Multiple Binding with Identical Linkage: A Mechanism That Explains the Effect of Lipoprotein(a) on Fibrinolysis

Abstract

We have previously shown that both recombinant apo(a) and native Lp(a) inhibit the binding of Glu-plasminogen to fibrin surfaces [Fleury & Anglés-Cano (1991) Biochemistry 30, 7630-7638; Rouy et al. (1992) Biochemistry 31, 6332-6339]. The aim of the present study was to characterize the mechanism of this inhibition and to define the parameters governing binding when two different Lp(a) species compete with plasminogen for fibrin, a situation that may be found in vivo in subjects heterozygous for the apo(a) trait. The Kd for the binding of plasminogen to fibrin was 660 nM whereas the affinity of Lp(a) was inversely related to apo(a) size (Kd range: 50 to > 500 nM). To determine the effect of plasminogen on Lp(a) binding and reciprocally, competition experiments were performed. The Kd of either Lp(a) or plasminogen for fibrin remained unchanged in the presence of the other competitor whereas Bmax, the maximal amount bound, was importantly decreased. In a similar fashion, competition for fibrin binding among Lp(a) isoforms was shown with the use of Lp(a) density fractions containing varying proportions of isoforms B (approximately 460 kDa) and S3 (approximately 640 kDa); variations in Kd values (from 141 nM to 460 nM) as a function of the relative content in isoform S3 were observed. Altogether, these results are indicative of multiple binding by ligands that bind with different affinities to equivalent but independent sites. Thus, in plasma from heterozygous subjects, the influence of each Lp(a) isoform on fibrinolysis will depend on their affinity for fibrin and on their concentration relative to each other and to plasminogen.

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

“…2). The dissociation constants calculated for plasminogen, K d = 1.16 F 0.22 Amol/l, and for apo(a), K d = 54 F 5 nmol/l, are in agreement with previously published data [12,27,29] in which fibrin was immobilised on multi-well plates using polymerised glutaraldehyde as a linker. This procedure has been previously proven to result in a fibrin monolayer that mimics a fibrin clot surface with regard to plasminogen binding and activation in a static system [27,40,41].…”

“…Among them, the lipoprotein Lp(a) [10], a highly atherogenic lipoprotein particle, which possesses multiple copies of plasminogen-like kringle 4 in its apolipoprotein(a), apo(a), moiety [11]. Lp(a) inhibits plasmin formation by competing with plasminogen for binding sites on fibrin and cell surfaces [12,13]. On the other hand, it has been demonstrated that the interaction of monoclonal antibodies with the low-or high-affinity LBSs of plasminogen may result in conformational changes yielding the plasminogen more susceptible to activation by plasminogen activators [14,15].…”

Effect of Individual Plasma Lipoprotein(a) Variations In Vivo on Its Competition With Plasminogen for Fibrin and Cell Binding

“…28,29,31 Furthermore, we have previously shown that in subjects heterozygous for the apo(a) trait, the influence of the various apo(a) isoforms on fibrinolysis depends on their affinity for fibrin and on their concentrations relative to each other and to plasminogen. 50 According to these data, high levels of Lp(a) and low plasminogen concentrations, conditions that were present in a majority of the nephrotic children included in the present study, may promote the deleterious effect of Lp(a). Indeed, congenital hypoplasminogenemia and high plasma levels of Lp(a) were recently associated with thrombosis.…”

“…50 In the present study, the binding assays were performed in plasma. Therefore, we sought to rule out potential confounding plasma factors that might influence the assay.…”

“…To simplify, the graph data shown represent specific binding obtained by subtracting binding in the presence of 6-aminohexanoic acid, a lysine analogue, from total binding. The Lp(a) tested contained an 18-kringle apo(a) isoform with high affinity for fibrin (K d 12 nmol/L) as calculated from the binding isotherms according to Hervio et al 50 …”