Coagulation factor XI induces Ca 2+ response and accelerates cell migration in vascular smooth muscle cells via proteinase-activated receptor 1

Abstract

Activated coagulation factor XI (FXIa) is a serine proteinase that plays a key role in the intrinsic coagulation pathway. The analysis of FXI-knockout mice has indicated the contribution of FXI to the pathogenesis of atherosclerosis. However, the underlying mechanism remains unknown. We hypothesized that FXIa exerts vascular smooth muscle effects via proteinase-activated receptor 1 (PAR 1 ). Fura-2 fluorometry revealed that FXIa elicited intracellular Ca 2+ signal in rat embryo aorta smooth muscle A7r5 cells. The influx of extracellular Ca 2+ played a greater role in generating Ca 2+ signal than the Ca 2+ release from intracellular stores. The FXIa-induced Ca 2+ signal was abolished by the pretreat-ment with atopaxar, an antagonist of PAR 1 , or 4-amidinophenylmeth-anesulfonyl fluoride (p-APMSF), an inhibitor of proteinase, while it was also lost in embryonic fibroblasts derived from PAR 1-/- mice. FXIa cleaved the recombinant protein containing the extracellular region of PAR 1 at the same site (R45/S46) as that of thrombin, a canonical PAR 1 agonist. The FXIa-induced Ca 2+ influx was inhibited by diltiazem, an L-type Ca 2+ channel blocker, and by siRNA targeted to Ca V 1.2. The FXIa-induced Ca 2+ influx was also inhibited by GF109203X and rottlerin, inhibitors of protein kinase C. In a wound healing assay, FXIa increased the rate of cell migration by 2.46-fold of control, which was partly inhibited by atopaxar or diltiazem. In conclusion, FXIa mainly elicits the Ca 2+ signal via the PAR 1 /Ca V 1.2-mediated Ca 2+ influx and accelerates the migration in vascular smooth muscle cells. The present study provides the first evidence that FXIa exerts a direct cellular effect on vascular smooth muscle.