mTOR and S6K1 inhibition blocks Rac1 activation and platelet spreading. (A) Purified human platelets were treated with apyrase (2 units/mL) and vehicle (DMSO), Rapamycin (1μM), RAD001 (1 μM), WYE-354 (10μM), or Ku-0063794 (10μM) before spreading on a surface of fibrinogen. Lysates from surface-bound platelets were analyzed for S6K1 activation by Western blotting for S6K1-pThr389. (B) Western blot analysis of Syk-pTyr323, FAK-pTyr576/577, and GSK3β-pSer9 phosphorylation of fibrinogen-activated platelets pretreated with Rapamycin, RAD001, WYE-354, or Ku-0063794. (C) Lysates from surface-bound platelets were incubated with PAK-CRIB-GST to capture activated Rac1. Captured GTP-Rac1 and total Rac1 levels were determined by Western blot. (D) Representative DIC images of human platelets spread on coverglass coated with 100 μg/mL fibrinogen after treatment with apyrase and vehicle (DMSO), Rapamycin, RAD001, or WYE-354. Scale bar = 10 μm. Results are representative of 3 independent experiments.