Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

The interaction of human factor VIIa with tissue factor.

The interaction of factor VIIa with tissue factor (TF) results in an increase in the catalytic efficiency for the hydrolysis of several synthetic peptidyl p-nitroanilide substrates by factor VIIa. The binding of human recombinant factor VIIa to recombinant human TF incorporated into vesicles containing phosphatidylcholine (TF/PC) or phosphatidylcholine/phosphatidylserine (TF/PCPS) was studied using the increased rate of H-D-phenylalanyl L-pipecoyl L-arginine p-nitroanilide (S2238) hydrolysis as a signal for the interaction. The saturable dependence of rate on increasing concentrations of factor VIIa or TF/PCPS yielded no obvious evidence for cooperativity and could be analyzed according to the interaction of factor VIIa with independent noninteracting sites (Kd = 259 +/- 60 pM, n = 1.05 +/- 0.12 mol of factor VIIa/mol of TF at saturation). Identical titration curves and equilibrium parameters were derived from titrations using TF/PC or TF in the absence of phospholipids, indicating that possible protein-membrane interactions do not further stabilize the extrinsic Xase complex. The dissociation constant for the interaction of factor VIIa with TF/PCPS inferred from measurements of factor X activation (Kd = 197 +/- 38 pM) was comparable with the values obtained from measurements of S2238 hydrolysis. In contrast to the membrane-independent nature of the enzyme-cofactor interaction, the rate of factor X activation was reduced by approximately 50-fold when the enzyme complex was assembled using solution-phase TF. Collectively, the result indicate that the membrane dependence of extrinsic Xase function primarily results from an influence of the membrane surface on factor X utilization.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Localization of the human tissue factor recognition determinant of human factor VIIa

Tissue factor is an integral membrane glycoprotein that serves as an essential cofactor for the blood coagulation factor VIIa. Recent studies have attempted to localize the tissue factor recognition determinant of human factor VIIa. While several regions of factor VIIa have been implicated as important for tissue factor binding, the high affinity tissue factor recognition determinant of human factor VIIa is unknown. In order to define the determinant, we constructed a set of six chimeric proteins composed of portions of factor VII and factor IX. We then utilized the chimeras in competition experiments with 125I-labeled factor VIIa for recombinant tissue factor bound to an Immobilon-P membrane. The data indicate that the high affinity tissue factor recognition determinant of human factor VIIa is within the epidermal growth factor domains.     

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.     

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.     

基因工程菌生产重组人组织因子的发酵研究

根据表达重组人组织因子基因工程菌的自身特点,在30L发酵罐上,通过控制发酵pH、葡萄糖浓度、诱导物磷酸浓度、搅拌速度及采用分批补料等方法,对重组人组织因子基因工程菌高密度发酵和高效表达的条件进行了研究。实验结果表明:诱导物终浓度低于0.1mmol/L,菌体密度OD60014,发酵周期10.5h,基因工程菌批发酵平均产量为37.41g/L,重组人组织因子表达量为6.56 mg/L。关键词:基因工程菌;高密度发酵;组织因子     

Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Calpha atoms of VIIa or sTF derived from NMA in the VIIa-sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Calpha atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa-sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.     

Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley-Liss, Inc.     

Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.     

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.     

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.     

The evaluation of complex-dependent alterations in human factor VIIa.

Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.     

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Profiling the enzymatic properties and inhibition of human complement factor B

Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.     

Expression of human alpha-fetoprotein in yeast.

Human alpha-fetoprotein (AFP) was expressed in Saccharomyces cerevisiae, with a plasmid containing the cDNA sequence for human AFP fused with the rat AFP signal peptide. The recombinant AFP was purified from the yeast lysate by DEAE-cellulose and immunoaffinity chromatography. The amino acid composition and the molecular weight of the recombinant AFP were similar to those of hepatoma AFP. N-terminal amino acids sequence analysis indicated that the signal peptide had been processed. The recombinant and hepatoma AFP reacted identically in Ouchterlony immunodiffusion and radioimmunoassay tests. These observations indicated that the yeast recombinant protein had the properties of native AFP.     

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.     

Formation and enzymatic properties of the UvrB.DNA complex

The UvrA, UvrB, and UvrC proteins collectively catalyze the dual incision of a damaged DNA strand in an ATP-dependent reaction. We previously reported (Orren, D. K., and Sancar, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5237-5241) that UvrA delivers UvrB to damaged sites in DNA; upon addition of UvrC to these UvrB.DNA complexes, the DNA is incised. In the present study, we have further characterized both the delivery of UvrB to DNA and the subsequent incision process, with emphasis on the role of ATP in these reactions. The UvrA-dependent delivery of UvrB onto damaged DNA is relatively slow (kon approximately 6 x 10(4) M-1 s-1) and requires ATP hydrolysis (Km = 120 microM). Although ATP enhances the stability of UvrB.DNA complexes (koff = 8.5 x 10(-5) s-1), the isolated UvrB.DNA complexes do not contain any covalently attached or stably bound nucleotide. However, ATP binding is required for the UvrC-dependent dual incision of DNA bound by UvrB. Interestingly, adenosine 5'-(3-O-thio)triphosphate can substitute for ATP at this step. The Km for ATP during incision is 2 microM, but ATP is not hydrolyzed at a detectable level during the incision reaction. The incisions made by UvrB-UvrC are on both sides of the adduct and result in the excision of the damaged nucleotide.