| Part I:Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems. In our previous study, we reported a chimeric human P2Y12receptor (cP2Y12) with high level of constitutive activity and identified a potent P2Y12inverse agonist AR-C78511. To investigate the role of constitutive activation of P2Y12receptor in platelet activation and thrombosis, transgenic mice conditionally and platelet-specifically expressing cP2Y12were generated using the tetracycline/doxycycline (Tet)-on system. In doxycycline-dosed transgenic mice, we detected high expression of cP2Y12in platelets and enhanced platelet reactivity, as evidenced by increased platelet aggregation in response to ADP,2MeSADP, AYPGKF, thrombin and U46619compared with wild type mice. The constitutive activity of cP2Y12in platelets of transgenic mice was confirmed by decreased intracellular cAMP in platelets in the absence of agonists; furthermore, AR-C78511reversed the decrease of cAMP. In addition, transgenic mice displayed shortened bleeding time, much more rapid and stable thrombus formation. Overall, these findings provide evidence supporting the prothrombotic role of constitutive activity of P2Y12receptor and validate the inverse agonist activity of AR-C78511on platelets expressing constitutively active P2Y12. Though constitutive activity of P2Y12receptor associated with receptor mutation has not been reported clinically, we have shown that P2Y12receptor expression is significantly enhanced in type â…¡ diabetes mellitus, which is associated with increased thrombogenicity. As a potent inverse agonist, whether or not AR-C78511has therapeutic advantage as antiplatelet agent in the prevention and treatment of thrombotic diseases in conditions associated with increased P2Y12receptor expression, such as type â…¡ diabetes mellitus, remains to be determined. Part â…¡:The roles of Gi and G12/13pathways in platelet aggregationPlatelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis. Arterial thrombotic diseases, such as heart attack and stroke, are the leading cause of morbidity and mortality in industrilized countries. Platelet activation triggered by atherosclerotic plaque disruption or endothelium injury caused by percutaneous coronary intervention (PCI) and the consequent intravascular arterial thrombogenesis is the common pathological basis of heart attack and stroke; therefore antiplatelet drugs are effective for prevention and treatment of coronary artery disease and stroke.G protein coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, constitute to a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. Numerous platelet agonists, such as adenosine diphosphate (ADP), thrombin, thromboxane A2, epinephrine, PAR1activating peptide SFLLRN, PAR4activating peptide AYPGKF, collagen, activate platelets directly or indirectly through the activation of GPCR, including Gq, Gi, and G12/13. The previous studies suggest that simultaneous activation of Gi and G12/13signaling pathway is sufficient to cause platelet aggregation. In this study we found that coactivation of G12/13and Gi pathways by stimulating platelets with SFLLRN, AYPGKF or thromboxane A2analogue (U46619) in combination with ADP,2MeSADP or epinephrine in the presence of Gq inhibitor YM-254890, is not sufficient to induce platelets aggregation in human, mouse and rat platelets when the agonists concentration were lower. In this study we found that coactivation of G12/13and Gi pathways only induces platelet aggregation under higher concentrations of agonists with higher levels of Akt and Erk phosphorylated. Our studies also indicated that platelet activation induced by coactivation of Gi plus G12/13pathway is independent of Ca2+, Src and Rho kinase. |
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