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.