Oxidized low-density lipoprotein impairs the anti-coagulant function of tissue-factor-pathway inhibitor through oxidative modification by its high association and accelerated degradation in cultured human endothelial cells

We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant r egulator in the extrinsic pathway of coagulation, in cultured human umbilic al vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC wit h ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one spe cific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rap id, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixe d with 10 mug of ox-LDL for 30 min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time -dependent manner. It did not affect the mRNA level, but ox-LDL-dependent r eduction of the TFPI antigen level in HUVEC was reversed by the simultaneou s treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal pr oton pump. These results indicate that ox-LDL lessens the anti-coagulant fu nction of TFPI through both oxidative modification and accelerated degradat ion of the molecule outside and inside HUVEC respectively.