Why α2-Antiplasmin Must Be Converted to a Derivative Form.

Human α2-antiplasmin (α2AP), the primary inhibitor of plasmin, is secreted from liver to plasma as a 464-residue protein with Met as the N-terminus (Met-α2AP). As it circulates, antiplasmin-cleaving enzyme, a possible derivative of fibroblast activation protein, cleaves the Pro12-Asn13 bond of Met-α2AP to yield Asn-α2AP. The Asn-α2AP form becomes crosslinked to fibrin by activated factor XIII (FXIIIa) ~13X faster than Met-α2AP, and consequently, fibrin clot stability increases in direct proportion to the ratio of Asn-α2AP/Met-α2AP in plasma. FXIIIa, a transglutaminase catalyzes isopeptide bond crosslinking between a primary amine donor Lys in fibrin and an acceptor Gln in α2AP. Using recombinant Asn-α2AP mutants, we reported multiple FXIIIa-catalyzed fibrin crosslinking sites in Asn-α2AP: the previously reported Gln14 (Gln2 in Asn-α2AP) site and our discovery of three other Gln residues. This current study was performed to determine whether the major crosslinking site in Met-α2AP is also Gln14 or whether steric hindrance shifts the predominant crosslinking site to one of the other three identified Gln residues. Native Asn-α2AP and two different forms of Met-α2AP defined by an Arg6Trp polymorphism were labeled with 5-(biotinamido)pentylamine (BPA) by FXIIIa catalysis. To identify FXIIIa-reactive sites in Met-α2AP(Arg6), Met-α2AP(Trp6) and Asn-α2AP, each BPA-labeled α2AP was reduced, alkylated, and digested by trypsin. The BPA-labeled peptides were isolated from the tryptic digest mixture by avidin affinity chromatography, and further separated by reverse-phase HPLC. Using N-terminal sequence analysis of BPA-labeled peptides, we identified only the Gln14 residue as a FXIIIa-reactive site in all three forms of α2AP. Although four Gln sites in recombinant Asn-α2AP were involved in crosslinking, the Gln14 site appears to function as the only significant crosslinking site in the native form of the protein, whether either polymorphic form of precursive Met-α2AP, or the derivative, Asn-α2AP. The finding of Gln14 as the significant crosslinking site in native Asn-α2AP, as opposed to the four sites in recombinant Asn-α2AP, is likely related to differences in glycosylation, since the native protein is glycosylated while the recombinant protein is not. Using liquid chromatography/mass spectrometry analysis of tryptic digested BPA-labeled Asn-α2AP, another FXIIIa-reactive site (Gln33; Gln21 in Asn-α2AP) was identified, but the reactivity of this residue was ~200-fold less than the Gln14 residue. Finally, the BPA-labeling efficiencies of α2AP were determined by avidin blot analysis. BPA was incorporated into all three forms of α2AP by FXIIIa catalysis, but Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA far more slowly than Asn-α2AP, which is consistent with the difference in fibrin-crosslinking rates. Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA at a similar rate despite the Arg6Trp polymorphism being located close to the Gln14 used for crosslinking to fibrin. These data suggest that the Gln14 site is cryptic in Met-α2AP, being sheltered by the 12-residue N-terminal peptide, which when cleaved, yields Asn-α2AP that becomes crosslinked to fibrin more quickly to provide greater protection from plasmin.