Upregulation of functionally active P2Y12 ADP receptor by thrombin in human vascular smooth muscle cells

Dr. Rauch, Bernhard H.1, Driessen, Julia1, Ermler, Swen1, Böhm, Andreas1, Dr. Rosenkranz, Anke C.1, Prof. Dr. Fischer, Jens W.1, Dr. Jakubowski, Joseph A.2, Dr. Sugidachi, Atsuhiro3, Prof. Dr. Schrör, Karsten1. 1Universitätsklinikum Düsseldorf Institut für Pharmakologie und Klinische Pharmakologie, Universitätsstr. 1, 40225 Düsseldorf, Germany ; 2Eli Lilly and Company, Indianapolis, United States; 3Daiichi Sankyo Co., Ltd., Tokyo Japan.

ADP contributes to activation of platelets upon vascular injury via activation of the P2Y12 receptor - the molecular target of the thienopyridine antiplatelet drugs. Recently, expression of P2Y12 has been observed in vascular smooth muscle cells (SMC). However, its function remains unclear. Here, we describe the transcriptional regulation of P2Y12 by thrombin and the functional consequences in human SMC.

Experiments were performed in cultured SMC prepared from human saphenous vein. Expression of P2Y12 was determined by RT-PCR/qPCR, Western blotting, flow cytometry, and immunohistochemistry. DNA synthesis was assayed by [3H]-thymidine incorporation. A marked upregulation of P2Y12 mRNA expression (about 3-fold) was observed by thrombin (3 U/ml). Total protein and cell surface expression of P2Y12 were increased within 6h by thrombin (2.5 and 1.5 fold, respectively) and remained elevated over 24h. This regulation was dependent on NFκB. The P2Y receptor agonist 2-methyl-thio-ADP (MeS-ADP; 1 µM) did not affect basal or thrombin-induced mitogenesis if given simultaneously with thrombin. Addition of MeS-ADP after 6h preincubation with thrombin resulted in a 30% increase in mitogenesis which was completely prevented by the selective P2Y12-antagonist R-138727, the active metabolite of prasugrel, suggesting that this effect was P2Y12-mediated. Furthermore, MeS-ADP-induced enhanced expression of IL-6 mRNA P2Y12-dependently after pretreatment with thrombin. In addition, positive P2Y12 immunostaining was observed in human carotid artery plaques and co-localize with tissue factor, the rate-controlling step in thrombin generation.

In conclusion, thrombin induces expression of P2Y12 receptor in human SMC resulting in an enhanced mitogenic and proinflammatory response. P2Y12 receptors are not only important for ADP mediated platelet function but may be involved in control of vascular events under conditions of enhanced endogenous thrombin formation, such as acute coronary syndromes.