Biomedical subjects

M L Koschinsky

Publications and source records attributed to M L Koschinsky.

At least 19 recordsLinked to original sources

PubMed1999

Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B).

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.

3' Untranslated Regions↗

PubMed1998

Lipoprotein(a) as a risk factor for coronary artery disease.

Since its identification by Kåre Berg in 1963, lipoprotein(a) [Lp(a)] has become a focus of research interest owing to the results of case-control and prospective studies linking elevated plasma levels of this lipoprotein with the development of coronary artery disease. Lp(a) contains a low-density lipoprotein (LDL)-like moiety, in which the apolipoprotein B-100 component is covalently linked to the unique glycoprotein apolipoprotein(a) [apo(a)]. Apo(a) is composed of repeated loop-shaped units called kringles, the sequences of which are highly similar to a kringle motif present in the fibrinolytic proenzyme plasminogen. Variability in the number of repeated kringle units in the apo(a) molecule gives rise to different-sized Lp(a) isoforms in the population. Based on the similarity of Lp(a) to both LDL and plasminogen, it has been hypothesized that the function of this unique lipoprotein may represent a link between the fields of atherosclerosis and thrombosis. However, determination of the function of Lp(a) in vivo remains elusive. Although Lp(a) has been shown to accumulate in atherosclerotic lesions, its contribution to the development of atheromas is unclear. This uncertainty is related in part to the structural complexity of the apo(a) component of Lp(a) (particularly apo(a) isoform size heterogeneity), which also poses a challenge for standardization of the measurement of Lp(a) in plasma. The fact that plasma Lp(a) levels are largely genetically determined and vary widely among different ethnic groups adds scientific interest to the ongoing study of this enigmatic particle. Most recently, the identification of proteolytic fragments of apo(a) in both plasma and urine has fueled speculation about the origin of these fragments and their possible function in the atherosclerotic process.

Coronary Disease↗

PubMed1998

Sequences within apolipoprotein(a) kringle IV types 6-8 bind directly to low-density lipoprotein and mediate noncovalent association of apolipoprotein(a) with apolipoprotein B-100.

Lipoprotein(a) [Lp(a)] particle formation is a two-step process in which initial noncovalent interactions between apolipoprotein(a) [apo(a)] and the apolipoprotein B-100 (apoB-100) component of low-density lipoprotein (LDL) precede disulfide bond formation. To identify kringle (K) domains in apo(a) that bind noncovalently to apoB-100, the binding of a battery of purified recombinant apo(a) [r-apo(a)] species to immobilized human LDL has been assessed. The 17K form of r-apo(a) (containing all 10 types of kringle IV sequences) as well as other truncated r-apo(a) derivatives exhibited specific binding to a single class of sites on immobilized LDL, with Kd values ranging from approximately 340 nM (12K) to approximately 7900 nM (KIV5-8). The contribution of kringle IV types 6-8 to the noncovalent interaction of r-apo(a) with LDL was demonstrated by the decrease in binding affinity observed upon sequential removal of these kringle domains (Kd approximately 700 nM for KIV6-P, Kd approximately 2000 nM for KIV7-P, Kd approximately 5100 nM for KIV8-P, and no detectable specific binding of KIV9-P). Interestingly, KIV9 also appears to participate in the noncovalent binding of apo(a) to LDL since the binding of KIV5-8 (Kd approximately 7900 nM) was considerably weaker than that of KIV5-9 (Kd approximately 2000 nM). Finally, it is demonstrated that inhibition of Lp(a) assembly by proline, lysine, and lysine analogues, as well as by arginine and phenylalanine, is due to their ability to inhibit noncovalent association of apo(a) and apoB-100 and that these compounds directly exert their effects primarily through interactions with sequences contained within apo(a) kringle IV types 6-8. On the basis of the obtained data, a model is proposed for the interaction of apo(a) and LDL in which apo(a) contacts the single high-affinity binding site on apoB-100 through multiple, discrete interactions mediated primarily by kringle IV types 6-8.

Aminocaproic Acid↗

PubMed1998

Expression of apolipoprotein(a) kringle IV type 9 in Escherichia coli: demonstration of a specific interaction between kringle IV type 9 and apolipoproteinB-100.

A number of studies have provided evidence that lipoprotein(a) [Lp(a)] assembly is a two-step process in which initial non-covalent interactions between apolipoprotein(a) [apo(a)] and apolipoproteinB-100 (apoB-100) precede specific disulfide bond formation. We have designed a construct encoding apo(a) kringle IV type 9 (KIV9) in which the unpaired cysteine at position 67 in this kringle is replaced with a tyrosine. The single kringle was expressed in bacteria and purified to homogeneity from cell homogenates. The purified derivative (designated KIV9deltaCys) was assessed for its ability to bind to purified human LDL. This interaction was detected either by ELISA using immobilized LDL or by column chromatography in which LDL binding to KIV9deltaCys immobilized on Ni2+-Sepharose was determined. In both cases, the interaction of KIV9deltaCys and LDL was observed. Further, we demonstrated that the binding interaction was sensitive to the addition of amino acids including lysine, the lysine analogue epsilon-aminocaproic acid, arginine, phenylalanine and proline, with arginine and lysine having the greatest inhibitory effect. Binding of KIV9deltaCys to an immobilized apoB peptide spanning residues 3732-3745 of apoB was also demonstrated by ELISA. As was the case for LDL, this binding interaction was sensitive to the addition of arginine and lysine. Computer modeling of KIV9 demonstrated an excellent fit with residues 3732-3738 (PSCKLDF) of the apoB peptide. The modeling predicts the presence of overlapping lysine and phenylalanine-binding pockets in KIV9 which explains the inhibitory effects of lysine, arginine and phenylalanine which were observed in the binding assays. In summary, this study represents the first demonstration that KIV9 can interact directly with LDL through non-covalent interactions which may contribute to the first step of Lp(a) formation.

Amino Acid Sequence↗

PubMed1998

Sequences within the amino terminus of ApoB100 mediate its noncovalent association with apo(a).

Although sequences within the C terminus of apolipoprotein B (apoB) have been implicated in the formation of covalent lipoprotein(a) [Lp(a)] particles, sequences in apoB that mediate initial noncovalent interaction with apo(a) remain to be characterized. To address this question, we have used an affinity chromatography method in which 2 recombinant forms of apo(a) [r-apo(a); either a 17-kringle form (17K) or a derivative containing apo(a) kringle IV types 5-8] have been immobilized onto Sepharose beads. Conditioned media from rat hepatoma (McA-RH7777) cell lines stably expressing various carboxyl-terminally truncated forms of human apoB (ranging from full-length apoB to apoB15) were applied to the r-apo(a) affinity columns; the columns were subsequently washed and eluted with epsilon-aminocaproic acid (epsilon-ACA). Specific binding was quantified by Western blot analysis of column fractions. Of the apoB truncations examined, apoB94, apoB42, apoB37, and apoB29 exhibited complete specific binding to 17K r-apo(a). Only approximately 50% binding was observed for apoB18, whereas essentially no detectable binding was observed with apoB15. In all cases, similar results were obtained when the r-apo(a) kringle IV types 5-8-Sepharose column was used. Additionally, substitution of proline for epsilon-ACA as the eluent resulted in similar column profiles with either r-apo(a) affinity column. We also demonstrated that apoB48 present in chylomicrons bound completely to the 17K column in an epsilon-ACA-dependent manner. Taken together, these results represent the first demonstration that N-terminal sequences in apoB between amino acid residues 680 (apoB15) and 781 (apoB18) are essential for noncovalent association with apo(a) and that these sequences interact with domain(s) present within apo(a) kringle IV types 5-8.

Amino Acid Sequence↗

PubMed1998

Apolipoprotein(a) enhances platelet responses to the thrombin receptor-activating peptide SFLLRN.

Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [14C]serotonin and suspended in Tyrode's solution, to ADP and the thrombin receptor-activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 micromol/L) or Lp(a) (up to 0.1 micromol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 micromol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 micromol/L 17K r-apo(a) increased aggregation from 15+/-4% to 58+/-6%, release of [14C]serotonin from 9+/-3% to 36+/-6%, and formation of thromboxane A2, measured as its stable metabolite thromboxane B2, from 7+/-1 to 29+/-5 ng/10(9) platelets (n=3; P<0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 micromol/L. Purified Lp(a) (0.25 to 0.1 micromol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 micromol/L) and low density lipoprotein (0.025 to 0.1 micromol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo(a) derivatives or Lp(a). The enhanced responses of platelets via the protease-activated receptor- thrombin receptor in the presence of Lp(a) may contribute to the increased risk of thromboembolic complications of atherosclerosis associated with this lipoprotein.

Adenosine Diphosphate↗

PubMed1997

Apolipoprotein(a) attenuates endogenous fibrinolysis in the rabbit jugular vein thrombosis model in vivo.

BACKGROUND: In many case-control as well as epidemiological studies, increased lipoprotein(a) [Lp(a)] levels are considered to constitute an independent risk factor for premature coronary artery and cerebrovascular disease. Lp(a) resembles an LDL particle with an additional linked protein [apolipoprotein(a), apo(a)], whose molecular structure has been demonstrated to be homologous to the fibrinolytic proenzyme plasminogen. Because of the high similarity between plasminogen and apo(a), apo(a) may potentially interfere in the fibrinolytic system by competing with plasminogen for fibrin binding sites. In vitro studies have demonstrated that Lp(a) indeed competes with plasminogen binding to fibrin and inhibits tissue plasminogen activator (TPA)-mediated activation of plasminogen. No direct in vivo studies to test this hypothesis have been performed. METHODS AND RESULTS: To test this hypothesis, we studied the effect of a recombinant form of apo(a) on endogenous and TPA-mediated thrombolysis in an in vivo model of experimental venous thrombosis. Thrombi containing either 16 microg r-apo(a), 8 microg r-apo(a), or vehicle (HEPES-buffered saline, control) were formed in the jugular veins of a rabbit and showed significantly reduced endogenous thrombolysis after 60 minutes in a dose-dependent fashion, ID 2.7+/-0.9% and 4.6+/-1.8%, respectively, versus 7.4+/-1.6% of that of the control. High concentrations of incorporated apo(a) significantly reduced TPA-induced thrombolysis (12.2+/-2.5% versus 22.2+/-2.6% in the control thrombi), but no effect of lower concentrations of incorporated r-apo(a) was demonstrated on the exogenous TPA-induced thrombolysis. CONCLUSIONS: The present study demonstrates the attenuation of endogenous fibrinolysis by apo(a) in an in vivo model of experimental venous thrombosis, lending support to the proposed mechanism of impaired fibrinolysis by which Lp(a) may contribute to atherothrombotic disorders.

Animals↗

PubMed1997

The solution phase interaction between apolipoprotein(a) and plasminogen inhibits the binding of plasminogen to a plasmin-modified fibrinogen surface.

In the present study, we assessed the binding of recombinant forms of apolipoprotein(a) [r-apo(a)] to plasminogen. Apo(a)-plasminogen interactions were demonstrated to be lysine-dependent, as they were abolished by the addition of epsilon-aminocaproic acid. Binding of r-apo(a) and plasma-derived Lp(a) to Glu-plasminogen was assessed in solution using a mutant form of recombinant plasminogen [Plg(S741C)] labeled at the active site with 5'-(iodoacetamido)fluorescein. High-affinity binding of apo(a) to plasminogen was observed with the 17-kringle r-apo(a) (Kd = 20.1 +/- 3.3 nM) as well as with plasma-derived Lp(a) (Kd = 5.58 +/- 0.08 nM). Binding studies using various truncated and mutant forms of r-apo(a) demonstrated that sequences within apo(a) kringle IV types 2-9 and the strong lysine binding site (LBS) in apo(a) kringle IV type 10 are not required for high-affinity binding to plasminogen. In all cases, the binding stoichiometry for the apo(a)-plasminogen interaction was determined to be 1:1. Binding data obtained using a 17-kringle r-apo(a) derivative lacking the protease-like domain (17KDeltaP; Kd = 3158 +/- 138 nM) indicate that sequences within the protease-like domain of apo(a) mediate its interaction with LBS in plasminogen. We determined that r-apo(a) and plasminogen bind to distinct sites on plasmin-modified fibrinogen with the concentration of plasminogen binding sites exceeding the concentration of r-apo(a) sites by a factor of 10. Furthermore, r-apo(a) is capable of inhibiting the binding of plasminogen to plasmin-modified fibrinogen surfaces, an effect which we show is attributable to the formation of a solution phase apo(a)/plasminogen complex which exhibits a greatly reduced affinity for plasminogen binding sites on plasmin-modified fibrinogen. The results of this study provide new insights into the mechanism by which apo(a) and Lp(a) may inhibit fibrinolysis, thus contributing to the atherothrombotic risk associated with this lipoprotein.

Aminocaproic Acid↗

PubMed1997

Lipoprotein(a): structural implications for pathophysiology.

The assembly between a low-density lipoprotein particle and apolipoprotein(a), a highly carbohydrate-rich protein, gives origin to a peculiar class of lipoproteins, only found in the hedgehog, primates, and humans, termed lipoprotein(a). Apolipoprotein(a), which shares a high degree of sequence homology with the fibrinolytic proenzyme plasminogen, is linked to the apolipoprotein B-100 component of low-density lipoprotein via a disulfide bond and confers distinct biochemical and metabolic properties to lipoprotein(a). Because of its peculiar structural features and the observed correlation between high lipoprotein(a) levels and the development of a variety of atherosclerotic disorders, this lipoprotein has become the focus of an intense research effort. Although accumulation of lipoprotein(a) in the vessel wall at sites of vascular injury has been clearly evidenced, the mechanism(s) by which lipoprotein(a) exerts its pathogenic effect in this milieu remain largely unknown. It has been hypothesized that the pathological effect of lipoprotein(a) is related either to its similarity to low-density lipoprotein (i.e., a pro-atherogenic effect) or to the apolipoprotein(a) similarity to plasminogen (i.e., a pro-thrombotic/anti-fibrinolytic effect). However, it is probable that both components contribute to the pathogenicity of lipoprotein(a). The fact that lipoprotein(a) levels are largely genetically determined, varying widely among individuals and racial groups, adds additional elements to the scientific interest that surrounds this lipoprotein. Both clinical and biochemical studies of lipoprotein(a) have been complicated by the high degree of structural heterogeneity of apolipoprotein(a), which is considered the most polymorphic protein in human plasma. Our aim in this paper is to provide an overview of the most salient structural features of lipoprotein(a) and their possible pathophysiological implications.

Apolipoproteins A↗

PubMed1997

Analysis of the mechanism of lipoprotein(a) assembly.

We have assessed the ability of a battery of purified recombinant apolipoprotein(a) (r-apo(a)) derivatives to bind to immobilized low-density lipoprotein (LDL) by ELISA. Removal of the apo(a) kringle IV type 8 and type 9 sequences dramatically reduced apo(a) binding to LDL. The binding of apo(a) to LDL was effectively inhibited by arginine, lysine, the lysine analogue epsilon-aminocaproic acid and proline; comparable inhibition was observed using the 17K and KIV5-8 r-apo(a) derivatives, suggesting a direct role for sequences contained in the latter species in mediating the initial non-covalent interactions which precede specific disulfide bond formation. We also determined that r-apo(a) binds directly to a synthetic apoB peptide spanning amino acid residues 3732-3745; this interaction appeared to be mediated by sequences present in apo(a) kringle IV types 8 and 9, and could be inhibited by arginine, lysine and proline. The results of this study indicate that the efficiency of Lp(a) assembly is a direct function of the initial non-covalent interactions between apo(a) and LDL; in addition, these studies suggest that Cys3734 in apoB mediates covalent linkage with apo(a) by virtue of the ability of the apoB sequences surrounding this residue to directly interact with apo(a) KIV type 9.

Apolipoproteins A↗

PubMed1997

Lipoprotein[a] is not present in the plasma of patients with some peroxisomal disorders.

Peroxisomal disorders arise either from defects in the biogenesis of peroxisomes or from the defective synthesis of one or more peroxisomal enzymes. These defects result in metabolic disturbances in peroxisomal beta-oxidation of various fatty acids and derivatives and/or in the biosynthesis of ether lipids. In the current study, lipoprotein levels were determined in plasma samples from patients diagnosed with one of four different peroxisomal disorders. While low density lipoprotein (LDL) levels were found to be within the normal range, lipoprotein[a] (Lp[a]) could not be detected by enzyme-linked immunosorbent assay (ELISA) in plasma from patients with cerebro-hepato-renal (Zellweger) syndrome (ZS) and rhizomelic chondrodysplasia punctata (RCDP). Conversely, Lp[a] was clearly present in control plasma obtained from healthy newborns and from patients affected with one of two other peroxisomal disorders, X-linked adrenoleukodystrophy (X-ALD) and Refsum disease (RD) as determined by ELISA. The lack of Lp[a] in plasma of patients with ZS may result from defective secretion of apolipoprotein[a] (apo[a]) (the distinguishing protein component of Lp[a]), as apo[a] mRNA transcripts were clearly present in ZS livers as assessed by PCR, and intracellular apo[a] protein was detected in total liver homogenates from ZS patients as determined by Western blot analysis. Furthermore, LDL present in the plasma of ZS patients was able to associate with recombinant apo[a] in an in vitro Lp[a] assembly assay.

Adolescent↗

PubMed1996

The binding activity of the macrophage lipoprotein(a)/apolipoprotein(a) receptor is induced by cholesterol via a post-translational mechanism and recognizes distinct kringle domains on apolipoprotein(a).

Elevated plasma levels of lipoprotein(a) (Lp(a)) can be a risk factor for atherosclerosis, and the interaction of Lp(a) with cholesterol-loaded macrophages (foam cells) in atheromata may be important in Lp(a)-induced atherogenesis. We have previously shown that when cultured macrophages are loaded with cholesterol, they acquire the ability to internalize and lysosomally degrade Lp(a) via interaction between a novel cell-surface receptor activity and the apolipoprotein(a) (apo(a)) moiety of Lp(a). Herein we explore the cell-surface binding of recombinant apo(a) (r-apo(a)) by foam cells. Whereas the induction of degradation of r-apo(a) by cholesterol loading of macrophages depended on new protein synthesis, the induction of binding of r-apo(a) did not. Furthermore, J774 macrophages bound r-apo(a) in a cholesterol-regulatable and specific manner but degraded r-apo(a) poorly. Thus, the binding and internalization/degradation functions of the receptor activity are distinct. To explore which domains on r-apo(a) interact with the foam cell receptor, we conducted a series of competitive and direct binding and degradation experiments using 12 r-apo(a) constructs that differed in their content of specific kringle subtypes. These data, as well as complementary data with anti-apo(a) monoclonal antibodies, indicated that the region centered around kringle type IV, subtypes 6-7 (KIV6-7) is important in receptor binding. Remarkably, a cholesterol-induced receptor activity with similar structural specificity was also found on Chinese hamster ovary cells. In conclusion, the foam cell Lp(a)/apo(a) receptor consists of a cholesterol-regulatable binding activity and a short-lived component necessary for internalization or lysosomal degradation; the binding activity interacts with a distinct region of apo(a) that is different from that involved in competition for plasminogen binding.

Animals↗

PubMed1996

The complete cDNA sequence encoding plasminogen from the European hedgehog (Erinaceus europaeus).

Using a PCR-based strategy, we have determined the complete cDNA sequence encoding hedgehog plasminogen (Plg). The 2700-nucleotide cDNA (corresponding to a 2.9-kb liver-derived transcript) encodes an open reading frame of 811 amino acids which shares 74-76% identity with Plg characterized from mouse, human and rhesus monkey. Residues corresponding to the catalytic triad, tPA-cleavage site, as well as seven of the eight lysine-binding residues in kringle IV are conserved in the hedgehog. However, potential N-linked glycosylation sites which have been reported in human and rhesus Plg are not present in analogous positions in the hedgehog Plg sequence.

Amino Acid Sequence↗

PubMed1996

Lipoprotein [a].

Explore the source record for details and available documents.

Alleles↗

PubMed1996

Lipoprotein(a) assembly. Quantitative assessment of the role of apo(a) kringle IV types 2-10 in particle formation.

We have developed a system for the quantitative assessment of the efficiency of lipoprotein(a) [Lp(a)] formation in vitro. Amino-terminally truncated derivatives of a 17-kringle form of recombinant apo(a) [r-apo(a)] were transiently expressed in human embryonic kidney cells. Equimolar amounts of r-apo(a) derivatives were incubated with a fourfold molar excess of purified human low density lipoprotein, and r-Lp(a) formation was assessed by densitometric analysis of Western blots. Although r-Lp(a) formation was observed with each r-apo(a) derivative, both the rate and extent of particle formation were greatly lower on removal of kringle IV type 7. Additional substantial decreases in these parameters were observed on removal of kringle IV type 8, thereby suggesting a major role for these two kringles in Lp(a) assembly. We directly demonstrated that the lysine-binding sites (LBSs) within kringle IV types 5-9 are "masked" in the context of the Lp(a) particle and are consequently unavailable for interaction with lysine-Sepharose. Using site-directed mutagenesis, we also demonstrated that the previously described LBS in kringle IV type 10 is not required for r-Lp(a) formation: r-Lp(a) formation using a mutated form of apo(a) that lacks this LBS is comparable in efficiency to that of wild-type r-apo(a) and can be inhibited to a similar extent by epsilon-amino-n-caproic acid. In summary, the results of our study indicate that apo(a) kringle IV types 7 and 8 are required for maximal efficiency of Lp(a) formation, likely by virtue of their ability to mediate lysine-dependent non-covalent interactions with apoB-100 that precede disulfide bond formation.

Base Sequence↗

PubMed1996

Interaction of a recombinant form of apolipoprotein[a] with human fibroblasts and with the human hepatoma cell line HepG2.

We have studied the binding, uptake, and degradation of a recombinant form of apolipoprotein[a] (r-apo[a]) using a cultured cell model. In HepG2 cells and in human fibroblasts, r-apo[a] complexed with low density lipoprotein(LDL) is bound and internalized via high affinity (Kd = 10 nM) receptors; in both cell types, low affinity (Kd = 200-300 nM) sites also mediate free apo[a] uptake. Using competition studies, we found that the high affinity binding component corresponds to the LDL receptor. Involvement of the LDL receptor in r-apo[a] uptake by fibroblasts was confirmed using fibroblasts derived from an individual homozygous for familial hypercholesterolemia; in contrast to normal fibroblasts, these cells lacked the high affinity r-apo[a] binding component. Cell association of 125I-labeled r-apo[a] was increased and decreased concomitantly with the up- and down-regulation of the LDL receptor in response to a number of compounds. The addition of alpha 2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease total specific binding of r-apo[a], suggesting that neither the low density lipoprotein receptor-related protein nor cell surface proteoglycans are involved in r-apo[a] clearance. The low affinity binding component present in both fibroblasts and HepG2 cells likely corresponds to the plasminogen receptor, as binding of r-apo[a] to these sites was specifically decreased by the addition of plasminogen or the lysine analogue epsilon-aminocaproic acid, but not by the addition of tissue-type plasminogen activator. Heparin abolished uptake of r-apo[a] by the LDL receptor component only; this indicates that apo[a] must be associated with LDL to be cleared by this receptor. In contrast, free apo[a] can be effectively cleared by the plasminogen receptor which may represent a significant route of clearance for free apo[a] in vivo.

Aminocaproic Acid↗

PubMed1995

Antifibrinolytic effect of recombinant apolipoprotein(a) in vitro is primarily due to attenuation of tPA-mediated Glu-plasminogen activation.

The effect of a 17-kringle form of recombinant apo(a) [r-apo(a)] on in vitro fibrin clot lysis was studied. In these assays, fibrin clots were formed in the wells of microtiter plates, and lysis of the clots was monitored by measurement of the turbidity at 405 nm. The results indicate that r-apo(a) produces a dose-dependent antifibrinolytic effect in clots formed using either purified components or barium-adsorbed plasma. This effect was found to be independent of clot structure, since lysis of clots formed using both high and low concentrations of thrombin was prolonged by r-apo(a) to the same extent. The two components of the antifibrinolytic effect of r-apo(a) were determined to be (i) attenuation of tPA-mediated plasminogen activation (the major component) and (ii) inhibition of plasmin degradation of fibrin, although r-apo(a) did not directly attenuate plasmin activity, as measured by S-2251 hydrolysis. r-Apo(a) interfered most substantially with tPA-mediated activation of Glu-plasminogen and less substantially with tPA-mediated Lys-plasminogen activation and urokinase-mediated activation of plasminogen. In summary, we have demonstrated that apo(a) is able to attenuate fibrin clot lysis in vitro, primarily as a consequence of the interference by apo(a) with tPA-mediated Glu-plasminogen activation. These studies illuminate possible mechanisms by which Lp(a) may contribute to the development of vascular disease in vivo.

Amino Acid Sequence↗

PubMed1995

Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a).

Lipoprotein(a) [Lp(a)] has been measured in numerous clinical and epidemiological studies by a variety of immunochemical methods. However, little, if any, consideration has been given to the confounding effect of the size heterogeneity of apolipoprotein(a) [apo(a)] on the measurement of Lp(a). We developed three direct-binding enzyme-linked immunosorbent assays (ELISAs) with detecting antibodies of different specificities to evaluate the effect of apo(a) size on Lp(a) measurement. The three assays used the same monoclonal antibody to capture the apo(a)-containing particles and were calibrated (in nanomoles per liter) with a serum containing apo(a) with 21 kringle 4 domains. Using all three ELISAs, we measured Lp(a) in a group of 723 subjects selected to have a single apo(a) band, as determined by a high-resolution phenotyping system. Essentially identical results were obtained by the two methods that measured Lp(a) by use of either a polyclonal antibody against apo B or a monoclonal antibody against apo(a) that does not recognize the kringle 4 type 2 repeats. In contrast, the ELISA using a monoclonal antibody specific for apo(a) kringle 4 type 2 repeats overestimated Lp(a) concentration in samples containing apo(a) with more than 21 kringle 4 domains and underestimated Lp(a) samples containing apo(a) with fewer than 21 kringle 4 domains. Thus, these differences in Lp(a) values varied as a function of apo(a) size. We conclude that antibody specificity and apo(a) size heterogeneity can significantly affect Lp(a) measurements.

Animals↗