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

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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 μmol/L CuCl2). The inactivation of TFPI was strongly negatively correlated with both an increase in the net electrical charge of LDL (r=−.80,P≤.0001) and with the production of thiobarbituric acid–reactive substances (r=−.78,P≤.0001) and lipid peroxides (r=−.80,P≤.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°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=−.93,P≤.0001) and the derivatization of free amino acid residues of LDL (r=−.90,P≤.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. The oxidative inactivation of LDL-associated TFPI will therefore effectively neutralize its inhibitory action on tissue factor activity, resulting in a disequilibrium in favor of coagulation.