Abstract 15821: Mitogenic and Proinflammatory Actions of Thrombin-regulated P2Y12 ADP Receptor in Human Vascular Smooth Muscle Cells
The platelet ADP receptor P2Y12 contributes to activation of platelets upon vascular injury and is the molecular target of the thienopyridine anti-platelet drugs. Here, we describe for the first time that thrombin induces the transcription of P2Y12 in human vascular smooth muscle cells (SMC). The functional consequences involve an enhanced mitogenic and inflammatory response to thrombin in human SMC. P2Y12 was determined by RT-PCR/qPCR, Western blotting, flow cytometry, and chromatin immunoprecipitation (ChIP) in cultured human SMC from saphenous vein and aorta. Immunohistochemistry was performed in human carotid artery plaques. DNA synthesis was assayed by [3H]-thymidine incorporation, interleukin-6 (IL-6) expression by qPCR. Thrombin (3 U/ml) stimulated a 3-fold transcription of P2Y12 mRNA. Total protein and cell surface expression of P2Y12 were increased by 2-fold within 6 hours after thrombin addition and sustained over 24 hours. The specificity of the used P2Y12 antibodies was confirmed by comparison with platelet samples and by gene-specific knockdown with small inhibitory RNA (siRNA) in human SMC. Thrombin-induced transcription of P2Y12 involved activation of NFκB as determined by Chip assay and confirmed by the use of a specific NFκB activation inhibitor. Addition of the P2Y receptor agonist 2-methyl-thio-ADP (MeS-ADP; 1 μM) MeS-ADP after 6 hour preincubation with thrombin resulted in a significantly enhanced SMC mitogenesis compared to thrombin alone. This increased mitogenic response to thrombin was prevented by the active metabolite of the P2Y12-inhibitor prasugrel and by siRNA suggesting that this effect was P2Y12-mediated. Furthermore, MeS-ADP enhanced expression of IL-6 mRNA P2Y12-dependently in human SMC after pretreatment with thrombin. In human carotid artery plaques, P2Y12 immunostaining was positive and co-localized with smooth muscle actin and with tissue factor, the rate-controlling step in thrombin generation. In conclusion, thrombin increases transcription of the P2Y12 receptor in human SMC. P2Y12 may not only mediates ADP signaling in platelets but could be involved in mitogenic and inflammatory response in the vessel wall under conditions of enhanced thrombin formation such as acute coronary syndromes.
- © 2010 by American Heart Association, Inc.