G6B cross-linking inhibits platelet aggregation in a Ca²⁺-independent manner. (A) SHP-1 coimmunoprecipitates with G6B in platelets after stimulation with G6B antisera. G6B was coimmunoprecipitated (IP) with SHP-1 and is tyrosine phosphorylated after incubation of platelets with G6B antisera (lane 2). G6B was absent when platelets were incubated with preimmune sera (lane 3) or nothing (lane 1). SHP-1 coimmunoprecipitation was equivalent for all conditions. WB indicates Western blot. Platelet aggregation following treatment with CRP-XL (B) or ADP (C). In each panel, the black line represents agonist alone, the blue line represents agonist and the anti-G6B polyclonal antibody, and the red line represents agonist and preimmune sera. 1 corresponds to the addition of 5 μL of either the preimmune sera or the anti-G6B polyclonal antibody; 2 corresponds to addition of either CRP-XL (final concentration 0.1 μg/mL) or ADP (final concentration 5 μM). Traces are representative of at least 3 independent experiments. (D) Graph showing platelet aggregation in response to ADP and CRP-XL as a function of percentage total aggregation in the presence of the G6B polyclonal or the preimmune sera. The number of repeats for each condition is indicated in the parentheses. (E) Calcium flux in the presence of ADP and G6B polyclonal (blue) or preimmune sera (red). The arrows indicate the addition of the antisera (1) and agonist (2). (F) As for panel B but using ionomycin to induce aggregation. (G) The corresponding Ca⁺ trace after incubation with ionomycin. (H) Graph showing platelet aggregation as a function of percentage total aggregation in response to ionomycin. Each experiment was carried out the number of times shown in parentheses. Error bars in panels D and H indicate standard deviation about the mean.