title: Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces.
abstract: Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 10⁵. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²⁺. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²⁺ and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²⁺ concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.
authors.affiliation: *Laboratory of Biophysics, Condensed Matter Physics Department, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Tilen
authors.initials: T
authors.lastname: Koklic
authors.affiliation: †Department of Biochemistry and Biophysics & Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
authors.forename: Rima
authors.initials: R
authors.lastname: Chattopadhyay
authors.affiliation: †Department of Biochemistry and Biophysics & Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
authors.forename: Rinku
authors.initials: R
authors.lastname: Majumder
authors.affiliation: †Department of Biochemistry and Biophysics & Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, U.S.A.
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lentz
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism*; D020013:Calcium Signaling; D020134:Catalytic Domain; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D005165:Factor V / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010718:Phosphatidylserines / Q000378:metabolism; D015539:Platelet Activation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization
chemical_list: D010718:Phosphatidylserines; C022475:prothrombinase complex; D006639:Histidine; D005165:Factor V; D015951:Factor Xa