CHARACTERIZATION OF THE N-TERMINAL AND C-TERMINAL DOMAINS OF HUMAN APOLIPOPROTEIN(A) - RELEVANCE TO FIBRIN BINDING

The structural domains of human apolipoprotein(a) (apo(a)) in the lipo protein(a) (Lp(a)) particle have been recently investigated by limited proteolysis [Huby, T., Doucet, C., Dieplinger, H., Chapman, J., & Thi llet, J. (1994) Biochemistry 33, 3335-3341]. We have shown that apo(a) can be cleaved into two structural domains: one was of constant size (170 kDa) and corresponded to the C-terminal (C-ter) domain of apo(a). This domain was linked by a disulfide bond to apo B100. By contrast, the N-terminal (N-ter) domain, whose size varied according to the dige sted apo(a) isoform, was not linked to apo B100. We now describe the p urification of these apo(a) domains and their interaction with fibrin surfaces in an in vitro binding assay. The N-ter domain of apo(a) was purified as a soluble protein in a two-step procedure which involved s equential use of a heparin-Sepharose column and a lysine-Sepharose col umn. The C-ter domain of apo(a), which remained in disulfide linkage w ith apo B100 of Lp(a), was isolated as a lipoprotein particle by a com bination of chromatographic steps on heparin-Sepharose and Q-Sepharose columns. This particle, termed ''mini-Lp(a)'', appeared homogeneous i n nondenaturing polyacrylamide gels and exhibited a particle size (285 Angstrom) which was intermediate between that of Lp(a) (300 Angstrom) and LDL (265 Angstrom). The cleavage site between the respective apo( a) domains was determined by N-terminal sequencing of the purified C-t er domain. Such cleavage occurred between residues 3532 and 3533, whic h are located in the interkringle region between apo(a) kringles 4(4) and 4(5). Consequently, the C-ter domain of apo(a) was composed of kri ngles 4(5) to 4(10), kringle V, and the protease domain. The binding p roperties of the purified N-ter domains and mini-Lp(a) were investigat ed on intact and on plasmin-modified fibrin and compared to those of L p(a). We demonstrated that the C-ter domain, in the form of mini-Lp(a) , binds to fibrin in a lysine-specific manner that approached saturati on, in contrast to the N-ter domains which did not bind either to fibr in or to plasmin-degraded fibrin. The apparent K-d for the mini-Lp(a) (380 +/- 30 nM) was slightly different from that of Lp(a) (150 +/- 15 nM) in binding to either plasmin-degraded or intact fibrin. This findi ng indicates that the C-ter domain of apo(a) mediates the interaction of Lp(a) with fibrin. On intact fibrin, the B-max values for Lp(a) and for mini-Lp(a) were 8.5 and 25 fmol/well, respectively; these values increased, upon plasmin digestion of fibrin, to 25 and 100 fmol/well, respectively. The increased number of accessible binding sites in the case of mini Lp(a) may result from a decreased steric hindrance as com pared to Lp(a).