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Knowledge on single platelet behavior and intracellular mechanisms during thromboembolism in vivo is scarce. We developed a new method that allows real-time detection and quantification of activation of individual platelets participating in a thromboembolic process in vivo, using their intracellular free Ca2+ concentration as a marker of activation. Blood platelets were isolated, labeled with the calcium-sensitive fluorescense probe fluo-3 and injected into anesthetized rabbits, in which a thromboembolic reaction was induced in mesenteric arterioles by wall puncture. Upon adherence to the stationary thrombus, platelets exhibited a prolonged incease in [Ca2+], accompanied by shape change and degranulation, indicating a high level of activation. Strong platelet agonists like subendothelial collagen, which is exposed during puncture, are probably responsible. In contrast, when platelets adhered to a growing embolus their [Ca2+] rise was transient and they hardly showed shape change and degranulation, suggesting a role for weaker agonists like ADP. Indeed, blockade of one of the platelet ADP receptors, P2Y1, led to a reduction in the platelet Ca2+ response, and also to reduced aggregate formation during embolization. These data illustrate the relation between single platelet activation patterns, which appear to be different during thrombus growth and embolus formation, and their behavior in a thromboembolic process in vivo. Current and future experiments will further elucidate the mechanisms underlying thromboembolism, as well as the contribution of activating and inhibiting mediators involved.
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J. Vasc. Biol. 42, Sup:2 (2005) p79