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Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1529-1531
doi: 10.1161/01.ATV.0000177041.47444.e2
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1529.)
© 2005 American Heart Association, Inc.

Editorials

Getting a GRP on Tissue Factor Activation

Lindsay M. Pozza; Richard C. Austin

From the Department of Pathology and Molecular Medicine, McMaster University and the Henderson Research Centre, Hamilton, Ontario, Canada.

Correspondence to Richard C. Austin, Henderson Research Centre, 711 Concession Street, Hamilton, Ontario L8V 1C3. E-mail raustin{at}thrombosis.hhscr.org

Atherothrombosis, defined as atherosclerotic lesion disruption with superimposed thrombosis, is the underlying cause of cardiovascular disease that accounts for the majority of deaths in North America.1 Thrombosis at these sites is triggered by tissue factor (TF), an integral membrane glycoprotein essential for the initiation of blood coagulation.2 TF-dependent procoagulant activity occurs on the surface of cells and is induced by a wide range of physiological agents/conditions, including endotoxin,3 viral infection,4 hypoxia,5 apoptosis,6 homocysteine,7 and reactive oxygen species.8 Furthermore, tumor cells express TF and the prothrombotic state observed in cancer patients has been largely attributed to increased TF procoagulant activity.9 Thus, the ability to inhibit TF-dependent procoagulant activity after plaque disruption would likely alleviate many of the acute clinical manifestations of cardiovascular disease.

See page 1737

Although TF expression is regulated in a cell-specific manner,10 the presence of TF does not necessarily correlate with procoagulant activity given that only a small proportion of TF on the cell surface is active.11 Current thinking is that TF activation occurs at the cell surface when latent or "encrypted" TF is converted into an active or "de-encrypted" form.12–14 The complex formed between de-encrypted TF on the cell surface and circulating factor VII/VIIa acts as a catalyst for the conversion of factors IX and X to IXa and Xa, respectively, thereby triggering thrombin generation. Although previous studies have demonstrated that de-encryption of TF is associated with Ca2+-induced changes in the distribution of cell surface phosphatidylserine,14 it is not solely dependent on these changes. Cross-linking studies suggest another possible mechanism that de-encryption of TF results from the dissociation of TF dimers to monomers on the cell surface upon cell perturbation.12 This change in quaternary structure could potentially expose a macromolecular substrate-binding site on monomeric TF essential for its activation. Alternatively, procoagulant activity could be mediated by additional cellular factors that interact with cell surface TF.

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Bhattacharjee et al15 provide intriguing evidence that TF-dependent procoagulant activity is regulated by the 78-kDa glucose regulated protein (GRP78), an endoplasmic reticulum (ER)-resident molecular chaperone responsible for proper folding of newly synthesized proteins and the prevention of protein aggregates.16 Although previous studies have shown that alterations in GRP78 levels can influence the processing and secretion of several coagulation factors17,18 and that GRP78 overexpression attenuates cell surface TF activation,19 the mechanisms by which GRP78 directly affects components of the coagulation cascade, including TF, are unknown. The present report not only extends previous findings, but also provides a novel cellular mechanism as to how an ER lumenal molecular chaperone regulates TF-dependent procoagulant activity on endothelial cells and macrophages, cell types relevant to atherothrombotic disease. In a non-cell based biochemical assay, the addition of recombinant GRP78 was able to inhibit TF-dependent plasma clotting and factor Xa generation. Using a complementary strategy, antibodies directed against GRP78 blocked the GRP78-mediated inhibition of cell surface TF, as observed by increased procoagulant activity. The additional observation that only antibodies specific to the C-terminal domain of GRP78 enhance TF activity suggests that this structural region in GRP78 is important and necessary for interacting with TF. Further studies involving mutational analysis of the C-terminal domain of GRP78 should provide additional information on the role of this region of GRP78 in regulating the activation of TF.

Previous studies by Watson et al19 have shown that GRP78 overexpression inhibits TF-dependent procoagulant activity. Stable overexpression of GRP78 in the human bladder carcinoma cell line, T24/83, did not (1) impair TF synthesis, (2) decrease cell surface levels of TF, or (3) irreversibly inhibit TF structure and function. However, GRP78 overexpression did attenuate TF procoagulant activity induced by the Ca2+ ionophore, ionomycin, suggesting that alterations in ER Ca2+ stores could play an important role in the regulation of TF activity by GRP78. Given that GRP78 is a major Ca2+-binding protein16 and that TF activation is mediated by changes in intracellular levels of free Ca2+,11,12 overexpression of GRP78 could potentially inhibit TF activity indirectly by sequestering intracellular Ca2+. Although it is not completely understood how alterations in intracellular Ca2+ activate TF, the production of reactive oxygen species is likely involved. It is well established that efflux of Ca2+ from the ER into the cytosol enhances the peroxidase activity of cyclooxygenases and lipoxygenases, thereby leading to the production of reactive oxygen species.20 Furthermore, elevated levels of cytosolic Ca2+ lead to mitochondrial Ca2+ uptake, induction of oxidative stress, membrane peroxidation, and cell dysfunction/death.21,22 The observation that GRP78 overexpression prevents oxidant-induced Ca2+ increases and cell injury22 and attenuates TF procoagulant activity induced by hydrogen peroxide19 suggests a link between intracellular Ca2+ and oxidative stress. However, these studies examined overexpression of exogenous GRP78 and may not completely explain the role of endogenous GRP78 on TF activation.

Although a mechanism involving intracellular Ca2+ and/or reactive oxygen species has been proposed, several other potential mechanisms exist. Exposure of anionic phospholipids on the outer plasma membrane increases TF procoagulant activity and is regulated by Ca2+ levels.14,23 Thus, GRP78 could limit the accessibility of anionic phospholipids essential for TF activation. Because GRP78 directly interacts with numerous proteins transiting the ER, it could impair the processing of proteins that directly interact with and mediate the activity of TF. In addition, it is well established that apoptosis can increase TF activation and is associated with enhanced TF activity within atherosclerotic plaques.6 We as well as others have recently demonstrated that overexpression of GRP78 protects cells from apoptosis elicited by ER stress agents24 or topoisomerase inhibitors.25 Furthermore, ER stress induces apoptosis through the increased expression of proapoptotic mediators, including GADD15326 and TDAG51.27 Given that apoptosis is associated with an increase in TF activity in atherosclerotic plaques, it is possible that GRP78 overexpression could inhibit TF procoagulant activity by attenuating the apoptotic process.

GRP78 contains a C-terminal KDEL retention sequence and is considered to be an ER lumenal protein; however, recent studies have identified GRP78 on the cell surface as well as secreted in cell culture media and in the circulation.28–33 Although GRP78 contains 4 evolutionary conserved hydrophobic domains that can form possible transmembrane helices and a subpopulation of GRP78 can exist as an ER transmembrane protein,25 it is still unknown how GRP78 is oriented in the plasma membrane. Most interestingly, it remains unclear as to how GRP78 can escape the KDEL receptor-dependent ER retrieval mechanism given that GRP78 retains its KDEL sequence when expressed on the cell surface.28 In contrast to its chaperone activity, cell surface GRP78 has been postulated to play the role of a receptor or coreceptor involved in a number of processes including mediating virus internalization via interactions with MHC class I molecules,34 inducing {alpha}2-macroglobulin mitogenic signaling35,36 and as a molecular target on tumor cells possibly related to metastasis.32,33 These observations have allowed the development of novel strategies using cell surface GRP78 to target proapoptotic signals to tumor cells, thereby suppressing tumor growth.32

The findings by Bhattacharjee et al15 that cell surface GRP78 mediates TF procoagulant activity raises important biochemical and physiological questions. First, how is GRP78 translocated from the ER lumen to the cell surface, and is the GRP78–TF complex formed in the ER or at the cell surface? Could it be that GRP78 and TF form a complex in the ER lumen that is undetected by the KDEL receptor? This would imply that the C-terminal KDEL sequence on GRP78 is embedded in the complex, thereby allowing for translocation to the cell surface (Figure). Alternatively, formation of the GRP78–TF complex could occur at the cell surface possibly involving other membrane components. In terms of biological relevance, GRP78 could lock TF into a latent or encrypted conformation before its activation. Second, how do antibodies directed against the C terminus of GRP78 induce TF procoagulant activity? Immunoprecipitation experiments reported by Bhattacharjee et al15 indicate that anti-GRP78 antibodies do not disrupt the GRP78/TF complex, suggesting that antibody binding could alter GRP78 tertiary structure leading to enhanced TF procoagulant activity. Given these findings, the identification of additional epitopes on cell surface GRP78 that attenuate TF activation would be of great interest in mediating plaque thrombogenicity. Previous studies have demonstrated that other ER-resident molecular chaperones, including protein disulfide isomerase and calreticulin, have been identified on the cell surface.28,29 Therefore, is it possible that these chaperones also have inhibitory effects on TF procoagulant activity similar to those observed for GRP78? Finally, previous studies have shown that the binding of factor VIIa to TF leads to the activation of proinflammatory pathways (reviewed in 37). Thus, would GRP78 act to regulate these pathways?



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  		Transport and localization of GRP78–TF complex to the plasma membrane. Bhattacharjee et al15 have demonstrated the presence of a GRP78–TF complex on the cell surface. However, the mechanism by which GRP78 is localized to the plasma membrane and how the GRP78–TF complex is formed remains unknown. It is possible that GRP78 and TF form a complex in the ER, thereby masking the KDEL retention sequence of GRP78 and allowing transport of the complex to the cell surface (i). Alternatively, GRP78 and TF could be transported to the cell surface independently where they form a complex on the plasma membrane (ii). Whether other membrane components are involved is unknown. This model highlights only the GRP78–TF complex and does not reflect total cellular levels of GRP78 and TF.

In summary, the results presented by Bhattacharjee et al15 in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology provide valuable insights into the mechanism by which cell surface GRP78 mediates TF-dependent procoagulant activity. Based on these findings, we may finally get a GRP on treating atherothrombotic disease by developing novel therapies directed at increasing cell surface GRP78 and/or enhancing the binding of GRP78 to TF.


*    Acknowledgments
 
This work was supported by research grants to Richard Austin from the Heart and Stroke Foundation of Ontario (T-5385), the Canadian Institutes of Health Research (MOP-67116, MOP-74477), and the Ontario Research and Development Challenge Fund. Lindsay Pozza (nee Watson) is a recipient of a Canadian Institutes of Health Research Canada Graduate Scholarship Doctoral Award. Richard Austin is a Career Investigator of the Heart and Stroke Foundation of Ontario.


*    References
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