Abstract
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.
Highlights
It is generally accepted that assembly of blood coagulation factor complexes and initiation of coagulation requires aMol Cell Biochem (2013) 382:193–201 phospholipid membrane surface [1, 2]
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
The precise structural details of the bound clotting factor optimised for coagulation are still debated and a complete understanding of coagulation factor–membrane binding has been further complicated by the wide range of results obtained for equilibrium constants published over many years in studies using a variety of techniques with model membranes
Summary
It is generally accepted that assembly of blood coagulation factor complexes and initiation of coagulation requires aMol Cell Biochem (2013) 382:193–201 phospholipid membrane surface [1, 2]. The mechanism of binding of coagulation factors to model phospholipid membranes, let alone heterogeneous cell membranes [4], is not completely understood [5]. Ions which both maintain a conformation with an exposed hydrophobic x-loop ready for membrane insertion, and bridge Gla domains with anionic phospholipid, primarily phosphatidylserine (PS), which is exposed on the external leaflet of cell membranes following damage. The precise structural details of the bound clotting factor optimised for coagulation are still debated and a complete understanding of coagulation factor–membrane binding has been further complicated by the wide range of results obtained for equilibrium constants published over many years in studies using a variety of techniques with model membranes. The importance of the kinetics of binding and enzyme activation has been highlighted in a number of studies (e.g. [9])
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