The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels

Abstract

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y 1 and P2Y 12 . The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y 1 receptors are involved in the initiation of platelet aggregation, P2Y 12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y 12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y 12 antagonists (2-methylthioadenosine 5 -monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require G i signaling or functional P2Y 12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving G s . However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP 2 receptors, which are known to couple to G s . Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y 12 receptors.Upon damage to the endothelial layer of the blood vessel wall, the underlying subendothelium is exposed to platelets in the blood, initiating a cascade of signaling events resulting in the transformation of "resting" platelets into "activated" platelets (1). One significant characteristic associated with these signaling events is the secretion of ADP from the platelet-dense granules (2). This released ADP acts to further amplify the platelet activation response by interacting with its G-protein-coupled receptors on the platelet surface, namely P2Y 1 (coupled to G q ) and P2Y 12 (coupled to G i ) (3-5). The consequence of platelet activation through ADP is a conformational change in the platelet membrane glycoprotein ␣IIb␤3 (6, 7), which then binds to fibrinogen present in the plasma. The binding of fibrinogen with ␣IIb␤3 on the surface of adjacent platelets results in fibrinogen-platelet cross-linking and the formation of a hemostatic plug at the site of vascular injury (8).Consequently, ADP is thought to play an integral role in the normal process of hemostasis. Of the two ADP-receptor signaling pathways in platelets, evidence has indicated that ADPmediated P2Y 12 signaling appears to play a more prominent role in platelet activation than ADP-media...

Multiscale prediction of patient-specific platelet function under flow

“…cAMP levels have been shown to strongly correlate with platelet activation regardless of whether the increase of cAMP was because of P2Y 12 inhibition or IP receptor stimulation. 38 Our results indicate that cAMP elevation is more effective than COX-1 or P2Y 1 inhibition under these conditions.…”

“…cAMP levels have been shown to strongly correlate with platelet activation regardless of whether the increase of cAMP was because of P2Y 12 inhibition or IP receptor stimulation. 38 Our results indicate that cAMP elevation is more effective than COX-1 or P2Y 1 inhibition under these conditions. The multiscale and patient-specific modeling presented here extends earlier modeling efforts of platelet function and/or coagulation 20,21,[39][40][41][42] by including a highly robust description of intracellular platelet signaling through GPVI, P2Y 1 , TP, and IP.…”

New P2Y 12 Inhibitors

“…28,29 A recent study suggested that cangrelor may also interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. 30 …”

Racial Differences in Resistance to P2Y12Receptor Antagonists in Type 2 Diabetic Subjects

“…Future studies will specifically address the influence of coronary artery disease and if African-American T2DM subjects display normal inhibition of thrombin-mediated responses to oral P2Y 12 inhibitors. One study has demonstrated that 2-methylthio-AMP is structurally distinct from the oral thienopyridine class of P2Y 12 -antagonists (e.g., clopidogrel) and has a different mechanism of action (Srinivasan et al, 2009), which limits the direct application of our finding to clinical practice. However, more recently it was reported that 2-methylthio-AMP does inhibit the P2Y 12 receptor (Xiang et al, 2012).…”

“…In this study, we specifically address the pharmacodynamic aspects of resistance to P2Y 12 antagonists in T2DM. To do so, we studied the effect of a direct P2Y 12 antagonist, 2-methylthio-AMP (2-methylthioadenosine 59-monophosphate triethylammonium salt) (Cusack and Hourani, 1982;Srinivasan et al, 2009;Xiang et al, 2012), in platelet activation.…”