Measurement of factor IX activity in plasma-derived and recombinant concentrates: insights from thrombin generation and activation-based assays.
BACKGROUND
Hemophilia B, resulting from a deficiency of coagulation factor IX, is treated effectively with either recombinant FIX (r-FIX) or plasma-derived FIX (pd-FIX) concentrates, although differences in pharmacokinetics are observed. FIX is activated in vivo by both activated FXI (FXIa) and tissue factor (TF)-activated FVII (FVIIa); however, conventional activated partial thromboplastin time (APTT)-based assays assess only activation by FXIa.


OBJECTIVE
To examine the differences between pd-FIX and r-FIX concentrates with respect to their thrombogenicity and activation.


RESULTS
FIX ELISA was used to quantify antigenic FIX. Calibrated automated thrombography was performed to evaluate the effect of FIX on thrombin generation. FIXa was quantified by the cleavage of FIXa-specific chromogenic substrate. FIX activation was studied in a purified system.


RESULTS
We found that r-FIX had ~ 1.6-fold greater specific activity than pd-FIX. r-FIX generated a markedly higher thrombin peak than pd-FIX at an equivalent antigen level when coagulation was initiated by TF, but this was not seen in contact activation-triggered thrombin generation (TG). Interestingly, the amount of FIXa in r-FIX concentrate was 10 times higher than that in pd-FIX concentrate. In a purified system, the amount of r-FIXa generated by FXIa in the first 10 min of activation was 1.37-fold that of pd-FIXa, whereas no difference between the concentrates was observed when triggered by TF-FVIIa.


CONCLUSIONS
Clear differences were observed between pd-FIX and r-FIX concentrates, including the proportion of FIXa and the activation by FXIa. These may explain some of the discrepancies observed clinically, and suggest that the APTT may not reflect their resultant in vivo properties.
Centre for Haematology, Department of Medicine, Imperial College London, London, UK.
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Y
Yu
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Millar
D004797:Enzyme-Linked Immunosorbent Assay; D005164:Factor IX / Q000378:metabolism*; D002836:Hemophilia B / Q000097:blood; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D011994:Recombinant Proteins; D005164:Factor IX; D013917:Thrombin
DNA recombinant proteins; factor IX; hemophilia B; plasma; thrombin