Membrane-binding properties of the Factor VIII C2 domain

Novakovic, Valerie A.; Cullinan, David B.; Wakabayashi, Hironao; Fay, Philip J.; Baleja, James D.; Gilbert, Gary E.

doi:10.1042/bj20101797

Cited by 26 publications

(29 citation statements)

References 51 publications

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Section: Fibrin-related Binding Sites For Fviii On Platelets 1239mentioning

confidence: 51%

“…We note that the competition studies with fVIII-C2 were performed in a low-salt buffer, similar to buffer conditions required for fVIII-C2 to bind phospholipid membranes. 19 These results indicate that fVIII binding to fibrin is similar to binding to platelets with regard to affinity, prevention by VWF, and participation of the C2 domain.Because fVIII binds to SF, we asked what effect fibrin has on the activity of fVIII (Figure 4). SF increased activity of the factor Xase complex ;2.7-fold with a half-maximal increase at 5 to 10 mg/mL fibrin ( Figure 4A).…”

mentioning

confidence: 51%

“…The fVIII C2 (fVIII-C2) domain was produced, purified, and stored as described. 19 fVIII was labeled with fluorescein maleimide as described. 11 The ratio of fluorescein/fVIII ranged from 0.6/1 to 1.2/1 for studies in this report.…”

Section: Proteinsmentioning

confidence: 99%

“…14 The C1 and C2 domains mediate membrane binding. [15][16][17][18][19] The fVIII C domains share similar sequence and structure with the C domains of factor V [20][21][22] and lactadherin, 23 a milk fat globule membrane protein. Like fVIII, both factor V and lactadherin bind to PS-containing membranes.…”

Section: Introductionmentioning

confidence: 99%

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Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert

Novakovic

Shi

et al. 2015

Blood

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• Coagulation fVIII binds to a protein complex, including fibrin, on stimulated platelets rather than to membrane PS.• Anti-fVIII antibodies inhibit function on platelets differently than on phospholipid vesicles used in clinical assays.Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three-to sixfold when soluble fibrin (SF) binds the a IIb b 3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two-to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. (Blood. 2015;126(10):1237-1244 IntroductionFactor VIII (fVIII) binds to platelet membranes where it serves as a cofactor for the enzyme, factor IXa, in the intrinsic factor Xase complex, 1,2 converting the zymogen factor X to factor Xa. 3,4 The importance of the factor Xase complex is illustrated by the disease hemophilia, in which deficiency of fVIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. In spite of the central importance of the platelet membrane, the platelet fVIII binding sites have been only partially characterized.fVIII circulates in plasma in a noncovalent complex with von Willebrand factor (VWF). Binding to VWF is mediated by the same motifs that bind platelet and phospholipid membranes. 5,6 After dissociation from VWF, fVIII binds specifically to membranes containing phosphatidylserine (PS), which is exposed on the platelet membrane in response to stimulation by several agonists. 1,6 The residual uncertainty about the identity of platelet binding sites relates to the quantity of PS exposed following stimulation by physiologic agonists and the availability of specific reagents to block the exposed PS. Thrombin stimulates platelets to expose limited PS, resulting in an outer membrane composition of 1% to 4% PS. 7,8 This amount of PS may remain below the threshold to support the observed expression of 200 to 1600 binding sites per platelet. 9,10 In contrast, the combination of thrombin and collagen, or higher concentrations of the calcium ionophore, A23187, le...

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Section: Fibrin-related Binding Sites For Fviii On Platelets 1239mentioning

confidence: 51%

mentioning

confidence: 51%

Section: Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert

Novakovic

Shi

et al. 2015

Blood

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“…For example, the isolated C2 domain does not compete with factor VIII for membrane binding and does not interfere with the factor VIIIa-factor IXa complex. 2 Furthermore, factor VIII engineered to lack the C2 domain, retains functional activity, although with a requirement for a higher concentration of phospholipid vesicles with a high density of negative charge. 3 In short, recent biochemical studies suggest that the C2 domain could have only marginal importance.…”

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confidence: 99%

Factor VIII inhibitor epitopes and an enigma

Gilbert

2013

Blood

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Domain organization of membrane‐bound factor VIII

et al. 2013

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Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function.

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Membrane-binding properties of the Factor VIII C2 domain

Cited by 26 publications

References 51 publications

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Factor VIII inhibitor epitopes and an enigma

Domain organization of membrane‐bound factor VIII

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