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

Abstract

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...

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: A computational protein–protein docking and molecular dynamics refinement study

“…The mutations of the residues Lys2092/Phe2093 of the C1 domain and Met2199/Phe2200/Leu2251/Leu2252 residues of the C2 domain have been shown to reduce the binding affinity of FVIIIa co-factor on the PL surface, suggesting that these residues might be responsible for membrane interactions[25]. While a similar data is not available for the role of specific residues of GLA domain involvement in membrane interactions, it has been shown that the GLA domain interacts with FVIIIa light-chain during the tenase complex formation [26]. Our current model shows that the -loop region of the GLA domain is at nearly the same plane of the hydrophobic spikes of the C1-C2 interface.…”

Hepatocyte tissue factor activates the coagulation cascade in mice

“…Next, intact control HPCs were incubated with 15 mM sulfo-NHS-SS-biotin, a membrane-impermeable compound that reacts with free amines in proteins, including the side chain of lysine. Previous studies have shown that several lysine residues in the TF molecule are critical for activation of FX by the TF:FVIIa complex, [28][29][30] and sulfo-NHS-SSbiotin was previously used to inhibit surface TF PCA. [31][32][33] Similar to 1H1, sulfo-NHS-SS-biotin significantly reduced FXa generation by intact control HPCs and also dramatically reduced the lysis-dependent increase in FXa generation ( Figure 3C).…”

Structural biology of factor VIIa/tissue factor initiated coagulation

“…In addition, R37 of FIXa can make a hydrogen bond with either T167 or D204 of TF (Figure 3B). Mutagenesis studies support the role of TF residues R200, K201, T167 and K165 in its interaction with the Gla domain of FXa/FIXa (80-83). Thus, comparable interactions exist between the C-terminal helix of the Gla domains of FXa or FIXa with TF.…”

“…The C-terminal region of TF interacts with the Gla domains of FX/Xa (22,80-83) and FIX/IXa (66,67,69). Further, the Gla domains of FX/Xa (68,70-73) and FIX/IXa (66,69) also interact with the Gla domain of FVIIa.…”