Coagulation process proceeds on cultured human mesangial cells via expression of factor V

Background. In a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin d eposition in the active type of IgA nephropathy. The conversion of prothrom bin to thrombin by factor Xa is potently accelerated more than 10(4)-fold b y the presence of factor V, which is a membrane-bound cofactor. Another mem brane-bound cofactor, tissue factor, is known to play an initiating role in the coagulation cascade and to be synthesized in mesangial cells (MCs) by the stimulation of tumor necrosis factor-alpha (TNF-alpha). However, the sy nthesis of factor V, which plays on the terminating stage of prothrombin ac tivation, has not been reported previously in MCs by in vitro study. Our cu rrent study tested the coagulation process via expression of factor V by th e stimulation of proinflammatory cytokine, TNF-alpha, in cultured human MCs . Methods. To evaluate factor V protein expression, immunoperoxidase staining with densitometric evaluation and Western blot analysis were conducted aft er stimulation of TNF-a. To test factor V activity, stimulated MCs were inc ubated in combination with factor Xa, prothrombin, fibrinogen and factor XI II, and fibrin production on MCs was assessed after immunoperoxidase staini ng on the cell surface. In a blocking test using an antibody against factor V, suppression of fibrin production was evaluated to clarify the role of f actor V activity. For the evaluation of factor V mRNA expression in culture d human MCs, in situ hybridization and Northern blot analysis were performe d, Results. Factor V protein expression in MCs after TNF-alpha stimulation inc reased both time- and dose-dependently. As a marker of factor V activity wi th exogenous factor Xa, fibrin production on TNF-alpha -stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of a ntifactor V antibody. Factor V mRNA was identified in MCs by in situ hybrid ization and showed an increase after stimulation with TNF-alpha on Northern blot analysis. Conclusions. Our data suggest that the coagulation process proceeds on MCs as the result of increased expression of endogenous factor V activity on its cell surface in cooperation with exogenous factor Xa.