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 lipoprotein(a) (Lp(a)) particle have been recently investigated by limited proteolysis [Huby, T., Doucet, C., Dieplinger, H., Chapman, J., & Thillet, 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 digested apo(a) isoform, was not linked to apo B100. We now describe the purification 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 sequential use of a heparin-Sepharose column and a lysine-Sepharose column. The C-ter domain of apo(a), which remained in disulfide linkage with apo B100 of Lp(a), was isolated as a lipoprotein particle by a combination of chromatographic steps on heparin-Sepharose and Q-Sepharose columns. This particle, termed ''mini-Lp(a)'', appeared homogeneous in 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-ter domain. Such cleavage occurred between residues 3532 and 3533, which 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 kringles 4(5) to 4(10), kringle V, and the protease domain. The binding properties of the purified N-ter domains and mini-Lp(a) were investigated on intact and on plasmin-modified fibrin and compared to those of Lp(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 saturation, in contrast to the N-ter domains which did not bind either to fibrin 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 finding 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 compared to Lp(a).