title: Prothrombin activation on dioleoylphosphatidylcholine membranes.
abstract: Factor-Xa-catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers-Riemslag, J.W.P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967-7974]. In the present study, we have performed a detailed analysis of the prothrombin-converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating anionic phospholipids. This is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2-GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100-fold. At ionic strength (I) 0.06, prothrombin activation on 100 microM (Ole)2-GroPCho was characterized by a Km for prothrombin of 2 microM, a Vmax of 3020 IIa min-1.Xa-1 and a Kd for factor XaVa complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of gamma-carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and an anionic lipid-containing membranes.
authors.affiliation: Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
authors.forename: J W
authors.initials: JW
authors.lastname: Govers-Riemslag
authors.forename: M P
authors.initials: MP
authors.lastname: Janssen
authors.forename: R F
authors.initials: RF
authors.lastname: Zwaal
authors.forename: J
authors.initials: J
authors.lastname: Rosing
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004563:Electrochemistry; D015951:Factor Xa / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008567:Membranes, Artificial; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D015055:1-Carboxyglutamic Acid; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C042511:1,2-dioleoylphosphatidylserine; D011516:Prothrombin; D015951:Factor Xa; C017251:1,2-oleoylphosphatidylcholine; D002118:Calcium