Abstract
Sustained elevation of intracellular concentration of calcium ion ([Ca2+]i) in platelets induced by several stimuli such as collagen leads to the exposure of phosphatidylserine (PS) from the inner leaflet of the platelet plasma membrane to the outer leaflet. The exposed PS provides a catalytic surface for interacting coagulation factors, which in turn converts prothrombin into thrombin via prothrombinase complex, resulting in fibrin clot formation. However, we have not yet known whether these processes on a thrombus occur in the exactly same way inside blood vessel because of difficult manipulation of platelets outside blood vessel without provocation of platelet activation before an experiment. To overcome this drawback, we attempted to visualize thrombus formation induced by laser and examine its mechanisms in the GFP-expressing mouse mesenteric vein using intravital microscopy. We employed GFP-labeled platelets as a marker of platelet aggregation and the exposed PS as an initial maker of coagulation. We monitored change in fluorescence intensity of GFP-labeled platelets and of alexa-568 labeled annexin V (alexa-ANX), a specific binding protein for PS. The change in fluorescence intensity of alexa-ANX spatiotemporally increases at the site of a laser-induced thrombus, suggesting that the PS-exposed platelets propagate from the initial site of thrombus formation. Thus, sustained [Ca2+]i elevation in platelets leads to the exposure to PS, thereby promoting fibrin clot formation inside blood vessel. [J Physiol Sci. 2007;57 Suppl:S86]
