Fibrin translocation to DRM raft fraction of human platelets by thrombin stimulation. (A) Sucrose density gradient analysis of proteins in washed human platelets. Resting platelets (left panel) and platelets stimulated for 3 minutes with 1 U/mL thrombin (right panel) were lysed in Triton X-100, and sucrose gradients (5% to 30%) were formed over them. Ten fractions were collected from top to bottom after centrifugation. The proteins were subjected to SDS-PAGE and stained with Coomassie brilliant blue (a). Immunoblotting of raft marker proteins with anti-CD36 antibody (b) and anti-Lyn antibody (c). A 40-kDa major protein was identified as actin by immunoblotting. (B) Two-dimensional PAGE analysis of DRM raft fraction (fraction 5) in resting platelets (a) and thrombin-stimulated platelets (b). The asterisk indicates actin. The arrowhead indicates integrin αIIb. (C) Immunoblotting with antifibrinogen antibody of panel A. (D) Time-dependent translocation of fibrin(ogen) by thrombin for 0 seconds (lane 1), 30 seconds (lane 2), 1 minute (lane 3), 5 minutes (lane 4), and 15 minutes (lane 5). Immunoblotting under nonreduced condition by antifibrinogen antibody of releasates (a), nonraft fraction (b), and DRM raft fraction (c). The asterisk indicates the fibrin polymer. (E) Immunoblotting with antifibrin specific antibody of DRM raft fraction (lanes 1 and 3), nonraft fraction (lanes 2 and 4) of resting platelets (lanes 1 and 2), and thrombin-stimulated platelets at 3 minutes (lanes 3 and 4). (F) Association of fibrin fiber with DRM of thrombin-stimulated platelets in immunoelectron microscopy. The DRM of thrombin-stimulated human platelets was incubated with an antifibrinogen antibody and then anti-IgG-labeled with colloidal gold. Scale bar, 200 nm. (G) Magnification of gold-attached area on DRM in panel F. The gold-positive fibrin fiber directly associated with the surface of DRM.