Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Ortel, Thomas L.; Quinn-Allen, Mary Ann; Charles, Linda; Devore-Carter, Denise; Kane, William H.

doi:10.1172/jci116123

Cited by 55 publications

(59 citation statements)

References 26 publications

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“…Popular consensus is that the RVV-V protease activates factor V to produce a species with only a light chain, equivalent (by electrophoretic migration) and with similar clotting activity to factor V IIa (12,66,67). We have reported that APC-inactivated membranebound factor V (cleaved at Arg 306 , Arg 506 , Arg 679 , and Lys 994 ) is further cleaved by the RVV-V activator at Arg 1018 and Arg 1545 (65).…”

Section: Limited Proteolysis Of Factor V By N Nigricollis Nigricollimentioning

confidence: 99%

“…Activation of Factor V by Russell's Viper Venom Factor V Activator and the Importance of the Light Chain-In contrast to the NN protease, RVV-V activator is an efficient activator of factor V. Although several laboratories have used the RVV-V activator to activate factor V (12,66,67) no explicit description of the activation/cleavage sites has been reported. Popular consensus is that the RVV-V protease activates factor V to produce a species with only a light chain, equivalent (by electrophoretic migration) and with similar clotting activity to factor V IIa (12,66,67).…”

Section: Limited Proteolysis Of Factor V By N Nigricollis Nigricollimentioning

confidence: 99%

“…The factor Va-prothrombin interaction is promoted by the heavy chain of the molecule (15,18). The factor Va-membrane interaction, governed by a K d value of ϳ3 nM, occurs at diffusionally limited rates, involves both hydrophobic and Ca 2ϩ -dependent electrostatic interactions, and results in penetration of a portion of the light chain into the membrane bilayer (21)(22)(23)(24)(25). Two sites on the light chain of the cofactor appear to be responsible for the interactions of factor Va with the membrane surface.…”

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Structural Requirements for Expression of Factor Va Activity

Kalafatis

Beck

Mann

2003

Journal of Biological Chemistry

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Thrombin activated factor Va (factor V IIa , residues 1-709 and 1546 -2196) has an apparent dissociation constant (K d,app ) for factor Xa within prothrombinase of ϳ0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp 697 , Asp 1509 , and Asp 1514 to produce a molecule (factor V NN ) that is composed of a M r 100,000 heavy chain (amino acid residues 1-696) and a M r 80,000 light chain (amino acid residues 1509/1514 -2196). Factor V NN , has a K d,app for factor Xa of 4 nM and reduced clotting activity. Cleavage of factor V IIa by NN at Asp 697 results in a cofactor that loses ϳ60 -80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg 1018 and Arg 1545 to produce a M r 150,000 heavy chain and M r 74,000 light chain (factor V RVV , residues 1-1018 and 1546 -2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor V NN at Arg 1545 by ␣-thrombin (factor V NN/IIa ) or RVV (factor V NN/RVV ) leads to enhanced affinity of the cofactor for factor Xa (K d,app ϳ 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg 1545 and formation of the light chain of factor V IIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.The prothrombinase complex responsible for the generation of ␣-thrombin in the hemostatic process is composed of factor Va and factor Xa associated on a phospholipid membrane in the presence of Ca 2ϩ (1, 2). Although factor Xa alone can convert prothrombin to ␣-thrombin, the prothrombinase complex has a catalytic efficiency five orders of magnitude greater than factor Xa acting alone (3). Plasma factor V circulates as a large single chain protein of M r 330,000 (4 -6). The cDNA sequences for human, murine, porcine, and bovine factor V have been reported previously (7-11). The factor V molecule is composed of triplicated "A" domains, duplicated "C" domains, and a "B" region. Human factor V is cleaved by ␣-thrombin at Arg 709 , Arg 1018 , and Arg 1545 and generates the active cofactor factor Va, which is composed of a heavy chain (A1-A2 domains, M r 105,000, amino acid residues 1-709) non-covalently associated with the light chain (A3-C1-C2 domains, M r 74,000, amino acid residues 1546 -2196). The interaction between the two chains is promoted by divalent cations (12, 13).Activation of factor V by ␣-thrombin is required for the interaction of the cofactor with factor Xa and prothrombin. Factor Va and factor Xa interact stoichiometrically in the absence of phospholipids with a K d of 0....

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Section: Limited Proteolysis Of Factor V By N Nigricollis Nigricollimentioning

confidence: 99%

Section: Limited Proteolysis Of Factor V By N Nigricollis Nigricollimentioning

confidence: 99%

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confidence: 99%

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Structural Requirements for Expression of Factor Va Activity

Kalafatis

Beck

Mann

2003

Journal of Biological Chemistry

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“…Antibodies to the C2 domain of both factors V and VIII have been shown to interfere with membrane binding and inhibit cofactor function (18,19). Deletion of the entire C2 domain results in a complete loss of phosphatidylserine-specific membrane binding (20). Alaninescanning mutagenesis within the C2 identified several key polar and hydrophobic amino acids as necessary for achieving maximal cofactor function (21,22).…”

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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.

139

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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“…However, factor VIII requires more PS per binding site than factor V (13), and current evidence implicates different domains in membrane binding. While binding of factor V is apparently mediated by both the A3 domain (17) and the C2 domain (18) and is influenced by glycosylation within the C2 domain (19), binding of factor VIII is apparently mediated by the C2 domain (20,21) and is independent of glycosylation. Recent publication of two crystal structures for the C2 domain of factor V (22) and a single structure for the C2 domain of factor VIII (23) has provided the basis for a model membrane-binding mechanism.…”

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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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Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Cited by 55 publications

References 26 publications

Structural Requirements for Expression of Factor Va Activity

Structural Requirements for Expression of Factor Va Activity

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

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

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