Binding of Anti-GRP78 Autoantibodies to Cell Surface GRP78 Increases Tissue Factor Procoagulant Activity via the Release of Calcium from Endoplasmic Reticulum Stores

Ali A Al-Hashimi,Jennifer Caldwell,Mario Gonzalez-Gronow,Salvatore V Pizzo,Danya Aboumrad,Lindsay Pozza,Hiam Al-Bayati,Jeffrey I Weitz,Alan Stafford,Howard Chan,Anil Kapoor,Donald W Jacobsen,Jeffrey G Dickhout,Richard C Austin

doi:10.1074/jbc.m110.119107

Abstract

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells. Recent studies have shown that TF PCA can be modulated by GRP78, an endoplasmic reticulum (ER)-resident molecular chaperone. In this study, we investigated the role of cell surface GRP78 in modulating TF PCA in several human cancer cell lines. Although both GRP78 and TF are present on the cell surface of cancer cells, there was no evidence of a stable interaction between recombinant human GRP78 and TF, nor was there any effect of exogenously added recombinant GRP78 on cell surface TF PCA. Treatment of cells with the ER stress-inducing agent thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca(2+) pump that causes Ca(2+) efflux from ER stores, increased cytosolic [Ca(2+)] and induced TF PCA. Consistent with these findings, anti-GRP78 autoantibodies that were isolated from the serum of patients with prostate cancer and bind to a specific N-terminal epitope (Leu(98)-Leu(115)) on cell surface GRP78, caused a dose-dependent increase in cytosolic [Ca(2+)] and enhanced TF PCA. The ability to interfere with cell surface GRP78 binding, block phospholipase C activity, sequester ER Ca(2+), or prevent plasma membrane phosphatidylserine exposure resulted in a significant decrease in the TF PCA induced by anti-GRP78 autoantibodies. Taken together, these findings provide evidence that engagement of the anti-GRP78 autoantibodies with cell surface GRP78 increases TF PCA through a mechanism that involves the release of Ca(2+) from ER stores. Furthermore, blocking GRP78 signaling on the surface of cancer cells attenuates TF PCA and has the potential to reduce the risk of cancer-related venous thromboembolism.

Highlights

The increased risk of venous thromboembolism in cancer patients has been attributed to enhanced tissue factor (TF) procoagulant activity (PCA) on the surface of cancer cells
The engagement of anti-GRP78 autoantibodies with cell surface GRP78 may explain how TF is activated on cancer cells and contributes to the hypercoagulable state observed in cancer patients
GRP78 was observed throughout the cell surface; punctuate regions of high GRP78 expression were apparent above the nucleus

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cancer cell lines were purchased from the American Tissue Culture Collection (Manassas, VA). To examine surface TF, cells were incubated (1:100 dilution) in the presence of a rabbit anti-human TF antibody Indirect Immunofluorescence—T24/83 cells grown on coverslips were washed with Hanks’ balanced salt solution (HBSS) (Invitrogen) containing 1 mM CaCl2, 1 mM MgCl2, and fixed for 30 min at room temperature in 4% fresh formaldehyde in 1ϫ PBS. Excess blocking buffer was removed from the slide, and cells were incubated with a primary sheep anti-GRP78 antibody, goat anti-human tissue factor The cells were washed three times in 1ϫ PBST and incubated with a secondary antibody containing a 1:1000 dilution of an Alexa Fluor 568-conjugated donkey anti-sheep IgG, an Alexa Fluor 488-conjugated rabbit anti-goat IgG, or a mixture of the two for 90 min at 4 °C in the dark.

Reverse primer

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS