Addition of a sequence from α(2)-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

BACKGROUND: The plasma protein α(2)-antiplasmin (α(2)AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of α(2)-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein. RESULTS: Fusion protein α(2)AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer α(2)AP moieties were inefficiently secreted and/or degraded. The α(2)AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native α(2)AP. Excess α(2)AP(13-42)-HSA competed with α(2)AP for cross-linking to chemical lysine donors more effectively than a synthetic α(2)AP(13-42) peptide, and reduced the α(2)AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α(2)AP was found in in vivo clots in rabbits to a greater extent than α(2)AP(13-42), however. CONCLUSION: In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α(2)AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α(2)-antiplasmin predicted to act in a clot-localized manner.