Differentiation of the roles of the apolipoprotein(a) and LDL moiety in the binding of lipoprotein(a) to limited plasmin digested des AA fibrin

Abstract

Elevated plasma levels of lipoprotein (a) (Lp(a)) in humans represent a major inherited risk factor for atherosclerosis. Lp(a) consists of a LDL-like particle with an additional glycoprotein, apolipoprotein(a) (apo(a)), linked to apolipoprotein B100. The apo(a) moiety is highly homologous to plasminogen as it contains multiple repeats resembling plasminogen kringle IV, a lysine and fibrin binding domain. In the present study, the individual contribution of the two constituents of Lp(a), namely LDL and apo(a), in the binding of Lp(a) to limited plasmin digested des AA fibrin (Desafib-X) is examined.

Lp(a) was isolated by sequential ultracentrifugation followed by gel filtration chromatography. Lysine binding (Lp(a)lys⁺) and non-lysine binding (Lp(a)lys⁻) Lp(a) were obtained by affinity chromatography using lysine-Sepharose. Lp(a)-free LDL was isolated by ultracentrifugation followed by Lp(a)-free LDL was isolated by ultracentrifugation followed by chromatofocusing. ¹²⁵I-labelled Lp(a), LDL, Lp(a)lys⁻, and Lp(a)lys⁺ preparations were incubated with Desafib-X coated wells in the presence or absence of ɛ-aminocaproic acid (ɛACA, a lysine-analogue) and/or autologous LDL.

Total Lp(a) contained 86±8% Lp(a)lys⁺. The mean apparent dissociation constant (Kd in nM) for Lp(a), LDL, Lp(a)lys⁻, and Lp(a)lys⁺ binding to Desafib-X was 48±11, 28±4, 7±4, and 43±23, respectively. The binding of Lp(a) and Lp(a)lys⁺ to Desafib-X could be inhibited to a similar degree by 0.2m ɛACA (for 31±14% and 37±15% respectively), indicating lysine specific binding of these preparations. The binding of Lp(a)lys⁻ and LDL could not be inhibited by ɛACA. A ten times molar excess of LDL could inhibit the binding of Lp(a), Lp(a)lys⁻, and Lp(a)lys⁺ to Desafib-X by 60 to 80%. For Lp(a) and Lp(a)lys⁺, an additional binding-inhibition can be observed when adding 0.2M ɛACA.

In conclusion, the overall findings indicate that the binding of Lp(a) to fibrin(-ogen) is more complex than previously thought and imposes an influence of the LDL moiety and LDL, additional to the apo(a) moiety, in the binding of Lp(a) to lysine-containing proteins like Desafib-X.