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 diphenyleneiodonium, 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.

Background-The use of sex steroids in oral contraception or hormonal replacement therapy is associated with an increased risk of cardiovascular thromboembolic complications. Although both the estrogen and the progestin components have been involved, the underlying mechanisms responsible are unclear. Methods and Results-This study examined whether sex steroids promote hemostasis indirectly by increasing the procoagulant activity of blood vessels. Treatment of vascular smooth muscle cells with several progestins (progesterone, 3-keto-desogestel, gestodene, and medroxyprogesterone acetate) upregulated proteolytically activatable thrombin receptor (PAR-1) expression, resulting in a potentiated thrombin-induced tissue factor expression and surface procoagulant activity. In contrast, neither the progestins levonorgestrel, norethisterone, and norgestimate nor the synthetic estrogen 17␣-ethinylestradiol had such effects. The effect of the stimulatory progestins, which induce glucocorticoid-like effects in several cell systems, was mimicked by dexamethasone and inhibited by the progesterone and glucocorticoid receptor antagonist RU-38486. In addition, long-term administration of progesterone, 3-ketodesogestrel, or medroxyprogesterone acetate to ovariectomized rats increased PAR-1 protein level in the arterial wall, resulting in an increased responsiveness of isolated aortic rings to thrombin. Conclusions-These data demonstrate that several progestins markedly potentiate the vascular procoagulant effects of thrombin by increasing the availability of membrane thrombin receptors in the smooth muscle, an effect that is most likely due to their glucocorticoid-like activity. (Circulation. 2001;104:2826-2831.)

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.

Abstract-Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription-polymerase chain reaction and the release of VEGF protein by immunoassay. ␣-Thrombin time-and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF 164/165 , and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating-peptide. Upregulation of VEGF expression was also induced by conditioned medium from ␣-thrombin-stimulated VSMCs. Both the early and the delayed ␣-thrombin-induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. ␣-Thrombin-induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)-, a transforming growth factor (TGF)-␤-, and a basic fibroblast growth factor (bFGF)-neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-␤, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.