Analysis of platelet adhesion and integrin α₂β₁-dependent outside-in signaling in CalDAG-GEFI–deficient murine platelets. Platelets from wild-type (+/+) and CalDAG-GEFI–deficient (–/–) mice were incubated with immobilized monomeric collagen. Platelet adhesion (A) and spreading (B) were measured using collagen-coated glass coverslips and determined by fluorescence microscopy analysis upon staining of adherent platelets with TRITC-phalloidin. The reported results are the mean ± SD of 3 independent experiments. The statistical significance of the observed differences, calculated by the Student t test, is also shown. PLCγ2 tyrosine phosphorylation, pleckstrin phosphorylation, and Rap1b activation were analyzed in samples containing identical amount of proteins from platelets adherent to collagen-coated 60 mm–diameter dishes as well as from nonadherent platelets, indicated as A (adherent) and NA (nonadherent) on the top of panels C, D, and E. (C) PLCγ2 was immunoprecipitated with specific antibody and then analyzed by immunoblotting with antiphosphotyrosine antibody (P-Tyr), followed by immunoblotting with anti-PLCγ2 antibody, as indicated on the right. (D) Activation of PKC was analyzed on total platelet proteins from adherent and nonadherent platelets by immunoblotting with antiphosphoserine PKC substrates. The position of phosphorylated pleckstrin (47 kDa) detected in adherent but not in nonadherent platelets is indicated by the arrow on the left. On the right, the position of molecular mass markers is reported. (E) Analysis of Rap1b activation (Rap1-GTP) in the same number of adherent and nonadherent platelets, as well as the total amount of Rap1 in the cell lysate used (Rap1b-TOT), are reported. Results from panels C, D, and E are representative of 2 independent experiments. (F) The specific binding of biotinylated fibrinogen to the same number of wild-type (+/+) and CalDAG-GEFI–deficient (–/–) murine platelets adherent to monomeric collagen through integrin α₂β₁. Fibrinogen binding measured in adherent wild-type platelets is reported as 100%, and the data are the means ± SD of 3 separate experiments.