The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

Saleh, Mahasen; Wu, Peng; Quinn-Allen, Mary Ann; Macedo-Ribeiro, Sandra; Fuentes‐Prior, Pablo; Bode, Wolfram; Kane, William H.

doi:10.1160/th03-04-0222

Cited by 40 publications

(46 citation statements)

References 46 publications

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“…We conclude that although PE does promote Va binding to membranes, PS promotes tighter binding in a PE-independent fashion. As a control, we showed that the K d for bovine Va binding to 75:25 DOPC:DOPS membranes (0.35 nM) ( Table 2) was comparable with that reported for human recombinant factor Va to synthetic vesicles (K d ϭ 0.24 nM) (28), indicating that our results are not species-specific.…”

Section: Membranesupporting

confidence: 83%

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Majumder

Liang

Quinn-Allen

et al. 2011

Journal of Biological Chemistry

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Background: PS is an allosteric regulator lipid that appears on activated platelets along with PE. Results: We show that PE 1) increases the k cat and decreases K m of thrombin formation; 2) reduces membrane surface packing to promote Va binding; and 3) binds to Va and Xa to trigger their tight association. Conclusion: This suggests a role in initiating blood coagulation. Significance: PE may also be a regulator lipid.

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Section: Membranesupporting

confidence: 83%

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Majumder

Liang

Quinn-Allen

et al. 2011

Journal of Biological Chemistry

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“…As in FVIII, the C2 domain contains the primary membrane-binding site of FV. 31,32 The FV A3 33 and C1 34 domains also appear to interact with membranes. In FV and/or FVIII, the C domains may reorient during membrane attachment.…”

Section: Discussionmentioning

confidence: 99%

The factor VIII C1 domain contributes to platelet binding

Hsu

Pratt

Thompson

2008

Blood

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Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrinlabeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrinlabeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluoresceinconjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7000 C1C2-binding sites per platelet, approximately 1400 for C2, and approximately 3000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity. IntroductionFactor VIII (FVIII) circulates in plasma in a noncovalent complex with von Willebrand factor (VWF); this interaction is mediated by the FVIII C2 domain and an acidic sequence prior to the A3 domain. [1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation. 4,5 FVIIIa forms a complex with FIXa and calcium on negatively charged phospholipid membranes, enhancing FIXa catalysis 100 000-to 200 000-fold. [6][7][8] Although FVIII can bind to FIXa on phospholipids, 9 or directly to activated platelets, 10 FVIIIa is required for procoagulant activity. 9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.To address the potential role of the C1 domain in FVIII(a) attachment to platelets, a recombinant human FVIII C1C2 protein (residues 2020-2332) was produced in Escherichia coli, refolded, and characterized. The binding of C1C2 to platelet surfaces both before ...

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“…Unlike the tryptophans on the C2 spikes, the tyrosines would not insert into, but rather could interact favorably with, phospholipid membranes (39,40). A very recent report using alanine-scanning mutagenesis identified these leucine and tyrosine residues on the C1-3 spike as important in prothrombinase activity (41). Additionally, two arginine residues in human factor Va (Lys-2010 and Arg-2014 in the bovine molecule) were shown to have a significant impact on function.…”

Section: Domain Interfacesmentioning

confidence: 99%

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

Adams

Hockin

Mann

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

142

157

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In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1⅐A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copperbinding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

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The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

Cited by 40 publications

References 46 publications

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

The factor VIII C1 domain contributes to platelet binding

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

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