Monitoring Recombinant FVIIa in Hemophilic and Normal Plasma Using a Thrombin Generation Assay

• Published in: Blood 2011-11, Vol.118 (21), p.1207-1207
• Main Authors: Loubele, Sarah T.B.G., Spronk, Henri M.H., van Oerle, Rene, ten Cate, Hugo, Giesen, Peter L.A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V118.21.1207.1207

Abstract 1207 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. 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. 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 m