title: A dimeric form of prothrombin on membrane surfaces.
abstract: Blood coagulation requires the conversion of zymogens to active enzymes. These reactions are facilitated by Ca2+-dependent protein binding to membrane surfaces containing anionic phospholipids. Here it is shown that only in the presence of both Ca2+ and phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine can a prothrombin dimer be chemically cross-linked. A cross-linker containing evenly spaced reactive groups was prepared by activating the carboxy groups of a ten-residue glutamic acid peptide and allowed to react with physiological concentrations of prothrombin. When Ca2+ and anionic phospholipids were both present during exposure to the cross-linker, it was found that more than 50% of the prothrombin was trapped as a chemically defined dimer with reaction times of the order of 1 min. The dimer yield remained high even when reactions were performed at high phospholipid-to-protein ratios at protein concentrations an order of magnitude less than physiological. Amino acid sequencing of a CNBr peptide produced from the purified dimer localized the cross-link to residues Lys341 and Lys427 of prothrombin. The specificity and high yield under mild conditions of the cross-linking suggest that dimeric membrane bound prothrombin might be a physiologically relevant substrate for the formation of thrombin. Prothrombinase converts this modified protein to an enzyme that catalyses the hydrolysis of a thrombin chromogenic substrate as efficiently as thrombin and is inhibited by a thrombin active-site directed inhibitor, but is a thrombin dimer. The thrombin dimer has impaired activity compared with thrombin with respect to physiological functions requiring binding to exosite I. A model based on the known structure of thrombin is presented that can account for the prothrombin dimer and the properties of the dimeric thrombin formed from it.
authors.affiliation: Department of Biochemistry, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. panderso@uottawa.ca
authors.forename: P J
authors.initials: PJ
authors.lastname: Anderson
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008566:Membranes / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D012756:Sheep; D013499:Surface Properties; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D011516:Prothrombin; D013925:Thromboplastin