Paolo Golino

Specific platelet mediators and unstable coronary artery lesions: experimental evidence and potential clinical implications

We have speculated previously that the abrupt conversion from chronic stable to unstable angina and the continuum to acute myocardial infarction may result from myocardial ischemia caused by progressive platelet aggregation and dynamic vasoconstriction themselves caused by local increases in thromboxane and serotonin at sites of coronary artery stenosis and endothelial injury. Platelet aggregation and dynamic coronary artery vasoconstriction probably result from the local accumulation of thromboxane and serotonin and also relative decreases in the local concentrations of endothelially derived vasodilators and inhibitors of platelet aggregation, such as endothelium-derived relaxing factor (EDRF) and prostacyclin. With severe reductions in coronary blood flow caused by these mechanisms, platelet aggregates may increase, and an occlusive thrombus composed of platelets and white and red blood cells in a fibrin mesh may develop. When coronary arteries are occluded or narrowed for a sufficient period of time by these mechanisms, myocardial necrosis, electrical instability, or sudden death may occur. We believe that unstable angina and acute myocardial infarction are a continuum in relation to the process of coronary artery thrombosis and vasoconstriction. When the period of platelet aggregation or dynamic vasoconstriction at sites of endothelial injury and coronary artery stenosis is brief, unstable angina or non-Q wave infarction may occur. However, when the coronary artery obstruction by these mechanisms is prolonged for several hours, Q wave myocardial infarction results. Chronic endothelial injury and coronary artery stenosis are probably associated with the accumulation of platelets, white and red blood cells, and a fibrin mesh at the site of stenosis and endothelial injury.

Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury.

Cyclic variations in coronary blood flow (CFVs) in dogs with experimental coronary artery stenosis and endothelial injury appear to result primarily from the aggregation of platelets at the site of stenosis followed by dislodgement and distal embolization. Using this canine model, we tested the hypotheses: (a) that thrombin is an important mediator of CFVs in dogs with coronary stenoses and endothelial injury; (b) that inhibition of thrombin with heparin, or MCI-9038, a selective thrombin inhibitor, abolishes CFVs in this model; and (c) that abolition of CFVs by thrombin inhibition is time dependent. CFVs, produced in open-chest dogs by placing a flow-reducing plastic constrictor around the left anterior coronary artery, were monitored for either 30 min (group I) or 3 h (group II) before treatment with either heparin or 4-methyl-1-(N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl (MCI-9038). In group I, cyclic flow variations were abolished by heparin in 12 of 18 dogs and by MCI-9038 in 5 of 7 dogs. In group II, cyclic flow variations were not abolished by heparin in any of seven dogs and were abolished by MCI-9038 in only one of seven dogs. Thus, (a) thrombin appears to be an important mediator of cyclic flow variations in dogs with coronary artery stenosis and endothelial injury and (b) inhibition of thrombin abolishes CFVs after short but not prolonged periods of CFVs.

Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators.

The goal of the present study was to demonstrate that intracoronary platelet deposition may trigger intense vasoconstriction of large epicardial coronary arteries in vivo and that this is largely mediated by thromboxane A2 and serotonin released by activated platelets. Cyclic flow variations (progressive declines in blood flow followed by sudden restorations of flow) due to recurrent intracoronary platelet activation and thrombus formation were induced by damaging the endothelium and placing a cylindrical constrictor on the left anterior descending coronary artery (LAD) in open-chest, anesthetized dogs. Coronary diameters were measured in vivo by means of ultrasonic crystals sutured on the LAD immediately distal to the constrictor (LAD1) and 1 cm below (LAD2) and on the circumflex coronary artery (Cx). Coronary artery diastolic diameters were measured continuously before and during cyclic flow variations and after they were abolished by administration of LY53857, a serotonin-receptor antagonist (group 1, n = 7), or SQ29548, a thromboxane-receptor antagonist (group 2, n = 7). During cyclic flow variations, at the nadir of coronary flow, LAD1 (a site of maximal platelet accumulation) cross-sectional area decreased by 52 +/- 10% and 38 +/- 6% in group 1 and 2 animals, respectively (p less than 0.001 compared with values recorded during a brief LAD occlusion obtained by a suture snare), whereas LAD2 (a site of minimal or no platelet accumulation) cross-sectional area did not differ from that recorded during the brief LAD occlusion. SQ29548 abolished cyclic flow variations in seven of seven dogs and LY53857 in six of seven, but they affected the increased coronary vasoconstriction differently: LAD1 cross-sectional area increased by 32 +/- 6% of the control value in SQ29548-treated animals, whereas it returned to baseline dimension values in the LY53857-treated group as these interventions also abolished the cyclic flow variations. We conclude that a marked coronary vasoconstriction may be triggered by local platelet deposition and that thromboxane A2 and serotonin are mediators of this vasoconstriction.

Effect of thromboxane and serotonin receptor antagonists on intracoronary platelet deposition in dogs with experimentally stenosed coronary arteries.

We have reported previously that thromboxane A2 (TXA2) and serotonin (5-HT, 5-hydroxytryptamine) are important mediators of cyclic flow variations (CFVs) in a canine model of coronary artery stenosis and endothelial injury. The present study tested the hypothesis that a TXA2 receptor antagonist is more effective in reducing intracoronary platelet deposition at sites of endothelial injury and severe stenosis than a 5-HT2 receptor antagonist. CFVs developed after placing a plastic constrictor around the left anterior descending coronary artery (LAD) in 51 of 56 dogs. Autologous platelets labeled with 111In were injected in 48 animals. Ten control dogs (group 1A) were killed after CFVs were observed for 1 hour at the nadir of coronary blood flow. Five dogs (group 1B) did not develop CFVs after placement of the constrictor. CFVs were abolished with SQ 28668 (2.75 +/- 0.36 mg/kg, group 2) and SQ 29548 (0.45 +/- 0.1 mg/kg, group 3), two different TXA2 and PGH2 receptor antagonists, in eight of 10 and six of seven dogs, respectively. In eight of 10 dogs (group 4), CFVs were abolished with ketanserin (0.66 +/- 0.12 mg/kg), a 5-HT2 receptor antagonist. In group 2, 3, and 4 dogs, the respective drugs were given so that the minimal dose required to abolished CFVs was administered. In six of six dogs (group 5), a higher dose of ketanserin (i.e., 1.5 mg/kg) was used to abolish CFVs. At death, intracoronary platelet deposition was evaluated by calculating the LAD platelet accumulation ratio (111In activity in the LAD/111In activity in the circumflex coronary artery) in 43 dogs and, in 22 dogs, by microscopic examination of the LAD. A marked LAD platelet accumulation ratio was found in group 1A dogs at the stenotic site and in segments immediately distal to it. The LAD platelet accumulation ratio was significantly reduced by both the low and the high doses of ketanserin compared with group 1A dogs (p less than 0.001). However, the two TXA2 receptor antagonists further reduced the LAD platelet accumulation ratio compared with ketanserin-treated animals (p less than 0.01). Microscopic examination confirmed these findings. We conclude that SQ 28668 and SQ 29548, two different TXA2 receptor antagonists, reduce residual intracoronary platelet deposition associated with CFVs in this canine model more effectively than ketanserin, a 5-HT2 receptor antagonist.

Simultaneous administration of thromboxane A2- and serotonin S2-receptor antagonists markedly enhances thrombolysis and prevents or delays reocclusion after tissue-type plasminogen activator in a canine model of coronary thrombosis.

Dynamic changes of the thrombus after its formation due to platelet activation may affect the speed of thrombolysis. In the present study, we wanted to evaluate the role played by thromboxane A2 (TXA2) and serotonin (5HT) in mediating platelet activation during lysis of intracoronary thrombi with human recombinant tissue-type plasminogen activator (t-PA). Coronary thrombi were induced in 26 anesthetized, open-chest dogs by inserting a copper coil into the left anterior descending coronary artery (LAD). LAD blood flow was monitored throughout the experiment by means of a Doppler flow probe placed proximally to the coil. Presence of the thrombus was documented for 30 minutes. The dogs were then assigned to one of four groups as follows: group 1 dogs (n = 8), serving as controls, received a bolus of heparin (200 units/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg/min) for up to 90 minutes or until reperfusion was achieved; group 2 dogs (n = 10) received, immediately before heparin and t-PA, an intravenous bolus of SQ29548 (SQ) (0.4 mg/kg, a selective TXA2-receptor antagonist) and LY53857 (LY) (0.2 mg/kg, a selective serotonin S2-receptor antagonist); group 3 dogs (n = 7) received, before heparin and t-PA, an intravenous bolus of SQ alone (0.4 mg/kg); and group 4 dogs (n = 7) received, before heparin and t-PA, an intravenous bolus of LY alone (0.2 mg/kg). After thrombolysis, all dogs were monitored for 90 minutes or until a persistent reocclusion occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin S2and thromboxane A2-prostaglandin H2receptor blockade provide protection against epinephrine-induced cyclic flow variations in severely narrowed canine coronary arteries☆☆☆

The object of this study was to test the hypothesis that administration of both serotonin S2 and thromboxane A2-prostaglandin H2 (PGH2) receptor antagonists provides significant protection against epinephrine-induced cyclic coronary artery flow variations in open chest, anesthetized dogs with severe proximal coronary artery stenosis and endothelial injury. Three groups of dogs were studied. In Group 1 (n = 7) and Group 2 (n = 6), cyclic coronary flow variations were initiated after placement of a concentric constrictor around the left anterior descending coronary artery and were abolished by administration of either a thromboxane A2-prostaglandin H2 receptor antagonist, SQ29,548 (SQ) (Group 1), or a serotonin S2 receptor antagonist, LY53,857 (LY) (Group 2). Cyclic flow variations were restored with an epinephrine infusion and the second antagonist (LY for Group 1; SQ for Group 2) was administered to abolish epinephrine-induced cyclic flow variations. The rate of epinephrine infusion was increased until cyclic coronary flow variations returned (n = 8) or significant hemodynamic changes occurred. Plasma epinephrine concentrations were determined during a control period of cyclic coronary flow variations, after epinephrine restored cyclic flow variations in the presence of either SQ or LY, and again after epinephrine restored cyclic flow variations in the presence of both SQ and LY. A third group of dogs (Group 3, n = 9) required both SQ and LY to eliminate the initial cyclic coronary flow variations and infused epinephrine restored cyclic flow variations (n = 8). Plasma epinephrine concentrations were determined during a control period and after cyclic coronary flow variation restoration with epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential enhancement of postischemic segmental systolic thickening by diltiazem

Prolonged depression of segmental systolic thickening after brief coronary artery occlusion may result principally from events during reperfusion rather than during the ischemic interval. Thus, cellular calcium overload at reperfusion may be a mediator of contractile dysfunction after brief ischemia, and reduction of calcium entry by diltiazem, a calcium channel antagonist, may enhance recovery of systolic thickening after brief periods of ischemia. Thirteen awake unsedated dogs instrumented with hemodynamic catheters, left anterior descending coronary artery occluders and five to six pairs of intramyocardial sonomicrometers underwent two 15 min coronary artery occlusions with 24 h reperfusion. The order of infusion of diltiazem (15 micrograms/kg per min) or saline solution was alternated. Systolic thickening, hemodynamic variables and regional myocardial blood flow were measured serially over 24 h. Despite equally severe ischemic dysfunction during coronary occlusion, diltiazem-treated segments with systolic thinning during ischemia recovered control segmental thickening significantly earlier than saline solution-treated segments (at 30 versus 180 min of reperfusion). Blood pressure was mildly decreased during diltiazem treatment; therefore, a second group of 10 dogs underwent a similar occlusion and reflow period during infusion of nitroprusside to lower mean arterial pressure equivalently. Decreases in blood pressure in this group resulted in some improvement in segmental systolic function; however, this did not reach statistical significance at any time. Regional myocardial blood flows were similar in the saline solution- and diltiazem-treated groups during ischemia and reflow. Thus, it is concluded that 1) diltiazem infusion significantly enhanced recovery of segmental systolic thickening after 15 min of ischemia and 24 h of reperfusion; 2) the enhancement in segmental systolic function could not entirely be attributed to decreased mean arterial pressure; 3) improvement in postischemic segmental ventricular function was seen only in those segments with systolic thinning during ischemia; thus, segments with the most severe ischemic dysfunction benefited most; and 4) there were no important differences in regional myocardial blood flow during ischemia and reperfusion between saline- and diltiazem-treated animals.

Failure of nitroglycerin and diltiazem to reduce platelet-mediated vasoconstriction in dogs with coronary artery stenosis and endothelial injury: Further evidence for thromboxane A2 and serotonin as mediators of coronary artery vasoconstriction in vivo☆

This study was designed to test the efficacy of nitroglycerin and diltiazem in inhibiting in vivo platelet aggregation and reducing platelet-mediated vasoconstriction in a canine model of coronary artery stenosis and endothelial injury. Coronary artery diameter was measured in vivo by means of ultrasonic crystals sutured on the left anterior descending coronary artery (LAD) immediately distal to an external constrictor (LAD1), 1 cm below (LAD2), and on the left circumflex coronary artery. Coronary diameter was continuously measured before, during cyclic flow variations (progressive declines in blood flow followed by sudden restorations of flow due to recurrent intracoronary platelet aggregation), during cyclic flow variations and intravenous infusion of nitroglycerin (5 micrograms/kg per min) or diltiazem (15 micrograms/kg per min), and after cyclic flow variations were abolished by administration of LY53857, a serotonin receptor antagonist (n = 7), or SQ29548, a thromboxane A2 receptor antagonist (n = 7). During control cyclic flow variations, at the nadir of coronary flow (6% to 11% of the nonstenosed values), LAD1 cross-sectional area decreased by 43 +/- 8% and 44 +/- 3% in the two groups of dogs subsequently treated with LY53857 and SQ29548, respectively. Neither nitroglycerin nor diltiazem caused changes in cyclic flow variation frequency or severity. Furthermore, neither drug significantly reduced the vasoconstriction associated with cyclic flow variations, whereas they significantly increased circumflex artery cross-sectional area. In contrast, LY53857 and SQ29548 were very effective in abolishing cyclic flow variations and the coronary vasoconstriction related to them. Five additional dogs received an intracoronary infusion of nitroglycerin (21 +/- 5 micrograms/kg per min) and later diltiazem (15 micrograms/kg per min).(ABSTRACT TRUNCATED AT 250 WORDS)

Combined thromboxane A2synthetase inhibition and receptor blockade are effective in preventing spontaneous and epinephrine-induced canine coronary cyclic flow variations☆

The purpose of this study was to test the hypothesis that combined thromboxane A2 synthetase inhibition and receptor blockade is superior to either action alone in preventing cyclic flow variations in stenosed and endothelially injured canine coronary arteries. Forty-five dogs developed coronary cyclic flow variations after a plastic constrictor was placed around the left anterior descending coronary artery at the site where the endothelium was injured and received different interventions. In Group I, 17 dogs were treated with SQ 29,548, a thromboxane A2-prostaglandin H2 receptor antagonist. In Group II, 11 dogs received dazoxiben, a thromboxane A2 synthetase inhibitor. In Group III, R 68,070, a dual thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor antagonist, was administered to 11 dogs. Group IV comprised six dogs that received aspirin before receiving R 68,070. Complete abolition of cyclic flow variations was achieved in 71% of dogs in Group I, 82% in Group II, 100% in Group III (p = 0.06 compared with Group I) and 50% in Group IV (p = 0.03 compared with Group III). Epinephrine was infused into dogs with abolished cyclic flow variations: all dogs in Group I had cyclic flow variations restored, 44% in Group II (p = 0.01 compared with Group I) and 64% in Group III (p = 0.04 compared with Group I). The plasma epinephrine levels required to restore cyclic flow variations were 2.2 +/- 0.5 ng/ml (control 0.04 +/- 0.01) in Group I, 8.7 +/- 4.5 ng/ml (control 0.05 +/- 0.02) in Group II and 7.4 +/- 2.6 ng/ml (control 0.07 +/- 0.02) in Group III.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1 alpha levels were significantly increased with respect to control and SQ 29548-treated dogs. Thus, simultaneous inhibition of TxA2 synthase and blockade of TxA2/PGH2 receptors is more effective than either intervention alone in this experimental model in enhancing thrombolysis and preventing reocclusion after t-PA administration.