HUMIC-ACID INDUCES EXPRESSION OF TISSUE FACTOR BY CULTURED ENDOTHELIAL-CELLS - REGULATION BY CYTOSOLIC CALCIUM AND PROTEIN-KINASE-C

Blackfoot disease is a thrombotic peripheral vascular disease causally related to the fluorescent humic acid (HA) found in the drinking wate r of wells in endemic areas in Taiwan. In this study we examined the e ffect of HA on tissue factor (TF) expression by vascular endothelial c ells. Incubation of cultured human umbilical vein endothelial cells (H UVEC) with HA isolated from endemic area drinking water or with a synt hetic humic acid polymer (SHA), resulted in enhanced cell surface expr ession of TF activity by HUVEC. The intracellular calcium level ([Ca2]i) was measured using a calcium-specific fluorescent probe, fura 2. C hanges in [Ca2+]i level were followed and quantitatively analyzed by s pectrofluorometric microscopy, after incubation of the fura 2-loaded H UVEC with HA or SHA in a medium containing 1.8 mM CaCl2. Both HA and S HA increased [Ca2+]i in the presence of extracellular calcium ions, bu t not in their absence, indicating that influx of extracellular Ca2+ o ccurred during incubation of HUVEC with HA or SHA. Verapamil, a potent calcium channel blocker, did not abolish the enhancement of [Ca2+]i i nduced by HA or SHA, indicating that specific calcium channels may not be involved in the HA/SHA-induced elevation of [Ca2+]i. The elevated [Ca2+]i level induced by HA or SHA returned to basal level following r emoval of HA or SHA and incubation of the washed cells in medium conta ining 1.8 mM CaCl2. All these changes occurred in the absence of signi ficant cytotoxic effects. The HA/SHA-induced enhancement of cell surfa ce TF activity was inhibited by a specific inhibitor of protein kinase C, H7, suggesting that protein kinase C is involved in the process le ading to the enhanced expression of TF activity induced by HA or SHA. In conclusion, this study demonstrates that HA and SHA enhance cell su rface expression of TF activity by permeabilization of the cell membra ne to extracellular Ca2+ ions, leading to elevation of [Ca2+]i that fu nctions as a second messenger to activate protein kinase C, leading fi nally to enhanced cell surface TF expression, Enhancement of vascular endothelial cell surface TF activity by HA may play a role in the HA-i nduced thrombotic vascular disorders of Blackfoot disease.