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Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m301006200

1083-351X

Lindsay M. Watson, Anthony K.C. Chan, Leslie R. Berry, Jun Li, Sudesh K. Sood, Jeffrey G. Dickhout, Ling Xu, Geoff H. Werstuck, Laszlo Bajzar, Henry J. Klamut, Richard C. Austin,

Heat shock proteins research

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca2+ and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells. Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca2+ and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells. endoplasmic reticulum 78-kDa glucose-regulated protein/immunoglobulin-binding protein von Willebrand factor tissue factor procoagulant activity activated partial thromboplastin time thrombin-antithrombin macrophage colony-stimulating factor fetal bovine serum phosphate-buffered saline bovine serum albumin radioimmune precipitation assay buffer plaque-forming unit horseradish peroxidase reactive oxygen species Chinese hamster ovary human aortic smooth muscle cells In eukaryotic cells, the endoplasmic reticulum (ER)1 is the cellular organelle where secretory proteins or proteins destined for the plasma membrane undergo a variety of modifications, including disulfide bond formation, glycosylation, folding, and oligomeric assembly. The inability of nascent polypeptide chains to fold into their native conformation generally leads to retention in the ER and degradation (1Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (562) Google Scholar). To assist in the correct folding of newly synthesized proteins and to prevent aggregation of folding intermediates, the ER contains a wide range of molecular chaperones such as the glucose-regulated proteins, calnexin, calreticulin, protein-disulfide isomerase, and Erp72 (2Gething M.-J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3606) Google Scholar, 3Ruddon R.W. Bedows E. J. Biol. Chem. 1997; 272: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). These chaperones are postulated to act as a quality control system by ensuring that only correctly folded proteins are processed prior to entering the Golgi apparatus for further processing and secretion (2Gething M.-J. Sambrook J. 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Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar) provides further evidence that GRP78/BiP plays a critical role in quality control processes during protein synthesis and folding. Recent studies have demonstrated that alterations in GRP78/BiP protein levels mediate selective changes in the abundance of membrane or secretory proteins that transit the ER. In mammalian cells, reduced levels of GRP78/BiP increase the secretion of factor VIII and a mutant form of tissue plasminogen activator lacking glycosylation sites (18Dorner A.J. Krane M.G. Kaufman R.J. Mol. Cell. Biol. 1988; 8: 4063-4070Crossref PubMed Scopus (134) Google Scholar, 19Dorner A.J. Wasley L.C. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7429-7432Crossref PubMed Scopus (116) Google Scholar, 20Dorner A.J. Wasley L.C. Kaufman R.J. EMBO J. 1992; 11: 1563-1571Crossref PubMed Scopus (301) Google Scholar). 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Belotti D. Consonni R. D'Orazio A. Robba L. Donati M.B. Barbui T. Blood. 1995; 86: 1072-1081Crossref PubMed Google Scholar, 53Rao L.V.M. Cancer Metastasis Rev. 1992; 11: 249-266Crossref PubMed Scopus (163) Google Scholar, 54Rickles F.R. Hair G.A. Zeff R.A. Lee E. Bona R.D. Thromb. Haemost. 1995; 74: 391-395Crossref PubMed Scopus (71) Google Scholar), and the prothrombotic state observed in cancer patients has been largely attributed to this expression (53Rao L.V.M. Cancer Metastasis Rev. 1992; 11: 249-266Crossref PubMed Scopus (163) Google Scholar). Given that TF transits the ER prior to its localization in the plasma membrane, we have postulated that GRP78/BiP overexpression could potentially alter the prothrombotic potential of cells. In this study, GRP78/BiP was stably overexpressed in a human bladder cancer cell line T24/83 having prothrombotic characteristics. Our findings indicate that overexpression of GRP78/BiP significantly decreases TF-mediated cell surface thrombin generation. The finding that GRP78/BiP overexpression fails to decrease cell surface levels of TF suggests that the inhibition of TF PCA by GRP78/BiP does not result from the retention of TF in the ER. The ability of GRP78/BiP overexpression to attenuate TF PCA induced by ionomycin, hydrogen peroxide, or adenovirus supports a mechanism involving cellular Ca2+ and/or oxidant stress. These findings show that GRP78/BiP overexpression decreases TF PCA and provides novel evidence that alterations in the levels of GRP78/BiP can have profound effects on the prothrombotic potential of cells. The human transitional bladder carcinoma cell line T24/83 was obtained from the American Type Culture Collection and cultured in M199 medium containing 107 fetal bovine serum (FBS), 100 ॖg/ml penicillin, and 100 ॖg/ml streptomycin in a humidified incubator at 37 °C with 57 CO2. Although previous studies classified T24/83 cells as an immortalized human umbilical vein endothelial cell line, designated ECV304 (55Takahashi K. Sawasaki Y. Hata J. Mukai K. Goto T. In Vitro Cell Dev. Biol. 1990; 26: 265-274Crossref PubMed Scopus (418) Google Scholar), recent genetic analysis and functional responses have demonstrated that ECV304 cells are indeed T24/83 (56Brown J. Reading S.J. Jones S. Fitchett C.J. Howl J. Martin A. Longland C.L. Michelangeli F. Dubrova Y.E. Brown C.A. Lab. Invest. 2000; 80: 37-45Crossref PubMed Scopus (179) Google Scholar). Human aortic smooth muscle cells (HASMC) were purchased from Cascade Biologicals (Portland, OR) and cultured in medium 231 containing 207 smooth muscle growth supplement and PSA solution (100 units/ml penicillin, 100 ॖg/ml streptomycin, and 0.25 ॖg/ml amphotericin) (Cascade Biologicals). Construction of the mammalian expression vector, pcDNA3.1(+)- GRP78/BiP, encoding human GRP78/BiP and the establishment of stable T24/83 cell lines overexpressing human GRP78/BiP have been described previously (57Werstuck G.H. Lentz S.R. Dayal S. Hossain G.S. Sood S.K. Shi Y.Y. Zhou J. Maeda N. Krisans S.K. Malinow M.R. Austin R.C. J. Clin. Invest. 2001; 107: 1263-1273Crossref PubMed Scopus (599) Google Scholar). As a vector control, T24/83 cells were transfected with pcDNA3.1(+) under the same conditions. Overexpression of GRP78/BiP was assessed by indirect immunofluorescence and immunoblot analysis, as described below. Monoclonal and polyclonal antibodies to human TF were purchased from American Diagnostica (Greenwich, CT). The anti-KDEL monoclonal antibody (SPA-827), which recognizes both GRP78/BiP and GRP94, was purchased from StressGen Biotechnologies(Victoria, BC). Total protein lysates from T24/83 cells were solubilized in Laemmli sample buffer (50 mm Tris, pH 6.8, 27 SDS, 107 glycerol, 17 औ-mercaptoethanol, 0.017 bromphenol blue), separated on SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad), as described previously (58Outinen P.A. Sood S.K. Liaw P.C.Y. Sarge K.D. Maeda N. Hirsh J. Ribau J. Podor T.J. Weitz J.I. Austin R.C. Biochem. J. 1998; 332: 213-221Crossref PubMed Scopus (212) Google Scholar). After incubation with the appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity Biologicals, Hamilton, ON), the membranes were developed using the Renaissance chemiluminescence reagent kit (PerkinElmer Life Sciences). Polyclonal antibodies to GRP78/BiP (sc-1050) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunofluorescence for GRP78/BiP was performed as described previously (58Outinen P.A. Sood S.K. Liaw P.C.Y. Sarge K.D. Maeda N. Hirsh J. Ribau J. Podor T.J. Weitz J.I. Austin R.C. Biochem. J. 1998; 332: 213-221Crossref PubMed Scopus (212) Google Scholar, 59Outinen P.A. Sood S.K. Pfiefer S.I. Pamidi S. Podor T.J. Li J. Weitz J.I. Austin R.C. Blood. 1999; 94: 959-967Crossref PubMed Google Scholar). For detection of cell surface levels of TF, cells were washed with ice-cold 1× PBS for 2 min, blocked with 37 BSA in 1× PBS for 1 h at 4 °C and incubated with an anti-human TF monoclonal antibody (5 ॖg/ml) for 1 h at 4 °C. After three washes with 1× PBS containing 0.57 BSA, cells were incubated with the appropriate Alexa-labeled secondary antibody (BioLynx, Brockville, ON) for 1 h at 4 °C, washed again, and fixed with 17 paraformaldehyde. Images were subsequently captured and analyzed using Northern Exposure image analysis/archival software (Mississauga, ON). Cell surface TF was immunoprecipitated essentially as described previously (42Penn M.S. Patel C.V. Cui M.-Z. DiCorleto P.E. Chisolm G.M. Circulation. 1999; 99: 1753-1759Crossref PubMed Scopus (57) Google Scholar). Briefly, anti-human TF polyclonal antibodies at 0.5 ॖg/ml in FBS-free M199 medium were added to cell monolayers at 4 °C for 2 h. After washing twice with ice-cold 1× PBS, cells were lysed in ice-cold RIPA buffer (50 mm Tris, 150 mm NaCl, 17 Nonidet P-40, 0.57 sodium deoxycholate, 0.17 SDS) containing protease inhibitors (Roche Diagnostics, Laval, QC), and cell surface TF protein was immunoprecipitated from total cell protein by overnight incubation with protein A-Sepharose-linked beads (Pierce). After washing with RIPA buffer, the beads were boiled in Laemmli sample buffer, and the immunoprecipitates were separated on 107 SDS-polyacrylamide gels under reducing conditions. Gels were transferred to nitrocellulose membranes and immunoblotted with anti-human TF polyclonal antibodies, as described above. Cell surface biotinylation was performed as described previously (60Xiao G. Chung T.-F. Fine R.E. Johnson R.J. J. Neurosci. Res. 1999; 58: 652-662Crossref PubMed Scopus (30) Google Scholar, 61Xiao G. Chung T.-F. Pyun H.Y. Fine R.E. Johnson R.J. Mol. Brain Res. 1999; 72: 121-128Crossref PubMed Scopus (97) Google Scholar). Briefly, cell monolayers were washed once with ice-cold FBS-free M199 medium and three times with ice-cold 1× PBS to remove residual FBS and other proteins from the culture medium. PBS containing 1 mg/ml EZ Link NHS-SS-Biotin (Pierce) was added to the monolayers, and the biotinylation reaction was carried out at room temperature for 30 min with gentle shaking. Following three washes with ice-cold 1× PBS, cells were solubilized in Laemmli sample buffer. Proteins were separated on 107 SDS-polyacrylamide gels and transferred to nitrocellulose membranes. To detect biotinylated cell surface proteins, immunoblot analysis was performed using HRP-conjugated ExtrAvidin (Sigma). Thrombin generation studies were conducted with either control pooled plasma from healthy adults or factor VII-deficient plasma (Affinity Biologicals). Factor VII levels in the deficient plasmas were ≤17, according to the manufacturer. Prior to use in the thrombin generation assay, plasmas were defibrinated using arvin and total amidolytic activity of thrombin generated on cell surfaces was measured as previously described (62Chan A.K.C. Berry L. Mitchell L. Baranowski B. O'Brodovich H. Andrew M. Am. J. Physiol. 1998; 274: L914-L921Crossref PubMed Google Scholar,63Ling X. Delorme M. Berry L. Ofosu F. Mitchell L. Paes B. Andrew M. Pediatr. Res. 1995; 37: 373-378Crossref PubMed Scopus (44) Google Scholar). Cell monolayers in 24-well plates were placed on a Thermolyne dri-bath set at 37 °C. After washing twice with 1 ml of acetate/barbital/saline (ABS) buffer (0.036 m sodium acetate, 0.036 m sodium diethylbarbitarate, 0.145m NaCl, pH 7.4), monolayers were incubated for 3 min with 100 ॖl of ABS buffer and 200 ॖl of defibrinated plasma, in the absence or presence of 107 activated partial thromboplastin time (APTT) reagent (Organon Teknika Corp., Durham, NC). At time periods up to 30 min following the addition of 100 ॖl of 40 mmCaCl2 in ABS buffer, 25-ॖl aliquots of the reaction mixture on the surface of the cells were removed and mixed with 475 ॖl of 5 mm EDTA on ice. 25 ॖl of each EDTA sample were then mixed with 775 ॖl of 0.16 mm S-2238 (KabiVitum, Stockholm, Sweden) in buffer and heated at 37 °C for 10 min prior to termination of the amidolytic reaction with 200 ॖl of 507 acetic acid. The absorbance at 405 nm was measured and the concentration of total thrombin determined by comparing results to a standard curve generated with purified thrombin in S-2238. EDTA samples were also used to measure the concentrations of prothrombin, thrombin-antithrombin (TAT) complexes and thrombin-heparin cofactor II complexes. Prothrombin, TAT, and thrombin-heparin cofactor II complexes were assayed using commercially available ELISA kits (Affinity Biologicals). Because thrombin bound to α2 macroglobulin (α2M) retains amidolytic activity against S-2238 (64Hemker H.C. Verstraete M. Vermylen J. Lijinen R. Anrout J. Thrombosis and Haemostasis. Leuven University Press, Leuven1987: 17-36Google Scholar), the contribution of thrombin-α2M to total thrombin activity was measured as previously described (63Ling X. Delorme M. Berry L. Ofosu F. Mitchell L. Paes B. Andrew M. Pediatr. Res. 1995; 37: 373-378Crossref PubMed Scopus (44) Google Scholar). Briefly, the amidolytic activity of total thrombin was measured as described above, except that the 25-ॖl reaction mixture taken at each time point was incubated with 3.5 ॖl of 0.15 m NaCl containing 0.25 units of standard heparin and 0.042 units of antithrombin (to inhibit any free thrombin) for 1 min on ice, followed by the addition of 475 ॖl of 5 mm EDTA. α2M-dependent thrombin activity was then subtracted from the total thrombin activity to give the amount of physiologically active free thrombin generated by the cell surface. EDTA samples were used to determine prothrombin consumption during the experiments. Prothrombin concentrations were determined for each time point during the thrombin generation experiments using a commercially available ELISA kit. Control plasma with a known concentration of prothrombin was used as a standard. Factor VIIa generation on the surface of T24/83 cells was performed by a one-step assay, which directly measures the conversion of factor VII to factor VIIa, using the chromogenic substrate S-2288 (65Wildgoose P. Foster D. Schiodt J. Wiberg F.C. Birktoft J.J. Petersen L.C. Biochemistry. 1993; 32: 114-119Crossref PubMed Scopus (61) Google Scholar). Cell monolayers were washed twice in ABS buffer and incubated for time periods up to 30 min in the absence (blank control) or presence of 108 nm recombinant human factor VII (Enzyme Research Laboratories, South Bend, IN) and 5 mm Ca2+ in ABS buffer. The generation of factor VIIa was assessed by subsampling cell surface supernatant into S-2288, followed by incubation at 37 °C for 30 min. After termination with 507 acetic acid, absorbance at 405 nm was determined for each sample. The rates of factor VIIa formation in the various samples were calculated based on a standard curve using known amounts of human factor VIIa (Enzyme Research Laboratories) and TF (Thromborel S, Dade Behring, Newark, DE). Cell surface TF activity, measured as the amount of factor Xa generated, was performed as described previously (66Hansen C.B. van Deurs B. Petersen L.C. Rao L.V.M. Blood. 1999; 94: 1657-1664Crossref PubMed Google Scholar, 67Nishibe T. Parry G. Ishida A. Aziz S. Murray J. Patel Y. Rahman S. Strand K. Saito K. Saito Y. Hammond W.P. Savidge G.F. Mackman N. Wijelath E.S. Blood. 2001; 97: 692-699Crossref PubMed Scopus (38) Google Scholar). Briefly, cells were seeded onto 24-well tissue culture plates and upon reaching confluence, the culture medium was removed, and the cells were washed once with 1× PBS and incubated with FBS-free medium for 1 h at 37 °C. Cells in FBS-free medium were untreated or treated with ionomycin (5–10 ॖm for 10 min) or hydrogen peroxide (10 mmfor 1 h). Cells were washed twice with 1× TBS (50 mmTris, 120 mm NaCl, 2.7 mm KCl, 3 mg/ml BSA, pH 7.4) and incubated with 300 ॖl of 1× TBS containing 0.5 nm human factor VIIa and 15 nm human factor X (Enzyme Research Laboratories). Activation of factor X was initiated by the addition of 5 mm CaCl2 for 30 min at 37 °C. 250-ॖl aliquots were removed and incubated with 25 ॖl of chromogenic substrate S-2765 (0.2 mm) (DiaPharma, West Chester, OH) for 3 min at 37 °C. The reaction was terminated using 20 ॖl of 507 acetic acid, and the absorbance at 405 nm was measured using a microplate reader. Standards containing the reaction mixture and various amounts of rabbit brain thromboplastin (Thromboplastin C Plus, Dade Behring) were also prepared and incubated with S-2765 as described above. The absorbance at 405 nm was determined and used to generate a standard curve where 100 units of TF activity was defined as the amount of activity in 1 ॖl of rabbit brain thromboplastin. Cells in the 24-well plates were lysed in RIPA buffer, and the amount of total protein was measured using the DC Protein Assay (Bio-Rad) according to the manufacturer's instructions. Absorbances were measured at 750 nm and the amount of total protein calculated by comparing the OD750 with ref