Abstract
BackgroundThe transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.ResultsExpression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1); H6DQMMLPWAVTLG4Y (XL2); H6WQHKIDLPYNGAG4Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.ConclusionsFusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.
Highlights
The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active a2-antiplasmin (a2AP) to fibrin
In order to address unmet clinical needs in the area of thrombosis, the pathological formation of clots within intact blood vessels, our laboratory previously investigated novel recombinant proteins comprised of portions of a2-antiplasmin fused to the N-terminus of human serum albumin [19]. a2AP is cross-linked to fibrin clots by activated factor XIII (FXIIIa), where it promotes clot stability by inhibiting the fibrinolytic enzyme plasmin [20,21]
While we demonstrated that a2AP(13-42)-human serum albumin (HSA) became a substrate of FXIIIa and competed for a2AP crosslinking to both small and macromolecular substrates, we could not demonstrate cross-linking to clots in vivo above background levels seen with recombinant HSA [19]
Summary
The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active a2-antiplasmin (a2AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising a2AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become incorporated into in vivo clots. Our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing a2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. Recombinant serum albumins provide an attractive scaffold for the delivery of attached therapeutic peptides or proteins [1,2]. Recombinant serum albumins provide an attractive scaffold for the delivery of attached therapeutic peptides or proteins [1,2] They are the most abundant proteins in the plasma of mammals, reaching concentrations of 4080 mg/mL (0.6-1.0 mM) in humans [3]. While we demonstrated that a2AP(13-42)-HSA became a substrate of FXIIIa and competed for a2AP crosslinking to both small and macromolecular substrates, we could not demonstrate cross-linking to clots in vivo above background levels seen with recombinant HSA [19]
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