Role of PI3Kβ and Pyk2 in integrin α2β1–mediated Rap1b stimulation and integrin αIIbβ3 activation. (A) Analysis of Rap1b activation. Active GTP-bound Rap1b was precipitated from platelets that had been allowed to adhere to monomeric collagen for 60 minutes after incubation with DMSO (none), wortmannin (100nM, 15 minutes), TGX-221 (0.5μM, 10 minutes), or AS252424 (0.5μM, 10 minutes), as indicated on the bottom. A representative immunoblot is reported in panel i, where the top row shows the active form of Rap1b and the bottom row the level of total Rap1b present in the platelet lysates. Quantification of Rap1b activity was performed by densitometric analysis of the immunoblots, and the results are reported in panel ii. The amount of active Rap1b in adherent DMSO-treated platelets was taken as 100%. Data are the means ± SD of 3 different experiments. *P < .05. (B) Comparative analysis of Rap1b activation in wild-type (WT) and Pyk2-deficient platelets (Pyk2 KO) after adhesion to monomeric collagen for 60 minutes. Both a representative immunoblot (i), and quantitative analysis (ii) of Rap1b activation are shown. Data in panel ii are the means ± SD of 3 different experiments. *P < .05. (C) Analysis of specific binding of biotinylated fibrinogen to adherent platelets. The effect of preincubation of platelets with wortmannin (100nM, 15 minutes), or TGX-221 (0.5μM, 10 minutes) is reported in panel i, where the binding of fibrinogen to DMSO-treated control platelets was taken as 100%. The comparative binding of fibrinogen to adherent platelets from wild-type (WT) or Pyk2-knockout (Pyk2 KO) mice is reported in panel ii. In both cases, data are the means ± SD of 4 different experiments. *P < .05; ***P < .001.