A novel inhibitor of blood coagulation has been isolated from the intima of bovine aorta. The inhibitor, vascular anticoagulant (VAC), has been purified to an active fraction that contains two Coomassie-blue-staining bands (Mr = 34000 and M , = 32000, as judged by sodium dodecyl sulfate/polyacrylamide electrophoresis). Both bands are single-chain proteins, having no glycoprotein features. Furthermore, they do not contain any detectable 4-carboxyglutamic acid residues. Both proteins have an identical isoelectric pH of approximately 4.5. VAC binds in the presence of calcium ions to a bilayer consisting of 20% dioleoylglycerophosphoserine and 80% dioleoylglycerophosphocholine with a Kd = 6 nM. The binding is dependent on the calcium concentration: halfsaturation of binding occurs at a calcium concentration of 0.8 mM. The binding is completely reversible with EDTA. Furthermore the phospholipid/VAC ratio at saturation was n = 112 and n = 32 mol/mol for 0.5 mM Ca2+ and 2 mM Ca2+, respectively. Binding does not occur between VAC and pure dioleoylglycerophosphocholine. In a system with purified coagulation factors VAC inhibits the activation of prothrombin by factor X, and calcium only in the presence of negatively charged phospholipids. VAC decreases the V,,, and increases the K, of the factor-X,-catalyzed prothrombin activation. Based on these results, we conclude that we have purified from bovine aortic intima an anticoagulant protein, which exerts its activity through a calcium-dependent binding to negatively charged phospholipids, and thus interferes with the assembly of prothrombinase on the phospholipid surface.The enzymes active in the process of blood coagulation belong to the class of serine proteases, and circulate in the blood as nonenzymatic precursors, the zymogens [l -31. The activation of such a zymogen proceeds in a proteolytic process in which an already activated coagulation factor cleaves a specific bond of its substrate, a zymogen [4]. The rate of this reaction is extremely low, but can be greatly enhanced by the presence of certain nonenzymatic cofactors, which can be divided into protein cofactors and negatively charged surfaces. The factor-X,-catalyzed activation of prothrombin, for Correspondence to C. P. M. Reutelingsperger, Laboratorium voor Biochemie, Rijksuniversiteit Limburg, Postbus 616, NL-6200-MD Maastricht, The Netherlands Abbreviations. MPT test, modified prothombin time test; PhCOIle-Glu-Pip-Gly-Arg-NH-Np . HCI (S2337), N-benzoyl-~-isoleucyI-~-glutamyl-L-piperidyl-glycyl-L-arginine p-nitroanilide hydrochloride; DPhe-Pip-Arg-NH-Np . 2 HCI (S2238), ~-phenylakanyl-L-pipecolyI-L-arginine p-nitroanilide dihydrochloride; NaCl/Tris/HCl, 100 mM NaCI, 50 mM Tris/HCI, pH 7.5; NaCl/Tris/HCi/EDTA, NaCI/Tris/ HCI with 2 mM EDTA; NaCl/Tris/HCl/albumin, NaCl/Tris/HCl with 0.5 mg/ml human serum albumin, pH 7.9; RVV-X, purified factor X activator from Russell's viper venom; Gla, 4-carboxyglutamic acid; Ole2Gro-P-Cho, 1,2-dioleoyl-sn-glycero-3-phosphocholine; Ole2Gro-P-Ser, 1,2-dioleoyl-sn-glycero-3-ph...
