Humic acid reduces protein‐C‐activating cofactor activity of thrombomodulin of human umbilical vein endothelial cells

Abstract

Humic acid in the drinking water of blackfoot disease endemic areas in Taiwan has been implicated as one of the aetiological factors of the disease. For this report we examined the effects of humic acid on the expression of thrombomodulin (TM) cofactor activity by cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with humic acid (HA) isolated from the drinking water, as a synthetic humic acid polymer (SHA) or with commercial HA, resulted in a dose-dependent reduction of cell surface thrombomodulin activity. Characterization of the mechanism by which humic acid reduced the protein C activation indicated that inhibition was not caused by production or release of a protein C inhibitor. Kinetic analysis showed that binding affinities of TM to thrombin and of TM-thrombin complex to protein C was unchanged upon humic acid treatment. However, the cell surface TM activity was reduced by humic acid, which functions as an irreversible noncompetitive inhibitor of thrombin binding. Down-regulation of TM was inhibited by non-selective protein kinase C inhibitors and a selective inhibitor. These results suggest that protein kinase C is intricately involved in HA-induced TM down-regulation. Down-regulation of TM was also inhibited by free radical scavengers. All these changes occurred in the absence of significant cytotoxic effect. In conclusion, our results suggest that HA induces down-regulation of TM by directly increasing permeability of the cell membrane, thus causing elevation in [Ca2+]i. This species functions as a second messenger to activate protein kinase C, and/or Ca-dependent enzymes eventually inducing down-regulation of TM. Attenuation of vascular endothelial cell TM cofactor activity by humic acid may play a role in the humic acid-induced thrombotic vascular disorders of blackfoot disease.