Role of PLC-derived second messengers on integrin α₂β₁-dependent activation of Rap1b. (A) Washed platelets were left untreated (none) or incubated with the PKC inhibitor Ro31-8220, with the intracellular Ca²⁺ chelating agent BAPTA-AM, or with both compounds, as indicated in “Materials and methods.” Platelet adhesion to monomeric collagen was evaluated after 30 minutes of incubation and is reported on the top of the panel, considering as 100% the adhesion of untreated platelets. Activated Rap1b in each sample was isolated by the pulldown assay using lysates from an identical number of adherent platelets and identified by immunoblotting (Rap1b-GTP). The bottom panel shows the level of total Rap1b present in identical aliquots of the platelet lysates used for the pulldown assay. (B) Analysis of PLC activation. ³²P-labeled platelets were incubated with immobilized monomeric collagen for 30 minutes. Adherent platelets were lysed and total proteins separated by SDS-PAGE on a 5% to 15% acrylamide gradient gel. Phosphorylation of pleckstrin, the main platelet substrate for PKC, was visualized by autoradiography. The migration of phosphorylated pleckstrin is indicated by the arrow on the right, while the migration of molecular mass markers is reported on the left. Some samples of ³²P-labeled platelets were preincubated with Ro31-8220, BAPTA-AM, or with the indicated concentrations of U73122, as indicated on the bottom, and analysis of pleckstrin phosphorylation was performed on the same number of adherent platelets. (C) Inhibition of PLC prevents Rap1b activation. Washed platelets were treated with increased concentrations of the PLC inhibitor U73122 or with 10 μM U73343, an inactive related compound, and then incubated with immobilized monomeric collagen. The effect of treatment with U73122 on the extent of platelet adhesion and on the accumulation of active Rap1b in the same number of adherent platelets (Rap1b-GTP), as well as the level of total Rap1b in adherent cells (Rap1b total), are reported.