Novel Agonists and Antagonists of Platelet Receptors

Abstract: Platelets are central for the primary haemostatis. The platelet has surface bound receptors that are vital for the platelet activation. One of these receptors, the purinergic ADP-activated G-protein coupled receptor (GPCR) P2Y12 is a key receptor for platelet activation, and thus for thrombus formation, which makes P2Y12 a target in pharmaceutical antiplatelet therapies. As a complement during revascularization surgery, mild hypothermia has been accepted, but its effect on platelet activation is unclear. We performed a comparative ex vivo study on platelets, treated with mild hypothermia and normothermia in the presence of P2Y12 inhibitors. We found that mild hypothermia increased platelet P2Y12 activation and that the P2Y12 inhibitor, clopidogrel, lost in efficiency. The P2Y12 receptor is also present on vascular smooth muscle cells (VSMC), promoting contractions. We initiated a comparative study on human and murine artery tissue to evaluate if any of the antiplatelet substances, clopidogrel and ticagrelor (AZD6140) a reversibly non-prodrug P2Y12 inhibitor, could inhibit P2Y12 signaling also on VSMC. The results showed that ticagrelor was able to inhibit P2Y12 stimulated vasoconstriction, while clopidogrel had no effect. The succinate GPCR (SUCNR1) mRNA has been found at high levels in platelets. We performed a study on SUCNR1 to investigate its function in platelets. The study showed that the succinate receptor was expressed as a membrane bound Gi-protein. Succinate per se could mediate platelet activation. It was dependent on P2Y12 activation, tromboxane A2 release and PI3K signaling. By screening intermediates in the mevalonate pathway, i.e. the cholesterol synthesis, we found that one of them, farnesyl pyrophosphate (FPP), had an antagonistic effect on ADP induced platelet activation. Further investigation on FPP with 35SGTPS binding and docking studies confirmed that FPP is an endogenous antagonist on the platelet P2Y12 receptor.