Ultrastructural Research of Platelet Raft

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Previous studies have shown critical and functional role of membrane rafts in initiating signaling through platelet activating collagen receptor, the GPVI/FcRγ complex upon collagen stimulation. However, little is known about how GPVI-anchoring membrane rafts are regulated upon collagen stimulation. Since platelet cytoskeleton has been known to be involved in localization and accumulation of signaling molecules to critical locations within activated platelets, we examined effects of cytochalasin D (CytD) on morphological changes of membrane rafts in collagen-stimulated platelets. Washed platelets were pretreated with or without 100μM CytD for 1 hr at room temperature followed by addition of 1 mM RGDS and 1 mM CaCl_2 before stimulation and then stimulated with 2 mg/ml collagen for 30 sec at 37℃ under stirring conditions. After the reaction was stopped, membrane rafts were isolated as Brij98 detergent-insoluble membranes by a modification of previously described method to ensure more acc … More urate isolation of physiological raft structure. Immunogold Electron microscopic analyses of Brij98-insoluble rafts were performed and revealed vesicle structure of individual raft using negative-staining.When the diameters of approximately 200 vesicles of rafts in each sample were measured, those after collagen stimulation were significantly (p<0.01) enlarged than those before stimulation (160.97±65.38 vs 105.91±34.94 nm). Immunogold-stained particles of GPVI were also significantly (p<0.01) increased in rafts isolated after collagen stimulation than in those before stimulation (4.0±3.8 vs 1.6±2.1). Pretreatment of platelets with CytD before collagen stimulation almost completely inhibited all those changes observed in GPVI-anchoring membrane rafts after stimulation. Thus, present study indicates that GPVI-anchoring membrane rafts are enlarged possibly by fusing each other in cytoskeleton-dependent manner after collagen stimulation, thereby resulting in accumulation of GPVI in individual membrane raft. Less