Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles.
Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.
Hogg
P J
PJ
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Stenflo
J
J
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism*
D001798:Blood Proteins; C026080:plasma protein Z; D013917:Thrombin; D000581:Amidohydrolases