Platelet-Dependent Activity of GlycoPEGylated rFVIIa.

Introduction: Prophylaxis is currently not available for hemophilia patients who have developed inhibitors. rFVIIa (NovoSeven®) is approved for on-demand treatment of inhibitor patients; however, due to a half-life of only 2–4 hr it might not represent the optimal option for prophylactic treatment of an inhibitor patient. Consequently, an effective and safe prophylactic treatment option for the inhibitor patient represents a clear medical need and a long-acting rFVIIa molecule could fulfill this need. Pegylation is an established method for prolonging the circulation half life of proteins. Site specific pegylation of the N-glycan of rFVIIa can be achieved by the Neose GlycoPEGylation™ technology. Here we report on the characterization of the activity of two GlycoPEGylated rFVIIa variants using a thrombin generation assay employing activated platelets as well as whole blood coagulation potential as determined by thrombelastography.

Methods: PEG molecules of 10 kDa or 40 kDa are attached to the N-glycans of rFVIIa using GlycoPEGylation™ technology. Thrombin generation was evaluated under haemophilia A-like conditions in 1) platelet-rich plasma with a neutralizing anti-FVIII IgG included and 2) a reconstituted model system containing purified platelets and purified coagulation factors VII, IX, X, XI, II, V, ATIII, and TFPI at their plasma concentrations. In both assays, platelets were activated using a PAR-1 agonist peptide in combination with collagen-related peptide. Thrombelastography analysis was performed using citrate-stabilized human whole blood pre-incubated with neutralizing anti-FVIII IgG. Coagulation was initiated by addition of calcium and either kaolin or low amounts of TF (Innovin diluted 1:200,000). Concentrations of rFVIIa, rFVIIa-10K PEG, and rFVIIa-40K PEG were added to 0.005–500 nM.

Results: Compared to unmodified rFVIIa, the thrombin generation activity of rFVIIa-10K PEG and rFVIIa-40K PEG was reduced in both the reconstituted model system and in the plasma-based model system. In the reconstituted model system the rate of thrombin generation obtained with rFVIIa-10K PEG was 82 ± 7% of that obtained with rFVIIa, while the rate of thrombin generation obtained with rFVIIa-40K PEG was 65 ± 13% of that obtained with rFVIIa. In the plasma-based model system 100 nM of the two GlycoPEGylated rFVIIa variants gave similar thrombin generation curves as 25 nM unmodified rFVIIa. rFVIIa-10K PEG was slightly more active than rFVIIa-40K PEG. In thrombelastography analyses clot time (R) and clot-development (MTG) were improved in a concentration-dependent manner by adding rFVIIa. The concentration-response curves for rFVIIa-10K PEG and rFVIIa-40K PEG showed the same maximal effect as rFVIIa, however, higher EC50 values indicated a 3–6 fold lower potency of the two GlycoPEGylated rFVIIa variants in this assay.

Conclusions: The study demonstrates that GlycoPEGylation reduces the potency of rFVIIa in plasma and whole blood assays; however, the maximal effect on clot formation and strength are maintained for both derivatives. In vivo studies are needed to further characterize these molecules.