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Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A2 (Lp-PLA2), a reaction that generated that predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lysoderivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2 moles of oxPtdPC/mole of Plg distributed between the kringles 1-4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (Por= 1.3 x 10(-7)) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P = 7.3 x 10(-12) and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27.

Lipoprotein(a) [Lp(a)] represents a class of lipoprotein particles having as a protein moiety apoB-100 linked by a single disulfide bond to apolipoprotein(a) [apo(a)], a multikringle structure with a high degree of homology with plasminogen. A recognized feature of Lp(a) is its instability on storage caused by attendant protein and lipid modifications that affect the structural, functional, and immunological properties of this lipoprotein. Here we present data showing that, under appropriate conditions of cryopreservation, Lp(a) retains the properties of the freshly isolated product, and we provide examples supporting the stability of this cryopreserved product as a primary standard in immunoassay settings and in cell culture systems.

A VLDL-like particle containing the apo B100-apo(a) complex was isolated from the post-prandial plasma of subjects fed a fat meal enriched in saturated fatty acids. The abundance of this lipoprotein particle, that we call TG-Lp(a), varied among subjects but not in the same subject. TG-Lp(a), but not the classic Lp(a), contained apo E; this apolipoprotein may cause divergence in cellular uptake and degradation between these two classes of lipoproteins.

Plasma Lp[a] levels and apo[a] isoform distribution among lipoproteins isolated by density gradient ultracentrifugation were studied in subjects with one-band or two-band apo[a] phenotypes as assessed by gradient gel electrophoresis before and after an oral fat load. There were no significant differences in the ultracentrifugal profile between fasting plasma and postprandial plasma that was freed of triglyceride-rich particles (TRP). One-band phenotypes exhibited a single symmetrical peak in the density gradient, whereas two-band phenotypes exhibited a multi-modal distribution. Low molecular weight apo[a] isoforms were preferentially associated with low density Lp[a] whereas high molecular weight apo[a] isoforms were found with high density Lp[a] particles. Feeding a high fat meal caused no significant increase in the total plasma level of Lp[a]. However, the isolated TRP contained the apoB-100-apo[a] complex in a quantity that represented only about 1% of its total amount in the fasting plasma. In all cases the apo[a] isoforms present in TRP were also present in the fasting plasma; however, in the two-band apo[a] phenotypes the ratio of the slow over the fast migrating band was in all cases about eightfold higher in TRP than in the fasting plasma. These observations indicate that postprandially a small percentage of apoB-100-apo[a] associates with TRP and suggest that this complex may derive from de novo synthesis rather than from a pre-existing Lp[a] plasma pool. The liver would be the source of the complex due to the presence in the latter of apoB-100.

We previously described a family of rhesus monkeys in which three out of six members had a spontaneous hypercholesterolemia related to a decrease in number of low density lipoprotein receptors (LDL-R) (Scanu et al. 1988. J. Lipid Res. 29: 1671-1681). During the current work an additional female normocholesterolemic offspring was generated from the mating of the original dam and sire. Moreover, from the breeding of one of the affected male offspring with six unrelated normocholesterolemic female monkeys, eight offspring were generated of which three were hypercholesterolemic on a cholesterol-free diet and exhibited the same degree of LDL-R deficiency as shown by studies in skin fibroblast cultures. All of the animals studied had levels of plasma lipoprotein[a] protein ranging between 1.0 mg/dl and 57.5 mg/dl that were only weakly correlated with total plasma cholesterol, LDL cholesterol, and apoB. LDL-R deficiency correlated with plasma LDL but not Lp[a]. A 7 week fat challenge (16.5% lard, 0.64% cholesterol) that raised the plasma LDL levels markedly had no effect on plasma Lp[a]. Animals with the single band apo[a] phenotype moving on SDS-PAGE faster than apoB-100 exhibited a tendency for high plasma Lp[a] levels which, however, varied widely. Wide variations in Lp[a] levels were also noted with the other apo[a] phenotypes. Taken together our results demonstrate a successful transmission to second generation animals of the LDL-R deficiency phenotype and provide evidence that this phenotype correlates well with plasma LDL levels but not Lp[a]. Our data also suggest that the apo[a] gene is only partially involved in the regulation of the plasma Lp[a] levels.

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

The protein moiety of Lp[a] consisting of apoB and apo[a] covalently linked to each other, once freed of lipids by delipidation at pH 8.0 with mixtures of diethyl ether and ethanol, is freely water-soluble at pH values above 6.4. This is in contrast to apoB which, if prepared by similar delipidation techniques, is only soluble at alkaline pH, indicating that the coupling of the carbohydrate-rich apo[a] to apoB confers water solubility to this apolipoprotein that it does not possess on its own. When probed in a sandwich ELISA with antibodies specific to apo[a], the results suggest that some apo[a] epitopes in Lp[a] are masked by lipid but are freely accessible to antibodies in the lipid-free apoB-apo[a] complex. Examination of apoB-apo[a] with an ELISA specific for apoB showed a decreased and altered immunoreactivity of apoB when compared to either low density lipoprotein (LDL) or Lp[a]. These results are consistent with a model in which the hydrophobic lipid binding domains of apoB in apoB-apo[a] self-associate and are shielded from the aqueous environment by the hydrophilic portions of apoB and by an envelope of apo[a]. The apoB-apo[a] complex has lipophilic properties as shown by its interaction with the phospholipid-stabilized triglyceride emulsion, Intralipid. In addition, it has an avidity for all types of lipoproteins although displaying a preference for triglyceride-rich particles. In the presence of plasma, the interaction of apoB-apo[a] with all lipoproteins is reduced. Neither iodinated apo[a] nor iodinated Lp[a] nor LDL had an affinity for lipoproteins, suggesting that the lipophilic properties of apoB-apo[a] are probably due to apoB since apo[a] is rather hydrophilic and is unable to bind to lipids. Thus, the apoB-apo[a] complex has amphipathic properties with apo[a] providing the hydrophilic capacity to interact with the aqueous environment and apoB providing the hydrophobic interactions necessary to bind lipids.

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42–49) and Nilsson et al. (1981. Anal. Biochem. 110: 342–348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.

Lipoprotein (a) (Lp(a)) contains equimolar amou- nts of apoB-100 and apolipoprotein (a) (apo(a)). Both proteins are amenable to degradation in vivo by mecha- nisms yet to be clearly defined. In this study, we examined the in vitro susceptibility of LDL and Lp(a), obtained from the same donor, to oxidation by either Cu 2 1 or the combined Crotalus adamanteus phospholipase A 2 and soy- bean lipoxygenase system, monitoring the course of the re- action by the generation of conjugated dienes and fatty acids. In some experiments, treatment with leukocyte elastase (LE) or matrix metalloproteinase 12 (MMP-12) was admin- istered before and after the oxidative step. In the case of Lp(a) we found that with both oxidizing systems, condi- tions that caused the breakdown of apoB-100 did not degrade apo(a) although oxidation-mediated changes were detected in the latter by intrinsic tryptophan fluorescence spectro- scopy. Similar results were obtained with a reassembled Lp(a) obtained by incubating free apo(a) with LDL. Both apo(a) and apoB-100 were cleaved by LE and MMP-12 but the enzymatic cleavage was more marked when the pre- oxidized proteins were used as a substrate. Taken together, our in vitro studies indicate that apo(a) but not apoB-100 resists oxidative fragmentation, whereas both proteins are cleaved by enzymes of the serine and metalloproteinase families. We speculate that the fragments of apo(a) observed in vivo may be preferentially generated by proteolytic rather than oxidative events, whereas apoB-100 can be degraded by vivo studies (3, 8, 9). Regarding the apoB-100 constituent of authentic LDL, its breakdown has been shown to occur in "maximally" oxidized particles (10) and also following the action of biologically occurring enzymes of the elastase and metalloproteinase families (3, 9). In the case of apoB-100 as a constituent of Lp(a), information about the mechanisms underlying its degradation as a function of oxidative versus proteolytic events is comparatively lim- ited, and this also applies to apo(a). At present it is unclear whether the LDL constituent of Lp(a) is susceptible to oxi- dative and proteolytic changes as is authentic LDL and whether apo(a), shown to be cleaved by proteolytic en- zymes, can also be degraded by oxidative events. We rea- soned that an understanding of these processes in vitro would provide a useful background for investigating the mechanism(s) of formation of Lp(a) fragments in vivo. To this end, in the current study we used in vitro systems to study the effect of oxidative and proteolytic conditions on purified preparations of human LDL, Lp(a), and free apo(a), all obtained from the same subject. We show here that oxidative events that in Lp(a) cause the breakdown of apoB-100 do not affect the cleavage of apo(a), whereas both proteins are readily cleaved by the action of proteolytic en- zymes of the serine and metalloproteinase families.