Monitoring Recombinant FVIIa in Hemophilic and Normal Plasma Using a Thrombin Generation Assay
Authors: Loubele Sarah T.B.G. , Spronk Henri M.H. , van Oerle Rene , ten Cate Hugo , Giesen Peter L.A. 
Published: October 2019

Abstract
Abstract 1207

Background:
Although the recombinant factor VIIa (rFVIIa) has been registered for use in hemophilia patients with inhibitors, there is still no method to monitor the effects of rFVIIa in restoring the coagulation balance in plasma. Hence, information is lacking about the individual optimal dose needed to normalize thrombin generation.

Methods:
The calibrated automated thrombogram (CAT) method was modified to increase sensitivity for rFVIIa addition to plasma at concentrations of 0, 2.5, 5, 10, 20, 40 and 80 nM, which covers the expected plasma concentration of 26 nM reached after standard administration. Thrombin generation was triggered using combinations of TF concentrations between 0 and 4 pM, and phospholipids concentrations between 0 and 4 μM. Endogenous thrombin potential (ETP), peak height, and velocity index were calculated in platelet poor plasmas (PPP) of different donors. All blood was collected in citrated tubes containing corn trypsin inhibitor (CTI) to minimize any contact activation.

Results:
The optimal conditions for discriminating rFVIIa (0–80 nM) in the CAT assay were determined in PPP: 0 or 0.25 pM TF with 4 μM of phospholipids. Also at higher TF concentrations, the CAT method was able to detect varying rFVIIa concentrations. The optimal concentration of phospholipids was 4 μM for all TF concentrations. In plasma of 6 healthy volunteers, thrombin generation triggered with 0.25 pM TF dose dependently increased using varying rFVIIa concentrations between 0 and 80 nM (Figure 1, left panel). The mean values for ETP, peak height and velocity index are depicted in Table 1. On average, addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in a 146, 156, 161, 174, 206, and 285 % of the peak height compared to 0 nM rFVIIa, which was set at 100 %. At 4 pM TF the maximum ETP, peak height, and velocity index were reached at concentrations less than 20 nM rFVIIa for all donors. The mean values are depicted in Table 2. Surprisingly, at 80 nM rFVIIa, thrombin generation was decreased compared to lower rFVIIa concentrations (Figure 1, right panel). Addition of 2.5, 5, 10, 20, 40 or 80 nM of rFVIIa resulted in 107, 109, 107, 103, 100, or 94 % of the peak height without addition of rFVIIa (0 nM set at 100 %). In FVIII deficient patient plasma (PPP), this effect was also present and even more pronounced. Here, a dose dependent effect of rFVIIa addition was visible at low (0 or 0.25 pM) TF trigger, whereas at 4 pM TF trigger ETP, peak height and velocity index were maximal in the presence of 10 nM rFVIIa. Overall, the peak height was 136, 142, 126, 102, 94, and 81 % upon addition of 5, 10, 20, 30, 40, or 80 nM rFVIIa respectively compared to 0 nM rFVIIa (set to 100 %).

Discussion:
In hemophilic as well as normal plasma, the addition of rFVIIa dose dependently altered thrombin generation triggered with a low TF trigger (0 or 0.25 pM). At a higher trigger of 4 pM TF, maximal thrombin generation was obtained at rFVIIa concentrations of less then 20 nM. Remarkably, thrombin generation was attenuated in the presence of 80 nM rFVIIa. This paradox may be explained by assuming that the endogenously activated VIIa is more active than the rVIIa that was added. At higher rVIIa dosages the fraction of TF occupied by endogenous VIIa will decrease resulting in less active TF:VIIa complexes. This effect will be more pronounced when FXa formation is dependent on TF:FVIIa alone without the involvement of the tenase complex, which shows from our analysis in hemophilic plasma. Overall, these data suggest that the assay is most sensitive to added rVIIa when the contribution to Xa formation of TF:VIIa complex is small compared to that of rVIIa alone, i.e., in conditions where there is no or very little TF present.

Disclosures:
Giesen: Thrombinoscope bv: Employment.