Introduction Factor VIII (fVIII) is an essential component of the blood coagulation cascade, since its congenitally or acquired low activity in blood is associated with a tendency to severe bleed- ing, Haemophilia A (1). It is acknowledged that initiation of blood clotting is mediated by the extrinsic pathway in which a complex of tissue factor (TF) with activated factor VII generates small amounts of activated factor X (2-5). On the other hand, the intrinsic tenase complex consisting of membrane-associated activated fVIII (fVIIIa) and activated factor IX (fIXa) was shown to maintain the clotting process in vitro by generating activated factor X (fXa) approximately 50 times more efficiently (6-11). When clotting is initiated via the extrinsic pathway, there are two ways of fIX activation: (i) by the fVIIa:TF complex (6, 12, 13) or (ii) by fXI activated by thrombin (7, 14). At present, however, it remains unclear in what situations each of these pathways of intrinsic tenase formation is operative and what their relative contribution is. Analysis of accumulated experimental data and mathema- tical modeling of the spatial dynamics of blood coagulation (15, 16) led us to hypothesize that fVIII (or fIX) may be a limiting factor in clot formation if it proceeds in two spatially separated phases. The initiation phase takes place on the activa- 235 © 2003 Schattauer GmbH, Stuttgart Effect of factor VIII on tissue factor-initiated spatial clot growth Mikhail V. Ovanesov 1 , Elena G. Lopatina 2 , Evgueni L. Saenko 3, 4 , Natalya M. Ananyeva 3 , Ljudmila I. Ul’yanova 5 , Olga P. Plyushch 2 , Andrey A. Butilin 6 , Fazly I. Ataullakhanov 1, 6 1 Laboratory of Physical Biochemistry of Blood and 2 Haemophilia Center,Research Center for Hematology, Russian Academy of Medical Sciences, Moscow, Russia 3 Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland, USA 4 George Washington University School of Medicine,Washington, District of Columbia, USA 5 Department of Genetics and Histology, Institute of Immunology, Ministry of Health, Moscow, Russia 6 Moscow State University, Moscow, Russia Thromb Haemost 2003; 89: 235 – 42 plasma volume. While the initiation phase did not differ in normal and haemophilic plasmas, the rate of clot growth in the elongation phase in haemophilic plasma constituted only 30% of that in normal plasma. Supplementation of haemophilic plasma with 0.05 U/ml fVIII restored the normal clot growth rate (44.9 ± 2.5 m/min) at high but not at low fibroblast density. Our results indicate that the functioning of the intrinsic tenase complex is critical for normal spatial clot growth. Keywords Factor VIII, Haemophilia A, spatial clot growth, fibroblast, time-lapse light-scattering videomicroscopy Summary Using time-lapse videomicroscopy, we studied the role of coagulation factor VIII (fVIII) in tissue factor-initiated spatial clot growth on fibroblast monolayers in a thin layer of non-stirred recalcified plasma from healthy donors or patients with severe Haemophilia A. Analysis of temporal evolution of light-scatter- ing profiles from a growing clot revealed existence of two phases in the clot growth-initiation phase in a narrow (0.2 mm) zone adjacent to activator surface and elongation phase in Correspondence to: Evgueni L. Saenko, Ph.D., Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA Tel: 301-738-0743, Fax 301-738-0499, E-mail: saenko@usa.redcross.org Received March 4, 2002 Accepted after resubmission November 7, 2002 Blood Coagulation, Fibrinolysis and Cellular Haemostasis