FIG. 2.

Stimulation of platelets with α-thrombin (α-thr) and other platelet agonists leads to rapid activation of Ral. (A) Activation of Ral by α-thrombin. Human platelets were isolated as described previously (18) and stimulated with 0.1 U of α-thrombin per ml for different times. The platelets were lysed and incubated with 15 μg of GST-RalBD precoupled to glutathione beads to recover GTP-bound Ral. Beads were washed four times, and collected Ral was identified by Western analysis with a monoclonal anti-RalA antibody (upper panel). The lower panel shows the levels of Ral in the whole lysates. (B) Determination of RalGTP induction. Platelets were stimulated with α-thrombin for 1 min and Ral activation was determined as described for panel A. In order to estimate the increase in RalGTP induced by α-thrombin, the α-thrombin-induced sample (far left) was diluted severalfold so it could be compared to the resting platelets (––). The gel shows that the signal obtained after a 6- to 10-fold dilution of the RalGTP obtained from α-thrombin-stimulated platelets matches the amount of Ral detected in resting platelets. (C) Ral activation by different platelet agonists. Platelets were stimulated for 1 and 3 min with 0.1 U of α-thrombin per ml, thrombin receptor-activating peptide (TRAP, 10 μM), PAF (200 nM), TxA₂ analog U46619 (1 μM), or ADP (10 μM). Ral activation was analyzed as described for panel A. (D) Ral activation occurs independently of TxA₂ formation. Platelets were treated with 30 μM indomethacin for 10 min to inhibit TxA₂ formation, prior to stimulation with 0.1 U of α-thrombin per ml for the indicated times, or were stimulated with 0.1 U of α-thrombin per ml alone. At the concentration used, indomethacin inhibits TxA₂ formation (not shown). Activation of Ral was monitored as described for panel A.