Figure 2.

Stimulation of murine platelets on a fibrinogen-coated surface induces αIIbβ3-dependent tyrosine phosphorylation of SLP-76. Washed murine platelets were incubated for 45 min at 37°C on Petri dishes coated with 5 mg/ml BSA (lane 1) or 100 μg/ml human fibrinogen (lanes 2–4) in the absence (lanes 1 and 2) or presence of 1 unit/ml thrombin (lane 3) or 1 unit/ml thrombin plus 10 mM EDTA to block fibrinogen binding to αIIbβ3 (lane 4). Nonadherent platelets were isolated by centrifugation (5 sec, 16,000 × g) and then lysed (lanes 1 and 4). Fibrinogen-adherent platelets were lysed directly on the Petri dishes (lanes 2 and 3). Equal amounts of protein from each lysate were subjected to immunoprecipitation with anti-murine SLP-76 followed by Western analysis for phosphotyrosine. Blots were reprobed with anti-murine SLP-76 to demonstrate equal amounts of immunoprecipitated SLP-76 in each lane.