Immunoprecipitation and biotinylation of TF in platelet membranes. (A) Platelet membrane TF in nonstimulated and TRAP-activated platelets for 30, 60, and 90 minutes was immunoprecipitated with the polyclonal α-TF antibody, and revealed with the α-TF MoAb. A TF band of approximately 47 kDa was detected, which was notably enhanced after activation. Lane 1 shows the lack of reaction when an irrelevant, nonspecific IgG replaced the polyclonal anti-TF antibody. Control hrTF migrated as an approximately 30-kDa major band and a minor band of approximately 60 kDa, and the TF bands disappeared when the anti-TF antibody was quenched with hrTF for 30 minutes before addition to the PVDF membrane, demonstrating the specificity of the reaction (not shown). (B) Resting and activated platelets were incubated with biotin for 30 minutes. The platelet membranes were immunoprecipitated with the polyclonal α-TF, and after electrophoresis and protein transfer, the PVDF membranes were revealed with streptavidin-HRP. Nonstimulated platelets express modest and nondistinctive TF-immunoreactive bands, which are markedly increased after TRAP stimulation.