High plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.

The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower K(m) values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the K(m) values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH(2), was cleaved 60 times more efficiently and had a K(m) approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors.

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.

The specificity of plasmin is more tightly controlled than previously recognized; interactions with substrates at all subsites between S4 and S2' contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This 'negative selectivity' avoids short-circuiting regulation of the fibrinolytic system and other important biological processes, and might be an important general mechanism for controlling protease cascades.

Individuals heterozygous for the apolipoprotein(a) [apo(a)] trait have phenotypes combining two different lipoprotein(a) [La(a)] particle suspecies that are present in plasma at a different concentration. Evaluation of the ability of each of these isoforms to bind to fibrin and affect plasminogen binding is essential to assess the pathogenic role of Lp(a) in these subjects; therefore, fractions containing different ratios of Lp(a) with distinct apo(a) isoforms (e.g. B/S3, S1/S4) were prepared by density gradient ultracentrifugation of plasma, and tested. Lp(a) fractions containing mainly small apo(a) isoforms (either B or S1) showed the highest affinity for fibrin (Kd approximately 150 nmol L-1) and the best competitor activity for plasminogen, whereas fractions containing mainly the high molecular mass isoforms (either S3 or S4) showed the lowest affinities (Kd > or = 500 nmol L-1). An increase in Kd was observed as a function of the relative content in isoforms of high molecular mass in these fractions. This inverse relationship between affinity for fibrin and apo(a) size indicates that Lp(a) subspecies in heterozygotes may have different pathogenic potential. Thus, the antifibrinolytic effect of Lp(a) in heterozygous subjects would depend on the relative concentration of the isoform with the highest affinity for fibrin.