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PubMed2001

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity.

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.

Amino Acid Substitution↗
PubMed2002

Factor VII deficiency in pregnancy treated with recombinant factor VIIa.

BACKGROUND: Factor VII deficiency is an autosomal recessive bleeding disorder with an incidence of one in 500,000. Few cases have been reported in pregnancy, and only two patients were treated with recombinant factor VIIa. In the past, fresh frozen plasma or factor VII concentrate has been the conventional treatment for this disorder. CASE: We report a case of factor VII deficiency diagnosed during pregnancy with a factor VII level of 1%. After prophylactic treatment with recombinant factor VIIa, the patient did not manifest any signs or symptoms of excessive bleeding during labor or postpartum. CONCLUSION: Given the inherent risks of transmission of human immunodeficiency virus and hepatitis with blood products, recombinant factor VIIa treatment is an alternative that is safe and effective during labor, delivery, and the puerperium.

Delivery, Obstetric↗
PubMed1997

Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Two sites in the tissue factor extracellular domain mediate the recognition of the ligand factor VIIa.

Tissue factor (TF) binds the serine protease coagulation factor VIIa and initiates the coagulation protease cascade by forming a catalytic cofactor-enzyme complex. Using a photoactivatable crosslinking reagent coupled to factor VIIa, we have identified interactive sites in the amino-terminal (residues 44-84) and the carboxyl-terminal (residues 129-169) aspect of the extracellular domain of TF. Epitopes of inhibitory antibodies have previously indicated participation of these regions in TF function. The presence of the gamma-carboxyglutamic acid domain in factor VIIa appears to facilitate the interaction with the negatively charged, amino-proximate site, whereas crosslinking of TF with VIIa or des-(1-38)-VIIa at the positively charged carboxyl-proximate site was similar. Lack of alpha-helical secondary structure in the TF extracellular domain is consistent with the proposed structural similarity of TF with the cytokine receptor family. The interactive sites identified for TF are located in sequence spans that demonstrate a low degree of sequence conservation among the members of this receptor family. Regions with highly conserved residues, such as sequences encoded by exon 2 and 5 in TF, were not implicated in ligand recognition, suggesting that conserved residues in the receptor family may maintain the common beta-strand architecture, and variable regions provide a pair of nonidentical motifs for oriented ligand recognition.

Affinity Labels↗
PubMed1987

Regulation of factor VIIa activity in plasma: evidence that antithrombin III is the sole plasma protease inhibitor of human factor VIIa.

The inhibition of human factor VIIa by antithrombin III and normal human plasma was studied in the presence and absence of heparin. In the absence of heparin, no apparent inhibition of factor VIIa was observed in either system. In the presence of heparin, factor VIIa activity was inhibited 50% by purified antithrombin III and plasma in 90 min and 75 min, respectively. No inhibition of factor VIIa was observed in heparinized plasma previously depleted of antithrombin III by immunoaffinity adsorption. Incubation of factor VIIa with antithrombin III-heparin or heparinized plasma resulted in the formation of a covalent complex with an apparent molecular weight of 100 kilodaltons. These data indicate that antithrombin III appears to be the sole plasma protease inhibitor of human factor VIIa, and the expression of its inhibitory activity against factor VIIa is absolutely dependent upon the presence of exogenous heparin.

Antithrombin III↗
PubMed2000

Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa x tissue factor-induced signal transduction.

Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on cell surfaces trigger the intracellular signaling events. At present, it is unclear how these signals influence phenotype. To elucidate this, we have used cDNA microarray technology to examine changes in transcriptional program in human fibroblasts in response to exposure to FVIIa. cDNA microarrays revealed that FVIIa binding to TF up-regulated the expression of Cyr61 and CTGF (connective tissue growth factor), the genes that encode extracellular matrix signaling proteins Cyr61 and CTGF, respectively. Northern blot analysis confirmed that FVIIa binding to TF markedly increased the expression of Cyr61 and CTGF in a time- and dose-dependent manner. FVIIa catalytic activity is required for the gene induction. In addition to FVIIa, thrombin also induced the expression of Cyr61 and CTGF. Hirudin abolished the thrombin-induced expression of these mRNAs but not the FVIIa-induced expression. FVIIa-induced expression of Cyr61 appears not to involve the currently known protease-activated receptors (PARs), whereas thrombin-induced expression involves the activation of PAR1 and possibly an additional PAR. Various intracellular signaling pathway inhibitors exhibited different inhibitory pattern on FVIIa and thrombin-induced up-regulation of Cyr61. Cyr61 and CTGF could act as downstream mediators of FVIIa x TF in affecting various biological processes.

Cell Line↗
PubMed1996

Influence of the gamma-carboxyglutamic acid-rich domain and hydrophobic stack of factor VIIa on tissue factor binding.

Des (1-38)- and des(1-44)-factor VIIa (fVIIa) were inhibited with Phe-Phe-Arg-chloromethyl ketone (FFR-cmk). Des(1-38)-FFR-fVIIa inhibited tissue factor (TF)-enhanced fVIIa amidolytic activity with an IC50 value of 15 nM, whereas 3- and 6-fold higher values were obtained with des(1-44)-FFR-fVIIa using soluble and full-length TF, respectively. The value for FFR-fVIIa was 8-10 nM. Clotting time was prolongated with IC50 values for inactivated des(1-38)- and des(1-44)-fVIIa of 50 nM and 1 mu M, respectively, whereas FFR-fVIIa yielded a value of 10 nM. From binding experiments in a BIA-core instrument, dissociation constants for the complexes between TF1-218 and fVIIa, des(1-38)-fVIIa and des(1-44)-fVIIa of about 3, 20 and 70 nM, respectively, could be estimated.

1-Carboxyglutamic Acid↗
PubMed1997

Characterization of the interaction between the light chain of factor VIIa and tissue factor.

Factor VIIa (fVIIa) consists of a heavy chain (serine protease domain) and a light chain (gamma-carboxyglutamic acid (Gla)-rich and epidermal growth factor (EGF)-like domains). The light chain, primarily the first EGF-like domain, appears to provide most of the binding energy in the interaction with tissue factor (TF). The Ca2+-binding sites in the protease domain and in the first EGF-like domain influence activity and interaction with TF, but the contribution from the Ca2+-binding sites in the Gla domain has not been established. We have compared the soluble TF (sTF)-binding properties of intact fVIIa to those of a fragment comprising almost the entire light chain and a small disulphide-linked peptide from the protease domain. Half-maximal binding of fVIIa and the light chain to sTF occurred around 0.3 and 1 mM Ca2+, respectively. The Ca2+ dependence of light-chain binding indicates an influence of Ca2+ binding to the Gla domain on the interaction between fVIIa and sTF. Comparison of the sTF-binding properties of fVIIa and a truncated variant lacking the Gla domain suggests that this domain interferes with sTF association at suboptimal Ca2+ concentrations. The light chain of fVIIa associated 5-fold slower with sTF than did fVIIa at saturating Ca2+ concentrations, whereas the dissociation of its complex with sTF was at least 100-fold faster than that of fVIIa:sTF. This gave a dissociation constant of 1-2 microM for the interaction between the light chain and sTF compared to about 3 nM for the fVIIa:sTF interaction.

1-Carboxyglutamic Acid↗
PubMed1997

The effect of active site-inhibited factor VIIa on tissue factor-initiated coagulation using platelets before and after aspirin administration.

Active site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIIa (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 +/- 0.8 nM (n = 8) and 0.9 +/- 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes and Ki for FFR-FVIIa competing with factor VIIa were similar (11.4 +/- 0.8 pM and 10.6 +/- 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 +/- 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 +/- 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Adult↗
PubMed2005

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway.

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.

Blood Coagulation↗
PubMed2003

Regulation of tissue factor-factor VIIa expression on cell surfaces: a role for tissue factor-factor VIIa endocytosis.

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa (FVIIa) and the formation of TF-VIIa complexes on cell surfaces triggers the coagulation cascade. Further, TF-VIIa, either directly or indirectly, influences various biological processes, such as development, inflammation and tumor metastasis. Therefore, a proper regulation of TF-VIIa expression is critical for the maintenance of hemostatic balance and health in general. TF-VIIa functional expression on cell surfaces is regulated primarily by transcriptional regulation of TF gene or by specific plasma inhibitors, particularly tissue factor pathway inhibitor (TFPI). However, a number of other mechanisms that are yet to be well defined also regulate TF-VIIa functional expression. One such mechanism is the endocytosis of TF-VIIa. In this article, we provide a comprehensive review on the regulation of TF-VIIa functional expression by these other mechanisms, with a particular emphasis on TF-VIIa endocytosis, and our perspective of these studies.

Animals↗
PubMed1999

Probing the structural changes in the light chain of human coagulation factor VIIa due to tissue factor association.

The crystallographic structure of human coagulation factor VIIa/tissue factor complex bound with calcium ions was used to model the solution structure of the light chain of factor VIIa (residues 1-142) in the absence of tissue factor. The Amber force field in conjunction with the particle mesh Ewald summation method to accommodate long-range electrostatic interactions was used in the trajectory calculations. The estimated TF-free solution structure was then compared with the crystal structure of factor VIIa/tissue factor complex to estimate the restructuring of factor VIIa due to tissue factor binding. The solution structure of the light chain of factor VIIa in the absence of tissue factor is predicted to be an extended domain structure similar to that of the tissue factor-bound crystal. Removal of the EGF1-bound calcium ion is shown by simulation to lead to minor structural changes within the EGF1 domain, but also leads to substantial relative reorientation of the Gla and EGF1 domains.

Amino Acid Sequence↗
PubMed1996

The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor.

Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extracellular domain of tissue factor. In the complex, factor VIIa adopts an extended conformation. This structure provides a basis for understanding many molecular aspects of the initiation of coagulation.

Amino Acid Sequence↗
PubMed1997

Platelet activity of high-dose factor VIIa is independent of tissue factor.

High-dose recombinant factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high-dose factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on tissue factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which factor VIIa acts on available lipid independently of tissue factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that factor VIIa bound weakly to activated platelets (Kd approximately 90 nM). This factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, factor X, factor V, antithrombin III and tissue factor pathway inhibitor. The activity was not dependent on tissue factor. The concentration of factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of factor VIIa required for efficacy in patients. High-dose factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high-dose factor VIIa was haemostatically effective without causing systemic activation of coagulation.

Blood Platelets↗
PubMed1994

The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor VIIa.

Tissue factor (TF), an integral membrane protein, is the cofactor for the serine protease, coagulation factor VIIa (FVIIa). Previous studies of the isolated extracellular domain of TF (sTF) reported a kcat for factor X (FX) activation by the sTF.VIIa complex only 4% that of wild-type TF.VIIa and furthermore, a complete inability of sTF to support FVII autoactivation. We now report that in the presence of poly(L-lysine), sTF promoted both FX activation and FVII autoactivation, the latter with an apparent second-order rate constant higher than reported previously for wild-type TF in phospholipid vesicles. This led us to reexamine the cofactor ability of sTF, using high concentrations of phospholipid to promote nearly quantitative binding of sTF.VII(a) complexes to the phospholipid surface. Rate constants for the activation of FX or FVII by sTF.VIIa were similar to those of wild-type TF.VIIa, indicating that the apparent deficiency of sTF is largely attributable to kinetic consequences of relatively weak affinity of FVIIa (and sTF.VIIa) for phospholipid surfaces in these surface-dependent reactions, compared with TF being embedded in the membrane. This supports the notion that sTF recapitulates the protein-protein interactions of wild-type TF with high, and possibly full, catalytic activity. It also provides a biochemical explanation for the specificity of our recently described, sTF-based clotting assay for plasma FVIIa.

Factor VIIa↗
PubMed2003

The effect of temperature and pH on the activity of factor VIIa: implications for the efficacy of high-dose factor VIIa in hypothermic and acidotic patients.

BACKGROUND: Recombinant coagulation factor VIIa (FVIIa) is approved for treating hemophiliacs with inhibitors. High-dose FVIIa has also been used off-label to manage hemorrhage in trauma and surgical patients, many of whom also develop hypothermia and acidosis. METHODS: We examined the activity of FVIIa on phospholipid vesicles in the presence and absence of tissue factor (TF) and on platelets as a function of temperature and pH. RESULTS: FVIIa activity on phospholipids and platelets was not reduced at 33 degrees C compared with 37 degrees C. The activity of FVIIa/TF was reduced by 20% at 33 degrees C compared with 37 degrees C. A pH decrease from 7.4 to 7.0 reduced the activity of FVIIa by over 90% and FVIIa/TF by over 60%. CONCLUSION: FVIIa should be effective in enhancing hemostasis in hypothermic patients. However, because the activity of FVIIa is so dramatically affected by pH, its efficacy may be reduced in acidotic patients.

Acidosis↗
PubMed1998

Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor.

The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

Animals↗