Interaction of bovine coagulation factor X and its glutamic‐acid‐containing fragments with phospholipid membranes

Erb, Eva-Maria; Stenflo, Johan; Drakenberg, Torbjörn

doi:10.1046/j.1432-1033.2002.02981.x

Cited by 20 publications

(16 citation statements)

References 35 publications

(34 reference statements)

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“…A nonlinear least square fit of the experimental data to a decaying exponential model (the reaction following a pseudo‐first‐order kinetics) yielded kinetic association and dissociation parameters of k a = 0.017 ± 0.007 n m −1 ·min −1 and k Da = 1.50 ± 0.22 min −1 for fX alone ( n = 3). These values are close to those reported in a recent surface plasmon resonance study of fX binding to synthetic phospholipids membranes [6], although an earlier stopped‐flow light scattering study reported two‐orders of magnitude greater values for fXa [7]. In the presence of fVIII, these parameters were changed to k a = 0.026 ± 0.012 n m −1 ·min −1 and k Da = 0.55 ± 0.04 min −1 ( n = 3) indicating a 1.5‐fold increase of the association rate and a threefold decrease of the dissociation rate.…”

Section: Resultssupporting

confidence: 90%

“…Numerous studies have reported rates [6–8], equilibrium‐binding parameters [9–11], and mechanisms [12,13] for the individual binding of fIXa, fVIIIa, and fX to phospholipid membranes. Interaction of fIXa and fVIIIa within the fX‐activating complex and formation of the fIXa–fVIIIa complex have been also investigated by several groups [5,14–16], which identified interaction sites, association parameters, and contributions of different fVIIIa domains in the stimulation of the fIXa activity.…”

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Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex

et al. 2005

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Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXafVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membranebound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-lm phospholipid vesicles (25 ⁄ 75 phosphatidylserine ⁄ phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 ± 5000 (33 200 ± 14 100), 20 000 ± 4500, and 30 500 ± 1300 binding sites per vesicle with apparent K d values of 76 ± 23 (71 ± 5), 1510 ± 430, and 223 ± 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 ± 370, 20 ± 5 nm) and fX (12 630 ± 690, 14 ± 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 ± 67, 23 ± 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 ± 0.12 and an intrinsic K m value of 8.0 ± 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.Abbreviations BSA, bovine serum albumin; DiIC16(3), 1,1¢-dihexadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate; fVIII(a),

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Section: Resultssupporting

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Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex

et al. 2005

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“…47 Recently, the utility of BIAcore technique for evaluation of membrane-binding ability of vitamin K-dependent proteins has been demonstrated. 36,38 In the BIAcore, the K d for the QGNSEDY variant was found to be around 0.5 M, suggesting the equilibrium binding affinity to be considerably higher than that of WT protein C, which yielded a K d of 3.5 M. The corresponding values obtained in the light-scattering experiment were 2.2 M and 7.3 M, respectively. The K d values now obtained for WT protein C binding to phospholipid are in good agreement with results on record derived from light-scattering experiments.…”

Section: Discussionmentioning

confidence: 95%

“…The interaction between the protein C variants and immobilized phospholipid was also investigated using a BIAcore 2000 biosensor instrument (Biacore, Uppsala, Sweden) as described. 36 The phospholipid vesicles used for these experiments were prepared by extrusion. The phospholipid vesicles were then captured on the surface of L1 sensor chip.…”

Section: Protein Binding To Phospholipid Membranesmentioning

confidence: 99%

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Sun

Shen

Dahlbäck

2003

Blood

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Protein C is a member of the vitamin Kdependent protein family. Proteins in this family have similar ␥-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 ( IntroductionNegatively charged phospholipid membranes play an important role in blood coagulation, providing a suitable surface for the assembly of enzyme-cofactor complexes, which efficiently convert circulating zymogens to active enzymes. Phosphatidylserine (PS) is a key component in the negatively charged membrane that supports the clotting reactions. [1][2][3] In most cells, PS is normally mainly present in the inner leaflet of the cell membrane, but certain cellular processes such as platelet activation result in the exposure of PS on the cell surface. The vitamin K-dependent coagulation proteins in plasma bind to the surface of negatively charged phospholipid with a common mechanism involving the ␥-carboxyglutamic acid (Gla)-rich domains of these proteins. 4 Vitamin K is required in the posttranslational carboxylation of glutamic acid residues to Gla. 5 The Gla domains comprise approximately 45 amino acid residues, and they are structurally very similar in the different vitamin K-dependent proteins. 6 Despite the extensive structural similarity between different Gla domains, they bind negatively charged phospholipid with very different affinity, with dissociation constants (K d 's) ranging more than 1000-fold. 6 This variation in phospholipid binding ability is related to amino acid sequence differences at relatively few positions. This has made it possible to modulate the phospholipid binding abilities of this group of proteins by site-directed mutagenesis. [7][8][9] The nonvitamin K-dependent cofactors of blood coagulation also interact with the phospholipid membranes, certain proteins being membrane spanning, whereas others, such as factor V (FV) and factor VIII (FVIII), contain binding sites for negatively charged phospholipid membranes. 2,10 FV circulates as a procofactor to activated FV (FVa), which functions as a cofactor to activated FX (FXa) in the activation of prothrombin to thrombin. Activated FVIII (FVIIIa) serves a similar function in the FIXa-mediated activation of FX. 2,3 The protein C anticoagulant system, which includes the vitamin K-dependent proteins protein C and protein S, regulates the activities of FVa and FVIIIa by limited proteolysis (reviewed by Dahlbäck et al 11 and Esmon et al 12 ). Protein C is activated on the endothelial cell surface by the thrombin-thrombomodulin complex, and the serine protease activated protein C (APC) down-regulates coagulation by cleaving a limited number of peptide bonds in each of FVa and FVIIIa. Protein S serves as a cofactor to APC by increasing the affinity of APC for the membrane 13 and also by changing the distance of the activ...

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“…Interestingly, in this study [17], prothrombin-binding kinetics were complicated and not analysed fully, but the K D value with the DNA-tethered vesicles was estimated to be 68 nM, close to our tight-binding complex. Erb et al [24] have also studied Gla-dependent coagulation factor X (FX) binding to phospholipid membranes by SPR. Again a complex binding mechanism was observed with multiple dissociation phases, indicating multiple steps, and also some binding to pure PC membranes was reported.…”

Section: Discussionmentioning

confidence: 99%

Kinetic regulation of the binding of prothrombin to phospholipid membranes

Smith

Vekaria²,

Brown

et al. 2013

Mol Cell Biochem

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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 KD = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with KD 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 KD 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 KD in the 10−8 M range and (ii) a kinetically rapid, low-affinity interaction with KD 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 KD 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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Interaction of bovine coagulation factor X and its glutamic‐acid‐containing fragments with phospholipid membranes

Cited by 20 publications

References 35 publications

Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex

Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Kinetic regulation of the binding of prothrombin to phospholipid membranes

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