Enhanced Fibrin Clot Formation and Stability with a Superactive Analog of Factor VIIa in an In Vitro Model of Hemophilia.

Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrated its clinical efficacy in treating bleeding in hemophilia patients with inhibitors. Using in vitro assays, we and others have previously shown that high dose rFVIIa shortens the onset of fibrin clot formation and normalizes the fibrin structure and porosity of clots formed under hemophilic conditions (absence of factors VIII and/or IX (

In the current study, we have used a cell-based reconstituted model system of coagulation to compare the abilities of rFVIIa and one of the superactive analogues, NN1731, to modulate the onset and rate of fibrin clot formation, as well as the ability of these molecules to increase the stability of fibrin clots formed in the presence of a fibrinolytic challenge. The model system consists of tissue factor-bearing monocytes, purified pro- and anti-coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), freshly-isolated, unactivated platelets and fibrinogen. This system permits the inclusion of protein and cell concentrations at their physiologic levels. “Coagulation” is initiated by combining the components and following clot formation by optical density. “Hemophilia” is simulated by omitting factors IX and VIII. We observed that NN1731 shortened the clot time of hemophilic conditions to normal and did so at lower doses than were required of rFVIIa. Further, NN1731 increased the rate of clot formation to normal. To test the ability of clots to form in a fibrinolytic environment, we performed a Plasmin Challenge Assay, in which clot formation is initiated in the presence of a low concentration of plasmin. In this assay, clot formation is indicated as an increase in turbidity, and clot lysis is seen as a subsequent decrease in turbidity. There is no turbidity development (no clot formation) under hemophilic conditions. We observed that NN1731 increased the rate of clot formation and the length of time that fibrin was present under hemophilic conditions, and did so at lower concentrations than were required for rFVIIa. Moreover, in all of these assays, NN1731 normalized hemophilic conditions. Our results suggest that NN1731 promotes clot formation and stability to a greater degree than equal doses of rFVIIa and may possess clinical utility in patients that are refractory to current doses of rFVIIa.