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Updated: Jul 30, 2026
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Coagulation process proceeds on cultured human mesangial cells via expression of factor V
1Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan. ono@kuhp.kyoto-u.ac.jp
Background:
In a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin deposition in the active type of IgA nephropathy. The conversion of prothrombin to thrombin by factor Xa is potently accelerated more than 104-fold by the presence of factor V, which is a membrane-bound cofactor. Another membrane-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 synthesis of factor V, which plays on the terminating stage of prothrombin activation, has not been reported previously in MCs by in vitro study. Our current study tested the coagulation process via expression of factor V by the 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 after stimulation of TNF-alpha. To test factor V activity, stimulated MCs were incubated in combination with factor Xa, prothrombin, fibrinogen and factor XIII, and fibrin production on MCs was assessed after immunoperoxidase staining 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 factor V activity. For the evaluation of factor V mRNA expression in cultured human MCs, in situ hybridization and Northern blot analysis were performed.
Results:
Factor V protein expression in MCs after TNF-alpha stimulation increased both time- and dose-dependently. As a marker of factor V activity with exogenous factor Xa, fibrin production on TNF-alpha-stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of anti-factor V antibody. Factor V mRNA was identified in MCs by in situ hybridization 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.
Insights
Mesangial cells (MCs) express factor V (FV) and initiate coagulation when stimulated by tumor necrosis factor-alpha (TNF-alpha). This study demonstrates FV
Area of Science:
- Nephrology
- Hematology
- Cell Biology
Background:
- Mesangial cells (MCs) are implicated in IgA nephropathy pathogenesis.
- Previous studies noted mesangial factor V (FV) and fibrin deposition in IgA nephropathy.
- The role of FV synthesis in MCs, particularly its response to TNF-alpha, was previously uncharacterized.
Purpose of the Study:
- To investigate the expression and activity of factor V (FV) in cultured human mesangial cells (MCs) upon stimulation with tumor necrosis factor-alpha (TNF-alpha).
- To determine if MCs can initiate coagulation processes in response to inflammatory stimuli.
Main Methods:
- Human MCs were stimulated with TNF-alpha.
- Factor V protein and mRNA expression were assessed using immunoperoxidase staining, Western blot, in situ hybridization, and Northern blot analysis.
- Factor V activity was evaluated by measuring fibrin production in the presence of factor Xa and prothrombin, with and without anti-factor V antibody.
Main Results:
- TNF-alpha stimulation increased factor V (FV) protein and mRNA expression in MCs in a time- and dose-dependent manner.
- Stimulated MCs exhibited increased fibrin production, indicating FV activity, which was inhibited by anti-FV antibody.
- These findings confirm the presence and functional activity of FV in MCs.
Conclusions:
- Cultured human mesangial cells (MCs) express functional factor V (FV) upon TNF-alpha stimulation.
- The coagulation cascade can be initiated on the surface of MCs, suggesting a novel mechanism in kidney disease pathogenesis.
