title: The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C.
abstract: Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.
authors.affiliation: Department of Biochemistry, University of Limburg, Maastrict, The Netherlands.
authors.forename: H M
authors.initials: HM
authors.lastname: Bakker
authors.forename: G
authors.initials: G
authors.lastname: Tans
authors.forename: T
authors.initials: T
authors.lastname: Janssen-Claessen
authors.forename: M C
authors.initials: MC
authors.lastname: Thomassen
authors.forename: H C
authors.initials: HC
authors.lastname: Hemker
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
authors.forename: J
authors.initials: J
authors.lastname: Rosing
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D005165:Factor V / Q000378:metabolism; D015943:Factor Va / Q000037:antagonists & inhibitors*; D005170:Factor X / Q000378:metabolism; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010743:Phospholipids / Q000494:pharmacology*; D011486:Protein C / Q000378:metabolism*; D017293:Protein S; D011516:Prothrombin / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D008081:Liposomes; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D002118:Calcium