Platelet-mediated Thrombosis Research Articles

HMG-CoA Reductase Inhibitor Protects Against In Vivo Arterial Thrombosis by Augmenting Platelet-Derived Nitric Oxide Release in Rats

Acute coronary syndromes are caused by platelet-mediated thrombosis following rupture of a plaque. HMG-CoA reductase inhibitors (statins) reduce the incidence of events early after acute coronary syndromes, which are independent of its cholesterol-lowering effect. Accordingly, we investigated whether statins inhibit platelet-mediated arterial thrombus formation in vivo and, if so, the underlying mechanisms. Rats were divided into 4 groups. Group 1 was treated with the vehicle, whereas groups 2, 3, and 4 were treated with cerivastatin for 7 days (1, 2, and 5 mg/kg i.p., respectively). Cerivatatin did not change serum cholesterol levels. Carotid arterial thrombosis was created by perivascular FeCl3 delivery. Cerivastatin significantly prolonged the time to thrombotic occlusion of carotid artery. Cerivastatin significantly dose-dependently inhibited both ex vivo platelet P-selectin expression, a marker of platelet activation, and platelet aggregation. Cerivastatin significantly augmented platelet-derived nitric oxide (NO) release, and up-regulated platelet and endothelial nitric oxide synthase (NOS) mRNA expressions. N-nitro-L-arginine methylester abolished the effects of cerivastatin. This study demonstrates that in vivo administration of statin protects against platelet-mediated arterial thrombosis, possibly by augmenting platelet- and endothelium-derived NO releases via up-regulation of platelet and endothelial NOS.

Blockade of the platelet P2Y12 receptor by AR-C69931MX sustains coronary artery recanalization and improves the myocardial tissue perfusion in a canine thrombosis model.

Reperfusion therapy for myocardial infarction is limited by a significant reocclusion rate and less optimal myocardial tissue perfusion due to excessive platelet accumulation and recruitment at the sites of vascular injury. We assessed the influence of a selective P2Y(12)-receptor antagonist (AR-C69931MX), in conjunction with thrombolytic therapy, on the prevention of platelet aggregation and thrombus formation. A canine coronary electrolytic injury thrombosis model was used. Tissue-type plasminogen activator (t-PA; 1 mg/kg in phase I, 0.5 mg/kg in phase II in the AR-C69931MX group, and 1 mg/kg in the placebo group in phase I and II) was administered 30 minutes after thrombus formation; either saline or AR-C69931MX (4 micro g x kg(-1) x min(-1)) was given to all animals intravenously 10 minutes before t-PA administration for a total of 2 hours. All animals received heparin (80 U/kg) as an intravenous bolus followed by a continuous infusion of 17 U x kg(-1) x h(-1). Myocardial tissue perfusion was evaluated by use of the colored microsphere technique and real-time myocardial contrast echocardiography. The incidences of reocclusion and cyclic flow variation were significantly decreased in the AR-C69931MX group (P<0.05). Myocardial tissue flow with AR-C69931MX treatment improved significantly at 20 and 120 minutes after reflow, whereas tissue flow with placebo remained at a level similar to that during occlusion (P<0.05). The adjunctive administration of AR-C69931MX blocked ADP-mediated platelet aggregation and recruitment and prevented platelet-mediated thrombosis, resulting in prolongation of reperfusion time and a decrease in reocclusion and cyclic flow variations. Importantly, myocardial tissue perfusion was significantly improved in the P2Y(12) antagonist group.

Platelet glycoprotein IIb/IIIa inhibitors: recognition of a two-edged sword?

Glycoprotein (GP) IIb/IIIa antagonists are potent inhibitors of platelet aggregation that provide marked protection from ischemic events in patients undergoing percutaneous coronary intervention (PCI).1,2⇓ Abciximab, the prototypic GP IIb/IIIa inhibitor, has been studied in >8000 patients undergoing elective or high-risk PCI.2,3,4⇓⇓ In these patients, it produces a consistent 35% to 56% reduction in ischemic end points at 30 days, with a significant 22% reduction in the risk of death, as shown in a combined analysis of long-term (3-year minimum) follow-up of the Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC), Evaluation in PTCA to Improve Long-term Outcome with abciximab GP IIb/IIIa blockade (EPILOG), and Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT) trials.5 Similar results, albeit of a smaller magnitude, have been seen with the small-molecule antagonists tirofiban and eptifibatide, with a 16% to 35% reduction in ischemic events in patients undergoing PCI.1,6⇓ Attempts to expand the therapeutic indication of GP IIb/IIIa antagonists to other conditions associated with platelet-mediated thrombosis, however, have been less fruitful than expected. Instead of reducing major ischemic events, long-term oral GP IIb/IIIa inhibitor therapy7 has uniformly increased the fatality rate. Furthermore, the overall efficacy of a number of the intravenous antagonists in non–ST-segment elevation acute coronary syndromes (ACS), or in combination with thrombolysis in ST-segment elevation myocardial infarction (MI), has been less than anticipated.8 Of particular concern is the fact that there has been a paradoxical increase in adverse events in 2 of the trials of intravenous therapy in ACS.9,10⇓ Platelets are known to play an important role in the pathogenesis of ACS, yet the high levels of platelet inhibition attainable with GP IIb/IIIa antagonists have failed to dramatically improve clinical outcomes outside of PCI. In this article, we discuss these …

Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons.

The antithrombotic efficacy of the monoclonal antibodies 6B4-Fab and MA-16N7C2 against platelet glycoprotein (GP) Ib and GP IIb/IIIa, respectively, on acute platelet-mediated thrombosis was evaluated in a baboon model of femoral artery stenosis, which is a modification of the original Folts model: platelet thrombi form on the injured stenosed artery, producing cyclic flow reductions (CFRs). A dose of 0.6 mg/kg 6B4-Fab significantly reduced the CFRs by 59 +/- 15%, whereas 2 mg/kg 6B4-Fab completely abolished the CFRs without prolongation of the bleeding time. MA-16N7C2 inhibited CFRs by 43 +/- 8% at a dose of 0.1 mg/kg and abolished the CFRs at a dose of 0.3 mg/kg but with a significant prolongation of the bleeding time. Finally, the combination of 0.6 mg/kg 6B4-Fab and 0.1 mg/kg MA-16N7C2 fully prevented the CFRs without prolongation of the bleeding time. The present study demonstrates that the inhibition of platelet GP Ib function by 6B4-Fab is a powerful intervention to prevent platelet thrombus formation in injured arteries without prolongation of the bleeding time; the latter is in contrast to the result after the inhibition of GP IIb/IIIa. Moreover, we demonstrate that combining a GP Ib blocker with a GP IIb/IIIa blocker can achieve a strong antithrombotic effect without increasing the bleeding time. This provides new information that will be beneficial in designing clinical therapeutic approaches.

Ticlopidine Inhibits the Prothrombotic Effects of Thrombopoietin and Ameliorates Survival after Supralethal Total Body Irradiation

Thrombopoietin modulates the response of platelets to several agonists and, on the other hand, those agonists can be released following irradiation. Thus, we have determined the effects of thrombopoietin, on its own and in combination with ticlopidine, an anti-platelet drug, on platelet activation, thrombosis formation and survival of irradiated C57BL6/J mice. Administration of thrombopoietin 2 h after 9 Gy total body irradiation increased the 125I-fibrin deposition in mouse tissues and accelerated platelet consumption as revealed by an enhanced drop in platelet counts. Additionally, the number of activated platelets, i.e. those expressing P-selectin on their membrane, was higher in thrombopoietin-treated mice as compared to the placebo group, regardless ex vivo stimulation with agonists. These effects of thrombopoietin on platelet activation and consumption were reduced when mice were pretreated with ticlopidine. The combination of ticlopidine with thrombopoietin almost fully promoted 180-day survival, reaching the same efficacy as bone marrow transplantation, while only 30% of the mice treated with thrombopoietin alone survived. In summary, thrombopoietin induces long term-mortality of irradiated mice probably through platelet-mediated thrombosis and thus, ticlopidine efficiently counteracts these adverse effects of thrombopoietin.

Administration of Raw Onion Inhibits Platelet-Mediated Thrombosis in Dogs

A number of studies suggest that dietary intake of onions is of benefit to cardiovascular health. Onion juice inhibits in vitro human platelet aggregation. To study the in vivo effect of onion on platelet aggregation, 11 dogs were prepared with mechanically damaged and stenosed coronary arteries. Periodic platelet-mediated thrombus formation followed by embolization produced cyclic flow reductions (CFR). In five dogs, 0.09 +/- 0.01 mL/kg onion juice administered intravenously abolished CFR within 20 min. This was followed by a 60 +/- 14% (P = 0.002) reduction in collagen-induced ex vivo whole-blood platelet aggregation. Six dogs were given 2.0 g/kg raw onion homogenate intragastrically. CFR were eliminated within 2.5-3 h in five of the dogs. This was accompanied by a 44 +/- 24% (P = 0.04) reduction in ex vivo aggregation. These findings suggest that the consumption of raw onion may help prevent platelet-mediated cardiovascular disorders. However, in vitro incubations of onion juice demonstrated that the platelet inhibitory response was significantly greater in dog blood than in human blood.

From bench to bedside: GP IIb-IIIa inhibitors.

Our understanding of the pathophysiology of acute coronary syndromes (ACS) has now been substantiated by many studies showing that atherothrombosis plays a predominant role. This article reviews the molecular interactions in thrombosis that may serve as therapeutic targets for more effective management of these syndromes. In particular, the discovery of the fact that the glycoprotein (GP) IIb-IIIa receptor plays a central and common role in platelet-mediated thrombosis has focused the therapeutic efforts. Blockade of this receptor has emerged as a new and potent strategy for inhibition of platelet aggregation and thus of clot formation. A number of trials have now corroborated the need for such antithrombotic drugs in the management of patients with non-ST-segment elevation ACS.

Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release

Moderate red wine consumption is inversely associated with coronary ischemia, and both red wine and purple grape juice (PGJ) contain flavonoids with antioxidant and antiplatelet properties believed to be protective against cardiovascular events. Acute cardiac events are also associated with decreased platelet-derived nitric oxide (NO) release. In this study, the effects of PGJ and PGJ-derived flavonoids on platelet function and platelet NO production were determined. Incubation of platelets with dilute PGJ led to inhibition of aggregation, enhanced release of platelet-derived NO, and decreased superoxide production. To confirm the in vivo relevance of these findings, 20 healthy subjects consumed 7 mL. kg(-1). d(-1) of PGJ for 14 days. Platelet aggregation was inhibited after PGJ supplementation, platelet-derived NO production increased from 3.5+/-1.2 to 6.0+/-1.5 pmol/10(8) platelets, and superoxide release decreased from 29.5+/-5.0 to 19.2+/-3.1 arbitrary units (P<0.007 and P<0.05, respectively). alpha-Tocopherol levels increased significantly after PGJ consumption (from 15.6+/-0.7 to 17.6+/-0.9 micromol/L; P<0.009), and the plasma protein-independent antioxidant activity increased by 50.0% (P<0.05). Last, incubation of platelets with select flavonoid fractions isolated from PGJ consistently attenuated superoxide levels but had variable effects on whole-blood aggregation, platelet aggregation, and NO release. Both in vitro incubation and oral supplementation with PGJ decrease platelet aggregation, increase platelet-derived NO release, and decrease superoxide production. These findings may be a result of antioxidant-sparing and/or direct effects of select flavonoids found in PGJ. The suppression of platelet-mediated thrombosis represents a potential mechanism for the beneficial effects of purple grape products, independent of alcohol consumption, in cardiovascular disease.

Sildenafil citrate (Viagra) does not exacerbate myocardial ischemia in canine models of coronary artery stenosis

OBJECTIVESOur aim was to determine whether sildenafil citrate (Viagra) unfavorably alters coronary perfusion in canine models of coronary artery stenosis.BACKGROUNDConcern has been raised that sildenafil may exacerbate ischemia in patients with coronary artery disease. However, the effects of sildenafil on coronary perfusion are largely unexplored.METHODSUsing anesthetized dogs, a micromanometer constrictor was applied to either an intact coronary artery (model of stable hypoperfusion: Protocol 1) or a site of arterial injury (model of recurrent platelet-mediated thrombosis: Protocol 2). After monitoring coronary flow for 1 h, dogs received two escalating, clinically relevant doses of sildenafil or placebo. Perfusion was assessed during the initial hour pretreatment, for 1 h following dose 1 and 1 h following dose 2 by measuring the area of the flow-time profile, normalized to baseline flow × 60 min. Interaction between sildenafil and adenosine-mediated inhibition of platelet aggregation was evaluated by in vitro platelet aggregometry (Protocol 3).RESULTSIn Protocol 1, flow-time area was maintained at 50% to 60% of baseline in both placebo- and sildenafil-treated groups. In Protocol 2, controls exhibited an expected modest, temporal adenosine-mediated improvement in flow-time area (from 40 ± 5% to 61 ± 7%; p < .05) while in contrast, perfusion in sildenafil-treated dogs remained unchanged (37 ± 6% vs. 33% to 35% before vs. after treatment). In vitro aggregometry confirmed that sildenafil rendered platelets refractory to the inhibitory effects of adenosine receptor stimulation.CONCLUSIONSSildenafil did not exacerbate ischemia in canine models of coronary stenosis. However, in the setting of recurrent thrombosis, sildenafil-treated dogs were apparently unresponsive to the platelet inhibitory effects of endogenous adenosine.

Optimizing antiplatelet therapy in coronary interventions.

Percutaneous coronary intervention has had a dramatic impact on the current practice of cardiology. One of its important limitations, however, is the potential for producing unfavorable outcomes such as acute coronary closure following angioplasty or atherectomy or subacute thrombosis following stent implantation. These complications may lead to death, myocardial infarction, or the need for urgent bypass surgery. One mechanism underlying these clinical events is platelet-mediated thrombosis due to arterial trauma. Therapeutically, platelet activation by thromboxane and adenosine diphosphate (ADP), as well as platelet aggregation by glycoprotein IIb/IIIa (GPIIb/IIIa) receptors, has been inhibited with various pharmacologic agents. c7E3 (abciximab), a monoclonal antibody directed against GPIIb/IIIa, has been shown to have potent effects on reducing both acute and subacute complications. Other parenteral GPIIb/IIIa inhibitors, including peptide and small nonpeptide molecules, have also been found to be clinically effective. Oral versions of similar drugs are currently being evaluated, but several have resulted in disappointing efficacy and safety profiles and have failed to show advantages over aspirin. With all antiplatelet agents, in particular GPIIb/IIIa receptor inhibitors, bleeding and vascular complications must be addressed. Inhibition of thromboxane-induced platelet activation with aspirin has been standard therapy for angioplasty and in the global management of vascular disease. Newer agents that block ADP-mediated platelet activation, the thienopyridines, have been found to be synergistic to aspirin in their effects on the complications of coronary intervention. Ticlopidine and, more recently, clopidogrel, in conjunction with aspirin, have become standard therapies for preventing subacute thrombosis after stent implantation. Large-scale clinical trials are ongoing to optimize their use in combination with GPIIb/IIIa inhibitors.

Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups.

Vascular injury causes the muscularization of peripheral pulmonary arteries, which is more pronounced in the infant than in the adult lung. Although inhaled NO gas attenuates pulmonary artery remodeling in hypoxic rats, whether or not it protects the lung by mitigating vasoconstriction is unknown. This investigation tested whether inhaled NO decreases the muscularization of injured pulmonary arteries in rat pups by modulating vascular tone. One week after monocrotaline administration, the percentage of muscularized rat pup lung arteries was increased by >3-fold. Nevertheless, monocrotaline exposure did not cause right ventricular hypertrophy, pulmonary hypertension, or vasoconstriction. In addition, it did not increase the expression of markers of inflammation (interleukin-1beta, intercellular adhesion molecule-1, and E-selectin) or of platelet-mediated thrombosis (GPIbalpha). Continuous inhalation of 20 ppm NO gas prevented the neomuscularization of the pulmonary arteries in pups with lung injury. Moreover, a 3-fold increase in cell proliferation and 30% decrease in cell numbers in pulmonary arteries caused by monocrotaline exposure was prevented by NO inhalation. These data indicate that inhaled NO protects infants against pulmonary remodeling induced by lung injury by mechanisms that are independent of pulmonary tone, inflammation, or thrombosis.

Monophosphoryl lipid A: a novel nitric oxide-mediated therapy to attenuate platelet thrombosis?

Nitric oxide (NO) is a potent inhibitor of platelet aggregation. However, the benefits of NO-based therapies can be confounded by concomitant hypotension. Monophosphoryl lipid A (MLA) is a nontoxic derivative of endotoxin that purportedly increases nitric oxide synthase (NOS) activity and, presumably, NO production, yet has a hemodynamically benign profile. Thus our aims were to determine whether (a) MLA attenuates in vivo platelet aggregation in damaged and stenotic canine coronary arteries by a NO-mediated mechanism but without reductions in arterial pressure; and (b) the platelet inhibitory effects are manifest in vitro. To address the first aim, anesthetized dogs underwent coronary injury + stenosis, resulting in cyclic variations in coronary blood flow (CFVs) caused by the formation/dislodgement of platelet-rich thrombi. In protocol I, dogs received MLA (100 microg/kg + 40 microg/kg/h) or vehicle beginning 15 min before stenosis. Protocol II was identical, except the NOS inhibitor aminoguanidine was coadministered with MLA/vehicle. Coronary patency was assessed throughout the initial 3 h after injury + stenosis. Infusion of MLA did not result in hypotension. However, in protocol I, the median nadir of the CFVs was higher (2.1 vs. 0.8 ml/min; p < 0.05), median duration of total thrombotic occlusion tended to be reduced (0 vs. 10.4 min; p = 0.1), and mean flow-time area, expressed as a percentage of baseline flow, was increased (53 +/- 9% vs. 33 +/- 3%; p < 0.05) in MLA-treated versus vehicle-treated dogs. In contrast, in protocol II, vessel patency was comparable in both groups. Finally, whole blood impedance aggregometry (protocol HI) revealed a significant reduction in the in vitro platelet aggregation in blood samples receiving exogenous MLA, which was blocked by coadministration of exogenous aminoguanidine. Thus MLA attenuates platelet-mediated thrombosis in both damaged and stenotic canine coronary arteries and in vitro, possibly by an NO-mediated mechanism, but without concomitant hypotension.

Trials of glycoprotein IIb-IIIa inhibitors in non-ST-segment elevation acute coronary syndromes: applicability to the practice of medicine in the United States.

Platelet-mediated thrombosis has been recognized as the primary pathophysiologic mechanism of acute coronary syndromes (ACS) and acute complications of percutaneous coronary intervention (PCI). Despite the clinical efficacy of the two most widely used antithrombotic agents, aspirin and heparin, each of them has significant therapeutic limitations. As a result, thrombosis and clinical events may occur despite the use of aspirin and heparin. The discovery that the platelet glycoprotein (GP) IIb-IIIa represents the final common pathway to platelet aggregation and the growing recognition of the key role of platelets in the progression of thrombosis prompted the development of several GP IIb-IIIa inhibitors as a potentially more effective form of antithrombotic therapy. Numerous trials of various GP IIb-IIIa inhibitors as adjuncts to PCI have strongly supported this hypothesis. The subject of this supplement is the review of more recent evaluations of GP IIb-IIIa inhibitors in the context of various treatment strategies for the management of patients with unstable angina or non-ST-segment elevation myocardial infarction, collectively known as non-ST-segment elevation ACS. Appropriate translation of these trials into clinical practice requires not only the knowledge of the trials' results but also the understanding of the design of individual studies, most notably the entry criteria and patient management strategies.

Brief antecedent ischemia attenuates platelet-mediated thrombosis in damaged and stenotic canine coronary arteries: role of adenosine.

Recent studies suggest that patients with angina before myocardial infarction exhibit improved recovery of coronary perfusion after thrombolysis by an as-yet-unknown mechanism. We therefore proposed that brief antecedent ischemia/reperfusion may, via release of adenosine, improve vessel patency in damaged and stenotic coronary arteries. Anesthetized dogs underwent coronary injury + stenosis, resulting in repeated cyclic variations in coronary blood flow (CFVs) caused by the formation/dislodgment of platelet-rich thrombi. Vessel patency was assessed for 3 hours after stenosis by quantification of the nadir of the CFVs, duration of total thrombotic occlusion (flow=0), and area of the flow-time profile (expressed as percent of baseline flow x 180 minutes). In protocol 1, dogs received 10 minutes of coronary occlusion + 10 minutes of reflow or a comparable 20-minute control period before injury + stenosis. The median nadir of the CFVs was higher (4.0 versus 0.3 mL/min), median zero flow duration per 30-minute time interval was shorter (0.4 versus 15.1 minutes), and mean percent flow-time area was greater (54+/-8% versus 28+/-9%) in dogs that received antecedent ischemia versus controls (P<.05). These benefits of antecedent ischemia/reperfusion were largely mimicked by a 10-minute intracoronary adenosine infusion (400 microg/min) in lieu of brief ischemia (protocol 2) and were abolished by administration of the adenosine A1/A2 receptor antagonist PD 115,199 (3 mg/kg i.v.) before brief antecedent coronary occlusion (protocol 3). Brief antecedent ischemia attenuates subsequent platelet-mediated thrombosis in damaged and stenotic canine coronary arteries, due, in large part, to an adenosine-mediated mechanism.

Effect of Shear Stress on Acute Platelet Thrombus Formation in Canine Stenosed Carotid Arteries: An In Vivo Quantitative Study.

We investigated the in vivo effect of percent stenosis, trans-stenotic pressure, and shear stress (SS) on platelet accumulation (PA) in canine mechanically injured and stenosed carotid arteries. In 10 dogs, intimal damage and controlled variations in stenosis were produced on the carotid artery. Blood flow through the stenosis, trans-stenotic pressure, and stenosis geometry were measured. A NaI gamma detector was collimated and placed over the stenosis to detect gamma rays emitted by autologous radiolabeled platelets as they accumulated inside the stenosis. The SS was obtained from the finite difference solution of the Navier-Stokes equations. As the flow declined during thrombus formation, the radioactive count accumulated in an inverse fashion. The rate of flow decline directly correlated with the rate of PA during thrombus formation (r(2) > 0.9). Compared with the undamaged and unstenosed artery, the PA increased by 52 +/- 34% due to mild stenosis (40-60%). PA increased by 94 +/- 66% due to severe stenosis (60-70%) and by 145 +/- 56% due to critical stenosis (70-80%; P > 0.01). The platelet accumulation produced totally occlusive thrombus formation at levels of stenosis higher than 70 +/- 5% (diameter narrowing), and for trans-stenotic pressure gradients higher than 50 +/- 5 mmHg producing SS greater than 100 +/- 10 Pa. The PA was maximum at the stenotic portion of the vessel where the level of SS is the highest (P < 0.001). In vivo platelet-mediated thrombosis increases with SS and occurs at the stenotic portion of the stenosis where the SS is the highest. Severe stenoses produce critical levels of SS that potentiate thrombosis and lead to life-threatening arterial occlusion.

Randomised placebo-controlled trial of effect of eptifibatide on complications of percutaneous coronary intervention: IMPACT-II

<h2>Summary</h2><h3>Background</h3> Platelet-mediated thrombosis has been implicated in the development of ischaemic complications of percutaneous coronary intervention. We investigated whether inhibition of the platelet glycoprotein IIb/IIIa integrin with eptifibatide (Integrilin) could prevent such complications. <h3>Methods</h3> We undertook a double-blind, placebo-controlled trial at 82 centres in the USA, enrolling 4010 patients undergoing elective, urgent, or emergency coronary intervention. Patients were assigned one of three treatments: placebo (n=1328), a bolus of 135 μg/kg eptifibatide followed by an infusion of 0·5 μg kg⁻¹ min⁻¹ for 20–24 h (n=1349), or 135 μg/kg eptifibatide bolus with a 0·75 μg kg⁻¹ min⁻¹ infusion (n=1333). The coronary procedure was started within 10–60 min of the start of study treatment. The primary endpoint was the 30-day composite occurrence of death, myocardial infarction, unplanned surgical or repeat percutaneous revascularisation, or coronary stent implantation for abrupt closure (by intention to treat). The primary safety endpoint was major bleeding. <h3>Findings</h3> By 30 days, the composite endpoint had occurred in 151 (11·4%) patients in the placebo group compared with 124 (9·2%) in the 135/0·5 eptifibatide group (p=0·063) and 132 (9·9%) in the eptifibatide 135/0·75 group (p=0·22). By treatment-received analysis, the 135/0·5 regimen produced a significant reduction in the composite endpoint (11·6 <i>vs</i> 9·1%, p=0·035), but the 135/0·75 regimen produced a less substantial reduction (11·6 <i>vs</i> 10·0%, p=0·18). Eptifibatide treatment did not increase rates of major bleeding or transfusion. <h3>Interpretation</h3> In the 135/0·5 group, treatment with eptifibatide during coronary intervention reduced rates of early abrupt closure and ischaemic events at 30 days. Non-significant differences were seen with the 135/0·75 regimen. The doses studied thus appear to be at the low end of the efficacy-response curve. Further investigation to refine eptifibatide dosing during coronary intervention is warranted.

High-dose heparin decreases nitric oxide production by cultured bovine endothelial cells.

Abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischemia. Endothelial NO is an important endogenous inhibitor of platelet-mediated thrombosis, yet biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. In an attempt to address the effect of heparin on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAECs) on microcarrier beads were incubated in the presence or absence of heparin. Results indicate that BAECs incubated with heparin were less able to inhibit platelet aggregation than control cells (P<.005 by ANOVA) and that this effect correlated with a decrease in NO production (36% decrease for heparin compared with control, P<.05). Dextran sulfate evoked the same response (67% decrease, P<.0001 compared with control), suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific polysaccharide sequence. The decrease in NO production by heparin was accompanied by a 72% decrease in steady-state Nos 3 mRNA as well as a 49% decrease in immunodetectable endothelial NO synthase (eNOS) protein. These data show that high-dose heparin at concentrations achieved in some acute cardiovascular settings increases in vitro platelet aggregation in media conditioned by endothelial cells by decreasing endothelial NO production through a mechanism that involves a decrease in steady-state Nos 3 mRNA and eNOS protein. These observations suggest a possible mechanism by which to explain in part the prothrombotic effects of heparin.

The Antithrombotic Effect of Aurin Tricarboxylic Acid in the Guinea Pig Is not Solely due to Its Interaction with the von Willebrand Factor-GPIb Axis

Commercial aurin tricarboxylic acid (ATA) has been reported to interfere specifically with von Willebrand factor-glycoprotein Ib (vWF-GPIb) axis. This study was designed to explore the antithrombotic effects of ATA by examining its effects on guinea pig platelet function in vitro, in vivo and ex vivo. In vitro, addition of various concentrations of ATA to platelet-rich guinea pig plasma totally inhibited ristocetin-induced platelet aggregation, as expected. Unexpectedly, however, ATA similarly inhibited the aggregation induced by ADP, PAF, collagen, I-BOP (a thromboxane A2/prostaglandin H2 analogue) and arachidonic acid. In vivo, the antithrombotic action of ATA was assessed in a model of acute platelet-dependent guinea pig mesenteric artery thrombosis triggered by laser-induced intimal injury. As the thrombotic response of arteries to such injury is a spontaneous cyclic recurrent process, 5 arteries in each animal were consecutively studied for 15 min each after i.v. bolus injection of 5, 7.5 or 10 mg/kg of ATA, which reduced the number of recurrent thrombi per artery in a dose-dependent manner. The highest dose of 10 mg/kg induced maximal inhibition of thrombus formation (72%, p < 0.001) 5 min after injection. Ex vivo, platelet aggregation was assessed in blood samples taken before and after i.v. bolus injection of 10 or 15 mg/kg ATA. Ten mg/kg significantly inhibited collagen-induced aggregation, and 15 mg/kg, the aggregation induced by ristocetin, ADP, PAF, collagen, I-BOP and arachidonic acid. The results of the in vivo studies confirmed that ATA is an effective antithrombotic agent. In the in vitro and ex vivo studies, ristocetin-induced platelet aggregation confirmed that ATA interacts with the vWF-GPIb axis, and suggests that the final common pathway of the aggregation induced by other agents tested consists of fibrinogen binding to the platelet GPIIb/IIIa receptor. We conclude that ATA interferes with vWF binding to GPIb, that it may interact with fibrinogen binding to GPIIb/IIIa, and that it might possess potent antithrombotic properties in platelet-mediated thrombosis.

Aspirin therapy for cardiovascular disease.

Aspirin is a widely used and effective antithrombotic agent. Recent studies suggest that aspirin's anti-inflammatory effects are mediated via inhibition of an inducible isoform of cyclooxygenase in inflammatory cells (COX-2) and through blockade of the nuclear transcription factor, NF-kappa B. The optimal dose of aspirin for most clinical situations is 75 to 325 mg/d, but debate continues as to whether higher doses are needed to prevent cerebral ischemia. Aspirin is very effective for inhibition of platelet-mediated thrombosis at sites of vascular injury but does not reduce restenosis after coronary angioplasty or carotid endarterectomy, nor does it prevent a first stroke. Combined therapy with warfarin and aspirin has been shown to limit systemic embolic in high-risk patients with artificial heart valves, but at the cost of increased bleeding. A new aspirin derivative is currently being developed that appears to stimulate platelet nitric oxide release, inhibit thrombin-induced platelet aggregation, and lower gastric toxicity.

Sex Differences in Platelet Adherence to Subendothelium: Relationship to Platelet Function Tests and Hematologic Variables

Men have significantly more atherosclerotic disease than women. Platelet-mediated thrombosis plays a role in the initiation of myocardial infarction and stroke. Citrated whole blood from male and female donors was perfused through an annular system over everted human umbilical artery segments. Comparisons were made between platelet adherence and thrombus formation on subendothelium, platelet aggregation in citrated whole blood, hematologic variables, and the bleeding time. Platelet spreading and adherence were approximately 22% greater with male blood (P < 0.001), whereas thrombus formation on subendothelium and collagen- and arachidonic acid-induced platelet aggregation did not show sex-related differences. Platelet aggregation with adenosine diphosphate was greater in women, related to their lower hematocrit values. By contrast, in women hematocrit values showed a slight but significant positive correlation with platelet adherence on subendothelium. Fibrinogen was significantly correlated with collagen- and adenosine-diphosphate-induced platelet aggregation and with platelet adherence, spreading, and thrombus formation on subendothelium. The mean bleeding time was slightly longer in women than in men (P = 0.118). Platelet aggregation was not associated with the bleeding time except for collagen-induced platelet aggregation in males; the latter was significantly correlated with platelet adherence and spreading in both sexes, while arachidonic acid-induced platelet aggregation predicted platelet adherence and spreading in males. Male blood shows enhanced primary hemostatic activity; this may predispose men to atherosclerosis.