title: Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII-treatment in the cynomolgus monkey.
abstract: BACKGROUND
  In cynomolgus monkeys, suprapharmacological doses of clotting recombinant factor XIII (rFXIII) cause a generalized coagulopathy, associated with formation of circulating high molecular weight protein complexes (HMEX). HMEX consist of plasma protein substrates cross-linked by FXIII transglutaminase activity.
  
  
  OBJECTIVE
  To characterize HMEX, with a view to develop safety biomarker assays.
  
  
  METHODS
  Cynomolgus monkeys received single i.v. injections with vehicle or rFXIII at 1, 3 and 10 mg kg(-1). Plasma HMEX were analyzed by sodium dodecylsulfate-polyacrylmide gel electrophoresis, silver staining, Western blotting and quantitative dissociation-enhanced lanthanide fluoroimmunoassay. Plasma FXIII antigen was analyzed by quantitative ELISA. Human HMEX were made in vitro, by spiking plasma with thrombin-activated rFXIII.
  
  
  RESULTS
  Maximal circulating HMEX levels were reached within 1 h of rFXIII treatment, and remained stable over 24 h. HMEX above 250 kDa contained fibrinogen alpha-chains and fibronectin. Fibrinogen gamma-chain was detected only in HMEX below 250 kDa. The total plasma concentration of HMEX was in the low microg mL(-1) range, distributed on less than 20 main species. Human and cynomolgus HMEX were similar. HMEX formation increased with rFXIII dose in a disproportionate manner, with 3-fold and fortyfold increases in HMEX exposure associated with rFXIII dose increments from 1 to 3 and 3 to 10 mg kg(-1), respectively.
  
  
  CONCLUSIONS
  The disproportionate HMEX formation parallels the steep toxicity dose response previously reported for rFXIII in cynomolgus monkeys, supporting a mechanistical role for HMEX in the generalized coagulopathy seen in rFXIII toxicity. Our findings support that HMEX constitute candidate (potential) safety biomarkers in rFXIII treatment.
authors.affiliation: Novo Nordisk A/S, Molecular Toxiology, Maalov, Denmark.
authors.forename: R
authors.initials: R
authors.lastname: Schaal-Jensen
authors.forename: B
authors.initials: B
authors.lastname: Kiehr
authors.forename: H T
authors.initials: HT
authors.lastname: Boesen
authors.forename: J S
authors.initials: JS
authors.lastname: Krabbe
authors.forename: C
authors.initials: C
authors.lastname: Sommer
authors.forename: H
authors.initials: H
authors.lastname: Jacobsen
authors.forename: M B
authors.initials: MB
authors.lastname: Oleksiewicz
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D001779:Blood Coagulation Factors / Q000737:chemistry*; D001798:Blood Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005176:Factor XIII / Q000737:chemistry* / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D008252:Macaca fascicularis; D008297:Male; D008954:Models, Biological; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D001779:Blood Coagulation Factors; D001798:Blood Proteins; D011994:Recombinant Proteins; D005176:Factor XIII