Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Factor VIIa”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1993

Importance of substrate composition, pH and other variables on tissue factor enhancement of factor VIIa activity.

Tissue factor (TF) markedly enhances the ability of factor VIIa (FVIIa) to cleave both macromolecular and small peptidyl substrates. Using soluble mutant TF (sTF) to investigate TF-enhanced FVIIa amidolytic activity in solution, we screened thirty-four commercially available peptidyl-p-nitroanilide substrates and found that substrate hydrolysis rates were influenced by both the peptide sequence and the N-terminal blocking group (MeSO2 > MeO-CO or free N-terminus >> benzoyl). Two substrates (Chromozym t-PA: MeSO2-D-Phe-Gly-Arg-pNA; and CBS 34.47: H-D-cyclohexylglycyl-alpha-aminobutyryl-Arg-pNA) were cleaved at rates higher than those of previously reported chromogenic substrates for FVIIa. The pH range of FVIIa amidolytic activity toward Chromozym t-PA was 6.5 to 10 with an optimum at pH 7.8, while sTF.VIIa had a higher pH optimum (pH 8.4 to 8.5). The degree of enhancement of FVIIa activity by sTF varied from 12-fold at pH 7.5 to 73-fold at pH 9.9. The effect of a variety of agents on FVIIa amidolytic activity was surveyed: most decreased activity, while glycerol and ethylene glycol enhanced the activity of FVIIa but not sTF.VIIa. These results indicate that the effect of sTF on the catalytic center of FVIIa is pH-dependent, and that certain polyalcohols can partially substitute for TF.

Alcohols↗
PubMed1995

Determinants of plasma factor VIIa levels in humans.

Several enzymes can activate factor VII in vitro, but the protease responsible for generating factor VIIa in vivo has not been determined. Using recombinant tissue factor that has undergone a COOH-terminal truncation, a sensitive functional assay has been established for measuring plasma factor VIIa levels. To evaluate the mechanism responsible for the generation of factor VIIa in vivo, we measured the levels of this enzyme after administering purified concentrates of factor IX and factor VIII to patients with severe deficiencies of these clotting factors. In patients with hemophilia B, factor VIIa levels were initially reduced to 0.5 +/- 0.1 ng/mL and gradually increased to normal after infusing 100 U/kg of body weight (BW) of factor IX. Despite these increases, there were no significant changes in the generation of factor Xa or thrombin. In patients with hemophilia A, only a slight reduction in factor VIIa levels (2.5 +/- 1.3 ng/mL) was observed as compared with controls (3.3 +/- 1.1 ng/mL) and no significant changes were observed after factor VIII levels were normalized. The administration of recombinant factor VIIa (10 micrograms/kg BW) to patients with factor VII deficiency increased the mean circulating level of the enzyme to 118 ng/mL, but this only resulted in normalization of the levels of the activation peptides of factor IX and factor X. The above data indicate that factor IXa is primarily responsible for the basal levels of free factor VIIa generated in vivo (ie, in the absence of thrombosis or provocative stimuli) and that changes in the plasma concentrations of free factor VIIa in the blood do not necessarily lead to alterations in the extent of factor X activation.

Adolescent↗
PubMed1988

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

The role of catalytic cleft and exosite residues of factor VIIa for complex formation with tissue factor pathway inhibitor.

The extrinsic coagulation pathway is initiated by the binding of plasma factor VIIa (VIIa) to the cell surface receptor tissue factor (TF). Formation of the TF-VIIa complex results in allosteric activation of VIIa as well as the creation of an extended macromolecular substrate binding exosite that greatly enhances proteolytic activation of substrate factor X. The catalytic function of the TF-VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI). TFPI inhibition of the TF-VIIa complex was enhanced by the presence of Xa. This study investigates the relative contribution of catalytic cleft and exosite residues in VIIa for inhibitory complex formation with TFPI. VIIa protease domain residues Q176, T239 and E296 are involved in the formation of stable inhibitor complex with free TFPI. Kinetic analysis further demonstrated a predominant role of the S2' subsite residue Q176 for the initial complex formation with TFPI. In contrast, no significant reductions in inhibition by TFPI-Xa were found for each of the mutants in complex with phospholipid reconstituted TF. However, reduced rates of inhibition of the VIIa Gla-domain (R36) and Q176 mutant by TFPI-Xa were evident when TF was solubilized by detergent micelles. These data demonstrate docking of the TFPI-Xa complex with the macromolecular substrate exosite and the catalytic cleft, in particular the S2' subsite. The masking of the mutational effect by the presence of phospholipid shows a critical importance of Xa Gla-domain interactions in stabilizing the quaternary TF-VIIa-Xa-TFPI complex.

Amino Acid Substitution↗
PubMed2006

Recombinant factor VIIa, its clinical properties, and the tissue factor pathway of coagulation.

Recombinant factor VIIa (rFVIIa) is a synthetic coagulation protease that is structurally similar to human-derived plasma FVIIa. Pharmacologic doses of rFVIIa have been shown to enhance thrombin generation and assist in forming stable fibrin plugs at the site of injury. Recombinant factor VIIa appears to emerge as a valuable treatment alter-native for the treatment of bleeding episodes and for achieving hemostasis post surgery in those with bleeding disorders.

Blood Coagulation↗
PubMed2002

Factor VIIa induces tissue factor-dependent up-regulation of interleukin-8 in a human keratinocyte line.

Tissue factor (TF), a transmembrane receptor for the serine protease coagulation factor VII(a) (FVIIa), is the main initiator of the coagulation cascade. Through incompletely elucidated mechanisms, TF serves additional functions in tumor-associated angiogenesis and metastasis. We have studied interleukin-8 (IL-8) as a possible link between TF-FVIIa complex formation and subsequent processes. Recombinant human FVIIa induced the up-regulation of both IL-8 mRNA and protein in a FVIIa dose- and time-dependent fashion. A neutralizing antibody to TF reduced this induction by 93 +/- 5%. Active site-inhibited FVIIa had no stimulatory effect and completely blocked that of FVIIa. This confirms that the increased IL-8 production was dependent on the formation of TF-FVIIa complexes and the proteolytic activity of FVIIa. The IL-8 promoter contains DNA binding sites for nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In response to FVIIa, the DNA binding activity of both NF-kappaB and AP-1 was enhanced in an electrophoretic mobility shift assay. In addition, the IL-8 promoter was transcriptionally activated both in a luciferase reporter system and a nuclear run-off assay. Moreover, IL-8 mRNA stability was significantly enhanced by FVIIa-induced activation of the mitogen-activated protein kinases ERK1/2 and p38. Taken together, TF-FVIIa signaling induced increased transcription as well as mRNA stabilization leading to the significant up-regulation of IL-8 protein synthesis.

Cell Line↗
PubMed2000

Blocking the initiation of coagulation by RNA aptamers to factor VIIa.

The tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factor-dependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of approximately 15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

Base Sequence↗
PubMed2004

Factor VIIa replacement therapy in factor VII deficiency.

Factor (F)VII deficiency is an autosomal recessive disorder for which a replacement therapy is not universally available; recombinant FVIIa has been utilized as a therapeutic substitute. As FVII competes with FVIIa for binding to tissue factor in initiating the extrinsic pathway of blood coagulation, a lower dose of FVIIa replacement in cross-reacting material-negative (CRM-) individuals can achieve hemostasis. Three coagulation models (computational, synthetic and in vitro whole blood) were used to predict the FVIIa levels needed to provide apparent hemostasis in a non-bleeding state. Our whole blood results show that a 'normalized' coagulation profile for FVII-deficient individuals has an initiation phase that ends at 5.8 +/- 0.5 min (clot time) and the propagation phase of thrombin generation (thrombin-antithrombin III) yields a maximum concentration of 380 +/- 29 nmol L(-1). When CRM- FVII-deficient subjects were infused with a prophylactic dose of 23 micro g kg(-1) of recombinant FVIIa, 6-8 h postinfusion resulted in a comparable normalized whole blood profile. This FVIIa concentration (0.3-0.7 nmol L(-1)/equivalent dose: 0.8-1.8 micro g kg(-1)) is approximately 1/10 that currently used in treating FVII-deficient individuals and suggests that therapies should be altered relative to the concentration of the FVII zymogen.

Adolescent↗
PubMed2006

Traces of factor VIIa modulate thromboplastin sensitivity to factors V, VII, X, and prothrombin.

BACKGROUND: Thromboplastin reagents are used to conduct prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and screen for clotting factor deficiencies. Thromboplastins made from purified, recombinant tissue factor are generally more sensitive to changes in plasma factor (F) VII levels than are thromboplastins prepared from tissue extracts. This may be problematic as FVII's short plasma half-life can result in day-to-day fluctuation during oral anticoagulant therapy. We hypothesized that trace contamination of tissue-derived thromboplastins with FVII(a) blunts sensitivity to plasma FVII levels. METHODS: Traces of purified FVIIa were added to thromboplastin reagents prepared using recombinant human tissue factor and the effect on sensitivity to individual clotting factors was quantified in PT clotting assays. RESULTS AND CONCLUSIONS: Adding 5-100 pm FVIIa not only decreased thromboplastin sensitivity to plasma FVII, it surprisingly increased sensitivity to plasma levels of FV, FX and prothrombin. In addition, traces of FVIIa interacted with changes in the salt content and phospholipid composition of recombinant thromboplastins to further modulate their sensitivities to individual clotting factors. These results help explain how thromboplastin reagents of differing composition exhibit differing sensitivities to individual clotting factor levels. Implications of our results for monitoring oral anticoagulant therapy and other uses of the PT assay are discussed.

Anticoagulants↗
PubMed2000

Cellular consequences upon factor VIIa binding to tissue factor.

Tissue factor (TF) is a cell-surface-bound glycoprotein that binds the zymogen, factor (F) VII, and the active serine protease, FVIIa. The FVIIa/TF complex is the major activator of coagulation in vivo. Under normal physiological conditions, TF is expressed only on extravascular sites and perivascularly in the adventitial layer of blood vessels. Although not normally expressed by cells within the circulation, TF can be induced in monocytes and endothelial cells. Also, several malignant cells express high levels of TF. Recent reports have shown that FVIIa binding to TF can influence a number of biological functions, such as angiogenesis and cancer metastasis. TF also seems to play an important role in cell adhesion and migration. The intracellular signalling is independent of downstream activation of the blood coagulation cascade. FVIIa/TF seems to transduce signalling by two distinct mechanisms: one independent of the cytoplasmatic domain but dependent on the proteolytic activity of FVIIa, and one dependent on the cytoplasmatic domain of TF.

Animals↗
PubMed1995

Tissue factor interactions with factor VII: measurement and clinical significance of factor VIIa in plasma.

Certain epidemiological studies have implicated elevated factor VII coagulant activity as a risk factor for ischaemia heart disease. However, progress in understanding the clinical significance of elevated plasma factor VII levels has been hampered by: (1) differences between laboratories in the methodology for measuring factor VII; (2) the existence of multiple forms of factor VII in plasma (i.e. factor VIIa, zymogen factor VII, and a possible phospholipase-sensitive form of factor VII); (3) the resulting uncertainty regarding what is actually being measured in factor VII coagulant assays. Recent mutagenesis studies of tissue factor (the obligate protein cofactor for factor VIIa) have led to new assay technologies capable of quantifying trace levels of plasma factor VIIa without interference from zymogen factor VII. This review article focuses on the current status of measurement of plasma factor VII/VIIa levels and the relationship between various plasma forms of factor VII and risk of thrombotic disease.

Blood Coagulation Disorders↗
PubMed2000

Recombinant factor VIIa for the treatment of congenital factor VII deficiency.

Factor VII deficiency is a rare autosomal bleeding disorder with a highly variable hemorrhagic predisposition. Severe bleeding, including hemarthroses, may be encountered when plasma factor VII levels are below 1%. Patients have prolonged prothrombin times, and the final diagnosis is established by quantitative factor VII assays. Some patients have true deficiencies, that is, very low factor VII activity and low factor VII antigen (cross-reacting material) levels (CRM-); others have normal antigen levels but low activity (CRM+). Still others have reduced antigen levels (CRMR). There is a rather poor correlation between clinical symptoms and factor VII activity levels in plasma. Treatment of these patients consists of fresh frozen plasma, prothrombin complex concentrates, or factor VII concentrates. Recombinant activated factor VII (rFVIIa) is a very useful alternative, and several patients have been treated successfully. Because of the short half-life of factor VIIa, repeated doses have to be administered, and continuous infusion may be even better. Antibodies to factor VII have been reported but seem to be rather rare. From the available data it appears that rFVIIa is a safe and effective treatment modality for patients with congenital factor VII deficiency.

Factor VII↗
PubMed2000

The regulation of the factor VII-dependent coagulation pathway: rationale for the effectiveness of recombinant factor VIIa in refractory bleeding disorders.

We have explored the molecular basis of the clinical therapeutic effect of factor VIIa in hemophilia A using empirical reconstituted in vitro thrombin generation models. Tissue factor acts as a receptor and activator of preexistent but virtually inactive two-chain plasma factor VIIa. However, most of the factor VII circulates as a single-chain inactive zymogen (10 nM) and a trace (approximately 10-100 pM) circulates in the active two-chain form. Empirical reconstitution (purified factors VIIa, X, IX, VIII, V, prothrombin, and relipidated tissue factor) showed that plasma concentrations of factor VII (10 nM) prolong the initiation phase of thrombin generation significantly at low concentrations of tissue factor and 100 pM factor VIIa. Thus, we show for the first time that the zymogen factor VII may have a very significant inhibitory action on thrombin generation at physiologic ratios of factor VII to factor VIIa. The inhibition kinetics of factor Xa generation by low concentrations of tissue factor indicate that factor VII inhibits the reaction by competition for the initial binding of factor VIIa to tissue factor. Physiological concentrations of factor VII also inhibit the maximal rate of thrombin generation by 100 pM factor VIIa in the absence of factor VIII. Increasing the concentration of factor VIIa to 2 nM in this hemophilia A model overcame the inhibition of thrombin generation by 10 nM factor VII. Increasing the concentration up to 10 nM factor VIIa in the absence of factor VIII completely normalized the thrombin generation profile to that observed in the presence of factor VIII and 10 nM factor VII/100 pM factor VIIa. The levels of factor VIIa that overcome the inhibitory effect of factor VII and that normalize thrombin generation in our model are consistent with the observed plasma levels of factor VIIa needed to manage hemophilia A. Our data strongly indicate that the therapeutic mechanism of factor VIIa in the medical treatment of hemophiliacs with inhibitors is in large part based on overcoming the inhibitory effect of factor VII on thrombin generation.

Blood Coagulation↗
PubMed1996

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.

Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.

Calcium↗
PubMed2003

Potential role of recombinant factor VIIa as a hemostatic agent.

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.

Blood Platelet Disorders↗
PubMed2004

Inhibitors of Tissue Factor. Factor VIIa for anticoagulant therapy.

Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of an amino-terminal gamma-carboxyglutamic acid-rich (Gla) domain and two epidermal growth factor (EGF)-like domains in the light chain, and a chymotrypsin-like serine protease domain in the heavy chain. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII). Inhibition of TF.FVIIa activity may prevent the formation of fibrin clots and thus be useful in the management of thrombotic disease. The development of TF.FVIIa inhibitors to validate this target has been of great interest. A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF.FVIIa complex has been pursued. This has been greatly aided from our understanding of the structures for TF, FVII, FVIIa, and the TF.FVIIa complex. These approaches have resulted in inhibitors directed specifically towards either FVIIa or TF. Antagonists include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII/FVIIa antibodies, naturally-occurring protein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that may interfere with substrate, membrane, or cofactor binding. The rationale of TF.FVIIa as a target and the development, characteristics and biological uses of TF.FVIIa inhibitors are discussed.

Animals↗
PubMed1996

Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3.

In an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E. coli. Assays for procoagulant activity were carried out with the resulting E. coli lysates using (HVIIa) human and mouse (MVIIa). The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa. By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities. With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X. Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗