minutes; n = 9), low-dose TP-9201 (120 micrograms/kg plus 3 micrograms.kg-1.min-1, 240 minutes; n = 7), or high-dose TP-9201 (185 micrograms/kg plus 5 micrograms.kg-1.min-1, 240 minutes; n = 7). Ex vivo platelet aggregation responses to ADP or arachidonic acid were determined. RESULTS: TP-9201 produced complete inhibition of platelet aggregation in citrated platelet-rich plasma but a partial and dose-dependent inhibition in heparinized platelet-rich plasma. A twofold and eightfold increase in the template bleeding time was associated with the infusion of low-dose and high-dose TP-9201, respectively. There were frequent cyclic flow reductions in both the saline and low-dose TP-9201-treated groups after thrombolysis. However, the high-dose TP-9201-treated group exhibited a sustained flow with minimal evidence of cyclic flow reductions. At the conclusion of the protocol, patent vessels were found more frequently in the high-dose TP-9201 (5/7; P = .0048) than in the low-dose TP-9201 treatment group (2/7; P = .17) when compared with the saline group (0/9). Infusion of high-dose TP-9201 was associated with a significant reduction in the thrombus mass as compared with the control vessels. CONCLUSIONS: Administration of TP-9201 in conjunction with successful thrombolysis inhibited ex vivo platelet aggregation and prevented rethrombosis of the canine carotid artery. This study demonstrates that TP-9201, an inhibitor of the platelet GPIIb/IIIa receptor, can inhibit platelet-vessel wall interaction and thus prevent rethrombosis.

Antithrombotic effect of GYKI-14766 in a canine model of arterial and venous rethrombosis: a comparison with heparin.

We compared the antithrombotic effects of the thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766) with those of heparin in a canine model of arterial and venous rethrombosis. Thrombogenesis was induced by electrolytic injury to the endothelial surface of the carotid artery and jugular vein. Either heparin (300 U/kg, n = 7), GYKI-14766 (0.5 mg/kg/h, n = 7), or saline (n = 10) was administered intravenously (i.v.) immediately after the local administration of anisoylated plasminogen streptokinase activator complex (APSAC 0.1 U/kg). Supplemental doses of heparin (100 U/kg) were administered at 1-h intervals. Infusion of GYKI-14766 was maintained for 5 h throughout the experiment. Ex vivo platelet aggregation in response to ADP or arachidonic acid (AA) was not changed in any of the experimental groups. Both GYKI-14766 and heparin increased the activated partial thromboplastin time (aPTT) over their respective baseline values. Heparin, but not GYKI-14766, increased the bleeding time. After successful thrombolysis, arterial and venous rethrombosis occurred in all saline-treated dogs. GYKI-14766 prevented cyclic flow variations and reocclusion in the artery and the vein (p < 0.01). Heparin had only minimal effects on the artery and no effect on the vein. Arterial thrombus weights were reduced by GYKI-14766 [saline control = 24 +/- 4 mg, GYKI-14766 = 9 +/- 3 mg, (p < 0.05); heparin = 14 +/- 2 mg, p = NS]. The venous thrombus weights were reduced slightly by GYKI-14766 and were unchanged by heparin (saline = 25 +/- 5 mg, GYKI-14766 = 13 +/- 4 mg, heparin = 26 +/- 3 mg). The data suggest that GYKI-14766 is effective in preventing occlusive rethrombosis in both the arterial and venous circulation after thrombolysis without augmenting bleeding time. GYKI-14766 may represent an alternative to heparin as an adjunctive agent during thrombolytic therapy.

Thromboxane antagonism in experimental canine carotid artery thrombosis.

BACKGROUND AND PURPOSE: The two objectives of this study were to assess the potential of BAY U 3405 to prevent arterial thrombosis in response to vessel wall injury and to determine the ability of BAY U 3405 to prevent thrombotic reocclusion after thrombolysis with anisoylated plasminogen streptokinase activator complex. METHODS: Dogs were instrumented with a carotid flow probe, stimulating electrode, and a stenosis. Current (150 microA) was applied to the intimal surface of the right carotid artery, and time to occlusive thrombus formation was noted. BAY U 3405 was administered, and the procedure for thrombus formation was repeated for the left carotid artery. RESULTS: BAY U 3405 administration prevented occlusive arterial thrombosis formation. Ex vivo platelet aggregation was inhibited, bleeding time increased, and thrombus weight reduced after BAY U 3405 treatment. In a second group, thrombi were formed initially in both carotid arteries, BAY U 3405 was administered as before, and anisoylated plasminogen streptokinase activator complex was infused in the right carotid artery proximal to the occlusive thrombus. BAY U 3405 did not alter the incidence of rethrombosis compared with the lytic agent alone. CONCLUSIONS: BAY U 3405 prevented primary arterial thrombosis, corresponding to inhibition of platelet aggregation, and increased bleeding times. BAY U 3405, however, did not prevent rethrombosis after successful thrombolysis with anisoylated plasminogen streptokinase activator complex, despite the fact that platelet reactivity was inhibited. The data are consistent with the concept that the residual thrombus represents a more effective thrombogenic stimulus as compared with arterial wall injury alone and that the mechanisms associated with primary versus secondary thrombus formation may require separate therapeutic approaches.

High dose intravenous aspirin, not low dose intravenous or oral aspirin, inhibits thrombus formation and stabilizes blood flow in experimental coronary vascular injury.

OBJECTIVES: The purpose of this study was to assess the anti-thrombotic potential of various forms of aspirin administration. BACKGROUND: Platelet activation in response to endothelial injury has been implicated in acute coronary syndromes. METHODS: Delivering 100-microA anodal direct current to the intima of the left circumflex coronary artery in dogs at a site of moderate external stenosis provides a thrombogenic model of vascular injury. Animals were treated with aspirin (Group I, 20 mg/kg intravenously [n = 11]; Group II, 4.6 mg/kg intravenously [n = 6]; Group III, 4.6 mg/kg orally 18 h before the experiment [n = 7]) or vehicle (Group IV, control [n = 11]). RESULTS: The time required for thrombotic occlusion to occur was longer and the incidence of thrombosis was lower in Group I (Group I, 238 +/- 7 min [n = 2]; Group II, 127 +/- 25 min [n = 3]; Group III, 156 +/- 35 min [n = 6]; Group IV, 90 +/- 11 min [n = 11]) (p < 0.05). Thrombus mass was smaller in Group I (Group I, 5.0 +/- 0.8 mg; Group II, 12.2 +/- 2.6 mg; Group III, 11.6 +/- 3.9 mg; Group IV, 9.1 +/- 1.6 mg) (p < 0.05). Initial hemodynamic variables did not differ among groups. An increase in mean arterial pressure was noted for several hours after intravenous aspirin administration in Group I (99 +/- 5 to 110 +/- 4 mm Hg) (p < 0.05). Left circumflex coronary artery blood flow was stable for 5 h in Group I (Group I, 31 +/- 2 to 26 +/- 4 ml/min) but decreased in all the other groups (Group II, 26 +/- 4 to 10 +/- 5 ml/min; Group III, 27 +/- 5 to 7 +/- 7 ml/min; Group IV, 29 +/- 4 to 0 ml/min) (p < or = 0.05). The in vivo area of left ventricle perfused by the left circumflex coronary artery was not different among groups. Platelet counts were similar and did not change over the course of the protocol. Ex vivo arachidonic acid-induced platelet aggregation decreased in all groups after aspirin (p < or = 0.001). Indium-111-labeled platelet adherence to the coronary vasculature was decreased in distal vessel segments after all doses of aspirin (p < 0.05). Platelet deposition in thrombi was similar for all treatment groups. CONCLUSIONS: High dose intravenous aspirin has salutary effects. It stabilizes left circumflex coronary artery blood flow, prolongs the time to thrombosis, reduces the incidence of thrombotic occlusion, reduces thrombus mass and limits platelet adherence to sites of arterial injury. Low dose aspirin given intravenously or orally was ineffective. When persistent intracoronary thrombi precipitate unstable coronary syndromes, high dose intravenous aspirin may be useful in the acute period even though platelets continue to interact with injured vascular segments through aspirin-insensitive mechanisms.

Cardioprotective effects of amlodipine on ischemia and reperfusion in two experimental models.

The cardioprotective effect of amlodipine, a long-acting dihydropyridine derivative, was studied in 2 experimental models of ischemia and reperfusion. Isolated and blood-perfused feline hearts were made globally ischemic for 60 minutes and then reperfused for 60 minutes. Alterations of left ventricular developed pressure and compliance were monitored in both amlodipine-treated hearts and saline-treated control animals. Changes in perfusion pressure indicated that amlodipine significantly reduced myocardial oxygen consumption and coronary vascular resistance. Furthermore, a progressive increase in resting left ventricular diastolic pressure indicated that amlodipine, administered before the onset of global ischemia, attenuated the development of ischemic contracture. Return of contractile function 60 minutes after reperfusion and maintenance of tissue concentrations of electrolytes were significantly better in the amlodipine-treated group than in the control animals. In intact canine hearts, regional myocardial ischemia was induced for 90 minutes, followed by 6 hours of reperfusion. Although the hemodynamic variables and the size of the region of risk did not differ significantly between treated animals and control animals, the infarct size was significantly smaller in the amlodipine-treated group than in the control animals, and a gradual reduction in coronary blood flow was observed in the control group that was prevented in the amlodipine group. A comparison of these findings with those observed with oxygen radical scavengers also is discussed. A detailed report of these studies was published in The American Journal of Cardiology (1989;64:101I-116I). This review is included here to maintain continuity of the symposium for the convenience of the reader.

Cardiac electrophysiologic interactions of bepridil, a new calcium antagonist, with enflurane, halothane, and isoflurane.

Bepridil is an investigational calcium antagonist that also has fast sodium channel blocking and antidysrhythmic properties. In the present study, the potential interactions of bepridil with volatile anesthetics on cardiac electrophysiologic parameters were evaluated in open-chest dogs. Twenty-four dogs anesthetized with enflurane (n=6), halothane (n=6), isoflurane (n=6), or chloralose (n=6) received 2.5 mg/kg of bepridil intravenously (IV). Twenty-five additional dogs anesthetized with enflurane (n=7), halothane (n=6), isoflurane (n=6), or chloralose (n=6), received bepridil, 5.0 mg/kg, IV. Dogs anesthetized with cloralose served as controls. Cardiac electrophysiologic parameters were measured after the dogs were anesthetized and were repeated 5, 15, 30, 45, and 60 minutes after bepridil infusion. Plasma bepridil concentrations were also determined at the above time points. Synergy between bepridil and enflurane was demonstrated in the following cardiac electrophysiologic parameters: depression of sinus node function as evidenced by severe depression of sinus node automaticity and conduction; depression of atrioventricular function as evidenced by prolongation of the atrial-His bundle interval and the Wenckebach R-R interval; and, prolongation of the atrial effective refractory period. No synergy was demonstrated between bepridil and halothane or isoflurane when compared to bepridil's effects during chloralose anesthesia. It is concluded that significant synergistic cardiac electrophysiologic effects exist between bepridil and enflurane in dogs. It is recommended that caution be used when anesthetizing patients receiving bepridil with enflurane until human data on the use of this combination of pharmacologic agents is available.

Reduction in digitalis-associated postinfarction mortality with nadolol in conscious dogs.

Previously, we have demonstrated an increased incidence of lethal ischemic arrhythmias in postinfarction dogs with clinically observable serum digoxin concentrations, and a significant reduction in digitalis-related lethal ischemic arrhythmias after subacute left stellectomy. In the present study, the protective actions of acute beta-adrenoceptor blockade with nadolol, 1.0 mg/kg administered intravenously immediately preceding the induction of posterolateral myocardial ischemia, were assessed in conscious dogs with recent, small anterior myocardial infarctions pretreated with digoxin, 0.0125 mg/kg/day intravenously, for 5 to 7 consecutive days (total n = 11). A cohort of postinfarction dogs pretreated with digoxin alone served as a control group (total n = 26). Pre vs postdigoxin electrophysiologic testing indicated reductions in myocardial refractoriness in ventricular noninfarct and infarct zones in both treatment groups, whereas the administration of nadolol tended to reverse the reductions in ventricular refractoriness. Arrhythmia-related deaths in response to posterolateral myocardial ischemia were reduced from 12 of 20 (60%) in the digoxin control group to 2 of 10 (20%) in the digoxin + nadolol group (p = 0.039). Serum digoxin concentrations (1.29 +/- 0.14 ng/ml vs 1.39 +/- 0.24 ng/ml), underlying anterior myocardial infarct size (6.9 +/- 1.5% vs 4.6 +/- 0.9% of left ventricle), and developing posterolateral myocardial infarct size (22.8 +/- 2.5% vs 17.5 +/- 3.6% of left ventricle) did not differ significantly between the digoxin and digoxin + nadolol groups. Acute beta-adrenoceptor blockade with nadolol appears to reduce digitalis-mediated ischemic postinfarction mortality, possibly because of a salutary increase in ventricular refractoriness.

Effect of flecainide acetate on prevention of electrical induction of ventricular tachycardia and occurrence of ischemic ventricular fibrillation during the early postmyocardial infarction period: evaluation in a conscious canine model of sudden death.

The antiarrhythmic and antifibrillatory effects of flecainide acetate during the early postinfarction period were evaluated in a conscious canine model of sudden cardiac death. Ventricular tachycardia remained inducible early after infarction in eight of nine dogs receiving an intravenous loading dose of flecainide (2.0 mg/kg body weight) and seven of eight dogs receiving saline vehicle. In both the drug and vehicle groups, there was no significant change in the ventricular refractory period or in the cycle length of the induced ventricular tachycardia. With a maintenance intravenous infusion of flecainide, 1.0 mg/kg per h for 4 hours, the subsequent occurrence of acute posterolateral ischemia resulted in the development of ventricular fibrillation and sudden death in seven of eight flecainide-treated and eight of eight vehicle-treated dogs. Seven additional postinfarction dogs with noninducible tachycardia during pretreatment programmed stimulation, and thereby considered to be at "low risk" for the development of ischemic ventricular fibrillation, were also given flecainide in an intravenous loading and maintenance dosing regimen. The subsequent occurrence of posterolateral ischemia resulted in the development of ventricular fibrillation in three of these seven dogs. These findings suggest that flecainide acetate may not possess pharmacologic properties useful in managing ventricular tachycardia or in preventing ischemic ventricular fibrillation in the presence of recent myocardial damage.

Preclinical assessment of antiarrhythmic drugs.

The morbidity statistics for the United States alone would suggest that there are 400,000 victims each year who succumb to sudden coronary death, which suggests that there is a need for the development of pharmacological interventions capable of preventing ventricular fibrillation (VF) in patients identified as being at high risk. A major deficiency in this area of experimental investigation is the lack of an animal model that would permit preclinical studies to identify potentially useful therapeutic agents. We have developed an experimental canine model of sudden coronary death in which VF occurs in the chronically injured heart that is subjected to a period of transient ischemia in a coronary region remote from the site of a previous myocardial infarction. The experimental model is subject to the induction of ventricular tachyarrhythmias in response to programmed electrical stimulation, thereby permitting the investigator to correlate the effectiveness of a pharmacological agent in preventing electrically induced arrhythmias with its potential to prevent the development of VF in response to ischemia at a site remote from a previous infarct. Thus, acute myocardial ischemia at a site distant from a previous myocardial infarction enhances the likelihood of primary VF in the conscious dog. This model of sudden coronary death may stimulate more closely the clinical state in humans and might serve as an appropriate model for the study of electrophysiological mechanisms associated with the development of VF and for the evaluation of potential antifibrillatory drugs.

Inability of methylprednisolone sodium succinate to decrease infarct size or preserve enzyme activity measured 24 hours after coronary occlusion in the dog.

Methylprednisolone sodium succinate (50 mg/kg) was given 30 minutes before or after the start of a 90 minute occlusion of the left circumflex coronary artery (LCX) in one group of dogs. In a second group, methylprednisolone sodium succinate was given 15 minutes after permanent occlusion of the left anterior descending artery (LAD). Infarct size was determined by dehydrogenase staining after 24 or 96 hours. Heart slices were incubated with nitro-blue tetrazolium and nonstaining infarcted tissue was dissected and weighed. Myocardial depletion of creatine phosphokinase activity (CPK) and lactate dehydrogenase activity (LDH) were determined 24 hours after temporary LCX occlusion. When measured after 24 hours, methylprednisolone sodium succinate treatment did not reduce infarct size or decrease enzyme loss. After temporary LCX occlusion infarct size was 30.4 +/- 3.6% of left ventricular weight in control dogs and 30.0 +/- 2.3% in treated dogs. No significant difference in infarct size was observed in hearts examined 24 or 96 hours after myocardial infarction. After permanent LAD occlusion, infarct size in control dogs was 39.2 +/- 1.6% of left ventricular weight and 33.7 +/- 3.5% in treated dogs. CPK activity in the LCX area decreased by 26.5 +/- 7% in controls and by 28.1% +/- 7% in treated dogs. Treated dogs sustained a significantly greater fall in arterial blood pressure after LCX occlusion than did controls. During LCX occlusion and upon reperfusion, methylprednisolone sodium succinate treated dogs exhibited a significantly greater number of premature ventricular beats. Since infarct size and enzyme depletion were not reduced when measured after 24 hours, methylprednisolone sodium succinate treatment does not appear to have enhanced myocardial cell viability.