Biomedical subjects

G M Kostner

Publications and source records attributed to G M Kostner.

At least 109 records · Page 6Linked to original sources

PubMed1995

Immunoturbidimetric determination of lipoprotein(a): improvement in the measurement of turbid and triglyceride-rich samples.

Immunoturbidimetry (IT), a widely used method in clinical chemical laboratories, was checked for its suitability for lipoprotein(a) (Lp(a)) quantification. When the conventional sample diluents were used, turbidimetry gave false results particularly with frozen or lipemic sera which correlated poorly with electroimmunodiffusion (EID). L-Proline which is known to dissociate Lp(a) from other apo B-containing lipoproteins improved the results considerably. One hundred frozen sera were investigated in IT with and without the addition of L-proline to the sample diluent. EID served as a comparison method. In a method comparison (IT vs. EID) linear regression analysis improved from r = 0.793: y = 0.89x - 9.4 (without L-proline) to r = 0.949: y = 0.98x + 4.8 (with L-proline). The improvement of the correlation of the two methods was most pronounced in sera with triglyceride values exceeding 5.5 mmol/l. The IT assay described here was linear between 50 and 1100 mg/l. Total imprecision (coefficient of variation) was below 10%. The assay was not affected by the addition of LDL or plasminogen to the samples. The Lp(a) concentration of the calibrator, i.e. a secondary standard serum, was compared with that of a purified primary Lp(a) standard which consisted of a mixture of four apo(a) isoforms. Total Lp(a) mass (lipids, protein, carbohydrates) was determined chemically and was compared with the Lp(a) mass determined immunochemically by IT and EID. Recovery of the purified Lp(a) was 106% (range 90-116%) in IT and 102% (range 91-115%) in EID. Dose response curves from pure single isoforms (S1 and S4), calibrator and serum samples were parallel. We consider IT to be a simple and rapid method for Lp(a) quantification which is not biased by different apo(a) isoforms.

Blood Proteins↗

PubMed1995

Human plasma phospholipid transfer protein accelerates exchange/transfer of alpha-tocopherol between lipoproteins and cells.

alpha-Tocopherol (alpha-T), an important anti-oxidant of plasma lipoproteins and cell membranes, is secreted from liver together with very-low-density lipoproteins into the blood stream. Other serum lipoprotein classes gain alpha-T by exchange and transfer processes. We show here that the lipoprotein-free d > 1.22 g/ml fraction of human or pig serum increases the exchange rate of alpha-T by a factor of 2-4 as compared with spontaneous exchange/transfer. The alpha-T exchange/transfer (alpha-TET) activity was purified by multiple-step column chromatography. It gave a single band in PAGE with an apparent molecular mass of 75 kDa, and was found to be identical with the phospholipid transfer protein (PLTP). PLTP catalysed alpha-T exchange between different lipoprotein classes, as well as the transfer of alpha-T from artificial liposomes to high-density lipoproteins. The alpha-TET activity measured with a newly developed assay in ten healthy people was 2.45 +/- 0.88 nmol.ml-1.h-1.alpha-TET activity was negatively correlated with plasma low-density lipoprotein-cholesterol (r = -0.75; P < 0.01). It is concluded that human PLTP catalyses exchange/transfer processes of alpha-T between lipid compartments. This factor may be of relevance in atherogenesis and tumour initiation and growth.

Animals↗

PubMed1995

Quantification of 7-dehydrocholesterol in plasma and amniotic fluid by liquid chromatography/particle beam-mass spectrometry as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome.

The qualitative and quantitative determination of cholesterol and 7-dehydrocholesterol (7-DHC) in plasma as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome by liquid chromatography/particle beam interface-mass spectrometry (LC/PB-MS) is presented. Baseline separation of cholesterol and 7-DHC is achieved on a silica column with hexane+ethanol (99: 1 v/v) as mobile phase within a 10 min analysis. Recoveries of cholesterol and 7-DHC in a simple two-phase extraction system are nearly 100%. The absolute limit of detection using LC/PB-MS is approximately 10 ng. The method presented allows extraction, analysis and quantification of cholesterol and 7-DHC within 15 min without the necessity of sample derivatization.

Amniotic Fluid↗

PubMed1995

A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency.

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.

Adenosine↗

PubMed1995

Influence of n-3 fatty acids on the growth of human breast cancer cells in vitro: relationship to peroxides and vitamin-E.

Epidemiological studies suggest a causal relationship of dietary polyunsaturated fatty acids (PUFA's) with the morbidity and mortality from breast cancer. In order to reveal possible underlying mechanisms of these findings, we studied the influence of n-3 and n-6 PUFA's in comparison to oleic acid on the proliferation of well characterized estrogen dependent (MCF-7, ZR-75, T-47-D) and estrogen independent (MDA-MB-231, HBL-100) breast cancer cells in culture. The cell growth inhibitory effect was related to the formation of lipid peroxidation products. Normal human skin fibroblasts served as a control. In fibroblasts, the addition of 20 micrograms/ml of exogenous fatty acids either had no effect or caused an insignificant increase of proliferation. Similar results were obtained with MCF-7 cells. In all other breast cancer cell types, n-3 long-chain PUFA's, eicosapentaenoic and docosahexaenoic acids, were the most effective fatty acids in arresting the cell growth. Alpha-linolenic and gamma-linolenic acid exerted a variable effect on cell proliferation depending on the cell line investigated. Oleic acid significantly stimulated the proliferation of hormone-independent breast cancer cells while it had no effect on the proliferation of hormone-dependent cells. Viability studies by trypan blue excretion indicated that the arrest in cell growth was not due to major cytotoxic effects. The addition of PUFA's to breast cancer cells caused a significant increase in the formation of conjugated dienes and lipid hydroperoxides in the cellular lipids; their content was significantly correlated with the capacity of arresting cell growth. In contrast, the addition of PUFA's to fibroblasts did not increase lipid hydroperoxide formation. The addition of Vitamin E to cancer cells at a concentration of 10 microM to the PUFA-supplemented medium almost completely restored cell growth. Our data indicate that PUFA's significantly interfere with cell proliferation of breast cancer cells in vitro due to the formation of oxidation products. In addition to that, there must be other factors involved, most probably related to the differential metabolism of PUFA's in tumor cells. Our findings may have some impact on treatment and prevention of breast cancer.

Adult↗

PubMed1995

Lipoprotein(a) plasma levels and apo(a) isoforms are not associated with longevity or disability in a sample of Italian octo-nonagenarians. Associazione Medica Sabin.

Cardiovascular diseases are the leading cause of disability and mortality in western countries. Lipoprotein(a) [Lp(a)] is now considered an independent risk factor for atherosclerosis, and might consequently be related to longevity and/or disability. In the context of a study on metabolic and anthropometric parameters in a sample of Italian octo-nonagenarians, Lp(a) and apo(a) isoforms were evaluated. One-hundred and fifty Italian octo-nonagenarians were classified as free-living or disabled, according to Katz's index, and compared to 91 healthy control adults. All the study subjects were recruited from a valley (Val Vibrata valley) near Teramo, in the central part of Italy. The median Lp(a) concentration of the whole group was 17 mg/dL (range 1-161 mg/dL), which is much higher than the values observed in Caucasian populations. No differences were detected between the octo-nonagenarian group (median 16 mg/dL, range 1-126 mg/dL) and the control group (median 19.5 mg/dL, range 1-161 mg/dL), nor between the free-living and the disabled groups. Apo(a) isoforms were similarly distributed among free-living, disabled and control subjects. While our findings suggest that Lp(a) plasma levels and apo(a) isoforms are not factors associated with longevity or disability, we cannot exclude that the low incidence of other major risk factors for atherosclerosis in our free-living octo-nonagenarians hampered the full expression of the lipoprotein(a) atherogenic potential, and thus allowed the achievement of a very old age in a good healthy status, even in carriers of high Lp(a) levels or small apo(a) isoforms.

Adult↗

PubMed1995

Cholesterol biosynthesis in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin.

As peroxisomes possess some of the integral enzymes for cholesterol biosynthesis, the role of these organelles in cholesterol formation was studied in dermal fibroblasts with three types of peroxisomal defect: group I, characterized by the absence of intact peroxisomes (neonatal adrenoleukodystrophy, cerebrohepatorenal syndrome of Zellweger); group II, showing impaired activity of a single peroxisomal enzyme (X-linked adrenoleukodystrophy, adrenomyeloneuropathy); and group III, defective in more than one peroxisomal enzyme (rhizomelic chondrodysplasia punctata). Cells were incubated with three different radioactive precursors, namely [14C]-octanoate, [14C]-acetate, and [3H]-mevalonate, and incorporation of these radiolabels into cholesterol was determined. All fibroblasts with peroxisomal defects were able to form cholesterol at concentrations comparable or higher than those in controls dependent on the radioactive substrate. Binding properties (KD) and bmax values) of LDL to fibroblasts with peroxisomal defects and downregulation of intracellular cholesterol biosynthesis were similar to those found in fibroblasts from normolipidaemic controls, but different to those observed in LDL-receptor negative fibroblasts. As our studies revealed that cholesterol biosynthesis is not impaired in fibroblasts from patients with metabolic disorders of peroxisomal origin, we conclude that peroxisomes play little or no role in the pathway of cholesterol synthesis beyond mevalonate. In earlier steps of the cholesterol synthesis pathway, peroxisomal and mitochondrial defects in parallel may alter cholesterol synthesis indirectly.

Adrenoleukodystrophy↗

PubMed1995

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.

Adult↗

PubMed1995

Lysine modification of LDL or lipoprotein(a) by 4-hydroxynonenal or malondialdehyde decreases platelet serotonin secretion without affecting platelet aggregability and eicosanoid formation.

The effects of lysine-modified atherogenic plasma lipoproteins, known to be constituents of human atherosclerotic plaques, were studied on platelet function in vitro. LDL and lipoprotein(a) [Lp(a)] modified with secondary breakdown products of lipid peroxidation (4-hydroxy-2,3-trans-nonenal [HNE] 0.1 to 10 mmol/L or malondialdehyde [MDA] 1 to 50 mmol/L) induced neither spontaneous platelet aggregation nor secretion of 5-hydroxytryptamine (5-HT) from platelet aminestorage granules. Incubation of platelets with HNE- or MDA-modified LDL or Lp(a) (up to 1200 micrograms protein/mL) prior to thrombin (0.2 U/mL)- or collagen (2 micrograms/mL)-induced aggregation did not enhance platelet aggregability or formation of eicosanoids, ie, thromboxane A2 or prostaglandins E2 and F2 alpha. In contrast to native lipoproteins, HNE- or MDA-modified LDL and Lp(a) (approximately 20% to 30% of total apolipoprotein lysine residues modified) exerted a pronounced dose-dependent inhibition of 5-HT release from activated platelets in the following order: HNE LDL (50%) > HNE Lp(a) (40%) > MDA LDL (20%) > MDA Lp(a) (5%). Preincubation of human blood platelets with acetylated LDL or Lp(a) (approximately 60% to 70% of total lysine residues modified) prior to aggregation impaired serotonin secretion by 50% compared with native LDL or Lp(a). These findings suggest that the interaction of platelets with aldehyde-modified atherogenic plasma lipoproteins should not necessarily be considered as proatherogenic with respect to the effects observed in our in vitro studies.

Acetylation↗

PubMed1995

Transfer of phospholipase A-resistant pyrene-dialkyl-glycerophosphocholine to plasma lipoproteins: differences between Lp[a] and LDL.

1-O-Hexadecyl-2-O-pyrenedecanyl-sn-glycero-3-phosphocholine, a non-hydrolyzable fluorescent diether analog of phosphatidylcholine (PC), was synthesized as a probe for studying phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human plasma with the new probe at 37 degrees C for 4.5 h, a characteristic partition between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a measure for choline glycerophospholipid distribution between plasma lipoproteins. In low density lipoprotein (LDL) and high density lipoprotein-3 (HDL3) the fluorescent PC analog showed only monomer fluorescence, whereas in Lp[a] and HDL2 monomer and excimer fluorescence were observed, indicating that the fluorescent phosphatidylcholine analog was incorporated into the respective lipoproteins to a different extent. According to the increased pyrene excimer fluorescence in Lp[a] compared with LDL the labeled phosphatidylcholine must be enriched and/or clustered in Lp[a]. Data from phospholipid and total fluorescence analyses are compatible with the assumption of higher label concentration in Lp[a]. On the other hand, transfer rates for serum protein-catalyzed lipid transport into isolated Lp[a] were slower as compared to LDL. It is suggested that slower lipid transfer to Lp[a] under these conditions is due to the decreased lipid mobility in the Lp[a] surface, whereas the higher extent of label partition into Lp[a] as observed in total plasma might be due to the higher affinity of apolipoproteins for phosphatidylcholine in Lp[a] (Sommer, A., et al. 1992. J. Biol. Chem. 267: 24217-24222). The use of a fluorescent dialkyl- instead of diacyl-glycerophosphocholine for transfer studies was mandatory, as we found that lipoproteins contained phospholipase A2 activity toward long-chain phosphatidylcholine. The lipoprotein-associated phospholipase A2 was three times more active in Lp[a] than in LDL. The degradation products formed by the phospholipase, fatty acids, and lyso-PC may add to the high atherogenic potential of Lp[a].

Biological Transport↗

PubMed1994

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.

Adult↗

PubMed1994

Structural requirements of apo-a for the lipoprotein-a assembly.

Lipoprotein-a [Lp(a)], one of the most atherogenic lipoproteins, is composed of a low-density lipoprotein (LDL) core in addition to an apo-a of variable size which is linked to apoB by a disulphide bridge. Lp(a) synthesized in vitro by incubation of recombinant apo-a (r-apo-a) with LDL is physico-chemically indistinguishable from native Lp(a). The synthesis of Lp(a) in vitro proceeds in two steps. In the first step, one of the unique kringle-IVs (K-IVs) in apo-a binds to a Lys residue of apoB; in the second step, Cys-4057 of K-IV type-9 (T-9) forms a disulphide bridge with Cys-3734 of LDL. Here we have produced r-apo-a with different combinations of unique K-IVs and shown that K-IV T-6 is required for the first step of Lp(a) assembly. For the second step not only is K-IV T-9 essential, but also the distance between T-6 and T-9 requires a length of two K-IVs. These findings give additional insight into the mode of Lp(a) assembly and are of relevance in the search for apo-a mutants influencing Lp(a) levels and for the development of Lp(a)-lowering medications.

Blotting, Western↗

PubMed1994

High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein.

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation.

Animals↗

PubMed1994

Apoprotein-phospholipid interactions in Lp(a).

Lipoprotein (a) (Lp(a)) and low-density lipoprotein (LDL) are structurally related to each other. Both exhibit identical phospholipid compositions and possess one molecule of apoprotein B-100 (apoB). Lp(a) contains, in addition, apoprotein (a) (apo(a)), which localizes to the particle surface and interacts with the apoB component by non-covalent and covalent forces. Protein-protein interaction is probably interrelated with protein-lipid interaction. Fluorescent analogs of phosphatidylcholine and sphingomyelin were inserted into the surface layer of LDL and Lp(a). The obtained fluorescence data reflecting mobility and distributional heterogeneity of the labeled lipids provided evidence that apo-proteins discriminate between choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) because of the presence of apolipoprotein (a). Higher affinity for Lp(a) as compared with LDL was also observed with a fluorescent diether analog of phosphatidylcholine in native serum. In contrast, the time-dependent transfer of the same lipid into Lp(a) was slower compared with LDL, probably as a consequence of the more rigid surface of the former lipoprotein.

Apolipoproteins↗

PubMed1994

The interaction of Lp(a) with normal and LDL-receptor-deficient human skin fibroblasts.

The role of LDL receptors in the in vivo catabolism of Lp(a) is still a matter of controversy. Since Lp(a) binds LDL with high affinity, it was essential for this study to separate Lp(a) quantitatively from all other apo-B and apo-E-containing lipoproteins. This was achieved by the addition of proline as a dissociating agent to all buffers during Lp(a) preparation. Normal human skin fibroblasts pre-incubated with 40 mg/ml of Lp(a) downregulated cholesterol biosynthesis by approx. 35%; the same amount of LDL caused a 90% reduction. Cholesterol biosynthesis of LDL-receptor-deficient fibroblasts was not affected at all by LDL, yet Lp(a) exhibited a similar effect as in normal fibroblasts (32% reduction). An LDL-receptor-independent uptake of Lp(a) into fibroblasts must therefore be postulated. We also studied the degradation of Lp(a) in normal fibroblasts in comparison with LDL. Pure Lp(a) was only slightly degraded in relation to LDL. If fibroblasts were pre-incubated with small amounts of LDL, Lp(a) degradation was enhanced by a factor of 3-5. This effect was even more pronounced in fibroblasts pre-incubated with mevinolin. Thus the LDL receptor may play an indirect role in Lp(a) catabolism. The significance of these findings for the in vivo metabolism of Lp(a) remains to be established.

Cells, Cultured↗

PubMed1994

Identification of glycoprotein IIb as the lipoprotein(a)-binding protein on platelets. Lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a).

Lipoprotein(a) [Lp(a)] plays an important role in atherosclerosis. The amino acid sequence of apolipoprotein(a) [apo(a)] reveals an arginyl-glycyl-aspartate (RGD) tripeptide that is the consensus sequence for binding of adhesive plasma proteins of the fibrinolytic system, such as fibrinogen and von Willebrand factor, to the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex. Therefore, we undertook the present study to further investigate the role of Lp(a) in hemostasis. Binding of 125I-Lp(a) to a single platelet membrane-associated protein (137 +/- 6 kD) comigrating with platelet GPIIb (140 kD) was found to be specific, saturable, and Ca2+ independent. Binding of 125I-Lp(a) to resting human blood platelets was saturable, insensitive to temperature, and independent of the apo(a) isoform (B, S1 through S3). Scatchard analysis revealed a Kd of 7.2 +/- 1.8 x 10(-9) mol/L, with 729 +/- 313 Lp(a) molecules bound per platelet. Monoclonal anti-GPIIb IgG diminished Lp(a) binding by approximately 80%, monoclonal anti-GPIIb-IIIa IgG by 60%, and anti-GPIIIa IgG by just 15%. 125I-Lp(a) binding was competitively inhibited to the same extent by either unlabeled Lp(a) or fibrinogen. Low- and high-density lipoproteins were much weaker competitors. A polyclonal antibody raised against the RGDGQSYRGT sequence of apo(a) was used to verify the presence of an RGD sequence in the different Lp(a) preparations investigated. However, two lines of evidence indicated that the RGD sequence is not the binding domain mediating Lp(a) binding to platelets. First, incubation of platelets with isolated RGD tripeptide did not influence Lp(a) binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

The role of lecithin: cholesterol acyltransferase for lipoprotein (a) assembly. Structural integrity of low density lipoproteins is a prerequisite for Lp(a) formation in human plasma.

The composition of lipoproteins in the plasma of patients with LCAT deficiency (LCAT-D) is grossly altered due to the lack of cholesteryl esters which form the core of normal lipoproteins. When plasma from LCAT-D patients and their relatives was examined we found that nine heterozygotes had plasma Lp(a) levels of 2-13 mg/dl whereas none of 11 affected homozygous individuals from different families contained detectable amounts of Lp(a) in their plasma. Therefore, the binding of apo(a) to LDL density particles was studied in vitro using LDL density fractions prepared from patients, and recombinant apo(a) [r-apo(a)], which was expressed and secreted by transfected COS-7 cells. The LDL from heterozygotes were chemically indistinguishable from normal LDL and homogeneous with regard to morphology, whereas the crude LDL floating fraction from homozygotes consisted of a heterogeneous mixture of large vesicles, and small spheres resembling normal LDL. The LDL density fraction from the LCAT-D patient lacked almost completely cholesteryl esters. Incubation of LCAT-D plasma with active LCAT caused a substantial augmentation of the original subfraction which morphologically resembled normal LDL. Using r-apo(a) and normal LDL or LDL of heterozygous individuals, apoB:r-apo(a) complexes were formed when incubated at 37 degrees C in vitro for 20 h. In contrast, the total LDL floating fraction from a homozygous LCAT-D patient failed to form apoB:r-apo(a) complexes. After treatment with active LCAT, a significant apoB:r-apo(a) association was observed with LCAT-D LDL-density particles. Our data emphasize the importance of the integrity of LDL structure and composition for the formation of Lp(a). In addition, we demonstrate that the absence of LCAT activity has a fundamental impact on the regulation of plasma Lp(a) levels.

Apolipoproteins A↗

PubMed1994

[Hypercholesterolemia ].

This review article describes the types, pathophysiology, differential diagnosis and therapy of hypercholesterolemia. Special attention has been directed towards familiar hypercholesterolemia and familiar apolipoprotein B 100 defect. Clinically, both forms present very similar and may cause severe coronary arteriosclerosis and death before the age of 40 years. In the homozygote form CHD even develops prior to adolescence. With a frequency of 1:500 these forms of hypercholesterolemia are among the most common genetic disorders worldwide. The poor prognosis and their monogenetic dominant character emphasize the need for an exact distinction from the poligenetic forms of hypercholesterolemia and moreover underscore the necessity to screen for affected members in the family of already diagnosed cases and call for more aggressive therapeutic interventions. By dietary measures and monotherapy only 25% of patients reach the recommended cholesterol values. HMG-CoA reductase inhibitors proved to be the most effective. The remainders have to be treated with combinations of up to 3 lipid-lowering drugs. Alternative treatment such as extracorporal cholesterol elimination may become necessary.

Adolescent↗