Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Lipid transfer protein-catalyzed exchange of cholesteryl ester between high density lipoproteins and apoB-containing lipoproteins

Low density lipoproteins (LDL), lipoprotein (a)(Lp(a)), and lipoprotein(a) after removal of the a-protein (Lp(a-)) were compared with respect to their ability to accept cholesteryl ester from high density lipoproteins (HDL). The incubations were performed at constant concentrations of HDL and various concentrations of either LDL, Lp(a), or Lp(a-). Lp(a) exchanged cholesteryl ester with HDL, but at a rate that was only 48.5 +/- 3.8% of the exchange rate found in the presence of autologous LDL. Cleavage of the apo(a) from Lp(a) resulted in Lp(a-), an LDL-like particle, with characteristics of cholesteryl ester exchange very similar to LDL.     

The linkage with apolipoprotein (a) in lipoprotein (a) modifies the immunochemical and functional properties of apolipoprotein B

Lipoprotein (a) [Lp(a)] was isolated from several donors and its apolipoprotein (a) [apo(a)] dissociated by a reductive treatment, generating the apo(a)-free form of Lp(a) [Lp(a--)] that contains apolipoprotein B (apo B) as its sole protein. Using anti-apo B monoclonal antibodies, the properties of apo B in Lp(a), Lp(a--), and autologous low-density lipoprotein (LDL) were compared. Marked differences in apo B immunoreactivity were found between these lipoproteins, due to the presence of apo(a) in Lp(a). Apo(a) enhanced the expression of two epitopes in the amino-terminal part of apo B while it diminished the immunoreactivity of three other epitopes in the LDL receptor binding domain. Accordingly, the binding of the lipoproteins to the LDL receptor was also decreased in the presence of apo(a). In a different experimental system, the incubation of antibodies that react with 27 distinct epitopes distributed along the whole length of apo B sequence with plastic-bound Lp(a) and Lp(a--) failed to reveal any epitope of apo B that is sterically hindered by the presence of apo(a). Our results demonstrate that the presence of apo(a) modified the organization and function of apo B in Lp(a) particles. The data presented indicate that most likely the modification is not due to a steric hindrance but that some more profound conformational changes are involved. We suggest that the formation of the disulfide bridge between apo B and apo(a) in Lp(a) alters the system of disulfide bonds present in apo B and thereby modifies apo B structure.     

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.     

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).     

Isolation of apolipoprotein(a) from lipoprotein(a)

An easy method was developed for the rapid and selective isolation of apo(a) from human plasma Lp(a). This procedure was applied to a "low density" Lp(a) subspecies (usually found in the density interval 1.050 to 1.070 g/ml) from a single individual whose apo(a) was of a size smaller than apoB-100. After reduction with 0.01 M dithiothreitol, apo(a) was separated from the Lp(a) particle by rate zonal centrifugation on a 7.5-30% NaBr density gradient. Two completely water-soluble products were recovered: apo(a), which contained less than 1% each of phospholipid and cholesterol, remained at the bottom of the gradient, and a lipid-rich floating LDL-like particle which contained apoB but not apo(a) and which we referred to as Lp(a-). The separation of these two components was also achieved by subjecting reduced Lp(a) to electrophoresis on 2.5-16% polyacrylamide gradient gels. However, dissociation of reduced Lp(a) could not be achieved by gel filtration in either low or high salt solutions. These observations indicate that apo(a) is associated to Lp(a) by non-covalent interactions in addition to its disulfide linkage to apoB. The latter is sensitive to chemical reduction whereas the former are broken through the action of a gravitational or electrical field.     

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.     

Characterization of the basis of lipoprotein [a] lysine-binding heterogeneity

Although elevated plasma concentrations of lipoprotein [a] (Lp[a]) are considered to be a risk factor for atherosclerosis, the mechanisms by which Lp[a] mediates its pathogenic effects have not been conclusively determined. The apolipoprotein [a] (apo[a]) component of Lp[a] confers unique structural properties to this lipoprotein, including the ability to bind to lysine residues in biological substrates. It has been shown, however, that only a fraction of plasma Lp[a] (Lp[a]-Lys(+)) binds to lysine-Sepharose in vitro. The nature of the non-lysine-binding Lp[a] fraction in plasma (Lp[a]-Lys(-)) is currently unknown. In the present study, the Lp[a]-Lys(+) fraction was determined in the plasma of six unrelated individuals; the Lp[a]-Lys(+) fraction in these plasma samples ranged from approximately 37 to approximately 48%. Interestingly, purification of the Lp[a] by density gradient ultracentrifugation followed by gel filtration and ion-exchange chromatography resulted in progressive increases in the Lp[a]-Lys(+) fraction. Addition of either purified low density lipoprotein (LDL) or fibronectin to the purified Lp[a] at a 1:1 molar ratio reduced the Lp[a]-Lys(+) fraction (maximal decrease of 34 and 20%, respectively) whereas addition of both fibronectin and LDL to the purified Lp[a] resulted in a further decrease (45% maximally) in this fraction. Similar results were obtained by using a recombinant expression system for apo[a]: addition of a 4-fold molar excess of either LDL or fibronectin to conditioned medium containing metabolically labeled recombinant apo[a] reduced the Lys(+) fraction by 49 and 23%, respectively.Taken together, our data suggest that the lysine-binding heterogeneity of plasma Lp[a] is not primarily an intrinsic property of the lipoprotein, but rather results in large part from its ability to noncovalently associate with abundant plasma components such as LDL and fibronectin. These interactions appear to mask the lysine-binding site in apo[a] kringle IV type 10, which mediates the interaction of Lp[a] with lysine-Sepharose. The contribution of these interactions to the function of Lp[a] in vivo remains to be investigated.     

Lipoprotein[a] is the major apoB-containing lipoprotein in the plasma of a hibernator, the hedgehog (Erinaceus europaeus)

We have undertaken studies aimed at elucidating the interrelationships existing between the seasonal modifications in endocrine status (already demonstrated by Saboureau, M., and J. Boissin. 1978. C.R. Acad. Sci. (Paris) 286D: 1479-1482) and plasma lipoprotein metabolism in the male hedgehog. During the course of these studies, we discovered that a lipoprotein comparable to human Lp[a] was a prominent component of the plasma lipoprotein spectrum in the hedgehog. This lipoprotein was present in the 1.040-1.100 g/ml density range (approximately), exhibited pre beta mobility upon agarose gel electrophoresis, and its Stokes diameter was 275 A. Its apolipoprotein moiety consisted of two proteins with molecular weights and amino acid compositions similar to those of human apoB-100 and apo[a], respectively. These two apolipoproteins were present in hedgehog Lp[a] as a complex that could be dissociated using dithiothreitol and whose stoichiometry could be 1:1. Lp[a] polymorphism due to size heterogeneity of apo[a] appeared to be present in the hedgehog as in man. The chemical composition of hedgehog Lp[a], obtained from animals bled during spring and summer, differed from that of its human counterpart in that the proportion of triglycerides was approximately three times higher in the hedgehog particle (13% vs. 4%), to the detriment of cholesteryl esters. Dissociation of the apoB:apo[a] complex has allowed us to obtain Lp[a] devoid of its specific polypeptide (Lp[a-]), a particle that retained the characteristics of Lp[a] as regards its lipid composition but whose Stokes diameter decreased by 30 to 40 A. The plasma concentration of LDL particles, defined as lipoproteins containing apoB-100 as their sole apolipoprotein constituent, was considerably lower than that of Lp[a]. These findings suggest that the hedgehog could be a unique animal model for studies regarding Lp[a] metabolism.     

Stimulation of interleukin-8 production in human THP-1 macrophages by apolipoprotein(a). Evidence for a critical involvement of elements in its C-terminal domain

In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a-)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a-), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3-4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5-2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis.     

Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.     

Assembly of lipoprotein (a) in transgenic rabbits expressing human apolipoprotein (a)

The study of human lipoprotein (a) [Lp(a)] has been hampered due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)] is found only in primates and humans. In addition, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this study, we generated three independent transgenic rabbits expressing human apo(a) in their plasma at 1.8-4.5 mg/dl. In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was covalently associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp(a). These results suggest that transgenic rabbits expressing human apo(a) exhibit efficient assembly of Lp(a) and can be used as an animal model for the study of human Lp(a).     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

Tissue-type plasminogen activator binds to and is inhibited by surface-bound lipoprotein(a) and low-density lipoprotein

Elevated levels of lipoprotein(a) [Lp(a)] are associated with an increased risk of atherothrombotic disease, but the mechanism(s) by which Lp(a) potentiates atherogenesis is unknown. The extensive homology of apolipoprotein(a) [apo(a)] to plasminogen has led us and others to postulate that Lp(a) may impair fibrinolysis. We have previously shown that Lp(a) inhibits fibrin stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA); however, we and other investigators have been unable to demonstrate direct inhibition of t-PA by Lp(a) in solution. We now report that t-PA binds reversibly and saturably to surface-bound Lp(a) and to low-density lipoprotein (LDL) and that as a result of this binding activation of plasminogen by t-PA is inhibited. The catalytic efficiency (kcat/Km) of t-PA when bound to polystyrene surface-bound fibrinogen increased 2.9-fold compared to t-PA bound to control wells. When bound to surface-bound Lp(a), however, the catalytic efficiency of t-PA was reduced 9.5-fold compared to t-PA bound to control wells; likewise, by binding to surface-bound LDL, the catalytic efficiency of t-PA was reduced 16-fold compared to the control. Studies with defined monoclonal antibodies suggest that major determinants of t-PA binding are its active site, the LDL receptor binding domain of apolipoprotein B-100 (apoB-100), and apo(a). These data suggest a unique mechanism by which Lp(a) and LDL incorporated in an atheroma can inhibit endogenous fibrinolysis and thereby contribute to the genesis of atherothrombotic disease.     

Effect of tranexamic acid and delta-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or delta-aminovaleric acid (delta-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or delta-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1. 5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1. 4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

A PvuII polymorphism of the low density lipoprotein receptor gene is not associated with plasma concentrations of low density lipoproteins including LP(a)

Lipoprotein(a) [Lp(a)] is a low density lipoprotein (LDL), in which apolipoprotein B-100 (apo B-100) is attached to apolipoprotein(a) [apo(a)], a glycoprotein of variable size. Lp(a) may be as atherogenic as LDL. In normal populations, Lp(a) concentrations in plasma are largely determined by the apo(a) gene locus on chromosome 6, but regulation of synthesis and catabolism of Lp(a) is poorly understood. In some studies, a PvuII restriction fragment length polymorphism (RFLP) in the LDL receptor gene seems to affect concentrations of LDL in plasma, and other studies have indicated that Lp(a) catabolism could be mediated by the LDL receptor. We therefore expected that the PvuII polymorphism in the LDL receptor gene might be associated with Lp(a) levels in 170 Caucasian men aged 40 years, selected to have a high representation of low molecular weight apo(a) phenotypes. However, plasma concentrations of cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and Lp(a) were all unrelated to the LDL receptor gene PvuII polymorphism both in the group as a whole and when it was subgrouped by apo(a) phenotype. Therefore our data do not support the concept that this particular LDL receptor gene polymorphism is associated with LDL receptor function, and our data therefore neither support nor rule out a role for the LDL receptor in Lp(a) catabolism.     

Evaluation of lipoprotein(a) as a prothrombotic factor: progress from bench to bedside

PURPOSE OF REVIEW: Since the homology between apolipoprotein(a) (apo(a)) and plasminogen was discovered in 1987, the role of lipoprotein(a) (Lp(a)) as an inhibitor of the normal fibrinolytic role of plasmin(ogen) has been a major research focus. In this review we summarize recent basic research aimed at identifying mechanisms by which Lp(a) can either inhibit fibrinolysis or promote coagulation, as well as recent clinical studies of Lp(a) as a risk factor for thrombosis either in the presence or absence of atherosclerosis. RECENT FINDINGS: It has recently been reported that the inhibition of plasminogen activation by apo(a) results from the interaction of apo(a) with the ternary complex of tissue-type plasminogen activator, plasminogen and fibrin, rather than competition of apo(a) and plasminogen for binding sites on fibrin. Lp(a) species containing smaller apo(a) isoforms bind more avidly to fibrin and are better inhibitors of plasminogen activation. Recent clinical studies have provided strong evidence that Lp(a), either alone or in synergy with other thrombotic risk factors, significantly increases the risk of venous thromboembolism and ischemic stroke. SUMMARY: Lp(a) both attenuates fibrinolysis, through inhibition of plasminogen activation, and promotes coagulation, through alleviation of extrinsic pathway inhibition. Further basic and clinical studies are required to more clearly define the role of Lp(a) in thrombotic disorders, and to determine the extent to which thrombotic risk is dependent on apo(a) isoform size.     

Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein and lipoprotein(a) as determined by time-resolved fluorometry.

Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrienylpropionic acid (DPH) were prepared and incorporated into the surface layer of human low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluorescence anisotropy measurements of DPH-PC and DPH-SM in both lipoprotein classes were carried out at different temperatures ranging from 20 to 37 degrees C. DPH-PC as well as DPH-SM were shown to reside in more rigid domains in Lp(a) than in LDL according to higher anisotropy values in Lp(a). In both LDL and Lp(a), DPH-PC experienced a more rigid environment than DPH-SM, suggesting different environments of PC and SM in the surface shell of the lipoproteins. Fluorescence lifetimes of the labeled lipoproteins were determined by phase and modulation fluorometry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and Lp(a). Lifetime distribution centers for labeled lipids were very similar except for DPH-PC in Lp(a) which was shifted to longer lifetimes, suggesting a less polar environment of PC in Lp(a) than in LDL. The distributional width of DPH-PC in Lp(a) was broader than in LDL. Accordingly, phosphatidylcholine must be localized in a more homogeneous environment in LDL as compared with Lp(a). On the other hand, no difference in distributional widths was observed for DPH-SM in both lipoproteins, showing that SM organization in Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose that apoproteins discriminate between the choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) due to the presence of apolipoprotein(a).     

Comparative study of Ca2+ binding to lipoprotein(a) and low density lipoprotein by spin labeling

The effect of Ca2+ binding on the dynamic properties of various spin labeled fatty acids in lipoprotein(a) (Lp(a)) was studied in comparison with low density lipoprotein (LDL) isolated from human plasma. In contrast to LDL, binding of Ca2+ to Lp(a) induced broadening of the lines in the ESR spectra of the spin labeled stearic acids. In 1.6 M NaBr solutions the thermotropic change in the surface structure was observed in both lipoproteins at similar temperatures. Ten millimolar concentration of Ca2+ shifted the temperature of the thermotropic change in the surface structure of Lp(a) to considerably higher values. We conclude that Ca2+ binding to Lp(a) induces changes in the lipid structure of the particle surface.     

Interaction of apolipoprotein(a) with apolipoprotein B-containing lipoproteins

Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).