Role of the FcR γ-chain in GP Ib-IX-V signaling. (A) Human platelets were plated on dA1VWF, BSA, or collagen (Coll) for 30 minutes. Platelets were lysed, and FcR γ-chain was immunoprecipitated. Western blots were probed for phosphotyrosine and reprobed for FcR γ-chain. As a control, lysate was immunoprecipitated with normal rabbit serum (NRS). (B) Washed platelets were prepared from transgenic mice expressing human GP Ibα (hGP Ibα/FcR γ^+/+) or from the same transgenic mice with a targeted deletion of the FcR γ-chain (hGP Ibα/FcR γ^-/-). After loading with calcium indicator dyes, cells were plated on dA1VWF and intracellular calcium concentrations were determined as in Figure 1. Results are representative of 4 experiments. (C) Role of FcR γ-chain in ADAP tyrosine phosphorylation. Human GP Ibα/FcR γ^-/- platelets were plated for 20 minutes on dA1VWF, BSA, or collagen. Cells were lysed, immunoprecipitated for ADAP, and Western blots were probed for phosphotyrosine and then reprobed for ADAP. This result is representative of 3 experiments. (D) To determine if there is a role for FcR γ-chain in αIIbβ3 activation, POW-2 Fab binding to mouse platelets was determined in the absence of an αIIbβ3 antagonist. Quantitative representation, as in Figure 4F, of POW-2 Fab binding is based on 4 separate experiments.