Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells (1097.1)

Abstract

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases. Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445