Cyclic stretch-induced thrombin generation by rat vascular smooth muscle cells is mediated by the integrin αvβ3 pathway

Abstract

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a pivotal role in atherothrombotic diseases. Thrombin generation at the surface of VSMCs and activation of integrin mechanotransduction pathways represent potential mechanisms. Here, we examine whether mechanical stretch increases thrombin generation on cultured rat aortic VSMCs. The integrin α(v)β(3) antagonist peptide (cRGDPV) dose-dependently decreased thrombin generation without stretch. Static stretch (5%, 1 Hz) failed to modify the thrombin-forming capacity of VSMCs, whereas 10% cyclic stretch during 60 and 360 min enhanced integrin α(v)β(3) expression and thrombin generation at the surface of VSMCs by 30% without inducing apoptosis. Cyclic stretch also stimulated Src phosphorylation, cleavage of talin, and binding of prothrombin to VSMCs. Upregulation of α(v)β(3) expression, Src phosphorylation, and enhanced thrombin generation by cyclic stretch were abolished by cRGDPV and silencing RNA (siRNA) against α(v) as well as by selective inhibition of integrin α(v)β(3) inside-out signalling by a talin-siRNA. Complete abolition of stretch-induced VSMC-supported thrombin generation by the RGT peptide, which disrupts the interaction of Src with the β(3) cytoplasmic tail, demonstrates the link between outside-in pathways involving β(3)-Src interaction and thrombin activity dependent on inside-out signalling. These data show that the contribution of cyclic stretch to VSMC-supported thrombin generation is driven by the integrin α(v)β(3) signalling pathway and suggest a role for pulsatility-induced intramural thrombin in VSMC-dependent vascular remodelling.