Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X.

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 104-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 active-site 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 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 C-terminal heavy chain contains the trypsin-like catalytic domain of the protease (16,17). Both the light and heavy chains of fVIIa have extensive interaction with both the membrane distal N-terminal and the membrane proximal C-terminal domains of TF in the extrinsic Xase complex (7,12,18) and most of these interactive-sites on both the cofactor and the protease have been mapped by mutagenesis studies (7,10,14,18). On the other hand, with the exception of the Gla-domain of fX, which is known to interact with the C-terminal domain of TF, no other recognition site on TF for the substrate has been characterized. Thus, it has been demonstrated that several basic residues on the C-terminal domain of TF (in particular Lys-165 and Lys-166) interact with the Gla-domain of fX (and also fIX), thereby stabilizing the substrate in an extended conformation on the membrane surface (10,19,20). While it is known that the first EGF domain (EGF-1) of fX is essential for the recognition and the assembly of the substrate into the extrinsic Xase complex (21), its interactive-site(s) on the fVII-TF complex has not been identified. Molecular modeling based on the x-ray crystal structure of the fVIIa-sTF complex (Figure 1) has predicted that the membrane proximal TF residues Asn-199, Arg-200, and Lys-201 may have suitable height and topology on the membrane surface to interact with either the C-terminal residues of the Gla-domain or EGF-1 of fX in the extrinsic Xase complex (10,12). To test this hypothesis, we substituted all three residues individually with Ala in three different constructs and expressed them as soluble sTF2-219 forms in a bacterial periplasmic expression/purification vector system. All three mutants were purified to homogeneity and characterized with respect to their ability to function as cofactors in the enhancement of the amidolytic and proteolytic activity of fVIIa using Spectrozyme fVIIa, wild-type fX and two deletion derivatives of fX as substrates. All three TF mutants exhibited normal cofactor activity, thereby enhancing the amidolytic activity of fVIIa toward the chromogenic substrate with normal catalytic efficiency. On the other hand, the Arg-200 and Lys-201 mutants of TF each exhibited ~3-fold decreased cofactor activity with both wild-type and the mutant of fX in which the Gla-domain of the substrate was deleted (GD-fX). However, both mutants had normal cofactor activity with another mutant of fX in which both the Gla and EGF-1 (E2-fX) were deleted. Further kinetic analysis revealed that the decreased cofactor activities of the mutants are primarily due to impairments in the kcat of the activation reactions. These results suggest that both Arg-200 and Lys-201 of TF interact with the first EGF domain of fX to mediate the optimal docking of the activation peptide of the substrate into the active-site groove of fVIIa in the extrinsic Xase pathway. Figure 1 The space-filling model of the crystal structure of the fVIIa-sTF complex. The side chains of three residues Asn-199, Arg-200 and Lys-201 are shown by arrows. The side chains of Lys-165 and Lys-166 which are thought to interact with the Gla-domain of ...