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Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme–cofactor–substrate fIXa– fVIIIa–fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane-bound binary complexes of fIXa, fVIIIa, and fX. | iFEBS Journal Factor villa regulates substrate delivery to the intrinsic factor X-activating complex Mikhail A. Panteleev1 2 Natalya M. Ananyeva1 Nicholas J. Greco1 Fazoil I. Ataullakhanov2 3 4 and Evgueni L. Saenko1 1 Jerome H. Holland Laboratory for the BiomedicalSciences American Red Cross Rockville Maryland USA 2 Laboratory of PhysicalBiochemistry of Blood NationalResearch Center for Hematology Russian Academy of MedicalSciences Moscow Russia 3 Laboratory of Metabolic Modeling and Bioinformatics Institute of Theoreticaland ExperimentalBiophysics Moscow Russia 4 Faculty of Physics Moscow State University Russia Keywords blood coagulation factor VIIIa factor IXa factor X flow cytometry Correspondence M.A. Panteleev Laboratory of Physical Biochemistry of Blood National Research Center for Hematology Russian Academy of MedicalSciences Novozykovskii pr. 4a Moscow 125167 Russia Fax 7 095 212 8870 Tel 7 095 212 3522 E-mail mapanteleev@yandex.ru Website http physbio.hc.comcor.ru Present address Department of Biochemistry Molecular Biology University of Maryland Schoolof Medicine Baltimore USA tPresent address Department of Medicine Case Western Reserve University Schoolof Medicine Cleveland OH USA Portions of this work were presented at the 30th FEBS Congress-9th IUBMB Conference Budapest Hungary 2-7 July 2005 and published in abstract form in FEBS Journal 2005 272 Suppl. 1 405. MikhailA. Panteleev and Natalya M. Ananyeva contributed equally to this work. Received 8 August 2005 revised 19 October 2005 accepted 22 November 2005 doi 10.1111 j.1742-4658.2005.05070.x Activation of coagulation factor X fX by activated factors IX fIXa and VIII fVIIIa requires the assembly of the enzyme-cofactor-substrate fIXa-fVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry we explored formation of the intermediate membranebound binary complexes of fIXa fVIIIa and fX. Studies of the coordinate binding of coagulation factors to 0.8-pm phospholipid vesicles