Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

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.

P-Selectin mediates targeting of a self-assembling phototherapeutic nanovehicle enclosing dipyridamole for managing thromboses

“…As shown in Fig. 3 c, FU and DIP decreased the rate of fibrin-clot formation, according to the fibrin clot absorbance at 340 nm [ 48 ]. The lower absorbance of the FU- and DIP-containing samples in a dose-dependent manner signifies that FU and DIP had inhibitory effects against the formation of fibrin clots.…”

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…Mice were anesthetized with 1.5% isoflurane, the vessel exposed, and recombinant protein (12.7 nmol KPI (90 µg/mouse) or KPIHSA (940 µg/mouse, respectively) or saline vehicle was injected intravenously 2 or 60 minutes prior to application of the FeCl 3 -saturated filter paper to the vessel, for 3 minutes. Thirty minutes after removal of the filter paper, clots were excised and g-counted to determine their content of 125 I-fibrin and ( 125 I-fibrinogen ( 125 I-fibrin(ogen)) by g-counting as described above [31, 32]. …”