A kinetic assay to determine prothrombin binding to membranes.
Activation of prothrombin by multisquamase, the prothrombin activator from the venom of Echis multisquamatus (Central Asian sand viper), is inhibited by membranes containing negatively charged anionic phospholipids. This inhibition appears to be due to the fact that the venom activator cannot activate membrane-bound prothrombin. Initial steady state rates of prothrombin activation by multisquamase in the presence of phospholipids appeared to depend on the fraction unbound prothrombin only and this phenomenon was used to quantitate binding of prothrombin to membranes of varying phospholipid composition. In this method, the initial rate of prothrombin activation by multisquamase is measured in the absence (total prothrombin) and in the presence of a procoagulant surface (rate depending only on free prothrombin) and from the difference in activation rates the amount of membrane-bound prothrombin is calculated. The validity of the method was established by determination of the binding parameters for prothrombin binding to 100 microM phospholipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. The binding parameters obtained were Kd=0.84 microM and n=0.021 micromoles prothrombin bound per micromole phospholipid which is in agreement with literature. Due to the nature of the measurement the method is especially suitable to quantitate binding of prothrombin at concentrations as low as 5 nM prothrombin.
Govers-Riemslag
J W
JW
Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands. j.govers@bioch.unimaas.nl
Johnsen
L
L
Petrovan
R J
RJ
Rosing
J
J
Tans
G
G
D000818:Animals; D000838:Anions / Q000494:pharmacology; D001780:Blood Coagulation Tests / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D015943:Factor Va / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008567:Membranes, Artificial; D008666:Metalloendopeptidases / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism*; D014757:Viper Venoms / Q000494:pharmacology
D000838:Anions; D008567:Membranes, Artificial; D010743:Phospholipids; D014757:Viper Venoms; D015943:Factor Va; D011516:Prothrombin; D008666:Metalloendopeptidases; C103417:ecamulin