Lyn is required for PECAM-1 phosphorylation and SHP-2 binding in GPVI-stimulated platelets. Platelets isolated from wild-type (Lyn^+/+) or Lyn-deficient (Lyn^−/−) mice were left unstimulated (Resting) or were stirred in the presence of CRP to achieve 20% or 100% aggregation. (A) Lysates were prepared and immunoblotted with antibodies specific for the phosphorylated form of the C-terminal PECAM-1 ITIM tyrosine residue at position 686 (PECAM-1 pY686), PECAM-1 to show equal antigen loading, or Lyn to show the presence and absence of Lyn in wild-type and Lyn-deficient platelets, respectively. Note that PECAM-1 is tyrosine phosphorylated in CRP-aggregated wild-type but not in Lyn-deficient platelets. (B) Lysates were subjected to IP with normal mouse IgG (NMIgG) as a negative control or with anti–SHP-2. Lysates and IP lysates were immunoblotted with antibodies specific for PECAM-1 or SHP-2. Note that PECAM-1 coimmunoprecipitates with SHP-2 from CRP-aggregated wild-type but not Lyn-deficient platelets. Results shown are representative of 3 independent experiments.