Marlys L. Koschinsky

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

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

A Polymorphism in the Protease-Like Domain of Apolipoprotein(a) Is Associated With Severe Coronary Artery Disease

Objectives—The purpose of this study was to identify genetic variants associated with severe coronary artery disease (CAD). Methods and Results—We used 3 case-control studies of white subjects whose severity of CAD was assessed by angiography. The first 2 studies were used to generate hypotheses that were then tested in the third study. We tested 12 077 putative functional single nucleotide polymorphisms (SNPs) in Study 1 (781 cases, 603 controls) and identified 302 SNPs nominally associated with severe CAD. Testing these 302 SNPs in Study 2 (471 cases, 298 controls), we found 5 (in LPA, CALM1, HAP1, AP3B1, and ABCG2) were nominally associated with severe CAD and had the same risk alleles in both studies. We then tested these 5 SNPs in Study 3 (554 cases, 373 controls). We found 1 SNP that was associated with severe CAD: LPA I4399M (rs3798220). LPA encodes apolipoprotein(a), a component of lipoprotein(a). I4399M is located in the protease-like domain of apolipoprotein(a). Compared with noncarriers, carriers of the 4399M risk allele (2.7% of controls) had an adjusted odds ratio for severe CAD of 3.14 (confidence interval 1.51 to 6.56), and had 5-fold higher median plasma lipoprotein(a) levels (P=0.003). Conclusions—The LPA I4399M SNP is associated with severe CAD and plasma lipoprotein(a) levels.

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).

Structure-function relationships in apolipoprotein(a): insights into lipoprotein(a) assembly and pathogenicity

Purpose of review Lipoprotein(a) is a structurally and functionally unique lipoprotein consisting of the glycoprotein apolipoprotein(a) covalently linked to LDL. Lipoprotein(a) is assembled extracellularly by a two-step mechanism, still incompletely understood, in which initial non-covalent interactions between apolipoprotein(a) and apolipoprotein B precede specific disulfide bond formation. Elevated concentrations of plasma lipoprotein(a) are a risk factor for a variety of vascular diseases, including coronary heart disease, ischaemic stroke and venous thrombosis. Whereas many pathogenic mechanisms have been proposed for lipoprotein(a), it remains to be conclusively demonstrated which mechanisms are relevant to human disease. Recent findings Structural and functional studies have verified that apolipoprotein(a) kringle 4 types 6-8 contain lysine binding sites of a weaker affinity for lysine analogues than kringle 4 type 10. Recent evidence has conclusively shown a role for kringle 4 types 7 and 8 in lipoprotein(a) assembly. Moreover, apolipoprotein(a) has been shown to undergo a conformational change, from a closed to an open form, which accelerates the rate of covalent lipoprotein(a) assembly. Functional studies in vitro have identified the domains in apolipoprotein(a) that mediate its inhibitory effects on fibrin clot lysis, binding to fibrin and other biological substrates, and pro-inflammatory and anti-angiogenic properties. Summary Extensive structure-function studies of apolipoprotein(a) have begun to yield important insights into the domains in apolipoprotein(a) that mediate lipoprotein(a) assembly and the pathogenic effects of this lipoprotein. Continued investigations of these relationships will contribute critically to unravelling the many outstanding questions about lipoprotein(a) metabolism and pathophysiology.

Lipoprotein(a) Assembly Quantitative Assessment of the Role of Apo(a) Kringle IV Types 2-10 in Particle Formation

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

Lipoprotein(a) concentration and apolipoprotein(a) size: A synergistic role in advanced atherosclerosis?

Despite more than 3 decades of intense scientific research that has fostered our understanding of the structure and biochemistry of lipoprotein(a) [Lp(a)], the physiopathological role of Lp(a) is still poorly understood. Consequently, despite its recognition as a risk factor for coronary artery disease (CAD), the role of Lp(a) in atherogenesis and the extent to which Lp(a) levels should be assessed in clinical practice remain controversial. Lp(a), which is the most complex and polymorphic of the lipoprotein particles, is formed by an LDL moiety and a unique protein, apo(a), linked to apolipoprotein (apo) B-100 of LDL.1 The most intriguing feature of apo(a) is that it shares an extensive structural homology with plasminogen, a key proenzyme of the fibrinolytic cascade. Kringle V and the protease domains of apo(a) share >85% amino-acid identity with the corresponding plasminogen domains, even though the protease domain of apo(a) does not appear to have a catalytic function. Apo(a) contains 10 different types of a sequence with variable degrees of homology with plasminogen kringle IV. The number of kringle IV type 2 repeats, which is encoded by a varying number of copies in the apo(a) gene,2 varies both within and among individuals, and at least 35 apo(a) size isoforms have been detected in human plasma.3 Despite the presence of LDL, apo(a) imparts to Lp(a) unique properties with respect to synthesis and catabolism. In fact, apo B-100 in Lp(a) particles does not appear to mediate the catabolism of this lipoprotein via the LDL receptor, thus suggesting that the attachment to apo(a) produces a steric hindrance and/or a conformational change of apo B-100. Whereas the rate of removal from the circulation determines the level of LDL, evidence has been provided that the rate of synthesis is the primary determinant of Lp(a) levels. Plasma Lp(a) concentration is primarily …

Apolipoprotein(a) inhibits the conversion of Glu‐plasminogen to Lys‐plasminogen: a novel mechanism for lipoprotein(a)‐mediated inhibition of plasminogen activation

Summary.u2002 Background:u2002Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for thrombotic disorders. Lp(a) is a unique lipoprotein consisting of a low‐density lipoprotein‐like moiety covalently linked to apolipoprotein(a) [apo(a)], a homologue of the fibrinolytic proenzyme plasminogen. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue‐type plasminogen activator‐mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin‐dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin‐mediated conversion of Glu‐plasminogen to Lys‐plasminogen. Objective:u2002Using acid–urea gel electrophoresis to resolve the two forms of radiolabeled plasminogen, we determined whether apo(a) is able to inhibit Glu‐plasminogen to Lys‐plasminogen conversion. Methods:u2002The assays were performed in the absence or presence of different recombinant apo(a) species, including point mutants, deletion mutants and variants that represent greater than 90% of the known apo(a) isoform sizes. Results:u2002Apo(a) substantially suppressed Glu‐plasminogen conversion. Critical roles were identified for the kringle IV types 5–9 and kringle V; contributory roles for sequences within the amino‐terminal half of the molecule were also observed. Additionally, with the exception of the smallest naturally‐occurring isoform of apo(a), isoform size was found not to contribute to the inhibitory capacity of apo(a). Conclusion:u2002These findings underscore a novel contribution to the understanding of Lp(a)/apo(a)‐mediated inhibition of plasminogen activation: the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin‐mediated Glu‐plasminogen to Lys‐plasminogen conversion.

Apolipoprotein(a) Enhances Platelet Responses to the Thrombin Receptor–Activating Peptide SFLLRN

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

Binding of recombinant apolipoprotein(a) to extracellular matrix proteins.

Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and fibrinogen, weakly to laminin, and not at all to von Willebrand factor, vitronectin, or collagen type IV. In contrast to the binding of plasminogen to fibronectin, r-apo(a) binding does not appear to be mediated by lysine-dependent interactions, based on the inability of epsilon-aminocaproic acid concentrations up to 0.2 mol/L to significantly decrease r-apo(a) binding to fibronectin. Plasminogen competed weakly for the binding of r-apo(a) to fibronectin, whereas r-apo(a) completely abolished plasminogen binding. The 29- and 38-kd heparin-binding thermolysin fragments of fibronectin, previously identified as the lipoprotein(a) binding domains, were digested with trypsin, and a peptide that retained the ability to bind r-apo(a) was isolated; the sequence of the peptide (AVTTIPAPTDLK) corresponds to the amino terminus of the 29- and 38-kd domains. A synthetic peptide with this sequence was able to compete effectively with fibronectin for r-apo(a) binding.

Effects of lipoprotein(a) on the binding of plasminogen to fibrin and its activation by fibrin-bound tissue-type plasminogen activator

Molecular assembly of plasminogen and tissue-type plasminogen activator (t-PA) at the surface of fibrin results in the generation of fibrin-bound plasmin and thereby in the dissolution of a clot. This mechanism is triggered by specific interactions of intra-chain surface lysine residues in fibrin with the kringle domains of plasminogen, and is further amplified via the interaction of plasminogen kringles with the carboxy-terminal lysine residues of fibrin that are exposed by plasmin cleavage. By virtue of its marked homology with plasminogen, apo(a), the specific apolipoprotein component of Lp(a), may bind to the lysine sites available for plasminogen on the surface of fibrin and thereby interfere with the fibrinolytic process. A sensitive solid-phase fibrin system, which allows the study of plasminogen activation at the plasma fibrin interface and makes feasible the analysis of products bound to fibrin, has been used to investigate the effects of Lp(a) on the binding of plasminogen and its activation by fibrin-bound t-PA. Plasma samples from human subjects with high levels of Lp(a) were studied. We have established that Lp(a) binds to the fibrin surface and thereby competes with plasminogen (Ki = 44 nM) so as to inhibit its activation. We have further shown that Lp(a) blocks specifically carboxy-terminal lysine residues on the surface of fibrin. To further explore the role of apo(a) on the Lp(a) fibrin interactions, we have performed ligand-binding studies using a recombinant form of apo(a) that contains 17 kringle 4-like units. We have shown that recombinant apo(a) binds specifically to fibrin (Kd = 26 +/- 8 nM, Bmax = 26 +/- 2 fmol/well) and that this binding increases upon treatment of the fibrin surface with plasmin (Kd = 8 +/- 4 nM, Bmax = 115 +/- 14 fmol/well). Altogether, our results indicate clearly that binding of native Lp(a) through this mechanism may impair clot lysis and may favor the accumulation of cholesterol in thrombi at sites of vascular injury.