Abstract
Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant alphaC-fragment (Aalpha-(221-610)) corresponding to the alphaC-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19-21 nm) between the immobilized alphaC-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the alphaC-fragment allowed localization of the apo(a)-binding site to the Aalpha-(392-610) region. The presence of epsilon-aminocaproic acid only slightly inhibited binding of apo(a) to the alphaC-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the alphaC-fragment was minimal. These results indicate that the alphaC-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.
Highlights
Elevated plasma levels of lipoprotein(a) (Lp(a))1 and fibrinogen are independent risk factors for atherosclerotic cardiovascular diseases [1,2,3]
This Kd was very close to that obtained in the same assay for the interaction between apo(a) A10 and immobilized fibrin (Fig. 1 and Table I). This high affinity interaction was confirmed in surface plasmon resonance (SPR) experiments, in which apo(a) A10 bound to the immobilized ␣Cfragment in a dose-dependent manner with a Kd of 21 nM (Fig. 2 and Table I)
Because fibrin(ogen) plays a prominent role in accumulation of Lp(a) in the vessel walls and development of atherosclerosis [10], an understanding of the molecular mechanisms underlying this pathological process requires a better knowledge of the mechanism of interactions between these two plasma components
Summary
Proteins—Plasminogen-depleted human fibrinogen was purchased from Calbiochem. It did not contain factor XIII because its incubation with thrombin resulted in non-cross-linked fibrin as revealed by SDS-PAGE under reduced conditions. Expression and Purification of Recombinant Proteins—The recombinant ␣C-fragments corresponding to the human fibrinogen ␣C-domain (A␣-(221– 610)) and its NH2- and COOH-terminal halves (A␣-(221–391) and A␣-(392– 610), respectively) were produced in Escherichia coli using the pET-20b expression vector as described earlier [33]. They were purified and refolded according to the procedures described [31, 33]. Following washing with phosphate-buffered saline containing 0.02% Tween 20, the indicated concentrations of apo(a) in the same buffer were added to the wells and incubated for 1 h. The values were examined for self-consistency of the data as described [43]
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