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 regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with 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 specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixed with 10 microg 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 reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively.
Horie
S
S
Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-0195, Japan. shorie-v@pharm.teikyo-u.ac.jp
Hiraishi
S
S
Hirata
Y
Y
Kazama
M
M
Matsuda
J
J
D000925:Anticoagulants / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008077:Lipoproteins, LDL / Q000502:physiology*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D000925:Anticoagulants; D003470:Culture Media; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C051928:lipoprotein-associated coagulation inhibitor; C099252:oxidized low density lipoprotein