title: Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa.
abstract: BACKGROUND
  The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α(2)-antiplasmin (α(2)AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α(2)AP 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 α(2)AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.
  
  
  RESULTS
  Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H(6)NQEQVSPLTLLAG(4)Y (designated XL1); H(6)DQMMLPWAVTLG(4)Y (XL2); H(6)WQHKIDLPYNGAG(4)Y (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 α(2)AP(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 α(2)AP 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.
  
  
  CONCLUSIONS
  Fusion 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.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada. sheffiel@mcmaster.ca
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
authors.forename: Louise J
authors.initials: LJ
authors.lastname: Eltringham-Smith
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000704:Analysis of Variance; D000818:Animals; D000933:Antifibrinolytic Agents / Q000378:metabolism*; D001777:Blood Coagulation / Q000502:physiology*; D002712:Chlorides; D026122:Factor XIIIa / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D000933:Antifibrinolytic Agents; D002712:Chlorides; D005290:Ferric Compounds; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D005337:Fibrin; D026122:Factor XIIIa; C024555:ferric chloride