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

Elevated plasma concentrations of lipoprotein(a) Lp(a)] areassociated with an increased risk for the development of atheroscleroticdisease which may be attributable to the ability of Lp(a) toattenuate fibrinolysis. A generally accepted mechanism for thiseffect involves direct competition of Lp(a) with plasminogenfor fibrin(ogen) binding sites thus reducing the efficiencyof plasminogen activation. Efforts to determine the domainsof apolipoprotein(a) apo(a)] which mediate fibrin(ogen) interactionshave yielded conflicting results. Thus, the purpose of the presentstudy was to determine the ability of single KIV domains ofapo(a) to bind plasmin-treated fibrinogen surfaces as well todetermine their effect on fibrinolysis using an in vitro clotlysis assay. A bacterial expression system was utilized to expressand purify apo(a) KIV ₂ , KIV ₇ , KIV ₉ Cys (which lacks theseventh unpaired cysteine) and KIV ₁₀ which contains a stronglysine binding site. We also expressed and examined three mutantderivatives of KIV ₁₀ to determine the effect of changing criticalresidues in the lysine binding site of this kringle on bothfibrin(ogen) binding and fibrin clot lysis. Our results demonstratethat the strong lysine binding site in apo(a) KIV ₁₀ is capableof mediating interactions with plasmin-modified fibrinogen ina lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by ~43% at a concentrationof 10 µM KIV ₁₀ . The ability of the KIV ₁₀ mutant derivativesto bind plasmin-modified fibrinogen correlated with their lysinebinding capacity. Mutation of Trp ⁷⁰ to Arg abolished bindingto both lysine–Sepharose and plasmin-modified fibrinogen,while the Trp ⁷⁰ Phe and Arg ³⁵ Lys substitutions each resultedin 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 mediatingbinding to plasmin-modified fibrinogen, albeit with a lowerapparent affinity than apo(a) KIV ₁₀ . However, apo(a) KIV ₇ had no effect on fibrinolysis in vitro . Apo(a) KIV ₂ andKIV ₉ Cys did not bind measurably to plasmin-modified fibrinogensurfaces and did not affect fibrinolysis in vitro .