Managing drug dosage is critical to the overall success of pharmaceutical intervention, not only in the clinic but also in clinical trials. Inadequate dose management of certain drugs can have fatal consequences; therefore, optimizing the dose of a drug has become a necessary component of successful clinical trial management.
One example of the necessity of dose management is with a class of drugs that is administered to inhibit blood clotting. The most popular thienopyridine, clopidogrel (commercially known as Plavix), irreversibly inhibits clotting by targeting the platelet ADP/P2Y12 receptor. Adenosine diphosphate (ADP) is a major in vivo platelet agonist and binds to the platelet ADP/P2Y12 receptor. Clopidogrel competes with ADP binding to this receptor.
Cardiologists reported in several clinical trials that patients exhibit a variable response to clopidogrel dosage, resulting in the need to modulate the dosage in order to increase the antithrombotic effect of the drug and potentially the bleeding risk during various surgical procedures.
There are a number of assays designed to assess platelet responsiveness to clopidogrel, one of which is the vasodilator-stimulated phosphoprotein (VASP) flow cytometry assay developed by BioCytex/Diagnostica Stago. The use of such assays has demonstrated interpatient variability in drug responsiveness not only to clopidogrel but also to prasugrel
VASP Biology
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.