Mutating factor VIII: lessons from structure to function

Fay, Philip J.; Jenkins, Patrick

doi:10.1016/j.blre.2004.02.003

Cited by 54 publications

(40 citation statements)

References 84 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1 FXa is required for the generation of thrombin and fibrin, which are needed to limit blood loss during vascular injury. 2 A focus of drug development for HemA is to increase the effectiveness of FVIII treatment.…”

Section: Introductionmentioning

confidence: 99%

“…Cofactor subunit dissociation, particularly dissociation of the A2 domain, is thought to limit FVIIIa activity. 1 Stabilization can be achieved by a variety of means, including covalent A2-A3 subunit disulfide linkage alone 5 or removal of cleavage sites between A2 and A3 in combination with mutations conferring resistance to activated protein C. 6 Additional approaches include point mutations in the B domain, 7 in the activated protein C cleavage site(s), [8][9][10] and in the interfaces between the A2 and A1 or A3 domains. 11 The predicted outcome of FVIIIa stabilization would be sustained cofactor activity, increased FXa and thrombin generation, and, ultimately, enhanced clot formation and protection from bleeding.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models

Leong

Sim

Patel

et al. 2015

Blood

Self Cite

View full text Add to dashboard Cite

• Increasing FVIIIa by stabilizing the A2 domain association enhances its function in vitro and in vivo in hemophilia.• Stabilized FVIIIa improved efficacy in several vascular injury models, including laser injury, in which it was particularly effective.An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain. (Blood. 2015;125(2):392-398)

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models

Leong

Sim

Patel

et al. 2015

Blood

Self Cite

View full text Add to dashboard Cite

show abstract

“…A number of studies involving fluorescence, peptide, or antibody inhibition and analyses of point mutations in the hemophilia A data base have identified several sequences involved in this interaction (see Ref. 16 for a review). One region in A2, the 558-loop was identified as a factor IXa-interactive site by both peptide inhibition studies (17) and by the capacity of factor IXa to selectively protect factor VIIIa from activated protein C-catalyzed cleavage at Arg-562 (18).…”

mentioning

confidence: 99%

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Jagannathan

Ichikawa

Kruger

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...

show abstract

“…The resulting A1-a1/ A2-a2/A3-C1-C2 heterotrimer no longer interacts with VWF but binds to the serine protease, FIXa, on negatively charged phospholipid membranes provided by activated platelets. This calcium-dependent complex of FVIIIa, FIXa, and acidic phospholipids, also termed intrinsic Xase, activates FX via cleavage of a single peptide bond ( Figure 1A; recently reviewed by Fay and Jenkins 4 and Graw et al 5 ). In addition to its role in cofactor activation, the acidic region a1 is involved in important interactions with the Xase substrate, FX.…”

Section: Introductionmentioning

confidence: 99%

Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

Venceslá¹,

Corral-Rodríguez

Baena³

et al. 2008

Blood

View full text Add to dashboard Cite

Mutating factor VIII: lessons from structure to function

Cited by 54 publications

References 84 publications

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites

Contact Info

Product

Resources

About