Homology Models of the C Domains of Blood Coagulation Factors V and VIII: A Proposed Membrane Binding Mode for FV and FVIII C2 Domains

Pellequer, Jean‐Luc; Gale, Andrew J.; Griffin, John H.; Getzoff, Elizabeth D.

doi:10.1006/bcmd.1998.0214

Cited by 62 publications

(66 citation statements)

References 59 publications

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“…We altered amino acids of the C2 domain of factor VIII based upon their hypothetical function in membrane binding (22,23,39) (Table I, Fig. 1 (40).…”

Section: Resultsmentioning

confidence: 99%

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Gilbert¹,

Kaufman²,

Arena³

et al. 2002

Journal of Biological Chemistry

110

148

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Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...

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“…We altered amino acids of the C2 domain of factor VIII based upon their hypothetical function in membrane binding (22,23,39) (Table I, Fig. 1 (40).…”

Section: Resultsmentioning

confidence: 99%

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Gilbert¹,

Kaufman²,

Arena³

et al. 2002

Journal of Biological Chemistry

110

148

View full text Add to dashboard Cite

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“…13 The C domains are smaller (approximately 160 residues) and are more distantly related to various members of the discoidin protein fold family, such as galactose oxidase. [14][15][16][17] The B domain has no known structural homologs, is heavily glycosylated, and is relatively dispensible for procoagulant activity. fVIII is initially processed by proteolytic cleavage events that remove a large portion of the B domain, generating a heterodimer that circulates in a tight complex with von Willebrand factor (VWF).…”

Section: Introductionmentioning

confidence: 99%

The tertiary structure and domain organization of coagulation factor VIII

Shen

Spiegel

Chang

et al. 2008

Blood

232

258

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Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 do IntroductionThe principal mechanism used to stop the loss of blood in mammals following vascular injury consists of a pair of overlapping proteolytic cascades called the extrinsic and intrinsic pathways. [1][2][3][4] The process of blood coagulation requires extraordinary spatial and temporal regulation, which is accomplished by assembling and tethering the central proteolytic activities of these cascades at the location of transiently exposed biomolecules and cellular surfaces ( Figure 1A). This includes an integral membrane protein called "tissue factor" that initiates the rapid up-regulation of the short-lived extrinsic pathway, 5 and the surfaces of activated platelets, which modulate the activation of the longer-lived intrinsic pathway. 6 A total of 2 homologous procoagulants, factors V and VIII (fV and fVIII), are each localized on the surface of these platelets, where they nucleate the assembly of multiprotein proteolytic complexes.When fVIII is bound to activated platelets at the site of vascular injury, it recruits the serine protease fIXa into a complex that then catalyzes the proteolytic activation of fX. 1,4,7 The proteolytic activity of fIXa is enhanced by approximately 200 000-fold through its interaction with fVIII, calcium, and the phospholipid bilayer, 8 corresponding to an increase of approximately 10 9 in k cat /K M .The full-length, unprocessed fVIII protein consists of 2332 amino acid residues and has the domain structure A1-A2-B-A3-C1-C2 9-12 ( Figure 1B). The 3 A domains are each approximately 330 residues, and approximately 40% identical to each other and to the copper-binding protein ceruloplasmin. 13 The C domains are smaller (approximately 160 residues) and are more distantly related to various members of the discoidin protein fold family, such as galactose oxidase. [14][15][16][17] The B domain has no known structural homologs, is heavily glycosylated, and is relatively dispensible for procoagulant activity. fVIII is initially processed by proteolytic cleavage events that remove a large portion of the B domain, generating a heterodimer that circulates in a tight complex with von Willebrand factor (VWF). 18 This interaction is essential for maintaining stable levels of fVIII in circulation. 19 Upon vascular injury, further proteolytic processing generates activated factor VIIIa (fVIIIa), a heterotrimer (A1/A2/A3-C1-C2) that is released from VWF and binds to activated platelets. 18 The carboxy-terminal 159 amino acids of fVIII comprise its C2 domain, which is invo...

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“…The C domains are also homologous to each other and have a weak homology to the discoidin protein fold family (eg, the lipid-binding domain of galactose oxidase). 7,8 The circulating form of the factor VIII protein is a metal bridged heterodimer consisting of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). This form of factor VIII is bound tightly to von Willebrand factor (vWF).…”

Section: Introductionmentioning

confidence: 99%

Structure of a factor VIII C2 domain–immunoglobulin G4κ Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII

Spiegel

Jacquemin

Saint-Remy

et al. 2001

Blood

131

153

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Homology Models of the C Domains of Blood Coagulation Factors V and VIII: A Proposed Membrane Binding Mode for FV and FVIII C2 Domains

Cited by 62 publications

References 59 publications

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

The tertiary structure and domain organization of coagulation factor VIII

Structure of a factor VIII C2 domain–immunoglobulin G4κ Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII

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