Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. Although exogenously derived ROS are known to affect the regulation of platelet activation, recent data suggest that the platelets themselves generate ROS. Intracellular ROS signaling in activated platelets could be of significant relevance after transient platelet contact with the vessel wall, during the recruitment of additional platelets, and in thrombus formation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Reactive oxygen species: players in the platelet game.

Adhesion molecules and atherosclerosis

F. Vinyl Sulfones and Other Michael Acceptors 4683 G. Azodicarboxamides 4695 IV. Acylating Agents 4695 A. Aza-peptides 4695 B. Carbamates 4699 C. Peptidyl Acyl Hydroxamates 4700 D. â-Lactams and Related Inhibitors 4704 E. Heterocyclic Inhibitors 4714 1. Isocoumarins 4715 2. Benzoxazinones 4722 3. Saccharins 4725 4. Miscellaneous Heterocyclic Inhibitors 4728 V. Phosphonylation Agents 4728 A. Peptide Phosphonates 4728 B. Phosphonyl Fluorides 4734 VI. Sulfonylating Agents 4735 A. Sulfonyl Fluorides 4735 VII. Miscellaneous Inhibitors 4736 VIII. Summary and Perspectives 4737 IX. Acknowledgments 4740 X. Note Added in Proof 4740 XI. References 4740

Irreversible inhibitors of serine, cysteine, and threonine proteases.

Neutrophils respond to myocardial ischemia-reperfusion in a manner similar to the bacterial invasion of a host. The inflammatory-like response that follows the onset of reperfusion involves intense interactions with the coronary vascular endothelium, arterial wall, and cardiomyocytes in a very well-choreographed manner. Neutrophils have been implicated as primary and secondary mediators of lethal injury after reperfusion to coronary vascular endothelium and cardiomyocytes. The involvement of neutrophils in the pathogenesis of lethal myocardial injury has been inferred from (1) their presence and accumulation in reperfused myocardium in temporal agreement with injury induced, (2) the armamentarium of toxic agents such as oxidants and proteases that are released by neutrophils in reperfused myocardium, (3) responsivity to (recruitment by and/or activation by) inflammatory factors released by reperfused myocardium, and (4) inhibition of lethal post-ischemic myocyte or endothelial cell injury by strategies that interdict neutrophil interactions at any number of stages. However, whether neutrophils are directly involved in the pathogenesis of lethal reperfusion injury in the myocardium, are just pedestrian (first) responders to inflammatory signals released after the onset of reperfusion, or are important to an early but not clinically important phase of pathology are still points of controversy. As with the general area of myocardial protection itself, the failure to reproduce the salubrious effects of anti-neutrophil therapeutic strategies and to successfully translate these strategies into clinical practice has not only fueled the debate, but has jeopardized the further pursuit of myocardial protection therapeutics to improve post-ischemic outcomes. This review will describe the molecular responses of neutrophils to ischemia-reperfusion, discuss the cellular and tissue damage inflicted either directly or indirectly by these white cells, and discuss the physiological impact of interdiction of neutrophil-mediated interactions with myocardial cells at various levels on lethal post-ischemic injury. In addition, it will discuss the arguments for and against the involvement of neutrophils in responses to ischemia-reperfusion in experimental models, and the failure to translate experimentally successful therapy into clinical practice.

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Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury

.Whilst there is increasing realization that inappropri-ate platelet activation plays a prime role in the increas-ing heart disease burden of society, there is still nogenerally accepted ideal measure of platelet activationthat would indicate a state of ‘high risk’. There is also aneed for the objective assessment of the relative eﬀec-tiveness and safety of antiplatelet agents. Furthermore,despite the great success of aspirin, it is clearly far fromperfect

Platelet activation: assessment and quantification

BACKGROUND: P-selectin, an integral membrane glycoprotein of platelets and endothelial cells, is rapidly redistributed to the cell surface after cellular activation. The soluble form of P-selectin has been shown to be present in people with normal circulation. The purpose of the present study was therefore to examine the soluble form of P-selectin in patients with acute myocardial infarction (AMI). METHODS: Whole blood was obtained from nine patients with AMI and from 10 volunteers who made up the control group. Plasma concentrations of the soluble form of P-selectin were examined with a monoclonal antibody-based enzyme immunoassay. RESULTS: Plasma P-selectin levels in control volunteers were 178 +/- 44 ng/ml. In patients with AMI, plasma P-selectin levels on days 1, 2 and 3 were 743 +/- 374, 627 +/- 267, and 588 +/- 223 ng/ml, respectively (P < 0.001)--significantly higher than the levels in the control volunteers. CONCLUSION: Plasma concentrations of the soluble form of P-selectin were markedly higher in patients with AMI, suggesting the activation of platelets, or endothelial cells, or both. Thus, quantitative measurements of plasma P-selectin concentrations may help to assess the pathophysiology of inflammatory reactions in patients with AMI.

Soluble form of P-selectin in patients with acute myocardial infarction.

Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries.

Neutrophil activation after percutaneous transluminal coronary angioplasty

We investigated the hypothesis that xanthine oxidase (XO) mediates platelet aggregation and cyclic flow variations (CFVs) in stenosed canine coronary arteries. CFVs were produced by an external constrictor placed at the site of the coronary artery with the injured endothelium. The severity of CFVs was evaluated by a pulsed Doppler flow probe. If CFVs developed, dogs intravenously received allopurinol, a specific XO inhibitor. The transcardiac gradient (difference between coronary vein and left atrium) of purine metabolites was determined during CFVs and after allopurinol administration. Allopurinol significantly reduced CFVs (from 8 +/- 1 to 1 +/- 1 cycles/h, P < 0.01, n = 14), whereas saline did not (from 8 +/- 1 to 7 +/- 1 cycles/h, n = 7). In seven dogs with CFVs, the transcardiac gradient of xanthine and uric acid concentrations significantly increased after the establishment of CFVs and significantly decreased after the administration of allopurinol. In vitro platelet studies showed that XO enhanced (from 30.9 +/- 2.0 to 47.6 +/- 1.5%, P < 0.0001, n = 10) and allopurinol inhibited ADP-induced platelet aggregation (from 48.3 +/- 1.3 to 24.8 +/- 1.5%, P < 0.0001, n = 10). Our results indicate that allopurinol inhibits platelet aggregation in vitro and provides a protection against CFVs in vivo. Thus XO may be an important mediator in this model.

Xanthine oxidase mediates cyclic flow variations in a canine model of coronary arterial thrombosis

To explore the role of calcium-dependent protease in the stunned myocardium, open-chest dogs underwent 15 min of left anterior descending coronary artery occlusion followed by 2 h of reperfusion. Dogs received a single bolus intravenous injection of either the protease inhibitor NCO-700 (n=6) or saline (n=6) 1 min before reperfusion followed by a 30-min infusion at the same dose. Regional myocardial function was assessed in terms of systolic wall thickening with an epicardial Doppler probe. The two groups exhibited comparable systolic thickening under baseline conditions and similar degrees of dyskinesis during occlusion. After reperfusion, recovery of contractile function, expressed as a percentage of the baseline value, was significantly greater in NCO-700-treated dogs as than in control dogs: -14.3±10.6 vs -48.9±7.2 (p<0.05) at 15 min, 10.8±10.3 vs -31.1±9.0 (p<0.05) at 30 min, 42.5±10.1 vs -16.4±9.1 (p< 0.005) at 1 h, and 47.5±8.3 vs -14.9±9.4 (p<0.001) at 2 h. The data suggest that the protease inhibitor markedly improved contractile function in stunned myocardium by inhibiting intracellular protease activity.

Tameo Oda

Papers

Soluble form of P-selectin in patients with acute myocardial infarction.

Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries.

Neutrophil activation after percutaneous transluminal coronary angioplasty

Xanthine oxidase mediates cyclic flow variations in a canine model of coronary arterial thrombosis

A protease inhibitor, NCO-700, improves the contractile function in stunned canine myocardium.