Regulation of Functionally Active P2Y12 ADP Receptors by Thrombin in Human Smooth Muscle Cells and the Presence of P2Y12 in Carotid Artery Lesions

Abstract

Objective-The platelet P2Y12 ADP receptor is a well-known target of thienopyridine-type antiplatelet drugs. This study is the first to describe increased transcriptional expression of a functionally active P2Y12 in response to thrombin in human vascular smooth muscle cells (SMC). Methods and Results-On exposure to thrombin, P2Y12 mRNA was transiently increased, whereas total protein and cell surface expression of P2Y12 were markedly increased within 6 hours and remained elevated over 24 hours. This effect was mediated by activation of nuclear factor B. Preincubation with thrombin significantly enhanced the efficacy of the P2Y receptor agonist 2-methylthio-ADP to induce interleukin 6 expression and SMC mitogenesis. Effects induced by 2-methylthio-ADP were prevented by RNA interference-mediated knockdown of P2Y12 and a selective P2Y12-antagonist R-138727, the active metabolite of prasugrel. In addition, positive P2Y12 immunostaining was shown in SMC of human carotid artery plaques and was found to colocalize with tissue factor, the rate-limiting factor of thrombin formation in vivo. Conclusion-These data suggest that the P2Y12 receptor not only is central to ADP-induced platelet activation but also may mediate platelet-independent responses, specifically under conditions of enhanced thrombin formation, such as local vessel injury and atherosclerotic plaque rupture. Key Words: atherosclerosis Ⅲ thrombin Ⅲ P2Y12 receptor Ⅲ inflammation Ⅲ vascular smooth muscle P 2Y12 ADP receptor antagonists, such as the thienopyridines, are widely used as antiplatelet drugs. 1,2 Besides their role as inhibitors of platelet aggregation, there is increasing evidence that these compounds also exert antiinflammatory actions. Reduced plasma levels of inflammatory markers, such as CD40 ligand, C-reactive protein, P-selectin, and platelet-leukocyte aggregates, have been shown with P2Y12 antagonists in atherothrombotic patients, 3 and platelet P2Y12 has also been shown recently to influence vessel wall responses to injury and thrombosis. 4 Thus far, these effects have been attributed solely to the antiplatelet action and have led to the concept that antiplatelet agents exert their antiinflammatory actions via platelet-related mechanisms. 5 The ADP receptor P2Y12 was originally identified in platelets and in the brain. 6 It is a member of the P2 receptor family, which consists of the ion-channel P2X and the G-protein-coupled P2Y receptors. 7 P2 receptors are activated by the adenine nucleotides ATP and ADP, which can be released from platelets, endothelial cells, sympathetic nerve terminals, and immune cells. 8 P2 receptors have been shown to be involved in a variety of inflammatory processes, such as allergen-driven lung inflammation. 9 In addition, the P2Y12 receptor has been associated with enhanced cell growth in certain brain tumors. 10 Recently, P2Y12 was found to be expressed in vascular smooth muscle cells (SMC), 11 and a role in vessel contraction was suggested. 12 However, no functional changes were seen after thienopyridine tr...

P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1

“…In cultured human vascular smooth muscle cells pretreated with thrombin, the agonist 2-methylthio-ADP induced mitogenic effects by an action on P2Y 12 receptors [50]. In agreement with these findings, 2-methylthio-ADP also increased the proliferation of cultured human coronary smooth muscle cells in our present experiments.…”

“…In contrast to the inhibition of proliferation due to the activation of P2X 1 receptors as observed in the present study, P2Y receptor subtypes including P2Y 2 , P2Y 6 , and P2Y 12 receptors mediate increases in proliferation of vascular smooth muscle cells [5,11,29,30,[46][47][48][49][50][51]. In cultured human vascular smooth muscle cells pretreated with thrombin, the agonist 2-methylthio-ADP induced mitogenic effects by an action on P2Y 12 receptors [50].…”

Roles of Purinergic Receptor P2Y, G Protein–Coupled 12 in the Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

“…12 More recently, several studies showed that P2Y12 is also expressed in vascular SMCs. 23,24 We examined the expression levels of P2Y12 in platelets, peripheral white blood cells (WBC), and aortic SMCs using quantitative real-time reverse transcription polymerase chain reaction to confirm the expression of P2Y12 in SMCs and evaluate P2Y12 expression in nonplatelet white cells. The results were unambiguous, and they demonstrated a much weaker expression of P2Y12 in aortic SMCs than in platelets (≈7% of P2Y12 level in platelets), and almost no expression of P2Y12 in peripheral blood white cells (≈0.5% of P2Y12 level in platelets; Figure II in the online-only Data Supplement).…”

P2Y 12 Promotes Migration of Vascular Smooth Muscle Cells Through Cofilin Dephosphorylation During Atherogenesis

“…Nevertheless, we provided substantial evidence which indicates that P2Y 12 may contribute to the progression of atherosclerosis at least in part by enhancing VSMCs migration from media to intima. Of note, in the study of Rauch et al, 10 direct stimulation of P2Y 12 receptor by its agonist 2-MeSADP did not increase human VSMCs proliferation. DNA synthesis was significantly enhanced only when 2-MeSADP was added after preincubation of cells with thrombin for 6 hours.…”