The ADP/P2Y12 receptor is a seven-transmembrane domain receptor and is biophysically linked to an inhibitory G-protein (Gi). Signaling through this receptor leads to lower cyclic adenosine monophosphate (cAMP) levels and inactivation of protein kinase A (PKA), one of the substrates of which is VASP. Also feeding into this pathway is P2Y1 receptor-mediated activation of platelets. In contrast, the inhibiting prostanoid prostaglandin E1 (PGE1) leads to higher levels of cAMP and activation of PKA.
The mechanism of signaling through this pathway is as follows: when platelets that are not treated with an anti-P2Y12 drug are incubated in vitro with PGE1, VASP is phosphorylated and platelets are inhibited. In contrast, in platelets left untreated, but incubated with PGE1 plus ADP, VASP is dephosphorylated and the platelets are activated. However, in platelets treated with an anti-P2Y12 drug and then incubated with PGE1 alone or in combination with ADP, VASP is constitutively phosphorylated and the platelets are inactivated (Figure).
This occurs due to the fact that the anti-P2Y12 drug irreversibly or reversibly binds to the ADP/P2Y12 receptor, thus preventing ADP from binding to its receptor and inhibiting the dephosphorylation of VASP.
In other words, the binding of clopidogrel, or any other P2Y12-targeting drug, to the platelet P2Y12 receptor results in direct inactivation of the intracellular signaling cascade that ultimately inactivates the platelet’s ability to form a clot. VASP is a reliable reporter of the platelet’s ability to be activated by ADP, and the degree to which VASP is phosphorylated can be measured in a direct, cost-effective manner.