Association of Src with GPIb is mediated by interaction of Src-SH3 with PI 3–kinase.

(A-B) Platelets pretreated with 1 μM cytochalasin D (Cyto D) were stimulated with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds). The platelet lysates without (lane 1, control; lane 2, after stimulation) or with (lane 3) p85 immunodepletion were examined for the presence of p85 by anti-p85 immunoblotting (A), and subjected to immunoprecipitation with anti-GPIb MoAb (B). The immunoprecipitates were resolved on SDS-PAGE and immunoblotted with anti-GPIb, anti-p85, and anti-Src antibodies, respectively. (C) Platelets were stimulated with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds), the lysates were precipitated with 20 μg/mL GST-Src-SH3 or GST alone. The precipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-GPIb and anti-p85 antibodies, respectively.