A prothrombotic state and plasminogen activator inhibitor-1 (PAI-1) have been associated with oxidative stress and vascular remodeling. However, the mechanisms connecting thrombotic events with proliferation of smooth muscle cells (SMC) are unclear. p21 activated kinase (PAK) has been shown to mediate signaling cascades in response to thrombin. We therefore investigated if PAK may link thrombin to PAI-1 expression and proliferation of SMC. Thrombin stimulated phosphorylation of PAK and PAK-dependent ROS production, increased expression and activity of the hypoxia-inducible transcription factor HIF-1alpha and enhanced PAI-1 promoter activity and expression. All these responses were inhibited by antioxidants and the calcium chelator BAPTA-AM. Transfection of an active or a dominant-negative mutant of PAK increased or decreased, respectively, HIF1 and PAI-1 expression and proliferation of SMC. Finally, antioxidants and BAPTA-AM inhibited PAI-1-dependent proliferation of SMC. These data show that thrombin stimulates SMC proliferation via calcium- and ROS-dependent activation of PAK, subsequent ROS formation and induction of PAI-1 via activation of HIF-1. This mechanism may play an important role in promoting vascular remodeling.