The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.