Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Mechanisms of action of recombinant activated factor VII in the context of tissue factor concentration and distribution Mikhail V. Ovanesov a,b,1 , Mikhail A. Panteleev a , Elena I. Sinauridze a , Dmitry A. Kireev a , Olga P. Plyushch a , Konstantin G. Kopylov a , Elena G. Lopatina a , Evgueni L. Saenko b and Fazoil I. Ataullakhanov a,c,d Supraphysiological concentrations of recombinant activated factor VII (rVIIa, NovoSeven) are used to control bleeding in hemophilia. Current experimental evidence suggests that rVIIa may increase thrombin generation via two pathways: one being tissue factor (TF)-dependent and another being activated platelet-dependent. Contribution of TF to the rVIIa action may justify different administration profiles of rVIIa. In the present study, thrombin and fibrin generation and spatial clot formation assays in platelet-free hemophilia A and normal plasma were used to investigate this contribution. By varying the concentration of TF and the way it becomes available to plasma, we obtained the following results. Activation of clotting with less than 5 pmol/l of TF facilitates thrombin and fibrin generation at low, but not at supraphysiological rVIIa concentrations. Activation with more than 13 pmol/l of TF saturates thrombin and fibrin generation kinetics, making it insensitive to rVIIa. rVIIa minimally modulates clot growth on the surface of TF-expressing fibroblasts. On the contrary, rVIIa produces spontaneous clot formation in nonflowing platelet-free plasma far away from fibroblasts via plasma lipid particles. Therefore, both the concentration and the distribution of TF determine relevance of a particular experimental system for the studies of rVIIa action. The results indicate that 300 – 1600 nmol/l (megadoses) of rVIIa may deliver coagulation outside of the TF-rich areas of blood vessel damage via the platelet-derived microparticles. Therefore, rate and extent of platelet-derived microparticles generation might be important with regard to rVIIa treatment safety. Blood Coagul Fibrinolysis 19:743– 755 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2008, 19:743–755 Keywords: factor VIIa, NovoSeven, spatial clot propagation, thrombin generation, tissue factor a National Research Center for Hematology, Moscow, Russia, b University of Maryland School of Medicine, Rockville, Maryland, USA, c Moscow State University and d Center for Theoretical Problems of Physical and Chemical Pharmacology, Moscow, Russia Correspondence to Mikhail V. Ovanesov, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA Tel: +1 240 731 0308; e-mail: ovanesov@gmail.com Correspondence to Mikhail A. Panteleev, National Research Center for Hematology, 4a Novyi Zykovskii pr., Moscow, 125167 Russia Tel: +7 495 612 3522; fax: +7 495 612 8870; e-mail: mapanteleev@yandex.ru Received 27 July 2007 Revised 25 September 2007 Accepted 28 September 2007 Introduction Recombinant activated factor VII (rVIIa or NovoSeven; Novo Nordisk, Copenhagen, Denmark) administered at high doses (90–120 mg/kg each 2–3 h, which is approxi- mately equivalent to a peak rVIIa concentration in blood of 50 nmol/l [1]), has been successfully used for the treatment and prevention of bleeding in hemophilia A and B patients with inhibitors [2–5]. In recent years, use of rVIIa was extended to a number of inherited and acquired bleeding disorders [6] but the main scope of rVIIa application remains to be the bleeding arrest in patients with fVIII or fIX deficiencies. Clinical trials demonstrated high efficacy of rVIIa treatment as well as its safety as confirmed by a very limited number of documented thrombotic complications [6]. However, there is presently an ongoing debate on the rVIIa mech- anism of action in hemophiliacs [7–9]. The enzyme fVIIa is virtually inactive and can activate fX only in the presence of a cofactor, either tissue factor (TF) or phospholipid surface [10,11]. Accordingly, there are two possible mechanisms explaining the effect of rVIIa on thrombin generation. The TF-independent mechanism [12,13] is based on the finding that rVIIa can bind to activated platelets and activate fX directly on their surface. The hypothesis was put forward that this action of the platelet-bound rVIIa mediates the predominant therapeutic mechanism of rVIIa in hemo- philia [10,14,15]. The alternative mechanism [16,17] suggests that hemostatic effect of rVIIa is explained by its action in a complex with TF. In addition to the Original article 743 Portions of this work were presented at the XIX Congress of the International Society on Thrombosis and Haemostasis, 12–18 July 2003, Birmingham, UK [Journal of Thrombosis and Haemostasis 2003; 1(Suppl 1): abstract P1109]. 1 Present address: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. 0957-5235 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI:10.1097/MBC.0b013e3283104093