Platelet-dependent thrombin generation assay for monitoring the efficacy of recombinant Factor VIIa.
Although the administration of recombinant coagulation factor VIIa (rFVIIa) is a well established treatment in haemophilia with inhibitory antibodies, monitoring the therapeutic efficacy is still a problem. This is because the complete haemostatic effect in vivo depends on negatively charged surfaces provided by exposed lipids from activated platelets which are not present in standard clinical assays using plasma. The thrombin generation assay, however, measures the endogenous thrombin potential (ETP) in platelet-rich plasma (PRP) and this assay might be useful for monitoring the haemostatic response to rFVIIa. We characterized the in vitro concentration-response relationship of rFVIIa and the thrombin generation parameters ETP, PEAK and TIME TO PEAK using platelet-rich plasma (PRP) and rFVIIa at concentrations between one and five times the therapeutic dose. We also studied the effect of inhibiting tissue-factor and the intrinsic coagulation pathway using excess TF-neutralizing antibodies and corn trypsin inhibitor (CTI), respectively. There was a sigmoid relationship between ETP and PEAK and the dose of rFVIIa. Increasing rFVIIa concentrations between 100 and 500 U/ml resulted in a progressive increase in ETP, whereas supratherapeutical concentrations led to a plateau phase. The plateau phase differed between patients, suggesting a biological variation in the maximum ETP. Neutralization of plasma TF with TF-Ab partially decreased FVIIa efficacy. The inhibitory effect of CTI on rFVIIa-induced thrombin generation via the intrinsic pathway was negligible. The thrombin generation assay using PRP is a useful test for determining the sufficient efficacy of rFVIIa in blood. Once a plateau level is reached, higher doses of rFVIIa have no additional effect on haemostatic efficacy. The variation in plateau levels between subjects indicates that there are inter-individual differences in the level of thrombin activity that can be generated. High doses of rFVIIa are effective even in the absence of TF.
Wegert
W
W
Institute for Clinical Pharmacology, Pharmazentrum Frankfurt, University Hospital Frankfurt am Main, Germany.
Harder
S
S
Bassus
S
S
Kirchmaier
C M
CM
D000328:Adult; D001780:Blood Coagulation Tests / Q000379:methods; D001792:Blood Platelets / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D016903:Drug Monitoring / Q000379:methods*; D005167:Factor VII / Q000494:pharmacology*; D015942:Factor VIIa; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D008297:Male; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000494:pharmacology*; D013917:Thrombin / Q000096:biosynthesis*; D013925:Thromboplastin
D010940:Plant Proteins; D011994:Recombinant Proteins; C052160:trypsin inhibitor, Zea mays; D005167:Factor VII; D013925:Thromboplastin; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin