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Biomedical subjects

G M Fless

Publications and source records attributed to G M Fless.

At least 19 recordsLinked to original sources
PubMed2000

The relationship between the effect of lysine analogues and salt on the conformation of lipoprotein(a).

Lipoprotein(a) [Lp(a)] exhibits many of the same properties as plasminogen, owing to a similar structural makeup from a composite of multiple kringle domains. Shared behavior includes induction of an expanded conformation by lysine analogues, inhibition of this effect, and creation of a compact conformation by NaCl. Here, we examine in detail the independent and mutual effects of NaCl and 6-aminohexanoic acid (6-AHA) on the structure of Lp(a) and the relationship between the binding of the two ligands. We find that NaCl promotes the compact conformation while binding to Lp(a) homogeneously. In the absence of salt, 6-AHA leads to the complete unfolding of Lp(a), a process that is accompanied by cooperative binding. Reversal of conformation and weakening of binding occurred when one ligand was added to Lp(a) in the presence of the other, suggesting competitive binding. High concentrations of NaCl completely reversed the expansion of Lp(a) in 100 mM 6-AHA, and high concentrations of 6-AHA unfolded Lp(a) in the presence of 100 mM NaCl, but only by 30% in the case of the 15 kringle IV Lp(a) studied. Induction of the compact form of Lp(a) appears to be an effect in common with all salts examined and cannot be attributed solely to the anion, as in the case of plasminogen. The results were summarized in terms of a model of Lp(a) depicting the conformational alterations of apo(a) caused by the binding of the two ligands. In the compact conformation in NaCl, apo(a) is apposed to the particle surface. The fully expanded form in 6-AHA results from release of both the variable and constant kringle domains. In the intermediate form in water and in a solution containing both NaCl and 6-AHA, only the variable domain is released from the particle surface.

Aminocaproic Acid↗
PubMed1999

Effect of phospholipase A2 digestion on the conformation and lysine/fibrinogen binding properties of human lipoprotein[a].

In vitro hydrolysis of human lipoprotein[a] (Lp[a]) by phospholipase A2 (PLA2) decreased the phosphatidylcholine (PC) content by 85%, but increased nonesterified fatty acids 3.2-fold and lysoPC 12.9-fold. PLA2-treated Lp[a] had a decreased molecular weight, increased density, and greater electronegativity on agarose gels. In solution, PLA2-Lp[a] was a monomer, and when assessed by sedimentation velocity it behaved like untreated Lp[a], in that it remained compact in NaCl solutions but assumed the extended form in the presence of 6-amino hexanoic acid, which was shown previously to have an affinity for the apo[a] lysine binding site II (LBS II) comprising kringles IV5-8. We interpreted our findings to indicate that PLA2 digestion had no effect on the reactivity of this site. This conclusion was supported by the results obtained from lysine Sepharose and fibrinogen binding experiments, in the presence and absence of Tween 20, showing that phospholipolysis had no effect on the reactivity of the LBS-II domain. A comparable binding behavior was also exhibited by the free apo[a] derived from each of the two forms of Lp[a]. We did observe a small increase in affinity of PLA2-Lp[a] to lysine Sepharose and attributed it to changes in reactivity of the LBS I domain (kringle IV10) induced by phospholipolysis. In conclusion, the extensive modification of Lp[a] caused by PLA2 digestion had no significant influence on the reactivity of LBS II, which is the domain involved in the binding of apo[a] to fibrinogen and apoB-100. These results also suggest that phospholipids do not play an important role in these interactions.

Aminocaproic Acid↗
PubMed1998

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E.

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.

Amino Acid Sequence↗
PubMed1998

Genotype-specific transcriptional regulation of PAI-1 gene by insulin, hypertriglyceridemic VLDL, and Lp(a) in transfected, cultured human endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease. Variations in plasma PAI-1 levels have been attributed to variations in the PAI-1 gene, and associations between PAI-1 levels and PAI-1 genotypes suggest that PAI-1 expression may be regulated in a genotype-specific manner by insulin, hypertriglyceridemic (HTG) very low density lipoprotein (VLDL), or lipoprotein(a) [Lp(a)]. Polymerase chain reaction-amplified 1106-bp fragments of the promoter of the 1/1 and 2/2 PAI-1 genotypes were sequenced and showed 5 regions of small nucleotide differences in the 1/1 versus 2/2 PAI-1 promoters that consistently occurred with high frequency. These fragments were ligated into the luciferase reporter gene, and 1/1 and 2/2 PAI-1 genotype human umbilical vein endothelial cell (HUVEC) cultures were transiently transfected with their respective p1PAI110/luc and p2PAI110/luc constructs and vice versa. Insulin induced an approximately 12- to 16-fold increase in luciferase activity in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p1PAI110/luc construct. HTG-VLDL and Lp(a) induced luciferase activity by approximately 14- to 16- and approximately 8- to 11-fold, respectively, in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p2PAI110/luc construct. The positive control interleukin-1 showed an approximately 7- to 12-fold response in the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with either of the constructs. These cross-over results demonstrate that regulation of either the 1/1 or 2/2 PAI-1 genotype by its respective inducer is due to the promoter itself and not to some factor(s) expressed differently in the 1/1 or 2/2 PAI-1 genotype HUVEC cultures.

Cells, Cultured↗
PubMed1998

Phospholipase A2 modification enhances lipoprotein(a) binding to the subendothelial matrix.

Lipoprotein(a), Lp(a), is found in the extracellular matrix in atherosclerotic plaques, but with a different localization than LDL. A two-compartment system, with a monolayer of endothelial cells forming a barrier, was used to compare the transport, cell binding, and retention of Lp(a) and LDL into the subendothelial matrix. Baseline values for transport and retention of Lp(a) and LDL were not significantly different. Incubation with lipoprotein lipase or sphingomyelinase caused modest and similar increases in transport and retention of the two lipoproteins. In contrast, incubation with phospholipase A2 (PLA2) resulted in a marked (4-fold) increase in retention of Lp(a) on the subendothelial matrix, but a lesser (2-fold) increase in LDL retention. Moreover, PLA2 treatment of Lp(a) enhanced its binding to individual matrix proteins (fibronectin, laminin, or collagen) by 4-10 times above that of LDL. The enzymatic activity of PLA2 was responsible for its effect on Lp(a) binding. The lysine binding sites of Lp(a) contributed to the increased binding of PLA2-modified Lp(a) to the matrix, and the enhanced lysine binding functions of PLA2-modified Lp(a) was demonstrated by two independent approaches. Thus, PLA2 modification leads to enhanced interactions of lipoproteins with the extracellular matrix, and this effect is more pronounced with Lp(a).

Animals↗
PubMed1997

Specificity of ligand-induced conformational change of lipoprotein(a).

The conformation of Lp(a) was probed with a set of omega-aminocarboxylic acids and other analogs of 6-aminohexanoic acid (6-AHA). Using the viscosity-corrected sedimentation coefficient, six additional ligands were shown to induce a major conformational change in Lp(a), from a compact form to an extended form. These were trans-4-(aminomethyl)cyclohexanecarboxylic acid (t-AMCHA), proline, 4-aminobutyric acid, 8-aminooctanoic acid, Nalpha-acetyllysine, and glycine. Lysine, Nepsilon-acetyllysine, glutamic acid, and adipic acid were determined not to cause a conformational change. Urea and guanidine hydrochloride were ineffective at inducing this conformational change at concentrations at which the above ligands did unfold Lp(a). The conformational change was inhibited by 100 mM NaCl and to a lesser extent by 20 mM sodium glutamate. Despite the fact that these two salts have nearly the same ionic strengths, the greater inhibition of the unfolding by NaCl is consistent with a proposed stabilization of interkringle interactions by chloride ions. In 100 mM NaCl, which most closely resembles physiological conditions, only proline, 4-aminobutyric acid, 6-AHA, and t-AMCHA were effective ligands. By analyzing the dimensions of the conformation altering ligands, we propose that a critical variable in determining the effectiveness of a ligand in disrupting Lp(a) is the distance between the carboxyl and amine functions of the ligand. The optimal distance is approximately 6 A, which agrees with the observed 6.6-6.8 A separation of the cationic and anionic centers of known plasminogen and apo(a) lysine binding sites. These studies have implications for the mechanism of Lp(a) particle assembly.

Aminocaproic Acid↗
PubMed1997

Molecular weight determination of lipoprotein(a) [Lp(a)] in solutions containing either NaBr or D2O: relevance to the number of apolipoprotein(a) subunits in Lp(a).

Molecular weight determination of low-density lipoprotein (LDL) is usually performed in solutions containing high concentrations of salt (up to 13.4 M NaBr) by sedimentation velocity and diffusion experiments, because it does not preferentially bind salt or water. Considering that lipoprotein(a) [Lp(a)] is structurally similar to LDL, differing only by the presence of Apo(a), the molecular weight, M, of Lp(a) has also been measured in solutions containing high concentrations of NaBr. We questioned the suitability of this practice by comparing the apparent molecular weight, Mapp, and partial volume, phi', of Lp(a) determined by sedimentation and flotation equilibrium in a three-component system containing NaBr with the analogous parameters, M and partial specific volume, v, determined in a two-component system containing D2O. LDL served as a control. In agreement with previous findings obtained with different methods, our results indicate no significant differences in M and v of four different LDL samples and apparently no significant preferential binding of solvent components. In contrast, values of Mapp and phi' of Lp(a) evaluated in NaBr are significantly greater than M and v. Preferential binding of solvent components appeared to be a function of Apo(a) mass or the number of kringle IV domains, as expressed by increasing percentage differences between the two sets of parameters, ranging from 4 to 13% in M and 0.2 to 0.5% in v of Lp(a) species having Apo(a) with 15-27 kringle IV domains. Furthermore, our results indicate that the variable Apo(a) kringle IV domains are more involved in this process than the constant domain of Apo(a). These findings indicate that the Lp(a) molecular weight should be determined in D2O and that high concentrations of NaBr should be avoided as their use would lead to overestimated molecular weights and partial specific volumes. Application of this method to the question of how much Apo(a) is released upon the reduction of Lp(a) led to the conclusion that Lp(a) contains only one Apo(a) molecule.

Apolipoproteins A↗
PubMed1997

Genotype-specific transcriptional regulation of PAI-1 expression by hypertriglyceridemic VLDL and Lp(a) in cultured human endothelial cells.

The hypothesized relationships between plasminogen activator inhibitor (PAI-1) genotypes, PAI-1 levels, and their potential regulation by hypertriglyceridemic (HTG) very low density lipoprotein (VLDL) and lipoprotein(a) [Lp(a)] was examined in a PAI-1 genotyped human umbilical vein endothelial cell (HUVEC) culture model system. Individual human umbilical veins were used to obtain cultured ECs and were genotyped for PAI-1 by using the HindIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an [alpha-32P]dCTP-labeled 2.2-kb PAI-1 cDNA, yielded three RFLPs designated 1/1 (22-kb band only), 1/2 (22-plus 18-kb bands), and 2/2 (18-kb band only). Individual PAI-1 genotyped HUVEC cultures were incubated in the absence or presence of HTG-VLDL (0 to 50 micrograms/mL) or Lp(a) (0 to 50 micrograms/mL) at 37 degrees C for various times (4 to 24 hours), followed by analyses of PAI-1 antigen (by ELISA) and mRNA (by ribonuclease protection assay) levels, EC surface-localized plasmin generation assays, and nuclear run-on transcription assays. Secreted PAI-1 antigen levels were increased approximately 2- to 3-fold by HTG-VLDL and approximately 1.6 to 2-fold by Lp(a); mRNA levels were increased approximately 3- to 4.5-fold by HTG-VLDL and approximately 2.5- to 3.2-fold by Lp(a) compared with medium-incubated controls, primarily in the 2/2 PAI-1 genotype HUVEC cultures. Increases in PAI-1 mRNA induced by HTG-VLDL or Lp(a) could be abolished by coincubation with actinomycin D (2 x 10(-6) mol/mL) or puromycin (1 microgram/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by approximately 5- to 6-fold and approximately 4- to 5-fold, respectively, primarily in the 2/2 PAI-1 genotype HUVEC cultures compared with 1/1 PAI-1 genotype HUVEC cultures or medium-incubated controls. The positive control interleukin-1 increased both 2/2 and 1/1 PAI-1 mRNA levels by approximately 5- to 6-fold. Increased PAI-1 antigen and mRNA expression were associated with a concomitant 50% to 60% decrease in plasmin generation. These combined results demonstrate the genotype-specific regulation of PAI-1 expression by HTG-VLDL and Lp(a) and further indicate that these risk factor-associated components regulate PAI-1 gene expression at the transcriptional level in cultured HUVECs. Results from these studies further suggest that individuals with this responsive 2/2 PAI-1 genotype may reflect the additional inherent potential for later HTG-VLDL- or Lp(a)-induced fibrinolytic dysfunction, resulting in the early initiation of thrombosis, atherogenesis, and coronary artery disease.

Arteriosclerosis↗
PubMed1996

Ligand-induced conformational change of lipoprotein(a).

Lipoprotein(a) undergoes a dramatic, reversible conformational change on binding 6-amino-hexanoic acid (6-AHA), as measured by a decrease in the sedimentation rate, the magnitude of which is directly proportional to apo(a) mass. A similar reversible transition from a compact to an extended form has been shown to occur in plasminogen on occupation of a weak lysine binding site. The magnitude of the change in Lp(a) with large apo(a) is about 2.5 times that seen for plasminogen, however. Regardless of apo(a) size, binding analysis indicated that 1.4-4 molecules of 6-AHA bound per Lp(a) particle; the midpoint of the conformational change occurs at 6-AHA concentrations of 100-200 mM. Since rhesus Lp(a), which lacks both kringle V and the strong lysine binding site on kringle IV 10, also undergoes a similar conformational change, the phenomenon may be attributable to weak sites, possibly located in K-IV 5-8. Compact Lp(a), i.e., native Lp(a), had a frictional ratio (f/f0) of 1.2 that was independent of apo(a) mass, implying constant shape and hydration. For Lp(a) in saturating 6-AHA, f/f0 ranged from 1.5 to over 2.1 for the largest apo(a) with 32 K-IV, indicating a linear relationship between hydrodynamic volume and number of kringles, as expected for an extended conformation. However, only the variable portion of apo(a) represented by the K-IV 2 domains, participates in the conformational change; the invariant K-IV 3-9 domains remain close to the surface. These results suggest that apo(a) is maintained in a compact state through interactions between weak lysine binding sites and multiple lysines on apoB and/or apo(a), and that these interactions can be disrupted by 6-AHA, a lysine analog.

Aminocaproic Acid↗
PubMed1996

Evaluation of common electrophoretic methods in determining the molecular weight of apolipoprotein(a) polymorphs.

Five different gel systems were evaluated for their utility in determining the molecular weights of apolipoprotein(a) (apo(a)) polymorphs by SDS polyacrylamide or agarose gel electrophoresis. Three linear polyacrylamide gradient gels (2-16% from Isolab (Akron, OH), 4-15% from Pharmacia (Piscataway, NJ), and 2.5-6% homemade), a 4% polyacrylamide, and a 1.5% agarose gel were examined. Crosslinked phosphorylase B oligomers served as molecular weight standards. Molecular weights of four different apo(a) polymorphs were determined in each gel system and compared to values measured previously by sedimentation equilibrium. The results indicate that molecular weights obtained by gradient polyacrylamide gel electrophoresis were within 10% and often not statistically different from values acquired by sedimentation equilibrium. The use of homogenous 4% polyacrylamide and 1.5% agarose gels led to molecular weights that were overestimated by 20 and 60-70%, respectively. ApoB100, which is a commonly used molecular weight marker, was found to have anomalously fast mobility in each of the four polyacrylamide gel systems. Because its use would lead to overestimated apo(a) molecular weights, it was not useful as a molecular weight standard. Our results indicate that SDS-gradient polyacrylamide gel electrophoresis with cross-linked phosphorylase B as standard is a suitable gel system for evaluating apo(a) molecular weights.

Apolipoproteins↗
PubMed1995

Effect of glycation on the properties of lipoprotein(a).

Lipoprotein(a) [Lp(a)] was glycated by incubation in vitro with glucose (0 to 200 mmol/L), and its properties were compared with native Lp(a) and native and glycated LDL. Glucose was incorporated into Lp(a) in proportions that mirrored the distribution of lysines between apolipoprotein (apo) B-100 and apo(a). Because the kringle IV domains of apo(a) are lysine poor, only 10% of glucose bound to apo(a), whereas 90% was attached to the apoB-100 of Lp(a). Approximately 3% of the lysines of both Lp(a) and LDL were modified, which is a level comparable with that observed in LDL isolated from diabetic individuals. Glucose uptake by Lp(a) and LDL was almost identical and was linear as a function of concentration and time. Glycation increased the negative charge of Lp(a) and LDL as monitored by electrophoresis and ion-exchange chromatography and also reduced the affinity of Lp(a) and LDL for heparin-Sepharose. Glycation did not affect the lysine-binding property of Lp(a) or generate measurable malondialdehyde oxidation adducts. The catabolism of glycated Lp(a) by human monocyte-derived macrophages (HMDMs), like that of native Lp(a), was largely LDL receptor independent. Both glycated Lp(a) and LDL were degraded at a comparatively faster rate and stimulated greater cholesteryl ester formation than their unmodified counterparts. However, the degradation rate of glycated Lp(a) was approximately four- to fivefold slower and its stimulation of cholesteryl ester formation was ninefold lower than that of either form of LDL. These results show that Lp(a) can be glycated nonenzymatically in vitro, that the incorporation of glucose is dependent on the distribution of lysines between apo(a) and apoB-100, and that glycation does not affect the lysine-binding properties of Lp(a). Furthermore, glycation produced modest increases in the degradation rate of Lp(a) and associated cholesteryl ester synthesis by HMDMs. Based on these data, glycation does not appear to significantly enhanced the atherogenic potential of unmodified Lp(a).

Adult↗
PubMed1995

Cellular binding and degradation of lipoprotein (a).

Lp(a) is an important contributing factor to the development of atherosclerosis, and in structure is similar to LDL. Given the central role of the LDL receptor (LDL-R) in the metabolism of LDL, we felt that a study of the binding and degradation of Lp(a) facilitated by the LDL-R of human monocyte derived macrophages (HMDM) would be of value in understanding its pathological nature. In this study we compared equimolar amounts of Lp(a) and LDL and found that nearly equal amounts of Lp(a) and LDL bound to the LDL-R of HMDM at 4 degrees C, however the affinity of both lipoproteins was much lower than has been observed for the LDL-R of fibroblasts, being 0.80 muM for Lp(a) and 0.23 muM for LDL. The binding of Lp(a) to HMDM could be competed by 63% with a 50-fold excess of LDL. Degradation of Lp(a) at 37 degree C, unlike 4 degrees C binding, was mainly nonspecific (75% of total Lp(a) degradation) and when compared on an equimolar basis, nearly 6 times more LDL than Lp(a) was processed by the LDL-R pathway in 5 hr. Lower degradation of Lp(a) appears to be the result of lower binding at 37 degree C and a lower degradation rate when compared to LDL. It was not caused by increased intracellular accumulation or retroendocytosis. Degradation of both lipoproteins was only modestly affected by up and down regulation of the LDL-R. Because the binding of LDL at 4 degrees C and degradation at 37 degree C is mainly LDL-R specific, whereas only the 4 degree C binding of Lp(a) is so, suggests that the poor LDL-R dependent degradation of Lp(a) at 37 degree C is caused by a conformational change that is inducted in Lp(a) upon lowering the temperature to 4 degree C which allows better recognition of Lp(a) by the HMDM LDL-R.

Arteriosclerosis↗
PubMed1995

Interaction of Lp(a) with plasminogen binding sites on cells.

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Binding Sites↗
PubMed1994

Subunit composition of lipoprotein(a) protein.

We determined the molecular weight of four different apo(a) polymorphs by sedimentation equilibrium in 6 M guanidine hydrochloride in order to estimate the molar ratio of apo(a) to apoB in Lp(a). They had molecular weights of 289,000, 310,000, 341,000, and 488,000 and 15, 16, 18, and 27 kringle 4 domains, respectively. Their carbohydrate content was similar (23.2 wt %), as was their partial specific volume (0.682 mL/g). Knowing the mass of apo(a), we estimated the molar ratio of apo(a) to apoB from (1) the molecular weight of the protein moiety of the four respective parent Lp(a) particles as calculated from their mass and percentage composition and the mass of apoB, (2) the mass of apo(a) lost from Lp(a) upon its reduction and carboxymethylation, by determining the difference in mass between Lp(a) and Lp(a-), and (3) from the mass (measured by sedimentation equilibrium in 6 M guanidine hydrochloride) of the lipid-free apoB-apo(a) complex (1.06 x 10(6) daltons) of the Lp(a) particle with the smallest apo(a) polymorph by subtracting the mass of apoB. Our results obtained with each of the three different physicochemical methods indicated that the protein moiety of each of the four Lp(a) particles that was investigated consisted of a complex of two molecules of apo(a) and one molecule of apoB.

Apolipoproteins B↗
PubMed1994

Polymorphic forms of Lp(a) with different structural and functional properties: cold-induced self-association and binding to fibrin and lysine-Sepharose.

Two different Lp(a) polymorphs were isolated from the same individual and shown to have important differences both in their solution properties and in interaction with lysine Sepharose and fibrin. One Lp(a) particle (d-Lp(a)) with a large apo(a) isoform had a density of 1.087 g/ml and a molecular weight of 3.17 million, while the other Lp(a) particle with a small apo(a) isoform having a mobility faster than that of apoB was larger and had a molecular weight of 3.75 million and a density of 1.054 g/ml. D-Lp(a) underwent cold-induced self-association and also had a higher affinity for lysine Sepharose, whereas the other Lp(a) polymorph did not. Both Lp(a) particles bound fibrin via two different binding sites, one of which involved fibrin lysine residues which are also recognized by plasminogen. Lysine-mediated binding of d-Lp(a) by fibrin was ten times stronger than that of the other Lp(a) particle, whereas non-lysine-mediated binding of either Lp(a) species by fibrin was of equal strength. At saturation, 80% of d-Lp(a) bound fibrin at sites that did not involve lysine residues, whereas only 33% of the other Lp(a) polymorph bound to these sites. These findings indicate that the binding of Lp(a) to fibrin is more complex than previously thought and imposes another layer of difficulty on our understanding of how Lp(a) regulates and/or impairs fibrinolysis.

Aminocaproic Acid↗
PubMed1994

Lipoprotein (a) displays increased accumulation compared with low-density lipoprotein in the murine arterial wall.

Lipoprotein (a) (Lp(a)) is known to be an independent risk factor for cardiovascular disease, but the mechanisms by which it contributes to this disease remain unclear. Current evidence indicates that the closely related plasma particle, low-density lipoprotein (LDL), may initiate atherosclerosis through deposition in the arterial wall. This study has compared the ability of both lipoproteins to enter and accumulate within the arterial wall. Experiments were conducted in vivo with animals from two strains of mice: C57BL/6 mice, which develop fatty streak lesions upon challenge by a high-fat diet, and C3H/HeJ mice, which are resistant to lesion formation. Animals from both strains were maintained up to 16 weeks either on chow or high-fat diet. The mice were intravenously injected with 125I-labeled human Lp(a) or 125I-labeled human LDL in equimolar amounts and the lipoprotein allowed to circulate in vivo for 2 or 24 h. Transverse sections of the aortic root including sites of predilection for lesion formation at the commissures of the valve were prepared and examined after autoradiography. The autoradiographic grains over lesions and histologically uninvolved areas were enumerated and compared after normalization. Both Lp(a) and LDL demonstrated nearly ten times greater accumulation in lesions compared with histologically uninvolved areas from C57BL/6 mice. Analyses of histologically uninvolved areas from both strains of mice showed a significantly higher accumulation of Lp(a) than LDL. Finally, significantly higher accumulations of both Lp(a) and LDL occurred in the histologically uninvolved intima and subintima of lesion-prone C57BL/6 mice as compared with lesion-resistant C3H/HeJ mice after 5 weeks on the diets. We propose that enhanced accumulation of Lp(a) in the arterial wall accounts, in part, for the increased risk of cardiovascular disease.

Animals↗
PubMed1994

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Cells, Cultured↗