NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells.

BACKGROUND: Tissue factor (TF) initiates the extrinsic coagulation cascade leading to thrombin formation. Thrombin induces TF mRNA in vascular smooth muscle cells (VSMCs), thereby contributing to the prolonged procoagulant activity and enhanced thrombogenicity at sites of vascular injury. However, the signaling mechanisms mediating this thrombogenic cycle are unclear. Characteristically, vascular injury promotes the generation of reactive oxygen species (ROS). Because ROS exert signaling functions, we investigated whether the NADPH oxidase, an important source of ROS in VSMCs, contributes to upregulation of TF by thrombin. METHODS AND RESULTS: Thrombin not only stimulated TF mRNA expression, but also TF-dependent surface procoagulant activity in cultured human VSMCs. This response was attenuated by antioxidants; the flavin inhibitor diphenylene-iodonium, Clostridium difficile toxin B, which inhibits Rho GTPases, p22phox antisense oligonucleotides, or the dominant-negative RacT17N mutant. Inhibitors of p38 MAP kinase and phosphatidylinositol (PI) 3-kinase also prevented thrombin-stimulated TF mRNA expression. Furthermore, thrombin stimulated the phosphorylation of the PI 3-kinase target protein kinase B/Akt in a redox-sensitive and NADPH oxidase-dependent manner. CONCLUSION: These findings indicate that the NADPH oxidase is essentially involved in the redox-sensitive induction of TF mRNA expression and surface procoagulant activity by thrombin. This response is mediated by NADPH oxidase-dependent activation of p38 MAP kinase and the PI 3-kinase/protein kinase B/Akt pathway. Given that active TF promotes thrombin formation, the NADPH oxidase may play a crucial role in perpetuating the thrombogenic cycle in the injured vessel wall.

Rac regulates thrombin-induced tissue factor expression in pulmonary artery smooth muscle cells involving the nuclear factor-kappaB pathway.

Pulmonary hypertension is associated with enhanced thrombogenicity of the vessel wall contributing to vascular remodeling. However, the signaling mechanisms promoting this prothrombotic state are not resolved. Here we investigated the role of the GTPase Rac in the regulation of tissue factor (TF) expression and activity in response to thrombin in pulmonary artery smooth muscle cells (PASMC). TF mRNA and protein expression and surface procoagulant activity were increased by thrombin in PASMC. These responses were enhanced in the presence of the constitutively active Rac mutant RacG12V, but were abrogated in cells expressing dominant-negative RacT17N. Thrombin and RacG12V also increased human TF promoter activity primarily involving a sequence between -636 and -111 bp containing a distal, nuclear factor-kappaB (NFkappaB)-dependent enhancer element. Indeed, thrombin and RacG12V stimulated NFkappaB-dependent transcriptional activity, and overexpression of p50/p65 significantly increased human TF promoter activity. Moreover, in RacG12V-overexpressing cells, TF promoter activity was significantly decreased by coexpression of dominant-negative mutants of IkappaBalpha and IkappaBKalpha, which prevent NFkappaB activation. As enhanced NFkappaB activity has been observed in patients with pulmonary hypertension, Rac-dependent activation of the NFkappaB pathway may be a critical element promoting thrombin-induced TF expression and activity, and thus a prothrombotic state in pulmonary hypertension.

Insights into the redox control of blood coagulation: role of vascular NADPH oxidase-derived reactive oxygen species in the thrombogenic cycle.

Various cardiovascular diseases including thrombosis, atherosclerosis, (pulmonary) hypertension and diabetes, are associated with disturbed coagulation. Alterations in the vessel wall common to many cardiovascular disorders have been shown to initiate the activity of the coagulation system, but also to be the result of an abnormal coagulation system. The primary link between the coagulation and the vascular system appears to be tissue factor (TF), which is induced on the surface of vascular cells and initiates the extrinsic pathway of the blood coagulation cascade, leading to the formation of thrombin. Thrombin can also interact with the vascular wall via specific receptors and can increase vascular TF expression. Such a "thrombogenic cycle" may be essentially involved in the pathogenesis of cardiovascular disorders associated with an abnormal coagulation. Therefore, the identification of the signaling pathways regulating this cycle and each of its relevant connecting links is of fundamental importance for the understanding of these disorders and their putative therapeutic potential. Reactive oxygen species (ROS) and the ROS-generating NADPH oxidases have been shown to play important roles as signaling molecules in the vasculature. In this review, we summarize the data supporting a substantial role of ROS in promoting a thrombogenic cycle in the vascular system.