Activation of Protease-Activated Receptors 3 and 4 Accelerates Tissue Factor–Induced Thrombin Generation on the Surface of Vascular Smooth Muscle Cells

OBJECTIVE: To determine factors regulating human aortic smooth muscle cells (HASMC) supported tissue factor-induced thrombin generation. METHODS AND RESULTS: The addition of nonlipidated tissue factor and Ca(2+) to HASMCs maintained in reptilase-treated platelet-poor plasma resulted in the robust formation of thrombin after a lag phase of approximately 6 minutes. Pretreatment with low concentrations of α-thrombin before the addition of tissue factor and Ca(2+) accelerated the rate of thrombin generation (time to reach half of peak thrombin was reduced by [mean ± SD] 42.0 ± 2.2%; P<0.05) but had no effect on the amount of peak thrombin generated. Protease-activated receptor (PAR) 3 activating peptides (APs) or PAR-4 APs accelerated thrombin generation without affecting peak thrombin levels (time to half of peak thrombin decreased by 17.4 ± 5.6% and 21.7 ± 3.5%; P<0.05 with PAR-3 AP and PAR-4 AP, respectively). The addition of PAR-3 AP and PAR-4 AP together had an additive effect, with a reduction in time to half of peak thrombin of 43.9 ± 4.0%. PAR-3 AP or PAR-4 AP enhanced tissue factor-induced factor Xa production and phosphatidylserine exposure on the surface of HASMCs. PAR-1 activation had no effect on thrombin generation, factor Xa production, or phosphatidylserine exposure. CONCLUSIONS: Low concentrations of α-thrombin accelerate tissue factor-induced thrombin generation on the surface of HASMCs, and this effect is mediated by PAR-3 and PAR-4.