Full CVX-mediated Rap1 activation is dependent on ADP but not TXA₂ signaling and specifically requires the P2Y₁₂ ADP receptor in human platelets.

(A) Washed human platelets were pretreated with dimethyl sulfoxide (DMSO; control) or the indicated concentrations of the cyclooxygenase inhibitor indomethacin for 15 minutes. CVX (250 ng/mL) was then added for 1 additional minute. (B) Washed human platelets were pretreated for 15 minutes with Tyrode buffer (control and no inhibitor), the specific P2Y₁₂ ADP receptor antagonist 2-MeSAMP (50 μM), or the specific P2Y₁ ADP receptor antagonist A3P5P (100 μM) prior to 1 minute of stimulation with either 250 ng/mL CVX (top panel) or 20 μM ADP (bottom panel). Control lanes are without agonist stimulation. The presence of active Rap1 in treated platelet lysates in panels A and B was detected as described in Figure 1. Note the dramatic inhibition of Rap1 activation by 2-MeSAMP pretreatment. These results were confirmed in 4 separate experiments.