Activation of Rac and PAK2 in platelets.

(A) Human platelets were stimulated with thrombin (1 U/mL) or the GPVI agonist convulxin (5 μg/mL), and Rac assay lysis buffer added at the indicated time points. Active Rac was pulled down with a GST-fusion protein containing the Rac binding domain of PAK1 and detected by immunoblotting for Rac. (B) Human platelets were stimulated with 3 μg/mL CRP in the presence and absence of apyrase (2 U/mL) and indomethacin (10 μM) as indicated. Active Rac was pulled down and blotted as above. (C) Human platelets, where indicated, were incubated with the PI 3-kinase inhibitors, wortmannin (100 nM), or Ly294002 (20 μM), or the Src kinase inhibitor PP1 (10 μM) followed by thrombin (1 U/mL, 30 seconds). Activation of Rac was measured as described above. (D) PAK2 activity in human platelets was measured using an in-gel kinase assay with MBP as the substrate. Platelets were activated with CRP (1 μg/mL) or thrombin (1 U/mL) and lysis buffer added at the indicated times. PAK2 immunoprecipitates were separated on SDS-PAGE gels containing MBP, the gels dried, and PAK2 activity detected by autoradiography. (E) The effect of wortmannin, Ly294002, and PP1 on thrombin- (1 U/mL, 90 seconds) and CRP-induced (1 μg/mL, 180 seconds) PAK2 activity was determined as described above. The autoradiograph for CRP activation of PAK2 is a longer exposure due to the weaker level of activity. (F-G) Rac (F) and PAK2 (G) activity was measured from control and Vav1-deficient mouse platelets as described above. Each panel depicts one experiment representative of at least 3 independent experiments.