Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII-treatment in the cynomolgus monkey.
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.
Schaal-Jensen
R
R
Novo Nordisk A/S, Molecular Toxiology, Maalov, Denmark.
Kiehr
B
B
Boesen
H T
HT
Krabbe
J S
JS
Sommer
C
C
Jacobsen
H
H
Oleksiewicz
M B
MB
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*
D001779:Blood Coagulation Factors; D001798:Blood Proteins; D011994:Recombinant Proteins; D005176:Factor XIII