Abstract
Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).
Highlights
Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis
A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin
The domains in apo(a) that mediate inhibition of fibrinolysis are not known. We addressed these issues in the current study using a series of recombinant apo(a) variants and a system to measure tPAmediated Glu1-plasminogen activation kinetics in the absence of plasmin-mediated positive feedback reactions
Summary
Lp(a), lipoprotein(a); 17K, 17-kringle; A, activator; ⑀-ACA, ⑀-aminocaproic acid; apo(a), apolipoprotein(a); apoB, apolipoprotein B; dEGR-CK, 1,5-dansyl-Glu-Gly-Arg-chloromethylketone dihydrochloride; F, cofactor; FDP, fibrin-degraded products; HBS, HEPES-buffered saline; I, inhibitor; kcat, turnover number; KIV, kringle IV type(s); Km, Michaelis constant; P, substrate; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PPA-CK, D-Phe-ProArg-chloromethylketone; r-apo(a), recombinant apo(a); tPA, tissue-type disorders including coronary artery disease [1]. Studies demonstrated that apo(a)/Lp(a) can inhibit the binding of plasminogen and tPA to fibrin [13, 18], but the mechanism by which apo(a) inhibits plasminogen activation remains controversial as both competitive [23] and uncompetitive [18, 24] mechanisms have been reported. Plasminogen activator; VFK-CK, D-Val-Phe-Lys-chloromethylketone dihydrochloride; Flu-plasminogen, fluorescently labeled form of recombinant human plasminogen; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. We adapted a methodology previously described by Horrevoets and co-workers [29] that allows plasminogen activation to be measured directly in a fibrin clot and in the absence of positive feedback reactions catalyzed by plasmin. Using a series of recombinant apo(a) (r-apo(a)) variants in this system, we have delineated the domains in apo(a) required for inhibition of plasminogen activation and have described the inhibition mechanism as an equilibrium template model
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