Tissue Factor Pathway Inhibitor Activity Associated With LDL Is Inactivated by Cell- and Copper-Mediated Oxidation

Abstract

Human plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which is a specific inhibitor of the action of the factor VII(a)-tissue factor complex in coagulation. A major fraction of plasma TFPI is transported in association with LDL. Because LDL may undergo oxidation in the arterial wall during atherogenesis, we examined the effect of copper- and cell-mediated oxidative modification on TFPI activity associated with LDL. Oxidation mediated by copper ions resulted in a significant inactivation of LDL-associated TFPI (60% to 72% at 24 hours with 2.5 mumol/l CuCl2). The inactivation of TFPI was strongly negatively correlated with both an increase in the net electrical charge of LDL (r = -.80, P < or = .0001) and with the production of thiobarbituric acid-reactive substances (r = -.78, P < or = .0001) and lipid peroxides (r = -.80, P < or = .0001). Cell-mediated oxidation, involving incubation of LDL for 48 hours with either monocyte-like THP1 cells or human monocytes in Ham's F-10 medium, effected a significant decrease (64% and 75%, respectively) in LDL-associated TFPI activity. By contrast, prolonged exposure of LDL to purified soybean lipoxygenase (5000 U/mL) was less effective in inactivating TFPI (47% reduction after incubation for 72 hours at 37 degrees C). We subsequently investigated the mechanism(s) that may underlie such inactivation. Oxidation of LDL is accompanied by the generation of various aldehydes, including malondialdehyde and 4-hydroxynonenal. Chemical modification with these aldehydes revealed a significant inverse correlation between the progressive loss of TFPI activity and both the increase in net electrical charge (r = -.90, P < or = .0001) and the derivatization of free amino acid residues of LDL (r = -.90, P < or = .0001). Specific chemical modification of lysine amino groups by acetylation similarly led to inactivation of LDL-associated TFPI activity. TFPI activity was almost totally abolished (< 1.4%) when the TNBS reactivities of acetylated LDL, malondialdehyde-modified LDL, and 4-hydroxynonenal-modified LDL were 31%, 21%, and 43% that of native LDL, respectively. Our data demonstrate that expression of LDL-associated anticoagulant activity is markedly decreased as a consequence of the oxidative process, and suggest that the progressive aldehydic derivatization of apo B of LDL, and of the associated TFPI protein, may contribute to this phenomenon. Because tissue factor is overexpressed in the atheromatous plaque, it may exert a marked local procoagulant effect.(ABSTRACT TRUNCATED AT 400 WORDS)

High Levels of Systemic Myeloperoxidase Are Associated With Coronary Plaque Erosion in Patients With Acute Coronary Syndromes

“…Myeloperoxidase can exert prothrombotic activity via the generation of oxidant reactive species as it catalyzes lipid peroxidation in vivo, 36 thus leading to tissue factor activation 37 and tissue factor pathway inhibitor inactivation. 38 Myeloperoxidase reacts with hydrogen peroxide to form an enzyme substrate complex with strong oxidant activity and can bind firmly to negatively charged glycosaminoglycans and proteoglycans in the extracellular matrix. 39 In a previous postmortem study of patients dying suddenly of acute MI, Sugiyama et al 31 identified a novel subset of macrophages containing myeloperoxidase, infiltrating the subendothelium at sites of coronary plaque erosion or rupture, with few neutrophils in the same coronary lesions.…”

Dose-Dependent Modulation of Tissue Factor Protein and Procoagulant Activity in Human Monocyte-Derived Macrophages by Oxidized Low Density Lipoprotein

“…TFPI is a protease inhibitor that regulates TF-mediated coagulation by binding factor Xa and inactivating TF VII 37) . Most of TFPI activity in plasma is associated with lipoproteins where 40-80% of its activity is transported by apoB-containing particles, largely by LDL; however, cell-and copper-mediated oxidation, even when resulting in a low-oxidized product such as minimally modified LDL, significantly inactivated TFPI activity and neutralized its inhibitory influence on TF-PCA 38) . In addition, if TFPI-inhibited TF-PCA was truly the mechanism underlying the effect of higher-concentration oxLDL, then LPS-stimulated TF-PCA could not have been attenuated to the extent observed, and TF protein expression evaluated by Western blotting would not have shown the pattern demonstrated.…”

Comparison of the Inhibitory Effects of ApoB100 and Tissue Factor Pathway Inhibitor on Tissue Factor and the Influence of Lipoprotein Oxidation

“…21 The purpose of this part of the study was to investigate the effects of mildly oxidized LDL on different forms of TFPI. Aliquots of LDL-r, or lipids alone, were incubated with recombinant TFPI, plasma TFPI, and Hep G-2 TFPI for 30 minutes before mild oxidation by exposure to air as described in Methods.…”

“…20 It has also been shown that the inhibitory potential of TFPI may be compromised by lipoprotein oxidation. 21 The first 2 Kunitz domains within TFPI responsible for inhibiting clotting factors VIIa and Xa contain lysine and arginine residues at the inhibition sites. 22 The third Kunitz also contains a lysine residue at this site.…”