Real-Time Detection of Activation Patterns in Individual Platelets during Thromboembolism in vivo: Differences between Thrombus Growth and Embolus Formation

Abstract

Knowledge on single platelet behavior and intracellular mechanisms during thromboembolism in vivo is scarce. In the present study, we used a new method that enables real-time detection and quantification of activation of individual platelets participating in a thromboembolic process in vivo, using their intracellular free Ca²⁺ concentration ([Ca²⁺]_i) as a marker of activation. Isolated platelets were labeled with the Ca²⁺-sensitive fluorescent probe fluo-3 and injected into anesthetized rabbits so that 0.5–1% of their circulating platelets were labeled. Wall puncture of mesenteric arterioles resulted in thrombus formation followed by embolization. Fluorescence intensity changes of labeled platelets participating in this process were quantified. Within 30 min after injection, labeled platelets behaved similarly to native platelets, and fluorescence intensity was not influenced by dye leakage. Upon adherence to the stationary thrombus, platelets exhibited a prolonged [Ca²⁺]_i increase, accompanied by shape change and degranulation, which is consistent with a role for strong platelet agonists like collagen. In contrast, when platelets adhered to a growing embolus their [Ca²⁺]_i rise was transient, and they hardly showed shape change and degranulation, suggesting the involvement of weaker agonists like ADP. These results show, for the first time, the relation between single platelet activation patterns, which are different during thrombus growth and embolus formation, and their behavior in a thromboembolic process in vivo.