title: Phospholipid regulates the activation of factor X by tissue factor/factor VIIa (TF/VIIa) via substrate and product interactions.
abstract: Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of approximately 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.
authors.affiliation: Department of Medicine, Mount Sinai School of Medicine, Box 1269, One Gustave Levy Place, New York, New York 10029, USA. James.Hathcock@mssm.edu
authors.forename: James J
authors.initials: JJ
authors.lastname: Hathcock
authors.forename: Elena
authors.initials: E
authors.lastname: Rusinova
authors.forename: Rodney D
authors.initials: RD
authors.lastname: Gentry
authors.forename: Harry
authors.initials: H
authors.lastname: Andree
authors.forename: Yale
authors.initials: Y
authors.lastname: Nemerson
mesh_terms: D001667:Binding, Competitive / Q000502:physiology; D002384:Catalysis; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D017186:Titrimetry
chemical_list: D065427:Factor Xa Inhibitors; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; C042511:1,2-dioleoylphosphatidylserine; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa; C017251:1,2-oleoylphosphatidylcholine