Oxidation of lipoprotein(a) and low density lipoprotein containing density gradient ultracentrifugation fractions.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for atherosclerotic cardiovascular disease. It is thought that the atherogenicity of Lp(a) is mediated both through its LDL-like properties and its plasminogen-like properties. In this study we have investigated the LDL-like atherogenic properties of Lp(a) by comparing the susceptibility to in vitro oxidation of Lp(a) and LDL isolated from the same subject. The subjects studied varied widely in plasma Lp(a) concentration (331-1829 mg/l) and Lp(a) phenotype (from B to S4). Lipoproteins are notoriously unstable in vitro, consequently differences in in vitro handling could influence oxidizability. Therefore, the isolation and handling of Lp(a) and LDL were performed in an identical fashion. Lp(a) and LDL containing fractions were obtained by density gradient ultracentrifugation. Separate fractions containing various amounts of Lp(a) and LDL, quantitated by measuring both Lp(a) and apo B-100, were subsequently oxidized on equimolar apo B-100 basis. Despite large differences in the Lp(a)/apo B-100 ratio of the various fractions (ranging from 5.3 +/- 1.7 to 0.2 +/- 0.1) they showed quite similar oxidation characteristics. The most dense Lp(a) containing fractions showed an aberrant susceptibility to oxidation. Subsequent gel filtration and reconstitution experiments showed that this was due to protein (i.e., albumin) contamination. Removal of excess protein revealed an oxidation pattern similar to that of LDL. It is concluded that the susceptibility of Lp(a) to lipid-peroxidation is similar to that of LDL when isolated simultaneously and in the same way from the same subject. Thus, lipid-peroxidation of Lp(a) is not influenced by the presence of its distinguishing apolipoprotein(a).

Modification of lipoprotein(a) by oxidation or desialylation influences its ability to compete with plasminogen for binding to the extracellular matrix.

Lipoprotein (a) [Lp(a)], and to a lesser extent low-density lipoprotein (LDL), have been shown to compete with plasminogen for binding to the extracellular matrix (ECM). Evidence exists that modification of lipoproteins alters their atherogenic properties. Therefore in the present study the effect of modifying Lp(a) and LDL by copper-induced in vitro oxidation on their ability to compete with plasminogen for binding to the ECM was studied. Oxidation of Lp(a) resulted in increased competitiveness for plasminogen binding. This effect was dependent on the Lp(a) concentration used, as well as the extent of oxidation. In the highest Lp(a) concentration used (100 nmol/l apo B100), inhibition of plasminogen binding was further increased with almost 30% compared with native Lp(a). In contrast, oxidation of LDL resulted in an additional inhibition of plasminogen binding of about 10% at all concentrations used. In separate experiments Lp(a) and LDL were modified by neuraminidase treatment. After desialylation a strong tendency for better competitiveness of Lp(a) was observed. Desialylation of LDL had no effect on its ability to compete with plasminogen for binding to the ECM. Modification of the additional and distinguishing apolipoprotein [i.e. apo(a)] in Lp(a) by oxidation and desialylation most likely explains the difference in behaviour of Lp(a) and LDL. It is concluded that modification by oxidation, and to a lesser extent desialylation, increases the anti-fibrinolytic potential of Lp(a).

Effect of lipoprotein(a) and LDL on plasminogen binding to extracellular matrix and on matrix-dependent plasminogen activation by tissue plasminogen activator.

Lp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

Sulfhydryl compounds influence immunoreactivity, structure and functional aspects of lipoprotein(a).

Human plasma Lp(a) is susceptible to various sulfhydryl compounds. In this study we present evidence indicating that after treatment of Lp(a) with sulfhydryl compounds, immunoreactivity is changed, structural changes occur and functional characteristics regarding the numerous kringle structures in apo(a) disappear. Purified Lp(a) was subjected to variable concentrations (0.01-10 mM) of various sulfhydryl compounds: DTT, 2-mercapto-ethanol (BME), N-acetylcysteine (NAC) and homocysteine (HCys). Free SH groups were blocked by iodoacetamide. Reduced and alkylated Lp(a) was tested in two ELISAs, one detecting apo(a) alone and one detecting apo(a)-apoB complexes. In both ELISAs polyclonal antibodies were used. For comparison a commercial apo(a) IRMA utilizing two monoclonal antibodies was used. The results indicate that a similar decrease in response of both ELISAs is observed, whereas the IRMA response is less affected. Western blotting of "DTT treated" Lp(a) after SDS-PAGE under nonreducing conditions showed that separate apo(a) and apoB-100 bands became detectable at 1 mM DTT. Native PAGE (2.5-16%) indicated structural changes of Lp(a) beginning to occur at 0.03 mM DTT. Epsilon-aminocaproic acid-inhibitable binding of "DTT-treated" Lp(a) to Desafib-X decreased with increasing DTT concentrations in concert with a loss of the capacity of Lp(a) to inhibit plasminogen activation upon treatment with DTT. The observed immunological and functional changes of Lp(a) indicate that apo(a) kringle function is severely affected by sulfhydryl compounds.

Lysine-binding heterogeneity of Lp(a): consequences for fibrin binding and inhibition of plasminogen activation.

Lipoprotein(a) [Lp(a)] is recognized as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) has a high homology with plasminogen (Pg). In vivo, Pg is activated on a fibrin surface by tissue Pg activator (tPA). We prepared Lp(a) from plasma by sequential ultracentrifugation followed by lysine-sepharose affinity chromatography. We found that a changing (donor dependent) fraction of the Lp(a) did not bind to lysine-sepharose. This fraction, designated Lp(a)lys-, was further purified using gel filtration. Bound Lp(a) [Lp(a)lys+] was eluted with 0.2 M EACA. Apo(a) isoforms in both fractions were identical. In contrast Lp(a)lys+ inhibited Pg activation by tPA in vitro (IC50% 20 mg/l), whereas Lp(a)lys- did not. In addition Lp(a)lys- did not bind to CNBr-digested fibrinogen whereas Lp(a)lys+ did (Kd, app = 0.2 nM). Therefore we conclude that a changing donor dependent fraction of human plasma Lp(a) does not inhibit Pg activation in vitro and does not bind to CNBr-digested fibrinogen.