Thrombin Generation Assay: Use in Monitoring Treatment of Patients with Coagulation Factor Inhibitors.
Authors: McCormick Aine N. , Turecek Peter L. , Varadi Katalin , Savidge Geoffrey F. 
Published: April 2020

Abstract
Treatment of severe Haemophilia A relies upon the infusion of plasma derived or recombinant Factor VIII concentrates. The development of antibodies to these products is approximately 25–30% in severe cases and remains a major clinical challenge to management. Treatment in inhibitor cases with high antibody levels is based on the effect of products that bypass Factor VIII in the cascade and activate the common pathway either directly or through the extrinsic pathway. These include recombinant Factor VIIa and Factor VIII inhibitor bypassing activity (FEIBA®). At present, the use and dosage of bypassing products is severely constrained by the current inability to measure directly their beneficial effect using a standardised assay, and clinical management rests exclusively upon clinical assessment.
The thrombin generation assay (TGA) provides a convenient and reproducible method for quantifying thrombin produced following activation of the cascade, and is measured by means of monitoring a fluorescent residue that arises following cleavage of an artificial thrombin substrate. Through sequential measurements quantifiable kinetic data may be collated and assessed. We have used the TGA to assess thrombin generation in inhibitor patients using samples taken at specific time intervals from patients after administration of FEIBA (n=4) or rFVIIa (n=3) following informed consent. In a number of experiments, platelets from the patient’s pre-infusion platelet rich plasma (PRP) were added. Peakthrombin (nM), peaktime (min) and maximum initial rates of thrombin generation (nM/min) from each sample were quantified, and the maximum initial rates of thrombin generation were further processed using PKAnalyst® Pharamacokinetic Data Analysis software program to define one and two PK compartment elimination models following bolus injection.
Thrombin generation parameters were found to persist at levels greater than those measured for the pre infusion samples for periods beyond 6 hours post FEIBA and for 2 hours post rFVIIa, prior to reinfusion of rFVIIa. In PK evaluation studies FEIBA was found to have a relative thrombin generation T1/2 of 1–3.7 hr using the one compartment model, while rFVIIa was found to have a relative thrombin generation T1/2 of 0–2.8 hr following a single infusion. A second infusion of rFVIIa gave a relative thrombin generation T1/2 of 1.2–1.8 hr suggesting a cumulative effect. The addition of platelets from pre-infusion PRP to the assay enhanced thrombin generation by approximately 30% in all samples studied.
The TGA was also used to quantify the thrombin generation produced by FEIBA and rFVIIa in samples obtained at specific time intervals post treatment, using a pre infusion sample spiked with a range of FEIBA/rFVIIa concentrations to generate a standard curve. This standard curve was then used to estimate the relative FEIBA/rFVIIa concentrations in the post infusion samples with and without the addition of platelets.
We consider that the use of the TGA in this clinical setting demonstrates its value as a monitoring method highly effective for providing a rational approach to dosage adjustments of FEIBA/rFVIIa in the treatment of patients with FVIII inhibitors. This valuable analytical means for monitoring FVIII bypassing treatment is of considerable relevance in relation to surgery and the management of acute bleeds in this patient category.