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A kinetic assay to determine prothrombin binding to membranes
J W Govers-Riemslag1, L Johnsen, R J Petrovan
1Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands. j.govers@bioch.unimaas.nl
Abstract:
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.
Insights
Multisquamase, a snake venom activator, cannot bind to prothrombin on anionic phospholipid membranes. This study developed a method to quantify prothrombin binding to these membranes, crucial for understanding its activation.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Prothrombin activation is a critical step in the coagulation cascade.
- Snake venom activators, like multisquamase from Echis multisquamatus, offer insights into prothrombin activation mechanisms.
- Membrane phospholipids influence protein interactions and enzymatic activity.
Purpose of the Study:
- To investigate the mechanism of multisquamase inhibition by anionic phospholipid membranes.
- To develop and validate a novel method for quantifying prothrombin binding to membranes.
- To determine binding parameters of prothrombin to specific phospholipid compositions.
Main Methods:
- Measurement of initial prothrombin activation rates by multisquamase in the presence and absence of phospholipid vesicles.
- Calculation of membrane-bound prothrombin based on the difference in activation rates.
- Determination of binding constants (Kd) and stoichiometry (n) using phospholipid vesicles.
Main Results:
- Multisquamase-mediated prothrombin activation is inhibited by negatively charged phospholipid membranes.
- The developed method accurately quantifies prothrombin binding to membranes, even at nanomolar concentrations.
- Binding parameters (Kd=0.84 µM, n=0.021 µmol/µmol) for prothrombin to PS/PC vesicles were determined and validated.
Conclusions:
- Multisquamase cannot activate prothrombin when it is bound to anionic phospholipid membranes.
- The developed assay provides a sensitive and reliable method for studying prothrombin-membrane interactions.
- Understanding prothrombin binding is essential for elucidating its role in hemostasis and thrombosis.