CLEC-2 deficiency impairs GPIbα signaling. (A) Akt, (B) p38 MAPK, and (C) Lyn activation in platelets stimulated with human plasma VWF and botrocetin, which facilitates VWF binding to GPIbα. Washed platelets were preincubated with dimethyl sulfoxide (DMSO) or 10 μM of PP2, an inhibitor of Src family kinases, at room temperature for 30 minutes and then stimulated with 2 μg/mL of botrocetin with or without 10 μg/mL of VWF for 5 minutes. Then platelets were lysed, and activation of Akt and p38MAPK was analyzed by western blotting. P-Akt, P-p38, and P-Lyn are the activated kinases. (D) Aggregation of PP2-treated platelets in response to botrocetin and VWF. Washed platelets were pretreated with DMSO or 10 μM of PP2 at room temperature for 30 minutes, and then aggregation of WT and CLEC-2–deficient platelets in response to botrocetin and VWF was observed. (E) Quantification of platelet aggregation shown in (D). (F) Flotation assay of lipid raft localization of GPIbα. Washed platelets were lysed with 1% triton X-100, and the lysate was applied to the top of 5% to 40% gradient of Optiprep. After centrifugation, the distribution of GPIbα, Lyn, and β actin in fractions from top to bottom of the gradient was analyzed by western blot. (G) Quantification of GPIbα that located in lipid rafts shown in (F). The data are representative of 5 independent experiments.