CVX activates Rap1 in platelets in a concentration- and time-dependent manner.

(A) Equal numbers of washed human platelets were stimulated with the indicated concentrations of CVX (left panel) or fibrillar type I collagen (right panel) for 1 minute. The platelets were lysed and GTP-bound Rap1 was precipitated with GST-RalGDS RBD bound to glutathione-agarose beads. The bound Rap1 was separated by SDS-PAGE and detected by immunoblotting with an anti-Rap1 mAb. (B) Washed human platelets were stimulated with 250 ng/mL CVX and equal aliquots were removed and lysed at the indicated time points. Active Rap1 was precipitated from the platelet lysates and detected as described in panel A. In lanes containing both total and GTP-bound Rap1, the presence of a second band may be phosphorylated Rap1, although no change in the ratio of these 2 bands was detected with CVX or collagen treatment in this study. Additionally, Woulfe et al find that inhibition of the protein kinase A pathway, which can phosphorylate Rap1, had no effect on Rap1 activation.38 These results were confirmed in 4 separate experiments.