P2X(1) receptor inhibition and soluble CD39 administration as novel approaches to widen the cardiovascular therapeutic window.

Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclooxygenase inhibitor, and a thienopyridine such as clopidogrel, which inactivates the adenosine diphosphate-stimulated P2Y(12) receptor. Although these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial "resistance." Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X(1) receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at prothrombotic locations. Because both proteins affect platelet activation at an early stage in the events leading to thrombosis but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety.

Primary and secondary agonists can use P2X(1) receptors as a major pathway to increase intracellular Ca(2+) in the human platelet.

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca(2+) mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca(2+) responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca(2+)](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca(2+) response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca(2+) increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca(2+)](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.