Lipoprotein(a) binding to other apolipoprotein B containing lipoproteins

A method combining ligand dot blotting and digital imaging was used to determine the apparent dissociation constant (KD) for the binding of lipoprotein(a) to low-density lipoproteins (Lp(a)-LDL2). By use of this approach, the KD for the Lp(a)-LDL2 complex was shown to be in the nanomolar range [(1.05 +/- 0.21) x 10(-8) M, n = 4]. The Lp(a)-LDL2 interaction was both hydrophobic and ionic; however, hydrophobic forces predominated because the interaction was demonstrable at high salt concentration (greater than 2 M NaCl), while no complex was detectable at low salt concentration (less than 0.08 M NaCl). Consistent with the hydrophobic nature of this interaction, the Lp(a)-LDL2 complex was stable over a wide pH range (4-10). Plasminogen did not compete with Lp(a) binding to LDL2 even at a 2.2 X 10(3) molar excess of plasminogen over the LDL2 concentration. The only component identified in plasma and serum that inhibited the binding of LDL2 to Lp(a) was apolipoprotein B containing lipoproteins (apoB-Lp). These studies indicate that the Lp(a)-LDL2 complex could exist in plasma. In fact, up to 72% of purified Lp(a) added to an Lp(a)-negative hypertriglyceridemic plasma floated with apoB-Lp (d less than 1.063 g/mL) following ultracentrifugation, whereas only 9% of the purified Lp(a) added to the apoB-Lp-free 1.12 g/mL infranate floated at d less than 1.063 g/mL. The formation of a complex of Lp(a) with apoB-Lp could increase the amount of cholesterol ester bound per cellular receptor, e.g., LDL receptor, and thus potentially accelerate cholesterol removal from the vascular compartment.     

Lp(a) interactions.

In this report, we have summarized our recent studies on lipoprotein(a) (Lp(a)) and its interactions with apolipoprotein B-containing lipoproteins (ApoB-Lp). These findings implicate the kringle-4-like domains of Apo(a) in the binding of Lp(a) to other ApoB-Lp and point to proline as important in this interaction. Other studies have indicated that Lp(a) interacts with the subendothelial extracellular matrix (ECM) and that Lp(a) is inversely related to plasma triglycerides. Since Apo(a) also has an affinity for ApoB-Lp, enhanced binding of Apo(a) to the arterial wall could increase the accumulation of LDL in the matrix and thus promote the development of cardiovascular disease.     

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

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

The linkage with apolipoprotein (a) in lipoprotein (a) modifies the immunochemical and functional properties of apolipoprotein B

Lipoprotein (a) [Lp(a)] was isolated from several donors and its apolipoprotein (a) [apo(a)] dissociated by a reductive treatment, generating the apo(a)-free form of Lp(a) [Lp(a--)] that contains apolipoprotein B (apo B) as its sole protein. Using anti-apo B monoclonal antibodies, the properties of apo B in Lp(a), Lp(a--), and autologous low-density lipoprotein (LDL) were compared. Marked differences in apo B immunoreactivity were found between these lipoproteins, due to the presence of apo(a) in Lp(a). Apo(a) enhanced the expression of two epitopes in the amino-terminal part of apo B while it diminished the immunoreactivity of three other epitopes in the LDL receptor binding domain. Accordingly, the binding of the lipoproteins to the LDL receptor was also decreased in the presence of apo(a). In a different experimental system, the incubation of antibodies that react with 27 distinct epitopes distributed along the whole length of apo B sequence with plastic-bound Lp(a) and Lp(a--) failed to reveal any epitope of apo B that is sterically hindered by the presence of apo(a). Our results demonstrate that the presence of apo(a) modified the organization and function of apo B in Lp(a) particles. The data presented indicate that most likely the modification is not due to a steric hindrance but that some more profound conformational changes are involved. We suggest that the formation of the disulfide bridge between apo B and apo(a) in Lp(a) alters the system of disulfide bonds present in apo B and thereby modifies apo B structure.     

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

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)     

Tissue-type plasminogen activator binds to and is inhibited by surface-bound lipoprotein(a) and low-density lipoprotein

Elevated levels of lipoprotein(a) [Lp(a)] are associated with an increased risk of atherothrombotic disease, but the mechanism(s) by which Lp(a) potentiates atherogenesis is unknown. The extensive homology of apolipoprotein(a) [apo(a)] to plasminogen has led us and others to postulate that Lp(a) may impair fibrinolysis. We have previously shown that Lp(a) inhibits fibrin stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA); however, we and other investigators have been unable to demonstrate direct inhibition of t-PA by Lp(a) in solution. We now report that t-PA binds reversibly and saturably to surface-bound Lp(a) and to low-density lipoprotein (LDL) and that as a result of this binding activation of plasminogen by t-PA is inhibited. The catalytic efficiency (kcat/Km) of t-PA when bound to polystyrene surface-bound fibrinogen increased 2.9-fold compared to t-PA bound to control wells. When bound to surface-bound Lp(a), however, the catalytic efficiency of t-PA was reduced 9.5-fold compared to t-PA bound to control wells; likewise, by binding to surface-bound LDL, the catalytic efficiency of t-PA was reduced 16-fold compared to the control. Studies with defined monoclonal antibodies suggest that major determinants of t-PA binding are its active site, the LDL receptor binding domain of apolipoprotein B-100 (apoB-100), and apo(a). These data suggest a unique mechanism by which Lp(a) and LDL incorporated in an atheroma can inhibit endogenous fibrinolysis and thereby contribute to the genesis of atherothrombotic disease.     

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate > arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL.These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.     

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).     

Nuclear magnetic resonance (NMR) solution structure, dynamics, and binding properties of the kringle IV type 8 module of apolipoprotein(a)

The plasma lipoprotein lipoprotein(a) [Lp(a)] comprises a low-density lipoprotein (LDL)-like particle covalently attached to the glycoprotein apolipoprotein(a) [apo(a)]. Apo(a) consists of multiple tandem repeating kringle modules, similar to plasminogen kringle IV (designated KIV1-KIV10), followed by modules homologous to the kringle V module and protease domain of plasminogen. The apo(a) KIV modules have been classified on the basis of their binding affinity for lysine and lysine analogues. The strong lysine-binding apo(a) KIV10 module mediates lysine-dependent interactions with fibrin and cell-surface receptors. Weak lysine-binding apo(a) KIV7 and KIV8 modules display a 2-3-fold difference in lysine affinity and play a direct role in the noncovalent step in Lp(a) assembly through binding to unique lysine-containing sequences in apolipoproteinB-100 (apoB-100). The present study describes the nuclear magnetic resonance solution structure of apo(a) KIV8 and its solution dynamics properties, the first for an apo(a) kringle module, and compares the effects of epsilon-aminocaproic acid (epsilon-ACA) binding on the backbone and side-chain conformation of KIV7 and KIV8 on a per residue basis. Apo(a) KIV8 adopts a well-ordered structure that shares the general tri-loop kringle topology with apo(a) KIV6, KIV7, and KIV10. Mapping of epsilon-ACA-induced chemical-shift changes on KIV7 and KIV8 indicate that the same residues are affected, despite a 2-3-fold difference in epsilon-ACA affinity. A unique loop conformation within KIV8, involving hydrophobic interactions with Tyr40, affects the positioning of Arg35 relative to the lysine-binding site (LBS). A difference in the orientation of the aromatic side chains comprising the hydrophobic center of the LBS in KIV8 decreases the size of the hydrophobic cleft compared to other apo(a) KIV modules. An exposed hydrophobic patch contiguous with the LBS in KIV8 and not conserved in other weak lysine-binding apo(a) kringle modules may modulate specificity for regions within apoB-100. An additional ligand recognition site comprises a structured arginine-glycine-aspartate motif at the N terminus of the KIV8 module, which may mediate Lp(a)/apo(a)-integrin interactions.     

Rhesus monkey apolipoprotein(a). Sequence, evolution, and sites of synthesis

Human lipoprotein(a) is a low density lipoprotein-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) (apo(a)) which is disulfide-linked to apolipoprotein B-100. Sequencing of rhesus monkey apo(a) cDNA suggests that this protein, like human apo(a), is highly similar to plasminogen. Sequence data suggests that a plasminogen-like protease activity and kringle 1-, 2-, 3-, and 5-like domains are unnecessary for apo(a) function, but a highly repeated kringle four-like domain is important. Liver is the major site of apo(a) RNA synthesis; reduced amounts of message were also found in testes and brain. Co-expression with apoB-100 and plasminogen in rhesus tissues is not mandatory.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a

The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr-/-), and apolipoprotein E-deficient (Apoe-/-) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr-/- mice and greatly accelerated in Apoe-/- mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr-/- mice was similar to that in wild-type mice and was only slightly accelerated in Apoe-/- mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance. In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.     

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.     

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.     

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.     

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.     

Stimulation of interleukin-8 production in human THP-1 macrophages by apolipoprotein(a). Evidence for a critical involvement of elements in its C-terminal domain

In the vessel wall, macrophages are among the cells that upon activation contribute to the atherosclerotic process. Low density lipoproteins (LDL) can mediate this activation but only after enzymatic or oxidative modification. Lipoprotein(a) (Lp(a)) is an LDL variant that has been shown to have an atherogenic potential by no clearly established mechanisms. In the present study we examined whether native Lp(a) can activate macrophages and, if so, identify the structural elements involved in this action. For this purpose, we utilized human THP-1 macrophages, prepared by treating THP-1 monocytes with phorbol ester, and we exposed them to Lp(a) and its two derivatives, apo(a)-free LDL (Lp(a-)) and free apo(a). We also studied apo(a) fragments, F1 (N terminus) and F2 (C terminus) and subfragments thereof, obtained by leukocyte elastase digestion. By Northern blot analyses, Lp(a), but not Lp(a-), caused up to a 12-fold increase in interleukin 8 (IL-8) mRNA as compared with untreated cells. Free apo(a) also induced the production of IL-8 mRNA; however, the effect was 3-4-fold higher than that of Lp(a). The increase in mRNA was associated with the accumulation of IL-8 protein in the culture medium. F1 had only a minimal effect, whereas F2 was 1.5-2-fold more potent than apo(a), an activity mostly contained in the Kringle V-protease region. A monoclonal antibody specific for Kringle V inhibited the apo(a)-mediated effect on IL-8. We conclude that Lp(a) via elements contained in the C-terminal domain of apo(a) causes in THP-1 macrophages an increased production of IL-8, a chemokine with pro-inflammatory properties, an event that may be relevant to the process of atherosclerosis.