Inhibitory effect of αIIbβ3 inhibitors on SIPA-associated protein tyrosine phosphorylation and p125FAK phosphorylation. (A) Platelets (2.8 × 10⁸/mL) were preincubated with MoAb 7E3 (20 μg/mL) or RGDS peptide (1 mM) for 5 minutes at room temperature, prior to exposure to 2B-rVWF (0.7 μg/mL) for 4.5 minutes at 37°C under high shear rates. Buffer indicates samples without 2B-rVWF. Lysates were prepared as described in “Materials and methods,” separated by an 8% polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with antiphosphotyrosine MoAbs 4G10 and PY20 (top). Membranes were then stripped and reprobed with anti–phospho-FAK polyclonal antibody (bottom). Data are representative of 3 experiments. (B) In separate experiments, platelets and 2B-rVWF, in the absence (top) or the presence (bottom) of the RGD-containing disintegrin kistrin at 50 nM, were exposed to shear and fixed with PFA. An aliquot was deposited in microtiter plates for microscopic observation of aggregates, as described in the legend to Figure 6C.