Assembly Of Lp Research Articles

Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Lipoprotein(a): when to measure, how to treat?

Abstract: Lipoprotein(a) [Lp(a)] is one of the most atherogenic lipoproteins consisting of a core low-density lipoprotein particle and the specific glycoprotein apo(a). Apo(a) is homologous to plasminogen yet in contrast exhibits a specific size polymorphism. This polymorphism is due to the fact that the number of kringle-IV (K-IV) repeats ranges between two and approximately 50. Apo(a) is synthesized almost exclusively in the liver, and there is still some discussion regarding whether the assembly of Lp(a) occurs intracellularly or in the circulating blood. The plasma Lp(a) concentration is markedly skewed to the right and extends from <1 mg/dL to more than 200 mg/dL. Up to 90% of the variance of Lp(a) concentrations may be genetically determined and the Lp(a) concentration correlates inversely with the number of K-IV repeats. In the apo(a) promoter there are numerous response elements for transcription factors and nuclear receptors, whereby the HNF4α binding sequence is the most important one. Activation of FXR causes the dissociation of HNF4α from its response element and in turn a significant down regulation of apo(a) transcription. Recent large epidemiological studies document beyond any doubt that Lp(a) is an independent causal risk factor for coronary heart disease and myocardial infarction. Hence, novel approaches to correct elevated Lp(a) are under investigation. Among the established lipid-lowering drugs, only nicotinic acid lowers Lp(a) in a consistent and clinically relevant fashion, and we recently elucidated the molecular mechanism underlying this effect. Novel medicines in clinical trials include CETP inhibitors, PCSK9 antibodies, the MTP inhibitor lomitapide and antisense oligonucleotides. APO(a)Rx ®, an antisense oligonucleotide, which is specifically directed against the mRNA for apo(a), has the strongest effect on Lp(a). It offers the opportunity to examine the impact of selective Lp(a) lowering on clinical events. Lp(a) emerged as an important screening parameter to assess the risk for atherosclerosis. Its quantitation in the clinical laboratory had not been standardized for a long period of time. New commercial methods, in particular enzyme immunoassays with monoclonal antibodies that recognize single epitopes in apo(a), or nephelometric and turbidimetric assays hold the potential to warrant comparable results in different laboratories.

In vivo kinetic studies to further understand pathogenesis of abnormal lipoprotein metabolism in chronic kidney disease

Patients undergoing hemodialysis (HD) have been shown to be at increased risk for cardiovascular disease (CVD) morbidity and mortality which are, at least in part, due to uremic dyslipidemia including increased triglyceride-rich lipoproteins, in particular remnants, decreased high-density lipoprotein (HDL), and increased lipoprotein(a) [Lp(a)]. In vivo kinetic studies using stable isotope revealed that apolipoprotein (apo)A-I, a primary apoprotein constitute of HDL, was catabolized at a faster rate in HD patients, leading to decreased apoA-I, and therefore reduced HDL cholesterol concentrations. Likewise, apoB catabolic rates were significantly lower in intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) apoB; the latter is also accompanied by a decreased production rate. In HD patients, IDL apoB levels were elevated, but LDL apoB levels remained within the normal range. Nonetheless, a prolonged residence time for LDL apoB of 2-5 days, made LDL more atherogenic. Atorvastatin completely ameliorated impaired LDL apoB catabolism. With regard to Lp(a) metabolism, both apoB and apo(a) were found to be slowly catabolized, indicating roles of normal kidney function on Lp(a) catabolism. Finally, a compartmental model suggests intracellular, rather than extracellular, assembly of Lp(a). This in vivo kinetic evidence will uncover the underlying mechanism for uremic dyslipidemia and provide strategies to reduce CVD in HD patients.

Mutation of lysine residues in apolipoprotein B-100 causes defective lipoprotein[a] formation

Lipoprotein[a] (Lp[a]) is assembled by a two-step process involving an initial lysine-dependent binding between apolipoprotein B-100 (apoB-100) and apolipoprotein[a] (apo[a]) that facilitates the formation of a disulphide bond between apoB-100Cys4,326 and apo[a]Cys4,057. Previous studies of transgenic mice expressing apoB-95 (4,330 amino acids) and apoB-97 (4,397 amino acids) have shown that apoB-100 amino acids 4,330-4,397 are important for the initial binding to apo[a]. Furthermore, a lysine-rich peptide spanning apoB-100 amino acids 4,372-4,392 has recently been shown to bind apo[a] and inhibit Lp[a] assembly in vitro. This suggests that a putative apo[a] binding site exists in the apoB-4,372-4,392 region. The aim of our study was to establish whether the apoB-4,372-4,392 sequence was important for Lp[a] assembly in the context of the full-length apoB-100. Transgenic mice were created that expressed a mutant human apoB-100, apoB-100K4-->S4, in which all four lysine residues in the 4,372-4,392 sequence were mutated to serines. The apoB-100K4-->S4 mutant showed a reduced capacity to form Lp[a] in vitro compared with wild-type human apoB-100. Double transgenic mice expressing both apoB-100K4-->S4 and apo[a] contained significant amounts of free apo[a] in the plasma, indicating a less-efficient assembly of Lp[a] in vivo. Taken together, these results clearly show that the apoB-4,372-4,392 sequence plays a role in Lp[a] assembly.

The Physiological Role of Lipoprotein (a).

Lipoprotein (a) (Lp(a)) is one of the most atherogenic lipoproteins, and, although we know plenty about the pathophysiology of Lp(a), its physiological function and metabolism remain elusive. From our previous results and more recent reports, the following model of Lp(a) metabolism emerges: apolipoprotein a (apo(a)) is biosynthesized in liver cells and the size of the isoform determines its rate of synthesis and excretion. In a first step, specific kringle IV domains in apo(a), mainly T-6 and T-7, bind to circulating low-density lipoproteins, followed by a second step in which stabilization of the newly formed Lp(a) complex is achieved by a disulfide bridge. Circulating Lp(a) interacts specifically with kidney cells, or possibly other tissues, causing cleavage of 2/3­3/4 of the N-terminal part of apo(a) by a collagenase-type protease. Part of these apo(a) fragments are found as excretory products of Lp(a) in urine, but there are indications that they, in fact, represent the biologically active form of apo(a) and are possibly responsible for the atherogenicity of Lp(a). Strategies for reducing this atherogenic lipoprotein with medication should, therefore, aim at interfering with either the assembly of Lp(a) or the stimulation of apo(a) fragmentation. (c) 2002 Prous Science. All rights reserved.

Effect of cardiopulmonary bypass on plasma levels of lipoprotein (a) in hypercholesterolemic patients.

Increments of lipoprotein (a) (Lp (a)) concentration during cardiopulmonary bypass (CPB) have not been justified in the literature yet. We have investigated whether Lp (a) levels remain constant or increase during CPB and if high plasma levels of low density lipoprotein (LDL; containing apolipoprotein (apo) B) in hypercholesterolemic patients affect the assembly of Lp (a) (containing apoB: Apo (a)). In this study, the change in plasma lipid and lipoprotein levels of 40 patients with hypercholesterolemia and 40 patients who have normal cholesterol values were determined and compared during CPB, and in the postoperative early stage. In our study, lipid and lipoproteins, except Lp (a), showed a falling trend and paradoxically, Lp (a) statistically showed a significant rising trend, like the acute phase reactant in two groups (p=0.011 for LDL, p=0.016 for high density lipoprotein (HDL) and p<0.001 for the others, in 80 patients). Concentrations of Lp (a) in plasma increased more sharply in the hypercholesterolemic group than the normocholesterolemic group during CPB. This difference was significant at the 60th minute of cardiopulmonary bypass with a nonparametric test (p<0.05 Mann-Whitney U test). High density lipoprotein values showed more decline in the hypercholesterolemic group patients than in the normocholesterolemic group patients (p<0.05). In conclusion, lipoprotein (a) levels increased more pronounced in patients with hypercholesterolemia during CPB. On the other hand, high LDL levels in hypercholesterolemic patients accelerated Lp(a)formation in the acute phase.

Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.

Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.

Transgenic rabbits expressing human apolipoprotein (a).

Elevated plasma levels of lipoprotein (a) [Lp(a)] constitutes an independent risk factor for coronary heart disease, stroke, and restenosis. Over the past years, our understanding of the genetics, metabolism and pathophysiology of Lp(a) have increased considerably. However, the precise mechanism(s) by which this atherogenic lipoprotein mediates the development of atherosclerosis remains unclear. This is partly due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)], a distinct component of Lp(a) is found only in primates and humans. Development of transgenic mice expressing human apo(a) has provided an alternative means to investigate many aspects of Lp(a). However, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this aspect, we generated transgenic rabbits expressing human apo(a). In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was 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). Our study suggests 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).

The seventh myth of lipoprotein(a): where and how is it assembled?

Our understanding of the genetics, metabolism and pathophysiology of the atherogenic plasma lipoprotein Lp(a) has considerably increased over past years. Nevertheless, the precise mechanisms regulating the biosynthesis and assembly of Lp(a) are poorly understood and controversially discussed. Lp(a) plasma concentrations are determined by synthesis and not by degradation. Transcriptional and post-translational mechanisms have been identified as regulating Lp(a) production in primary hepatocytes and transfected cell lines. Assembly of Lp(a) occurs extracellularly from newly synthesized apolipoprotein(a) and circulating LDL. This view has recently been challenged by in-vivo kinetic studies in humans which are compatible with an intracellular assembly event. Lp(a) assembly is a complex two-step process of multiple non-covalent interactions between apolipoprotein(a) and apolipoprotein B-100 of LDL followed by covalent disulfide linkage of two free cysteine residues on both proteins.

Synthesis, Secretion and Assembly of Lp(a) in Hepatocyte Cultures

Synthesis, Secretion and Assembly of Lp(a) in Hepatocyte Cultures

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).

Intracellular metabolism of human apolipoprotein(a) in stably transfected Hep G2 cells.

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.

Increased plasma concentrations of LDL-unbound apo(a) in patients with end-stage renal disease: Rapid Communication

Lipoprotein(a) [Lp(a)] and its characteristic glycoprotein apolipoprotein(a) [apo(a)] are risk factors for atherosclerosis in the general population. Patients with renal disease show an elevation of Lp(a). Recent studies have described an arteriovenous difference of Lp(a) in the renovascular bed as well as the plasma-derived fragmented LDL-unbound apo(a) in urine, suggesting that the kidney is involved in the metabolism of Lp(a). We therefore investigated whether patients with chronic renal failure have higher levels of LDL-unbound apo(a) and whether this could account for the increased Lp(a) concentrations in these patients. In addition, we studied the possible generation of apo(a) fragments in vitro by mimicking uremic plasma conditions and by investigating the assembly of Lp(a) in cell culture experiments. Patients treated by hemodialysis (N = 185) and by continuous ambulatory peritoneal dialysis (CAPD; N = 20) had markedly elevated absolute (1.22 +/- 1.55 mg/dl and 2.14 +/- 2.86 mg/dl) as well as relative (7.5% and 7.3%) amounts of LDL-unbound apo(a) in comparison to controls (0.46 +/- 0.48 mg/dl or 4.5%). Following renal transplantation the absolute amount decreased significantly. Lp(a) plasma concentration was the most important determining variable for the absolute amount of LDL-unbound apo(a) and showed a positive correlation in both hemodialysis patients (r = 0.85) and controls (r = 0.92). In vitro experiments demonstrated that "uremization" of plasma samples did not generate a higher amount of LDL-unbound apo(a). Although LDL of renal patients has different chemical and structural properties as compared to control LDL, the extracellular assembly of Lp(a) did not differ between patients and controls. Therefore, the higher amounts of LDL-unbound apo(a) found in renal disease are not caused by an impaired assembly of Lp(a), but rather indicate a catabolic role of the kidney for LDL-unbound apo(a) as was already shown for Lp(a). Despite a small contribution, these elevated levels cannot explain the higher Lp(a) values found in patients with end-stage renal disease.

Metabolism of Lp(a): assembly and excretion.

Lp(a) is one of the most atherogenic lipoproteins, and we know much more about the pathophysiology of Lp(a) than about its physiological function and metabolism. From our previous investigations and the new results reported here, we propose the following model of Lp(a) metabolism: apo(a) is biosynthesized in liver cells and the size of the isoform determines its rate of synthesis and excretion. Specific kringle-4 domains in apo(a), mainly T-6 and T-7, bind in a first step to circulating LDL, followed by the stabilization of the newly formed Lp(a) complex by a disulfide bridge. Circulating Lp(a) interacts specifically with kidney cells, or possibly other tissues, causing cleavage of 2/3-3/4 of the N-terminal part of apo(a) by a collagenase-type protease. Part of the apo(a) fragments is found in the urine, but there are indications that they in fact represent the biologically active form of apo(a). The core portion of Lp(a) in turn is cleared by the LDL-receptor or another specific binding system of the liver. Strategies for reducing plasma Lp(a) levels with medication should aim at interfering with the assembly of Lp(a) on one hand and the stimulation of apo(a) fragmentation on the other hand.

Characterization and modulation of LP(a) in human hepatoma HEPG2 cells

HepG2 cells have been widely used to study factors which affect the secretion of apoB100 lipoprotein particles. The objectives of this study were to determine if Lp(a) particles were present in conditioned medium from HepG2 cells and if so, was this accumulation affected by factors which alter apoB100 lipoprotein metabolism. The data demonstrate that Lp(a) accumulated in the medium in a time dependent manner over a 48 h incubation period. Ultracentrifugation fractionation and Western blot analysis demonstrated that lipoprotein particles containing apo(a) in complex with apoB100 were present at a density consistent with human plasma Lp(a). Incubation of the HepG2 cells with LDL or VLDL caused increases in Lp(a) accumulation in the medium (+33%±14%, P NS and 56%±21%, P<0.05, respectively). In contrast, apo(a) mRNA decreased (−17%±3%, P<0.01 for both LDL and VLDL incubation). Increasing concentrations of amino acids in the medium resulted in progressively less Lp(a) and apoB100 in the medium, the effect being greater on apoB100. ApoB100 mRNA levels decreased with incubation of HepG2 cells with amino acids (−22%±10%, P<0.06) whereas apo(a) mRNA levels increased significantly (+47%±14%, P<0.005). Taken together, our data show that HepG2 cells express mRNA for apo(a), and accumulate Lp(a) in the medium. The close correlation of medium Lp(a) levels with medium apoB100 levels, and not with apo(a) mRNA levels, suggests that medium Lp(a) accumulation may be a function of lipoprotein synthesis and secretion and is consistent with extracellular assembly of Lp(a) lipoprotein particles.

Transgenic Mice Expressing Human ApoB95 and ApoB97: EVIDENCE THAT SEQUENCES WITHIN THE CARBOXYL-TERMINAL PORTION OF HUMAN apoB100 ARE IMPORTANT FOR THE ASSEMBLY OF LIPOPROTEIN(a)

The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond. To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Inhibitors for the in vitro assembly of Lp(a).

Lp(a) is composed of an LDL-like core and the glycoprotein apo(a). Current evidence strongly suggests that the assembly of this atherogenic lipoprotein proceeds outside the liver cells in a two-step fashion. In the first step, a loose complex is formed involving kringle-4 motifs in apo(a) and one or more Lys side chains in apoB-100. In the second step, this complex is stabilized by a disulfide bridge. Indications are that Lp(a) assembly is critical in the determination of plasma apo(a) concentrations. Therefore, we searched for substances that interfere with the first step of Lp(a) assembly. epsilon-Aminohexoic acid (epsilon-AHA), known as an inhibitor from earlier assembly studies, had an IC50 of 4.8 mmol/L. The IC50 of Pro, HO-p-aminobenzene sulfonamide, Lys, N-epsilon-acetyl-Lys, taurine, Glu, serotonin, and benzamidine were all > 20 mmol/L. gamma-Aminobutyric acid, spermine, and spermidine exhibited IC50 on the same order of magnitude as epsilon-AHA. The substances with the highest inhibitory action were tranexamic acid and delta-aminovaleric acid. Seven of eight patients treated in a pilot study with tranexamic acid (Cyclocapron) responded with a decrease of plasma apo(a) of 18.5 +/- 8.2%. We suggest that substances that interfere with the Lp(a) assembly are worth pursuing further for their usefulness as therapeutic agents in reducing high plasma Lp(a) concentrations.

Decreased binding of apolipoprotein (a) to familial defective apolipoprotein B-100 (Arg3500–>Gln). A study of the assembly of recombinant apolipoprotein (a) with mutant low density lipoproteins.

In familial defective apolipoprotein B-100 (FDB), glutamine is substituted for arginine at position 3500 of the amino acid sequence. This mutation alters the structure of low density lipoproteins (LDL) and reduces their binding to LDL receptors. We studied the assembly in vitro of FDB-LDL with two recombinant apo(a) (r-apo(a)) isoforms containing 17 or 18 kringle IV-type repeats, respectively. R-apo(a) complexed to LDL in a concentration- and time-dependent manner. When we mixed normal LDL at protein concentrations from 1 to 10 mg/liter with 200 micrograms/liter r-apo(a) and incubated for 20 h, 15-44% of r-apo(a) were bound to LDL, forming an artificial Lp(a)-like particle. With LDL from a homozygous FDB patient, only 2-16% of r-apo(a) were complexed; heterozygous FDB-LDL bound 2-30% of r-apo(a). We also studied the effect of r-apo(a) on the interaction of the monoclonal antibody MB47 with normal and mutant apoB-100. FDB-LDL displayed enhanced binding of MB47. Adducts generated from normal LDL and r-apo(a) had an increased affinity for MB47, when compared to LDL alone. In contrast, r-apo(a) did not change MB47 reactivity when incubated with FDB-LDL. Altogether, our findings suggest that domains in apolipoprotein B which are important for the interaction with the LDL receptor play a role in the assembly of Lp(a) as well. They provide, in addition, an explanation for the observation that in Lp(a) of heterozygous FDB patients, the ratio of defective to normal apoB-100 is significantly smaller than in LDL from the same patients.