Demonstration of Covalent Binding of Lipoprotein(a) [Lp(a)] to Fibrin and Endothelial Cells

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Abstract
It has been well documented that Lp(a) binds noncovalently to fibrin or human umbilical vein endothelial cells. This binding is to lysines and is inhibited by lysine analogues such as ε-aminocaproic acid (EACA). In the present study, Lp(a) (0.006−0.6 μM) binding to immobilized fibrin and endothelial cells was evaluated by ELISA with an anti-Lp(a) antibody. A significant portion (≈65%) of the Lp(a) was found to resist dissociation by EACA (0.2 M). The EACA resistant binding of Lp(a) was time and concentration dependent. The addition of EDTA to the incubation mixture had no effect, thereby excluding cross-linking by transglutaminase as a mechanism. This portion of Lp(a) was also resistant to dissociation by acid (0.1 N HCl), 0.1% SDS, 1 M benzamidine, Tris-HCl (1 M, pH 12), or DTT (5 mM), but it was washed off by 0.1 N NaOH (which did not remove the immobilized fibrin). This suggested that the Lp(a) was covalently linked by an ester bond. Covalent binding was inhibited when Lp(a) was mildly oxidized by BioRad Enzymobeads, which may explain why it escaped recognition in experiments with radiolabeled Lp(a). Covalent binding was attenuated when Lp(a) was pretreated with DFP suggesting that the serine residue in the pseudo active site of Lp(a) was involved. Lp(a) also bound covalently to immobilized BSA, indicating some nonspecificity. However, binding to BSA was almost 3-fold less than to fibrin, suggesting that lysine binding may facilitate covalent binding. A similar proportion of EACA resistant binding of Lp(a) was found with endothelial cells. In conclusion, the findings demonstrate a novel, covalent binding by Lp(a) which is kringle independent and is postulated to involve the pseudo protease domain of Lp(a). This property may contribute to the deposition of Lp(a) on endothelial surfaces and its colocalization with fibrin in atheromas.