S6K1 is activated upstream of Rac1 in platelets. (A) Representative DIC images of purified human platelets (2 × 107/mL) treated with vehicle (DMSO), the Src inhibitor PP2 (20μM), the Syk inhibitor BAY 61-3606 (1μM) or the Rac1 inhibitor EHT 1864 (50μM), on a surface of fibrinogen (FG). Scale bar = 10 μm. (B) Lysates from quiescent platelets in solution (basal) or FG-surface-attached platelets were analyzed for Src, Syk and FAK activation by Western blotting (WB) for Syk-pTyr323, LAT-pTyr171 and FAK-pTyr576/577. (C) Lysates were incubated with glutathione-sepharose conjugated to GST-PAK-CRIB to capture activated GTP-bound Rac1. Captured GTP-Rac1 and total Rac1 inputs were analyzed by Western blotting. (D) Platelets were treated with inhibitors as above and analyzed for S6K1 activation by Western blotting for S6K1-pThr389. PP2 and BAY 61-3606 decreased S6K1 phosphorylation by 86.9% and 66.9%, respectively (n = 3, P < .05). EHT 1864 increased pS6K1 levels by 121% relative to vehicle (n = 3, P < .05).
