Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-ß due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibri

Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution

Study of apo(a) length polymorphism and lipoprotein(a) concentrations in subjects with single or double apo(a) isoforms.

Cardiovascular risk is associated with high lipoprotein(a) (Lp(a)) concentrations and low molecular weight apolipoprotein(a) (apo(a)) isoforms. We studied the relationship between these two biological parameters, particularly in subjects expressing two apo(a) isoforms. Plasma Lp(a) was measured by immunonephelometry in 530 unrelated Caucasian patients at high cardiovascular risk, and apo(a) size determined by immunoblotting using a recombinant standard. Two, one, or no apo(a) isoforms were detected in 258, 270, and 2 subjects, respectively. Lp(a) concentrations showed a non-Gaussian distribution, being higher in the 'double band' than in the 'single band' group (median 0.42 vs. 0.11 g/l, p < 0.0005). Apo(a) size distribution was bimodal, with two frequency peaks at 18 kringles (K) and 27 K. Small size apo(a) isoforms were more frequently found in the 'double band' group, where major isoforms were of lower size than minor isoforms (median 20 vs. 27 K). Regression analysis showed that apo(a) gene length accounted for 33% of Lp(a) variation, with a threshold effect at 20 K, no correlation being found over this value. The minor apo(a) isoform did not significantly influence Lp(a) concentration. These data confirm the relationship between apo(a) size and Lp(a) concentration and suggest that the assessment of cardiovascular risk should take into account the threshold effect at 20 K and the absence of influence of the minor apo(a) isoform