**5. Lp(a) and plasminogen**

746 Lipoproteins – Role in Health and Diseases

account for the atherogenic nature of Lp(a).

concentration 4 μg/ml).

subaggregant doses of thrombin receptor activation peptide (TRAP) and arachidonic acid while inversely platelet stimulation with arachidonic acid enhanced platelet binding of apo-a [54]. In both studies it turned out that the size of r-apo-a determined by the number of KIV

Summarizing, in vitro studies indicate that Lp(a) induced decreases, increases or no change at all in platelet aggregation [43, 45, 50, 51, 53, 54]. In all cases the mechanisms involved are quite unclear and only speculative. A recent work strongly supports the evidence to suggest that Lp(a) binds to platelets via its arginyl-glycyl-aspartyl (RGD) epitope of the apo(a) but not via apo(a)'s lysine binding region in both strong and weak agonist-stimulated platelets and inhibits the binding of fibrinogen thus reducing aggregation [55]. On the other side there are in vivo studies published quite recently suggesting that Lp(a) concentrations greater than 30 mg/dl are a frequent and independent risk factor for venous thrombosis [56] and that high levels of Lp(a) could be a more frequently thrombophilic risk factor in young women [57]. To date disagreement exists about increased arterial thrombosis due to elevated blood levels of Lp(a). The fact that this procedure is a result of collagen-exposed platelets in case of plaque rupture followed by clot formation argues against the proaggregatory nature of Lp(a) and maybe procedures others than platelet activation

**Figure 2.** Aggregation curves showing the influence of lipoproteins on collagen-induced platelet aggregation. Gel filtered platelets (200 μl; 2x108/ml) were incubated for 30 min at 37°C with a) LDL 5 mg/ml, b) Tyrodes's buffer or c) Lp(a) 0.5 mg/ml. Aggregation was triggered with 10 μl collagen (final

type 2 modules seems not to play a crucial role in its proaggregant effect.

The mechanism by which Lp(a) accelerates atherosclerosis could not yet been clarified. One possible explanation leads via the connection of Lp(a) to the fibrinolytic system as in 1987 it was found out that Lp(a) and plasminogen are immunochemically related [58] leading to speculations whether Lp(a) might interfere with fibrinolysis. Through partial amino acid sequencing it could be shown for the first time that apo-a has a striking homology of about 70% to plasminogen, the precursor of the proteolytic enzyme plasmin which dissolves fibrin clots [58]. This could be confirmed in our own studies demonstrating that polyclonal antisera from rabbit, sheep and horse as well as three monoclonal antibodies from mouse raised against apo-a reacted with plasminogen on immunoblots and similar to plasminogen, Lp(a) bound selectively but with somewhat lower affinity to lysine-Sepharose [59]. Plasminogen, a protein of 791 amino acids and a molecular weight of about 92 000 D is a plasma serine protease zymogen that consists of five cysteine-rich sequences of 80-114 amino acids each, called kringles, followed by a trypsin like protease domain [60]. The highly glycosylated apo-a exists in various polymorphic forms with molecular weights higher, lower or equal to apoprotein B (Mr ≈ 550 000 D) [61] which are covalently linked to apoprotein B via disulfide bridges [62]. It contains a hydrophobic signal sequence for secretion followed by up to >50 copies of kringle IV of plasminogen predicting the risk for coronary heart disease in the way that apo-a alleles with a low kringle IV copy number (<22) and high Lp(a) plasma concentration are significantly more frequent in the CHD group (p<0.001) [63]. Additionally one kringle V as well as protease domains of plasminogen are found in apo-a [58]. Later on cDNA sequencing revealed that human apolipoprotein(a) is homologous to plasminogen but despite the fact that apo-a contains a protease domain it does not act fibrinolytically like plasminogen because the arginine at the cleavage site for tissue plasminogen activator in plasminogen is changed to serine in apo-a [64].

Nevertheless Lp(a) might interfere with the fibrinolytic system in different ways due to its similarity to plasminogen as it may inhibit the binding of plasminogen to its receptor on endothelial cells thereby preventing generation of plasmin and increasing the thrombotic risk [65, 66]. Furthermore it could be demonstrated that Lp(a) accumulates in atherosclerotic lesions maybe via adherence to fibrinogen or fibrin incorporated in atherosclerotic plaques thereby inhibiting fibrinolysis [66]. Another mechanism by which Lp(a) is thought to attenuate fibrinolysis involves direct competition with plasminogen for fibrinogen or fibrin binding sites thus reducing the efficiency of plasminogen activation [67]. Fibrinolysis is initiated by binding of plasminogen to lysine residues on fibrin thereby initiating activation of plasmin and amplifying fibrinolytic processes [68]. Like plasminogen Lp(a) also binds to lysine residues [69] but without catalytical activity leading to interference with or inhibition of fibrinolysis resulting in hypofibrinolysis and accumulation of cholesterol included in the LDL-like component of Lp(a) [66]. The fact that low molecular weight isoforms of apo-a are associated with greater inhibition of fibrinolysis [70, 71] confirms the hypothesis that subjects with small apo-a phenotypes have a two-fold risk of CHD and stroke compared with those with larger isoforms of apo-a [34]. In contrast Knapp et al. [72] observed that the rate of plasmin formation was inversely related to Lp(a) but inhibition of plasmin generation increased with the size of apo-a using a standardized in vitro fibrinolysis model. From the fact that the inhibitory effect of free apo-a was much stronger than that of the complete Lp(a) particle they conclude that the apo-a component is responsible for the observed reduction of plasmin formation maybe due to the availability of additional lysine binding sites in the unbound apo-a which was formerly reported by Scanu et al. [73]. On the other hand there are also data showing that the plasma concentration of Lp(a) is inversely related to plasmin formation but that this relationship is not influenced by the size of apo-a isoforms [74]. Above all there are other reports explaining the inhibitory effect of Lp(a) on fibrinolysis not only by competition of Lp(a) with plasminogen for the binding sites on fibrin, endothelial cells and monocytes but also by reduction of tissue plasminogen activator or streptokinase-induced fibrinolytic activity [75, 76, 77].

A novel contribution to the understanding of Lp(a)/apo-a-mediated inhibition of plasminogen activation comes from results showing the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin-mediated conversion of Gluplasminogen to Lys-plasminogen an essential step for fibrin clot lysis [78]. Interestingly, 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).

In summary, the proposed mechanisms modulating the antifibrinolytic effects of elevated Lp(a) levels in vitro are manifold and emphasize the prothrombotic effects of this lipoprotein particle. The in vivo situation however seems to be much more complex the more that there is a strong positive correlation reported between bleeding time and fasting serum concentrations of Lp(a) [ 52].
