Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain

SummaryThe C-terminal C domains of activated coagulation factor VIII (FVIIIa) are essential to membrane binding of this crucial coagulation cofactor protein. To provide an overall membrane binding mechanism for FVIII, we performed simulations of membrane binding through coarsegrained molecular dynamics simulations of the C1 and C2 domain, and the combined C-domains (C1+C2). We found that the C1 and C2 domain have different membrane binding properties. The C1 domain uses hydrophobic spikes 3 and 4, of its total of four spikes, as major loops to bind the membrane, whereas all four of its hydrophobic loops of the C2 domain appear essential for membrane binding. Interestingly, in the C1+C2 system, we observed cooperative binding of the C1 and C2 domains such that all four C2 domain spikes bound first, after which all four loops of the C1 domain inserted into the membrane, while the net binding energy was higher than that of the sum of the isolated C domains. Several residues, mutations of which are known to cause haemophilia A, were identified as key residues for membrane binding. In addition to these known residues, we identified residues from the C1 and C2 domains, which are involved in the membrane binding process, that have not been reported before as a cause for haemophilia A, but which contribute to overall membrane binding and which are likely candidates for novel causative missense mutations in haemophilia A.

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The Role of Cleavage of the Light Chain at Positions Arg1689or Arg1721in Subunit Interaction and Activation of Human Blood Coagulation Factor VIII

Journal of Biological Chemistry ◽

10.1074/jbc.270.8.3648 ◽

1995 ◽

Vol 270 (8) ◽

pp. 3648-3655 ◽

Cited By ~ 58

Author(s):

Marie-José S. H. Donath ◽

Peter J. Lenting ◽

Jan A. van Mourik ◽

Koen Mertens

Keyword(s):

Factor Viii ◽

Blood Coagulation ◽

Light Chain ◽

Human Blood ◽

Coagulation Factor ◽

Coagulation Factor Viii ◽

Subunit Interaction ◽

Blood Coagulation Factor Viii ◽

Human Blood Coagulation

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Changes in the Factor VIII C2 domain upon membrane binding determined by hydrogen–deuterium exchange MS

Biochemical Journal ◽

10.1042/bj20140121 ◽

2014 ◽

Vol 461 (3) ◽

pp. 443-451 ◽

Cited By ~ 5

Author(s):

Dionysios Pantazatos ◽

Christopher R. Gessner ◽

Virgil L. Woods ◽

Gary E. Gilbert

Keyword(s):

Factor Viii ◽

Blood Coagulation ◽

Membrane Binding ◽

Coagulation Factor ◽

Coagulation Factor Viii ◽

Hydrogen Deuterium Exchange ◽

Factor Ixa ◽

Hydrogen Deuterium ◽

Binding Peptides ◽

Blood Coagulation Factor Viii

Blood coagulation Factor VIII binds to a membrane in order to function as a cofactor for Factor IXa, preventing haemophilia. The present study indicates that membrane-binding peptides of Factor VIII are largely protected from water exposure, indicating that they become immersed in the membrane.

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Disruption of Protein-Membrane Binding and Identification of Small-Molecule Inhibitors of Coagulation Factor VIII

Chemistry & Biology ◽

10.1016/j.chembiol.2004.08.006 ◽

2004 ◽

Vol 11 (10) ◽

pp. 1413-1422 ◽

Cited By ~ 23

Author(s):

P.Clint Spiegel ◽

Shari M. Kaiser ◽

Julian A. Simon ◽

Barry L. Stoddard

Keyword(s):

Factor Viii ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Membrane Binding ◽

Coagulation Factor ◽

Coagulation Factor Viii ◽

Protein Membrane

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Factor VIII C1 Domain Residues 2092–2093 Participate in Membrane Binding

Blood ◽

10.1182/blood.v112.11.587.587 ◽

2008 ◽

Vol 112 (11) ◽

pp. 587-587

Author(s):

Henriet Meems ◽

Alexander B Meijer ◽

Dave Cullinan ◽

Koen Mertens ◽

Gary E. Gilbert

Keyword(s):

Factor Viii ◽

Membrane Binding ◽

Phospholipid Membranes ◽

Ionophore A23187 ◽

C2 Domain ◽

Microtiter Plate Assay ◽

Surface Loop ◽

Lower Affinity ◽

C1 Domain

Abstract Background: Activated factor VIII (FVIIIa) assembles with factor IXa (FIXa) on the membranes of activated platelets and on synthetic phosphatidylserine(PS)-containing membranes. This membrane-bound complex catalyses the conversion of the zymogen, factor X, to factor Xa. Established membrane-binding amino acids of FVIII are in the C2 domain. However, the C2 domain alone binds with a lower affinity to membranes than intact FVIII or the FVIII light chain, suggesting a role of the A3 and/or C1 domains in membrane binding. Moreover, a possible role for the C1 domain in platelet binding has been recently reported. The position of the C domains in the FVIII crystal structure suggests a possible role for residues from surface loop K2092-S2094 of the C1 domain in membrane binding. The present study addresses the role of this loop in membrane binding. Methods: The role of the C1 surface loop K2092-S2094 was assessed by competition studies using KM33. This is a scFv fragment cloned from the antibody repertoire of a hemophilia A patient, with an epitope that comprises residues 2092–2094. In addition, FVIII mutants incorporating yellow fluorescent protein in place of the B domain and with K2092/F2093 changed to alanine (FVIIIYFP and FVIIIYFP K2092A/F2093A) were expressed and purified. Binding of recombinant FVIII labelled with fluorescein-maleimide (FVIIIfl), FVIIIYFP and FVIIIYFP K2092A/F2093A to phospholipid membranes (4% or 15% PS/20% PE/PC as balance) supported by glass microspheres and purified platelets was measured by flow cytometry. Lower affinity, non-equilibrium binding of sonicated vesicles to immobilized factor VIII was measured in a microtiter plate assay. The cofactor function of FVIII was measured in a factor Xase assay with limiting phospholipid. Results: KM33 inhibited >95% of FVIII binding to phospholipid membranes containing 15% PS, indicating that the C1 domain epitope is important for membrane binding. The affinity of FVIIIYFP K2092A/F2093A for the same membranes was reduced 3-fold compared with FVIIIYFP (Kd’s of 91 ± 6 vs. 31 ± 2 nM). KM33 decreased the overall activity for the factor Xase complex by 95% on vesicles with 15% PS and >99% on vesicles with 4% PS. The implied membrane affinity for FVIIIYFP K2092A/F2093A in the factor Xase complex was decreased 3-fold for vesicles with 15% PS but the Vmax was equivalent to FVIIIYFP. The implied affinity of FVIIIYFP K2092A/F2093A was reduced approximately 40-fold for vesicles with 4% PS confirming the importance of the C1 domain epitope for full factor VIII function. In the microtiter assay, mAb BO2C11, against the C2 domain, blocked approx. 80% of binding to vesicles containing 15% PS, KM33 blocked 5% of binding and both antibodies together blocked ~95% of binding. Binding to 4% PS vesicles was inhibited 70% by KM33 alone and B02C11 alone blocked all binding. Thus, the two membrane-binding motifs are required for detectable binding to membranes with 4% PS but can independently support some binding to membranes with 15% PS. KM33 inhibited approx 90% of FVIII binding to platelets. The binding of FVIIIYFP K2092A/F2093A to platelets stimulated with calcium ionophore A23187 was reduced 50% compared to FVIIIYFP; however binding to platelets stimulated with thrombin receptor activating peptide (TRAP) was comparable to FVIIIYFP. The cofactor function of FVIIIYFP K2092A/F2093A was reduced approximately 80% on platelets stimulated with either TRAP or A23187. Conclusion: The present study demonstrates that the FVIII C1 domain contributes to membrane binding and residues K2092 and/or F2093 participate in this interaction. The relative importance of these residues for membrane binding is dependent on the amount of PS present in synthetic membranes. On platelets K2092 and/or F2093 are necessary for full cofactor function of FVIII.

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Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity

Blood ◽

10.1182/blood-2009-01-197707 ◽

2009 ◽

Vol 114 (18) ◽

pp. 3938-3946 ◽

Cited By ~ 45

Author(s):

Henriët Meems ◽

Alexander B. Meijer ◽

David B. Cullinan ◽

Koen Mertens ◽

Gary E. Gilbert

Keyword(s):

Factor Viii ◽

Membrane Binding ◽

Antibody Fragment ◽

Normal Activity ◽

Microtiter Plate ◽

Binding Motif ◽

Synthetic Membranes ◽

Fold Reduction ◽

C1 Domain

AbstractBinding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33scFv, and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 → Ala/Ala (fVIIIYFP 2092/93) and 2 single mutants Lys2092 → Ala and Phe2093 → Ala. KM33scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIIIYFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33scFv inhibited binding to platelets and fVIIIYFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIIIYFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIIIYFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIIIYFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.

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