Apolipoprotein(a) stimulates vascular endothelial cell growth and migration and signals through integrin alphaVbeta3

Elevated plasma concentrations of Lp(a) [lipoprotein(a)] are an emerging risk factor for atherothrombotic disease. Apo(a) [apolipoprotein(a)], the unique glycoprotein component of Lp(a), contains tandem repeats of a plasminogen kringle (K) IV-like domain. In the light of recent studies suggesting that apo(a)/Lp(a) affects endothelial function, we evaluated the effects of apo(a)/Lp(a) on growth and migration of cultured HUVECs (human umbilical-vein endothelial cells). Two full-length r-apo(a) [recombinant apo(a)] variants (12K and 17K), as well as Lp(a), were able to stimulate HUVEC growth and migration to a comparable extent; 17K r-apo(a) also decreased the levels of total and active transforming growth factor-beta secreted by these cells. Using additional r-apo(a) variants corresponding to deletions and/or site-directed mutants of various kringle domains in the molecule, we were able to determine that the observed effects of full-length r-apo(a) on HUVECs were dependent on the presence of a functional lysine-binding site(s) in the apo(a) molecule. With respect to signalling events elicited by apo(a) in HUVECs, we found that 17K treatment of the cells increased the phosphorylation level of FAK (focal adhesion kinase) and MAPKs (mitogen-activated protein kinases), including ERK (extracellular-signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase). In addition, we showed that LM609, the function-blocking antibody to integrin alphaVbeta3, abrogated the effects of 17K r-apo(a) and Lp(a) on HUVECs. Taken together, the results of the present study suggest that the apo(a) component of Lp(a) signals through integrin alphaVbeta3 to activate endothelial cells.     

Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments.

Lipoprotein(a) [Lp(a)], but not low-density lipoprotein (LDL), was previously shown to impair the generation of fibrin-bound plasmin [Rouy et al. (1991) Arterioscler. Thromb. 11, 629-638] by a mechanism involving binding of Lp(a) to fibrin. It was therefore suggested that the binding was mediated by apolipoprotein(a) [apo(a)], a glycoprotein absent from LDL which has a high degree of homology with plasminogen, the precursor of the fibrinolytic enzyme plasmin. Here we have evaluated this hypothesis by performing comparative fibrin binding studies using a recombinant form of apo(a) containing 17 copies of the apo(a) domain resembling kringle 4 of plasminogen, native Lp(a), and Glu-plasminogen (Glu1-Asn791). Attempts were also made to identify the kringle domains involved in such interactions using isolated elastase-derived plasminogen fragments. The binding experiments were performed using a well-characterized model of an intact and of a plasmin-digested fibrin surface as described by Fleury and Anglés-Cano [(1991) Biochemistry 30, 7630-7638]. Binding of r-apo(a) to the fibrin surfaces was of high affinity (Kd = 26 +/- 8.4 nM for intact fibrin and 7.7 +/- 4.6 nM for plasmin-degraded fibrin) and obeyed the Langmuir equation for adsorption at interfaces. The binding to both surfaces was inhibited by the lysine analogue AMCHA and was completely abolished upon treatment of the degraded surface with carboxypeptidase B, indicating that r-apo(a) binds to both the intrachain lysines of intact fibrin and the carboxy-terminal lysines of degraded fibrin. As expected from these results, both r-apo(a) and native Lp(a) inhibited the binding of Glu-plasminogen to the fibrin surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein(a), a link between atherosclerosis and tumor angiogenesis

Lipoprotein (a) [Lp(a)] is a LDL-like particle with one apolipoprotein(a) [apo(a)] covalently bound to apolipoprotein B, the structural protein of Low Density Lipoprotein (LDL). Lewis Lung Carcinoma (LL/2) cells exhibited delayed growth and reduced angiogenesis in apo(a) transgenic mice, expressing a recombinant apo(a) [r-apo(a)] with 18 kringle 4 repeats. The mean microvessel density of subcutaneous LL/2 tumors from apo(a) transgenic mice was significantly lower than that of tumors from control wild type mice. CHO cells secreting a truncated apo(a) protein with only six kringle 4 repeats did not exhibit delayed tumor growth nor did it impair angiogenesis. These data point to an unappreciated role of human apo(a) in angiogenesis and cancer biology. As angiogenesis is necessary for reendothelialization following vascular injury, suppression of angiogenesis by apo(a) may also contribute to the atherogenicity of apo(a). The differences between the truncated apo(a) and r-apo(a) are consistent with the higher atherogenicity of higher molecular weight isoforms.     

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a)

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.     

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

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

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp^55/57→Ala^55/57 substitution of kringle (K)IV₁₀)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV₁₀ LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp⁵⁷→Ala⁵⁷ substitution in KIV₁₀ that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV₁₀ LBS and is unique to human apo(a), which may explain Lp(a)’s pro-atherogenic potential.     

Identification of sequences in apolipoprotein(a) that maintain its closed conformation: a novel role for apo(a) isoform size in determining the efficiency of covalent Lp(a) formation

We have previously demonstrated that, in the presence of the lysine analogue epsilon-aminocaproic acid, apolipoprotein(a) [apo(a)] undergoes a conformational change from a closed to an open structure that is characterized by a change in tryptophan fluorescence, an increase in the radius of gyration, an alteration of domain stability, and an enhancement in the efficiency of covalent lipoprotein(a) [Lp(a)] formation. In the present study, to identify sequences within apo(a) that maintain its closed conformation, we used epsilon-aminocaproic acid to probe the conformational status of a variety of recombinant apo(a) isoforms using analytical ultracentrifugation, differential scanning calorimetry, intrinsic fluorescence, and in vitro covalent Lp(a) formation assays. We observed that the closed conformation of apo(a) is maintained by intramolecular interaction(s) between sequences within the amino- and carboxyl-terminal halves of the molecule. Using site-directed mutagenesis, we have identified the strong lysine-binding site present within apo(a) kringle IV type 10 as an important site within the C-terminal half of the molecule, which is involved in maintaining the closed conformation of apo(a). Apo(a) exhibits marked isoform size heterogeneity because of the presence of varying numbers of copies of the kringle IV type-2 domain located within the amino-terminal half of the molecule. Using recombinant apo(a) species containing either 1, 3, or 8 copies of kringle IV type 2, we observed that, while apo(a) isoform size does not alter the affinity of apo(a) for low-density lipoprotein, it affects the conformational status of the protein and therefore influences the efficiency of covalent Lp(a) assembly. The inverse relationship between apo(a) isoform size and the efficiency of covalent Lp(a) formation that we report in vitro may contribute to the inverse relationship between apo(a) isoform size and plasma Lp(a) concentrations that has been observed in vivo.     

The apolipoprotein(a) component of lipoprotein(a) mediates binding to laminin: contribution to selective retention of lipoprotein(a) in atherosclerotic lesions

Lipoprotein(a) [Lp(a)] entrapment by vascular extracellular matrix may be important in atherogenesis. We sought to determine whether laminin, a major component of the basal membrane, may contribute to Lp(a) retention in the arterial wall. First, immunohistochemistry experiments were performed to examine the relative distribution of Lp(a) and laminin in human carotid artery specimens. There was a high degree of co-localization of Lp(a) and laminin in atherosclerotic specimens, but not in non-atherosclerotic sections. We then studied the binding interaction between Lp(a) and laminin in vitro. ELISA experiments showed that native Lp(a) particles and 17K and 12K recombinant apolipoprotein(a) [r-apo(a)] variants interacted strongly with laminin whereas LDL, apoB-100, and the truncated KIV(6-P), KIV(8-P), and KIV(9-P) r-apo(a) variants did not. Overall, the ELISA data demonstrated that Lp(a) binding to laminin is mediated by apo(a) and a combination of the lysine analogue epsilon-aminocaproic acid and salt effectively decreases apo(a) binding to laminin. Secondary binding analyses with 125I-labeled r-apo(a) revealed equilibrium dissociation constants (K(d)) of 180 and 360 nM for the 17K and 12K variants binding to laminin, respectively. Such similar K(d) values between these two r-apo(a) variants suggest that isoform size does not appear to influence apo(a) binding to laminin. In summary, our data suggest that laminin may bind to apo(a) in the atherosclerotic intima, thus contributing to the selective retention of Lp(a) in this milieu.     

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots.

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd approximately 1.2-1.6 microM). The total concentration of binding sites (Bmax) present on the fibrin surface was approximately 4-fold greater for the 12K than for the 17K (Bmax values of 0.81 +/- 0.09 nM, and 0.20 +/- 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmax values of 2.7 +/- 0.3 nM and 0.20 +/- 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmax was correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low microM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.     

Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

Effect of plasminogen activators on human recombinant apolipoprotein(a) having the plasminogen activation cleavage site.

The serine-proteinase domain in human apolipoprotein(a) [apo(a)] and plasminogen exhibit 89% sequence identity including the catalytic triad. Cleavage of the Arg(561)-Val(562) activation site in plasminogen by either tissue- or urokinase-type plasminogen activator results in formation of the fibrinolytic enzyme plasmin. Apo(a) does not contain measurable amidolytic activity nor can it be activated by plasminogen activators. It has been suggested that the latter finding might be explained by the substitution of the plasminogen Arg-Val activation site by Ser-Ile in apo(a). To investigate if introduction of the Arg-Val activation site in apo(a) might result in sensitivity towards plasminogen activators, we expressed wild-type and Arg-Val mutant recombinant apo(a) [r-apo(a)] in human embryonic kidney and hepatocyte cell lines. Free r-apo(a) and lipoprotein-like particles [r-Lp(a)] were obtained in the culture supernatants of transfected 293 and HepG2 cells, respectively. Incubation of mutant r-apo(a)/r-Lp(a) with plasminogen activators produced neither plasmin-like activity nor cleavage at the Arg-Val activation site, even in the presence of various stimulators of plasminogen activation. Our data suggest that the high selectivity of activators for plasminogen activation requires interactions with regions in plasminogen distant from the activation disulfide loop which are not present in apo(a).     

Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a)

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Properties of human free apolipoprotein(a) and lipoprotein(a) after either freezing or lyophilization in the presence and absence of cryopreservatives

Apolipoprotein(a), apo(a), the specific multikringle glycoprotein constituent of lipoprotein(a), Lp(a), occurs in the plasma mostly bound to apoB100-containing lipoproteins but also in a free form. Often the properties of these products are determined after storage in the cold; yet limited information is available on their stability at low temperatures. To shed light on this subject, we examined the effect of two parameters, freezing and lyophilization, in either the absence or the presence of cryopreservatives. Lp(a)s each having a single apo(a) size isoform containing either 14 or 17 kringle (K) IVs were isolated from the plasma of healthy donors by combining density gradient ultracentrifugation and lysine-Sepharose column chromatography using solutions containing both antioxidants and proteolytic inhibitors. Apo(a) was obtained from parent Lp(a) by a mild limited reductive procedure. Either freezing at -20 degrees C or lyophilization in the presence of 5% sucrose did not change the electrophoretic, immunochemical, and lysine-binding properties of Lp(a) including its ability to generate free apo(a). Irrespective of source, apo(a) remained stable when either frozen at -20 and -80 degrees C or lyophilized in the presence of 125 mM trehalose. In all cases, the absence of cryopreservatives caused the samples to aggregate irreversibly. Thawed or reconstituted samples of both free and bound apo(a) kept at 4 degrees C under sterile conditions in the presence of antioxidants, proteolytic inhibitors, and cryopreservative exhibited no significant changes in properties within the time of observation. Both apo(a) isoforms gave comparable results. We conclude that apo(a), either free or bound, can be kept stable at low temperatures in the presence of appropriate cryopreservatives.