Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse.

Platelet accumulation at sites of vascular injury arrests bleeding but also plays a critical role in the pathogenesis of thrombosis, leading to ischemia in myocardial infarction or stroke. Intracellular calcium mobilization in platelets is a critical step in the activation of platelets and formation of the platelet thrombus. Here we show the relationship of the dynamics of intracellular calcium mobilization with platelet accumulation into the developing thrombus in a living mouse. Following injection of 100 x 10⁶ fura-2 loaded platelets into a living mouse we used high speed intravital multi-channel digital fluorescence microscopy to monitor calcium status in circulating and thrombus-bound platelets during thrombus development. One population of platelets binds transiently to the developing thrombus but does not mobilize calcium. The mean duration of platelet-thrombus interaction for these platelets is 11 sec. Another population of platelets undergoes calcium mobilization after binding to the developing thrombus. The time interval from attachment to calcium mobilization for individual platelets varied from 1.0 to 12 sec, with a median of 3.5 sec. More than 90% of platelets that undergo calcium mobilization do so with in 5 sec of attachment. The calcium mobilization in the thrombus bound platelets is reversible. About two thirds of the platelets return rapidly to the basal Ca²⁺ state while the remaining thrombus bound platelets maintain an elevated Ca²⁺ level for an extended period. The mean duration of platelet-thrombus interaction is 35 sec with a range of 1.5 sec to 284 sec (median duration 39.5 sec) as calculated from multiple independent observations of single platelets. In each platelet studied, only one calcium peak is detected per platelet. There is a close correlation between the duration of calcium mobilization in an individual platelet and the time that the platelet remains attached to the developing thrombus, suggesting a relationship of calcium-dependent events and platelet-thrombus affinity. A population of platelets binds to the thrombus, mobilizes calcium and remains associated with the thrombus. Using widefield deconvolution techniques to obtain planar images and increased numbers of dye-loaded platelets, individual platelets could be observed undergoing sustained calcium elevation within the thrombus. As the platelet thrombus reaches maximal size at about 120 sec, calcium mobilization continues in the stable core of the thrombus for several minutes, then decreases. These studies describe thrombus formation in a living animal under conditions in which the endothelium and vessel wall, blood cells and plasma components, and flowing blood are preserved in the absence of anticoagulants. Our results indicate that stable platelet thrombus formation is dependent upon durable calcium mobilization, and that intracellular calcium regulates thrombus development and maturation in vivo.