During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV 6 -8 ), and two lysine residues (Lys 680 and Lys 690) within the NH 2 terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV 6 -8 and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV 6 , KIV 7 , and KIV 8 , as well as mutants that disrupt the WLBS in both KIV 6 and KIV 7 , or both KIV 7 and KIV 8 , were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV 7 and KIV 8 , but not KIV 6 , are required for maximally efficient noncovalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV 7 or KIV 8 resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV 7 and KIV 8 resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV 7 and KIV 8 specifically bind with high affinity to apoBderived peptides containing Lys 690 or Lys 680, respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV 7 and KIV 8 and Lys 680 and Lys 690 in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.

We have previously demonstrated that, in the presence of the lysine analogue epsilon-aminocaproic acid, apolipoprotein(a) [apo(a)] undergoes a conformational change from a closed to an open structure that is characterized by a change in tryptophan fluorescence, an increase in the radius of gyration, an alteration of domain stability, and an enhancement in the efficiency of covalent lipoprotein(a) [Lp(a)] formation. In the present study, to identify sequences within apo(a) that maintain its closed conformation, we used epsilon-aminocaproic acid to probe the conformational status of a variety of recombinant apo(a) isoforms using analytical ultracentrifugation, differential scanning calorimetry, intrinsic fluorescence, and in vitro covalent Lp(a) formation assays. We observed that the closed conformation of apo(a) is maintained by intramolecular interaction(s) between sequences within the amino- and carboxyl-terminal halves of the molecule. Using site-directed mutagenesis, we have identified the strong lysine-binding site present within apo(a) kringle IV type 10 as an important site within the C-terminal half of the molecule, which is involved in maintaining the closed conformation of apo(a). Apo(a) exhibits marked isoform size heterogeneity because of the presence of varying numbers of copies of the kringle IV type-2 domain located within the amino-terminal half of the molecule. Using recombinant apo(a) species containing either 1, 3, or 8 copies of kringle IV type 2, we observed that, while apo(a) isoform size does not alter the affinity of apo(a) for low-density lipoprotein, it affects the conformational status of the protein and therefore influences the efficiency of covalent Lp(a) assembly. The inverse relationship between apo(a) isoform size and the efficiency of covalent Lp(a) formation that we report in vitro may contribute to the inverse relationship between apo(a) isoform size and plasma Lp(a) concentrations that has been observed in vivo.

Plasminogen (Pg) activation to plasmin (Pn) by tissue-type Pg activator (tPA) requires fibrin as a cofactor. Intact fibrin up regulates tPA mediated Glu-Pg activation by approximately 1000-fold compared to no fibrin. Before lysis of the fibrin clot, Pn modifies the fibrin surface resulting in the exposure of carboxyl-terminal lysine and arginine residues. This Pn modified form of fibrin further up regulates Glu-Pg activation by approximately 3-fold over intact fibrin, creating a positive feedback loop for Pn generation. The carboxypeptidase B-like enzyme, activated thrombin activatable fibrinolysis inhibitor (TAFIa), removes these exposed lysine and arginine residues, decreasing the cofactor activity of fibrin to approximately 100-fold less than Pn modified fibrin, thereby attenuating fibrinolysis. While Glu-Pg activation by tPA on plasmin and TAFIa modified fibrin has been characterized, no such analysis has been done with Lys-Pg. Because a fibrin clot is insoluble, soluble high molecular weight fibrin degradation products (HMW-FDPs) were used as a model for plasmin modified fibrin. A fluorescently labeled recombinant Pg mutant, 5IAF-Pg, was used to simplify the kinetic analysis. The active site serine in this Pg derivative has been mutated to a cysteine (S741C), to which 5-iodoacetamidofluorescein has been covalently attached. 5IAF-Pg cleavage does not generate active Pn, thus eliminating the feedback cleavages. In addition, cleavage of this derivative results in an approximate 50% decrease in fluorescence intensity, which can be used to monitor the kinetics of cleavage. Both 5IAF-Glu-Pg and 5IAF-Lys-Pg cleavage by tPA were studied on untreated and TAFIa treated HMW-FDPs. The initial rates of cleavage were fit to a previously derived steady-state template model for tPA mediated Pg activation on fibrin. The catalytic efficiency (kcat/Km) for 5IAF-Glu-Pg cleavage decreased by 94% upon TAFIa treatment of the HMW-FDPs (0.90 microM^-1s^-1 to 0.05 microM^-1s^-1), whereas a 36% decrease in kcat/Km was observed for 5IAF-Lys-Pg (1.38 microM^-1s^-1 to 0.84 microM^-1s^-1). Thus, TAFIa treated HMW-FDPs are an approximately 17-fold better cofactor for 5IAF-Lys-Pg cleavage than 5IAF-Glu-Pg cleavage. This increase in cofactor activity is due to the higher affinity of 5IAF-Lys-Pg for HMW-FDPs than 5IAF-Glu-Pg. TAFIa treatment of the HMW-FDPs resulted in a 62-fold increase in the Kd for 5IAF-Glu-Pg (0.356 ± 0.251 microM to 22.120 ± 472.731 microM). However, TAFIa treatment resulted in only a 2-fold increase in the Kd for 5IAF-Lys-Pg binding to HMW-FDPs (0.356 ± 0.251 microM to 0.721 ± 2.311 microM). Furthermore, a 5-fold increase in the Kd for 5IAF-Glu-Pg binding to HMW-FDPs with bound tPA was observed upon TAFIa treatment (0.079 ± 0.022 microM to 0.387 ± 0.076 microM), but no change was observed for 5IAF-Lys-Pg (0.033 ± 0.017 microM to 0.030 ± 0.029 microM). These data rationalize previous results showing that although TAFIa increases the lysis time of purified clots in the presence of Glu-Pg, it does not do so in the presence...