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We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.

Abstract-Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogenactivated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity. Key Words: angiogenesis Ⅲ activated protein C Ⅲ endothelial protein C receptor Ⅲ protease-activated receptor-1 Ⅲ mitogen-activated protein kinase A ctivated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation. Activation of protein C occurs on the endothelial cell surface by thrombin bound to thrombomodulin. 1 The endothelial protein C receptor (EPCR), which is a glycoprotein on the vascular endothelium, binds to protein C, thereby augmenting protein C activation by the thrombin-thrombomodulin complex. 2 APC regulates the coagulation cascade by inactivating activated forms of factors V and VIII in the presence of protein S, 3 and it exerts a profibrinolytic effect by inactivating plasminogen activator inhibitor-1. 4 The importance of APC as a natural anticoagulant is illustrated by the development of severe thrombosis in patients with congenital protein C deficiency. 5 In addition to the anticoagulant effect, APC has been shown to be capable of activating protease activated receptor (PAR)-1 on endothelial cells, 6 thereby activating the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway is activated during mitosis, meiosis, and G0-G1 transition 7 and is implicated in regulation of the cell cycle, mitogen-induced cell growth, and proliferation. 8 The MAPK pathway plays an important role in signal transduction from growth factor receptor...

Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)-induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone-treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation,