IMMUNOCHEMICALLY DETECTABLE LIPID-FREE APO(A) IN PLASMA AND IN HUMAN ATHEROSCLEROTIC LESIONS

Although Lp(a) is an independent risk factor for cardiovascular diseas es in humans, the precise pathogenetic mechanisms are still unknown. W e have shown that Lp(a) accumulates in human atherosclerotic lesions, and some particles undergo oxidation. Since, following agarose electro phoresis of both plaque extracts and plasma, a region close to the ori gin immunostained intensely for apo(a) but was lipid-free, we sought t o identify whether such samples contained lipid-free apo(a), as previo usly reported to occur in plaque extracts. Immunochemically identifiab le apo(a) was found following density-gradient ultracentrifugation bot h in the 1.05 < d < 1.09 and the d > 1.21 density fraction from both p lasma and plaque extracts. However, because in a competitive binding R IA, displacement curves of apo(a) in plasma and the d > 1.21 were not parallel, it is premature to ascribe a relative amount of total apo(a) to this fraction. Whereas apo(a) immunoblots of SDS-PAGE under reduci ng conditions of the d > 1.21 fraction of a plaque extract with high a po(a) content showed high molecular weight bands consistent with apo(a ) isoforms, the corresponding d > 1.21 fraction showed multiple low mo lecular weight bands characteristic of fragmentation. Since the d > 1. 21 of arterial extracts contained all the material immunostaining for apo(a) migrating towards the cathode, characteristic of immunoglobulin s (IgG), we asked whether fragments of apo(a) might have associated wi th human IgG both in plasma and tissue extracts, or whether our anti-a po(a) reacted with epitopes on human IgG. Immunoblotting with our anti -apo(a) of samples of plasma and plaque extracts run on agarose electr ophoresis or SDS-PAGE further demonstrated intense staining of multipl e bands in the molecular weight range of human IgC. Furthermore, a fra ction of plasma and tissue extracts that bound to a protein G affinity column demonstrated immunostaining for apo(a) and was in the size ran ge of IgG. Although one polyclonal anti-apo(a) provided by another lab oratory showed the same findings as our antibody, two other polyclonal anti-apo(a) failed to demonstrate immunostaining of human IgG, either on agarose electrophoresis or SDS-PAGE. We speculate that the Lp(a) i mmunogen used to prepare our anti-apo(a) may have undergone modest oxi dation, thus exposing epitopes not normally expressed on apo(a) in nat ive Lp(a). Either antibodies to these epitopes could be recognizing ap o(a) fragments, possibly released during oxidation, which are then cov alently bound to IgG, or oxidation of apo(a) creates epitopes on apo(a ) that are homologous with IgG, thereby leading to cross-reactivity wi th IgG. Such a scenario could be consistent with recently published st udies showing recognition of oxidized LDL by the Fc receptor on macrop hages.