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Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylp...

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Autores principales: Smith, Emma, Vekaria, Rina, Brown, Katherine A., Longstaff, Colin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771376/
https://www.ncbi.nlm.nih.gov/pubmed/23812842
http://dx.doi.org/10.1007/s11010-013-1735-2
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author Smith, Emma
Vekaria, Rina
Brown, Katherine A.
Longstaff, Colin
author_facet Smith, Emma
Vekaria, Rina
Brown, Katherine A.
Longstaff, Colin
author_sort Smith, Emma
collection PubMed
description A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K (D) = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K (D) values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K (D) of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K (D) in the 10(−8) M range and (ii) a kinetically rapid, low-affinity interaction with K (D) in the 10(−6 )M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K (D) values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.
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spelling pubmed-37713762013-09-13 Kinetic regulation of the binding of prothrombin to phospholipid membranes Smith, Emma Vekaria, Rina Brown, Katherine A. Longstaff, Colin Mol Cell Biochem Article A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K (D) = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K (D) values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K (D) of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K (D) in the 10(−8) M range and (ii) a kinetically rapid, low-affinity interaction with K (D) in the 10(−6 )M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K (D) values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface. Springer US 2013-06-28 2013 /pmc/articles/PMC3771376/ /pubmed/23812842 http://dx.doi.org/10.1007/s11010-013-1735-2 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Article
Smith, Emma
Vekaria, Rina
Brown, Katherine A.
Longstaff, Colin
Kinetic regulation of the binding of prothrombin to phospholipid membranes
title Kinetic regulation of the binding of prothrombin to phospholipid membranes
title_full Kinetic regulation of the binding of prothrombin to phospholipid membranes
title_fullStr Kinetic regulation of the binding of prothrombin to phospholipid membranes
title_full_unstemmed Kinetic regulation of the binding of prothrombin to phospholipid membranes
title_short Kinetic regulation of the binding of prothrombin to phospholipid membranes
title_sort kinetic regulation of the binding of prothrombin to phospholipid membranes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771376/
https://www.ncbi.nlm.nih.gov/pubmed/23812842
http://dx.doi.org/10.1007/s11010-013-1735-2
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