Integrin α₂β₁-dependent platelet adhesion triggers Rap1b activation. (A) Rap1b activation in platelets adherent to different ligands for integrin α₂β₁ Human platelets were incubated with immobilized BSA, monomeric collagen, the small proteoglycan decorin, or the collagen-derived peptides CB8(II) and CB11(II), as indicated, for 30 minutes. Active Rap1b (Rap1b-GTP) was precipitated with immobilized GST-tagged RalGDS-RBD and identified by immunoblotting with anti-Rap1 antibody. An identical amount of proteins from each cell lysate was also subjected to immunoblotting analysis with the anti-Rap1 antibody to verify the level of the protein in the different samples (Rap1b TOT). (B) None of the analyzed ligands activate platelet GPVI. The GPVI-associated FcR γ-chain was immunoprecipitated from platelets adherent to decorin, CB8(II), CB11(II), monomeric collagen, and convulxin and analyzed by immunoblotting with antiphosphotyrosine antibody (P-Tyr). The same nitrocellulose membranes were then reprobed with anti-FcR γ-chain (bottom panel). (C) Time course of integrin α₂β₁-dependent platelet adhesion and Rap1b activation. Human platelets were incubated with the 4 different integrin α₂β₁ ligands for 15, 30, or 60 minutes, as indicated. The percentage of adherent platelets was determined by a colorimetric assay and is reported above the top panel. The immunoblots show active Rap1b (Rap1-GTP) isolated by the pulldown assay with GST-RalGDS-RBD and the level of total Rap1b (Rap1b TOT) present in the lysates used for the Rap1b activation assays.