Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF·VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF·VIIa with macromolecular substrate.     

Disulfide isomerization switches tissue factor from coagulation to cell signaling

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF.VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys186-Cys209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys186 retains TF.VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF.PAR2 complex and TF.VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF.VIIa signaling can be specifically inhibited with minimal effects on coagulation.     

Tissue factor-mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor

Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface-bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.     

Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages.

The potential for tissue factor (TF) to enhance inflammation by factor VIIa-dependent induction of proinflammatory changes in macrophages was explored. Purified recombinant human factor VIIa enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) was demonstrated in vivo by treatment with an anti-TF antibody. These data demonstrate that, in addition to its role in activation of coagulation, TF can directly augment macrophage activation. These effects are initiated by binding factor VIIa and are independent of other coagulation proteins. These studies provide the first demonstration of a direct proinflammatory role for TF acting as a cell-signaling receptor.     

Structure of human factor VIIa and its implications for the triggering of blood coagulation.

Factor VIIa (EC 3.4.21.21) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade. On injury, factor VIIa forms a complex with its allosteric regulator, tissue factor, and initiates blood clotting. Although the structure of the binary complex has already been determined [Banner, D. W., D'Arcy, A., Chène, C., Winkler, F. K., Guha, A., Konigsberg, W. H., Nemerson, Y. & Kirchhofer, D. (1996) Nature (London) 380, 41-46], the conformational effects of cofactor binding to factor VIIa are not known in detail because of a lack of structural information on free factor VIIa. Here we report the structure of gamma-carboxyglutamic acid-domainless human coagulation factor VIIa at a resolution of 2.8 A. The molecule adopts an extended conformation within the crystal similar to that previously observed for the full-length protein in complex with tissue factor. Detailed comparison of free and tissue factor-bound factor VIIa reveals several structural differences. The binding mode of the active-site inhibitor D-Phe-Phe-Arg methyl ketone differs in the two structures, suggesting a role for the cofactor in substrate recognition. More importantly, a surface-exposed alpha-helix in the protease domain (residues 307-312), which is located at the cofactor recognition site, is distorted in the free form of factor VIIa. This subtle structural difference sheds light on the mechanism of the dramatic tissue factor-induced enhancement of factor VIIa activity.     

Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor

Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa, is aberrantly expressed in human cancers. We demonstrated a significant correlation between TF and vascular endothelial growth factor (VEGF) production in 13 human malignant melanoma cell lines (r(2) = 0.869, P < 0.0001). Two of these cell lines, RPMI-7951, a high TF and VEGF producer, and WM-115, a low TF and VEGF producer, were grown s.c. in severe combined immunodeficient mice. The high-producer cell line generated solid tumors characterized by intense vascularity, whereas the low producer generated relatively avascular tumors, as determined by immunohistologic staining of tumor vascular endothelial cells with anti-von Willebrand factor antibody. To investigate the structure-function relationship of TF and VEGF, a low-producer melanoma cell line (HT144) was transfected with a TF cDNA containing the full-length sequence, a cytoplasmic deletion mutant lacking the coding sequence for the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length sequence produced increased levels of both TF and VEGF. Transfectants with the full-length sequence and the extracellular domain mutant produced approximately equal levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF plays a role in the regulation of VEGF expression in some tumor cells.     

Tissue factor-dependent factor VIIa signaling

Tissue factor (TF) is known primary as a cofactor for factor VIIa-mediated triggering of blood coagulation, which proceeds in a cascade of extracellular reactions. Recent investigations have, however, revealed that intracellular activities can also be induced by the proteolytic activity of factor VIIa bound to cell surface TF. Factor VIIa signal transduction has thus been reported to induce mobilization of intracellular Ca(2+) stores and p44/p42 MAPK phosphorylation and to result in expression of specific genes, which presumably affects a number of cellular functions. The factor VIIa-induced signal transduction is independent of the presence of the TF cytoplasmic domain and it is distinctly different from signaling involving presently known protease-activated receptors (PARs) including receptors for thrombin and factor Xa. This short review summarizes recent advances in our understanding of TF-dependent factor VIIa signaling.     

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)--factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF--VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF--VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF--VIIa--X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF--VIIa--Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF--VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.     

A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties

Tissue factor (TF) initiates blood coagulation, but its expression in the vascular space requires a finite period of time. We hypothesized that targeting exogenous tissue factor to sites of vascular injury could lead to accelerated hemostasis. Since phosphatidylserine (PS) is exposed on activated cells at sites of vascular injury, we cloned the cDNA for a chimeric protein consisting of the extracellular domain of TF (called soluble TF or sTF) and annexin V, a human PS-binding protein. Both the sTF and annexin V domains had ligand-binding activities consistent with their native counterparts, and the chimera accelerated factor X activation by factor VIIa. The chimera exhibited biphasic effects upon blood coagulation. At low concentrations it accelerated blood coagulation, while at higher concentrations it acted as an anticoagulant. The chimera accelerated coagulation in the presence of either unfractionated or low-molecular-weight heparins more potently than factor VIIa and shortened the bleeding time of mice treated with enoxaparin. The sTF-annexin V chimera is a targeted procoagulant protein that may be useful in accelerating thrombin generation where PS is exposed to the vasculature, such as may occur at sites of vascular injury or within the vasculature of tumors.     

The relationship between tissue factor and cancer progression: insights from bench and bedside

It is now widely recognized that a strong correlation exists between cancer and aberrant hemostasis. Patients with various types of cancers, including pancreatic, colorectal, and gastric cancer, often develop thrombosis, a phenomenon commonly referred to as Trousseau syndrome. Reciprocally, components from the coagulation cascade also influence cancer progression. The primary initiator of coagulation, the transmembrane receptor tissue factor (TF), has gained considerable attention as a determinant of tumor progression. On complex formation with its ligand, coagulation factor VIIa, TF influences protease-activated receptor-dependent tumor cell behavior, and regulates integrin function, which facilitate tumor angiogenesis both in vitro and in mouse models. Furthermore, evidence exists that an alternatively spliced isoform of TF also affects tumor growth and tumor angiogenesis. In patient material, TF expression and TF cytoplasmic domain phosphorylation correlate with disease outcome in many, but not in all, cancer subtypes, suggesting that TF-dependent signal transduction events are a potential target for therapeutic intervention in selected types of cancer. In this review, we summarize our current understanding of the role of TF in tumor growth and metastasis, and speculate on anticancer therapy by targeting TF.     

Properties of factor VIIa/tissue factor complexes in an umbilical vein model

An umbilical vein model was designed in which washed vein segments are filled with a reaction mixture containing factor VIIa, Ca(+)+, and a substrate, either 3H-factor IX or 3H-factor X. The vein wall provides the tissue factor (TF) for factor VIIa/TF complexes that activate the substrates as measured by activation peptide release. The model was developed to study TF induced on venous endothelium in situ. However, unlike previous studies with TF expressed on cultured umbilical vein endothelial cells, factors IX and X were activated without first having to expose the vein wall to a perturbing stimulus. Histologic studies revealed that washing the vein and mixing the reaction mixture before subsampling had disrupted the endothelium. Immunostaining with anti-TF antibodies revealed no staining of endothelium but intense staining in extensions of Wharton's jelly penetrating fenestrations of the muscularis media of the vein. Thus, the model provided data on factor VIIa/TF formed, not on endothelium, but within the mucoid connective tissue of Wharton's jelly. It is known that factor VIIa/TF formed with TF in suspension or with TF expressed on the surface of cultured cells activates factor X more rapidly than factor IX. In contrast, in the umbilical vein model, when each substrate was present in an 88 nmol/L concentration, factors IX and X were activated at equivalent rates (mean activation rate for factor IX, 18.8 +/- 3.6 nmol/L/h; for factor X, 17.8 +/- 2.9 nmol/L/h; n = 9 paired vein segments). These data strengthen the evidence that factor VIIa/TF activation of factor IX represents a key initial reaction of coagulation in tissues. These results also show that data obtained with factor VIIa/TF complexes formed on the surface of cultured cells need not hold for factor VIIa/TF complexes formed in extracellular matrix.     

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect. (Blood. 2000;95:1330-1335)     

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.     

Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor-factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor-factor VIIa interaction.     

Monoclonal antibodies against bovine tissue factor, which block interaction with factor VIIa

Two monoclonal antibodies that recognize bovine tissue factor (coagulation factor III) have been obtained following the fusion of hyperimmune mouse spleen cells with NS-1 plasmacytoma cells. Both antibodies, TF1-E2 and TF1-F7, have gamma 1 heavy chains and lambda light chains. TF1-E2 and TF1-F7 have each been used to purify bovine tissue factor from a crude detergent extract of bovine brain by immunoaffinity chromatography. Both antibodies inhibit tissue factor procoagulant activity and block the association of factor VIIa with tissue factor. The association of TF1-F7 and tissue factor solubilized in Triton X-100 was measured under equilibrium conditions. The Kd for this antibody-antigen interaction was 2.1 +/- 0.2 nmol/L. TF1-E2 effectively competes with TF1-F7 for tissue factor binding, indicating that the monoclonal antibodies recognize overlapping sites on the protein. These antibodies will be useful reagents for large-scale purification and for structure-function studies of bovine tissue factor. In particular, since they appear to bind to the same region of the tissue factor molecule as factor VIIa, they will be useful as specific probes for studying the kinetics of tissue factor-initiated coagulation and for immunocytochemical localization of tissue factor in bovine cells.     

Structural requirements for TFPI-mediated inhibition of neointimal thickening after balloon injury in the rat.

The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK(1), a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI(WT), the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI(K36I), an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI(13-161), a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK(1), TFPI(WT), and high levels of TFPI(K36I) begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI(13-161) was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK(1) approximately TFPI(WT)>TFPI(K36I)>TFPI(13-161). The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.     

Cellular localization and trafficking of tissue factor

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.     

Factor Xa binding to annexin 2 mediates signal transduction via protease-activated receptor 1

The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active factors VIII (VIIIa), IX (IXa), factor X, and Ca2+. Factor Xa has procoagulant activity by conversion of prothrombin to thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex. Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa. Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin 2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles when factor Xa is generated in a TF-depleted environment.     

Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.