Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

BackgroundThe plasma protein α2-antiplasmin (α2AP) 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 α2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.ResultsFusion protein α2AP(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 α2AP moieties were inefficiently secreted and/or degraded. The α2AP(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 α2AP. Excess α2AP(13-42)-HSA competed with α2AP for cross-linking to chemical lysine donors more effectively than a synthetic α2AP(13-42) peptide, and reduced the α2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α2AP was found in in vivo clots in rabbits to a greater extent than α2AP(13-42), however.ConclusionIn this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α2-antiplasmin predicted to act in a clot-localized manner.