Factor VIIa-Tissue Factor: Functional Importance of Protein-Membrane Interactions

Morrissey, James H.; Neuenschwander, Pierre F.; Huang, Qiuling; McCallum, Christine; Su, Bixia; Johnson, Arthur E.

doi:10.1055/s-0038-1657511

Cited by 45 publications

(39 citation statements)

References 15 publications

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“…In support of this proposal, recent fluorescence resonance energy transfer studies demonstrated that the activesite of fVIIa in complex with TF in the extrinsic Xase complex is also located far above the membrane surface (13). Thus, for effective recognition by fVIIa, both fIX and fX must also ‡To whom correspondence should be addressed: Alireza R. Rezaie, Ph.D., Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, Phone: (314) Fax: (314) assemble into the activation complex in the same extended conformations in order to maintain the activation peptides of the substrates at a similar distance to that of the active-site of fVIIa above the membrane surface (2,12,14,15).Like other vitamin K-dependent plasma serine proteases, the structure of fVIIa consists of a light and a heavy chain held together by a disulfide bond (16,17). The N-terminal light chain of fVIIa contains the Gla and two epidermal growth factor (EGF)-like domains and the Cterminal heavy chain contains the trypsin-like catalytic domain of the protease (16,17).…”

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Identification of a Basic Region on Tissue Factor that Interacts with the First Epidermal Growth Factor-like Domain of Factor X

2007

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Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa, however, with the exception of a basic recognition site for the Gla-domain of fX, no other interactive-site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues located at a proper height on the membrane surface to interact with either the C-terminus of the Gla-domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wildtype fX and its two mutants which lack either the Gla-domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX was impaired ~3-fold. Further kinetic analysis revealed that k cat with both TF mutants are impaired with no change in K m . These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.Tissue factor (TF) 1 is an integral membrane cofactor that upon exposure to circulating blood and subsequent binding to factor VIIa (fVIIa) catalyzes the rapid activation of factors IX (fIX) and X (fX) to their catalytically active forms, thereby initiating the blood clotting cascade (2-4). The structure of TF is composed of two fibronectin type III-like extracellular domains, a single membrane spanning domain and a short cytoplasmic tail (5,6). All three domains of TF are required for the physiological function of the cofactor, however, the two extracellular domains expressed by recombinant DNA methods as a soluble protein (sTF) can bind to fVIIa with a high affinity to enhance both the amidolytic and proteolytic activities of the protease (7-10). TF improves the proteolytic activity of fVIIa toward both of its natural substrates fIX and fX more than 10 4 -fold (11). Recent x-ray crystal structure of the fVIIa-sTF complex has provided some insight into the mechanism by which the cofactor may improve the catalytic efficiency of fVIIa in the extrinsic Xase complex (12). It has been noted that fVIIa makes extensive interactions with both the N-and C-terminal domains of sTF, and based on the elongated structure of the complex, it has been hypothesized that, similar to fVIIa, the cofactor would also interact with the natural substrates in a similar manner (10,12). In support of this proposal, recent fluorescence resonance energy transfer studies demonstrated that the activesite of fVIIa in complex with TF in the extrinsic Xase complex is also located far above the membrane surface (13). Thus, for effective recognitio...

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“…assemble into the activation complex in the same extended conformations in order to maintain the activation peptides of the substrates at a similar distance to that of the active-site of fVIIa above the membrane surface (2,12,14,15).…”

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confidence: 99%

Identification of a Basic Region on Tissue Factor that Interacts with the First Epidermal Growth Factor-like Domain of Factor X

2007

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“…[11][12][13][14] The activated platelets provide anionic surfaces for the assembly and catalysis of intrinsic tenase (VIIIa/IXa), prothrombinase (Va/Xa), and XIa complexes, leading to explosive generation of thrombin and consolidation of the fibrin-platelet plug. [15][16][17] Thus, rapid exposure of PS on the cell surfaces of platelets is the key thrombogenic stimulus initiating and propagating the clotting cascade. 9,10,17) Annexins (ANXs) are a family of calcium-dependent phospholipid binding proteins.…”

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confidence: 99%

Staphylokinase-Annexin XI Chimera Exhibited Efficientin VitroThrombolytic Activities

Chiou¹,

Woon²,

Cheng³

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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“…Tissue factor (TF) 1 is an integral membrane cofactor that upon exposure to circulating blood binds with high affinity to factor VIIa (fVIIa) to catalyze the rapid activation of procoagulant zymogens factors IX and X, thereby initiating the blood clotting cascade (1)(2)(3). The structure of TF is composed of two fibrinonectin type III-like extracellular domains, a single membrane-spanning domain and a short cytoplasmic tail (4,5).…”

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confidence: 99%

“…The structural data have indicated that the extracellular domains of TF make extensive interactions with both the light and heavy chains of fVIIa (10). It is believed that these interactions allosterically change the conformation of the active-site pocket of fVIIa, leading to a dramatic improvement in the catalytic efficiency of the protease toward both synthetic and natural macromolecular substrates (1). Most of the functionally critical residues of TF have been mapped by the Ala-scanning mutagenesis approaches (7,9,11).…”

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The Cofactor Function of the N-terminal Domain of Tissue Factor

Kittur

Manithody

Morrissey

et al. 2004

Journal of Biological Chemistry

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Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF 2-219 in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N-and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg 74 mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.Tissue factor (TF) 1 is an integral membrane cofactor that upon exposure to circulating blood binds with high affinity to factor VIIa (fVIIa) to catalyze the rapid activation of procoagulant zymogens factors IX and X, thereby initiating the blood clotting cascade (1-3). The structure of TF is composed of two fibrinonectin type III-like extracellular domains, a single membrane-spanning domain and a short cytoplasmic tail (4, 5). Although all three domains of TF are required for the physiological function of the cofactor, previous studies have indicated that the two extracellular domains expressed by recombinant DNA methods as a soluble protein (sTF) can bind to fVIIa with a high affinity to enhance both the amidolytic and proteolytic activities of the protease (6 -9). The crystal structure of sTF either alone or in complex with fVIIa has been determined (4, 10). The structural data have indicated that the extracellular domains of TF make extensive interactions with both the light and heavy chains of fVIIa (10). It is believed that these interactions allosterically change the conformation of the active-site pocket of fVIIa, leading to a dramatic improvement in the catalytic efficiency of the protease toward both synthetic and natural macromolecular substrates (1...

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Factor VIIa-Tissue Factor: Functional Importance of Protein-Membrane Interactions

Cited by 45 publications

References 15 publications

Identification of a Basic Region on Tissue Factor that Interacts with the First Epidermal Growth Factor-like Domain of Factor X

Identification of a Basic Region on Tissue Factor that Interacts with the First Epidermal Growth Factor-like Domain of Factor X

Staphylokinase-Annexin XI Chimera Exhibited Efficientin VitroThrombolytic Activities

The Cofactor Function of the N-terminal Domain of Tissue Factor

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