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Updated: Jul 13, 2026
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Characterization of high molecular weight plasma protein complexes induced by clotting factor rFXIII-treatment in the
R Schaal-Jensen1, B Kiehr, H T Boesen
1Novo Nordisk A/S, Molecular Toxiology, Maalov, Denmark.
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.
