Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV 2 , KIV 7 , KIV 9Δ Cys (which lacks the seventh unpaired cysteine) and KIV 10 which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV 10 to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV 10 is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by ~43% at a concentration of 10 μM KIV 10 . The ability of the KIV 10 mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp 70 to Arg abolished binding to both lysine–Sepharose and plasmin-modified fibrinogen, while the Trp 70→ Phe and Arg 35→ Lys substitutions each resulted in decreased binding to these substrates. None of the KIV 10 mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV 7 contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV 10 . However, apo(a) KIV 7 had no effect on fibrinolysis in vitro . Apo(a) KIV 2 and KIV 9Δ Cys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro .