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

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

doi:10.1074/jbc.m301006200

Abstract

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 Ca(2+) 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.

Highlights

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
The ability of GRP78/BiP overexpression to attenuate tissue factor (TF) procoagulant activity (PCA) induced by ionomycin, hydrogen peroxide, or adenovirus supports a mechanism involving cellular Ca2ϩ and/or oxidant stress
TF-dependent thrombin generation plays a critical role in hemostasis after tissue injury and in the pathogenesis of multiple thrombotic disorders associated with a wide range of diseases, including cardiovascular disease, sepsis and cancer [21,22,23,24,73]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatment Conditions—The human transitional bladder carcinoma cell line T24/83 was obtained from the American Type Culture Collection and cultured in M199 medium containing 10% fetal bovine serum (FBS), 100 ␮g/ml penicillin, and 100 ␮g/ml streptomycin in a humidified incubator at 37 °C with 5% CO2. Total protein lysates from T24/83 cells were solubilized in Laemmli sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 1% ␤-mercaptoethanol, 0.01% bromphenol blue), separated on SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad), as described previously [58]. 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 [62, 63]. 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. 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 Ca2ϩ in ABS buffer.

RESULTS

Total inhibitor

DISCUSSION