Abstract
Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. We have shown that high concentrations of rFVIIa improve parameters of thrombin generation and fibrin clot formation and stability in an in vitro model of hemophilia (Wolberg et al. 2005 Brit J Haem 131:645–55). Recently, new rFVIIa analogs with increased procoagulant activity have been described. One of these analogs, rFVIIa with mutations V158D/E296V/M298Q (NN1731), exhibits increased activity in in vitro and in vivo models. In the present study, we compared the abilities of rFVIIa and NN1731 to specifically modulate factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability in an in vitro model of hemophilia. RFVIIa and NN1731 similarly increased factor X activation on tissue factor−bearing monocytes; however, NN1731 exhibited 30−fold higher factor Xa generation rates on activated platelets than similar concentrations of rFVIIa. We employed a complete, cell−based, reconstituted model of coagulation to compare the activities of rFVIIa and NN1731 in hemophilic conditions. The model system consists of tissue−factor bearing monocytes, purified pro− and anti−coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), and freshly−isolated, unactivated platelets. Fibrinogen is included in certain wells to examine clot formation. “Hemophilia” is simulated by omitting factors VIII and IX. In this assay, NN1731 produced 4 – 10−fold higher maximal thrombin generation rates than equal concentrations of rFVIIa. In contrast to even very high concentrations of rFVIIa (up to 500 nM), NN1731 was able to normalize the rate of thrombin generation. NN1731 did not directly activate platelets and thrombin generation was not detected prior to platelet activation, suggesting that the presence of NN1731 would not lead to thrombin generation in the absence of injury. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at lower concentrations than were required of rFVIIa. In a plasmin challenge assay, in which clot formation is initiated in the presence of plasmin, both rFVIIa and NN1731 increased fibrin formation and the length of time that fibrin was present in the absence of factors IX and VIII; however, NN1731 was effective at lower concentrations than were required of rFVIIa. Our results indicate that NN1731 increases factor Xa and thrombin generation and promotes clot formation and stability to a greater degree than equal concentrations of rFVIIa. For these reasons, NN1731 may be especially efficacious in situations in which rapid formation of a firm fibrin clot with increased resistance to fibrinolysis is necessary to achieve hemostasis.
Disclosures: Three of the authors (EP, ME, UH) work for a company (Novo Nordisk) whose product was studied in this work.; Two of the authors (ASW, GAA) have received research funding (Novo Nordisk) from a company whose product was studied in this work.; One of the authors (ASW) received honoraria for a presentation at a meeting sponsored by a company (Novo Nordisk) whose product was studied in this work.
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