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1.
Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line 总被引:14,自引:1,他引:13
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Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings. 相似文献
2.
Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium 总被引:1,自引:0,他引:1
Ghosh S Pendurthi UR Steinoe A Esmon CT Rao LV 《The Journal of biological chemistry》2007,282(16):11849-11857
Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. (125)I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively (125)I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in (125)I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR. 相似文献
3.
Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF. 相似文献
4.
A Role for Tissue Factor in Cell Adhesion and Migration Mediated by Interaction with Actin-binding Protein 280 总被引:22,自引:0,他引:22
Ilka Ott Edgar G. Fischer Yohei Miyagi Barbara M. Mueller Wolfram Ruf 《The Journal of cell biology》1998,140(5):1241-1253
Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling. 相似文献
5.
Martin Müller Conrad Flössel Michael Haase Thomas Luther Sybille Albrecht Peter Paul Nawroth Youming Zhang 《Virchows Archiv. B, Cell pathology including molecular pathology》1993,64(1):265-269
Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours.
The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene
expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer
cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable
cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing
logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and
to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced
localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells
TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony
pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns
as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated
TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and
differentiation of breast cancer cells in culture. 相似文献
6.
Iakhiaev A Pendurthi UR Voigt J Ezban M Vijaya Mohan Rao L 《The Journal of biological chemistry》1999,274(52):36995-37003
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. 相似文献
7.
《The Journal of cell biology》1996,133(2):293-304
The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low- density, detergent-insoluble microdomains that inefficiently support TF- VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity. 相似文献
8.
Rand KD Jørgensen TJ Olsen OH Persson E Jensen ON Stennicke HR Andersen MD 《The Journal of biological chemistry》2006,281(32):23018-23024
Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases. 相似文献
9.
Oskar Eriksson Margareta Ramstr?m Katarina H?rnaeus Jonas Bergquist Dariush Mokhtari Agneta Siegbahn 《The Journal of biological chemistry》2014,289(47):32379-32391
Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2. 相似文献
10.
Dorfleutner A Hintermann E Tarui T Takada Y Ruf W 《Molecular biology of the cell》2004,15(10):4416-4425
In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility. 相似文献
11.
Ndonwi M Broze GJ Agah S Schmidt AE Bajaj SP 《The Journal of biological chemistry》2007,282(21):15632-15644
We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX. 相似文献
12.
Coagulation factor VIIa (FVIIa) is a key protease initiating the coagulation cascade in the presence of its receptor, tissue factor (TF). FVIIa elicits several cellular responses, probably involving other receptors(s) than TF. This study investigates the implication of recombinant FVIIa on the apoptosis of K562 erythroleukemia cells. These cells undergo apoptosis when induced to differentiate towards the erythroid lineage by hemin. They do not express TF, but can be transfected to do so. FVIIa treatment significantly reduced the degree of hemin-induced apoptosis in K562 cells, but not in TF+ derived transfectants. Induction of apoptosis by hemin also elicited decrease in intracellular Ca2+ concentration ([Ca2+]i), but FVIIa restored this [Ca2+]i close to that of non-treated cells. These results suggest that FVIIa acts via a TF-independent pathway to counteract apoptosis by a mechanism involving its Gla domain and linked to the maintenance of Ca2+ homeostasis in K562 cells. 相似文献
13.
Eigenbrot C Kirchhofer D Dennis MS Santell L Lazarus RA Stamos J Ultsch MH 《Structure (London, England : 1993)》2001,9(7):627-636
BACKGROUND: Coagulation factor VIIa (FVIIa) contains a Trypsin-like serine protease domain and initiates the cascade of proteolytic events leading to Thrombin activation and blood clot formation. Vascular injury allows formation of the complex between circulating FVIIa and its cell surface bound obligate cofactor, Tissue Factor (TF). Circulating FVIIa is nominally activated but retains zymogen-like character and requires TF in order to complete the zymogen-to-enzyme transition. The manner in which TF exerts this effect is unclear. The structure of TF/FVIIa is known. Knowledge of the zymogen structure is helpful for understanding the activation transition in this system. RESULTS: The 2 A resolution crystal structure of a zymogen form of FVII comprising the EGF2 and protease domains is revealed in a complex with the exosite binding inhibitory peptide A-183 and a vacant active site. The activation domain, which includes the N terminus, differs in ways beyond those that are expected for zymogens in the Trypsin family. There are large differences in the TF binding region. An unprecedented 3 residue shift in registration between beta strands B2 and A2 in the C-terminal beta barrel and hydrogen bonds involving Glu154 provide new insight into conformational changes accompanying zymogen activation, TF binding, and enzymatic competence. CONCLUSIONS: TF-mediated allosteric control of the activity of FVIIa can be rationalized. The reregistering beta strand connects the TF binding region and the N-terminal region. The zymogen registration allows H bonds that prevent the N terminus from attaining a key salt bridge with the active site. TF binding may influence an equilibrium by selecting the enzymatically competent registration. 相似文献
14.
Henriquez S Calderon C Quezada M Oliva B Bravo ML Aranda E Kato S Cuello MA Gutiérrez J Quest AF Owen GI 《Journal of cellular physiology》2011,226(12):3278-3285
Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits. 相似文献
15.
16.
Andersen LM Andreasen PA Svendsen I Keemink J Østergaard H Persson E 《The Journal of biological chemistry》2012,287(12):8994-9001
In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation. The antibodies could be classified into two groups based on their patterns of affinities for different conformations of FVIIa. Whereas one class of antibodies affected both the K(m) and k(cat), the other class mainly affected the K(m). The antibody-induced activity enhancement could be traced to maturation of the S1 substrate binding pocket and the oxyanion hole, evident by an increased affinity for p-aminobenzamidine, an increased rate of antithrombin inhibition, an increased rate of incorporation of diisopropylfluorophosphate, and an enhanced fraction of molecules with a buried N terminus of the catalytic domain in the presence of antibodies. As demonstrated by site-directed mutagenesis, the two groups of antibodies appear to have overlapping, although clearly different, epitopes in the 170-loop. Our findings suggest that binding of ligands to specific residues in the 170-loop or its spatial vicinity may stabilize the S1 pocket and the oxyanion hole, and they may have general implications for the molecular understanding of FVIIa regulatory mechanisms. 相似文献
17.
The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII. 总被引:1,自引:0,他引:1
Y H Kao G F Lee Y Wang M A Starovasnik R F Kelley M W Spellman L Lerner 《Biochemistry》1999,38(22):7097-7110
The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity. 相似文献
18.
Our previous study has demonstrated a unique biological function of compound 48/80 (48/80) in the downregulation of monocytic tissue factor (TF)-initiated hypercoagulation in response to bacterial endotoxin (lipopolysaccharide, LPS) [A. J. Chu et al. (1999) Biochim. Biophys. Acta 1472, 386-395]. The inhibition was not due to the blockade of LPS cell signaling as evidenced by the unaffected LPS-induced TF synthesis. In the present study, we investigate the direct inhibitory action of 48/80 on the extrinsic coagulation cascade. TF-initiated coagulation was assayed by a single-stage clotting assay. Chromogenic assays dissected the extrinsic pathway to measure the activities of FVII, FX, and prothrombin by monitoring the hydrolyses of nitroaniline-conjugated substrates, identifying the inhibitory site(s). We report that 48/80 in vitro instantaneously inhibited rabbit brain thromboplastin (rbTF)-initiated coagulation in a dose-dependent manner. 48/80 preferentially inhibited FVII activation without any detectable effect on FVIIa, FXa, and thrombin activities. Neither FX activation nor prothrombin activation was affected. The significant inhibition on FVII activation was found to be noncompetitive with a fourfold reduction in the apparent Vmax of FVIIa formation from 7.1 to 1.7 nM/min, while the apparent Km (approximately 365 nM) remained unaffected. Western blotting analysis further confirmed that FVIIa formation derived from FVII was significantly diminished by 48/80, which was accompanied by blocked FVII binding to rbTF. In conclusion, 48/80 readily blocked FVII binding to rbTF, leading to diminished FVII activation and FVIIa formation. As a result, TF-initiated extrinsic coagulation was downregulated. 相似文献
19.
Disse J Petersen HH Larsen KS Persson E Esmon N Esmon CT Teyton L Petersen LC Ruf W 《The Journal of biological chemistry》2011,286(7):5756-5767
Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage. 相似文献
20.
Coagulation factor VIIa (FVIIa) belongs to the chymotrypsin family of S1 peptidases, whose members require the cleavage of at least one internal peptide bond to attain an active conformation. FVIIa also requires association with tissue factor (TF) to attain full catalytic competency. Without this, FVIIa has very low activity toward peptide and physiologic substrates. Reregistration of beta strands has been suggested to play a part in the activation of FVII, and their positioning is possibly important for the active conformation of FVIIa. To scrutinize this hypothesis, we have designed FVIIa variants which prevent beta strand movement and lock FVIIa in the alleged active conformation. The V299M mutation, alone or combined with the L280I mutation, was introduced to alter the first of two Leu-X-Val motifs in beta strand B2 and thereby prevent reregistration. Along the same line, C164V/V299C-FVIIa has a new disulfide which would keep beta strand B2 in the registration of active FVIIa. The amidolytic and proteolytic activities of V299M-, L280I/V299M-, and C164V/V299C-FVIIa were indistinguishable from or lower than those of wild-type FVIIa, and none of the mutants displayed an altered exposure of the N-terminal amino group of the protease domain. Moreover, the affinities of mutant and native FVIIa for TF increased to a similar extent upon incorporation of an active site inhibitor, and the enzymatic activities were equally stimulated by TF. In conclusion, we found no evidence that the mutants were in a more active state than native FVIIa. Thus, the proposed beta strand reregistration, if part of the regulatory mechanism governing FVIIa activity, apparently does not suffice for the transformation of FVIIa into an enzymatically active conformation. Our data raise the possibility that the structural differences between enzymatically latent (zymogen-like) and active FVIIa resemble those between trypsinogen and trypsin. 相似文献