Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus.Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets.Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice.In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation.In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited.The kinetics of platelet activation and platelet accumulation were similar in FcRγ -/-mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice.Our results indicate that the tissue factor-mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed.In the tissue factor-mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo. Results Incorporation of fura-2-loaded platelets into the developing thrombus.Fura-2/AM is a calcium ion-binding fluorochrome used to measure intracellular calcium mobilization.Fura-2 is characterized by distinct spectral properties in the presence of low and high concentrations of Ca 2+ .The binding constant, K d , of fura-2 for Ca 2+ is 0.14 μM, and thus this fluorochrome is sensitive to changes in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) when, in the resting state, Ca 2+ concentrations are significantly lower than 0.14 μM while in the activated state, Ca 2+ concentrations are significantly higher than 0.14 μM.Ca 2+ binding to fura-2 results in a shift in the absorbance maximum from 380 nm to 340 nm.The magnitude of fluo-Nonstandard abbreviations used: [Ca 2+ ]i, intracellular Ca 2+ concentration; GPIb, glycoprotein Ib.