Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta- and gamma-chains

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombo tic disease in humans. The physiological function(s) of Lp(a) as well as th e precise mechanism(s) by which high plasma levels of Lp(a) increase risk a re unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, b ut the domains in fibrin(ogen) critical for interaction are undefined. We u sed apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that e merged from the screen, clones were identified as fibrinogen beta- and gamm a -chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the f ibrinogen beta- and gamma modules specifically interacts with apo(a)np(a) i n human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) fo r the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.