Hema Kothari

Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.

Cystine 186–cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption

Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TF(C186S), TF(C209S), or TF(C186S/C209S) expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TF(C186S), TF(C209S), or TF(C186S/C209S). Analysis of TF activity in HUVECs expressing similar levels of wild-type TF and TF(C186S/C209S) showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TF(C186S/C209S) with HgCl(2) or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation.

Tissue Factor encryption and decryption: Facts and controversies

Tissue factor (TF)-initiated coagulation plays a critical role in both hemostasis and thrombosis. It is generally believed that most of the tissue factor expressed on cell surfaces is maintained in a cryptic, i.e., coagulantly inactive state and an activation step (decryption) is required for the expression of maximum TF procoagulant activity. However, what exactly constitutes cryptic or procoagulant TF, molecular differences between these two forms and mechanisms that are responsible for transformation from one to the other form are not entirely clear and remain highly controversial, thus are a matter of ongoing debate. This brief review discusses pertinent literature on TF encryption/decryption with specific emphasis on the role of membrane phospholipids and reduction/oxidation of the TF Cys186-Cys209 disulfide bond in regulating TF activity at cell surfaces.

Bovine protein disulfide isomerase-enhanced tissue factor coagulant function : is phospholipid contaminant in it the real culprit?

To the editor: Tissue factor (TF) initiates coagulation after the binding of clotting factor VIIa (FVIIa). Only a small fraction of the total TF present on cell surfaces is coagulant-active; the majority is inactive or cryptic. At present it is not completely clear how the TF that is active in

Lipoprotein Receptor–Related Protein 1 Regulates Collagen 1 Expression, Proteolysis, and Migration in Human Pleural Mesothelial Cells

The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-α and IL-1β, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-α or IL-1β (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1-deficient cells demonstrated increased uPAR t(1/2) versus LRP-1-expressing PMCs. uPA enzymatic activity was also increased in LRP-1-deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-α and IL-1β regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.

Tissue factor: mechanisms of decryption.

It is generally believed that only a small fraction of the tissue factor (TF) found on cell surfaces is active whereas the vast majority is cryptic in coagulation. It is unclear how cryptic TF differs from the coagulant active TF or potential mechanisms involved in transformation of cryptic TF to the coagulant active form. Exposure of phosphatidylserine (PS) in response to various chemical or pathophysiological stimuli has been considered as the most potent inducer of TF decryption. In addition to PS, TF self-association and association with specialized membrane domains may also play a role in TF decryption. It has been suggested recently that protein disulfide isomerase regulates TF decryption through its oxidoreductase activity by targeting Cys186-Cys209 disulfide bond in TF extracellular domain or regulating the PS equilibrium at the plasma membrane. However, this hypothesis requires further validation to become an accepted mechanism. In this article, we critically review literature on TF encryption/decryption with specific emphasis on recently published data and provide our perspective on this subject.

Analysis of tissue factor expression in various cell model systems: cryptic vs. active.

Tissue factor (TF) encryption plays an important role in regulating TF coagulant activity. Potential differences in experimental cell model systems and strategies hampered our understanding of the TF encryption mechanisms.

Inactivation of Factor VIIa by Antithrombin In Vitro, Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg−1 body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40–50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF’s role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.

Influence of endothelial cell protein C receptor on breast cancer development.

Tumor cells of several cancers are known to constitutively express the coagulation initiating factor, tissue factor (TF). TF-mediated coagulation generates thrombin, platelet activation, and fibrin formation which altogether orchestrate cancer cell survival and proliferative pathways. Thrombin also induces activation of natural anticoagulant protein C. Activated protein C (APC) not only inhibits subsequent thrombin generation, but also induces cellular signaling through activation of protease activated receptor-1 (PAR1) [1,2]. Endothelial cell protein C receptor (EPCR) plays a key role in supporting APC-mediated cell signaling [1,2]. EPCR-APC-mediated cytoprotection and other cellular effects may accelerate cancer progression and metastasis. EPCR-dependent APC activation of PAR1 was shown to stimulate cell migration of breast cancer cells [3]. Recent studies have shown that EPCR-APC axis conferred a significant survival advantage to lung adenocarcinoma cells and favored their prometastatic activity [4]. Interestingly, our recent studies suggested that EPCR may also function as a negative regulator of cancer progression [5]. The present study was carried out to investigate the influence of EPCR on human breast cancer development. MDA-231t cells (tumor cells established from in vivo tumor developed by injection of MDA-MB-231 cells to a nude mouse) were stably transfected with a control vector (CV) or EPCR expression vector in pZeoSV plasmid vector. After 48 h of transfection, Zeocin (100 μg/ml) was added to the cells. After 3 weeks, stable transfectant colonies were isolated, expanded, and EPCR stable transfectants exhibiting similar TF activity as that of parental MDA-231t cells were selected for the present study. MDA-231t(+CV) and MDA-231t(+EPCR) cells expressed similar levels of TF antigen and activity (Fig. 1 panels A to C). MDA-231t(+CV) cells expressed very little EPCR, whereas EPCR expression levels in MDA-231t(+EPCR) cells was similar to that of HUVEC (Fig. 1A, ​,1B1B). Fig. 1 Influence of EPCR on tumor growth in a murine breast carcinoma model. TF and EPCR expression levels in MDA-231t cells stably transfected with a control or EPCR expression vector were analyzed by Western blot analysis (A) or immunofluorescence microscopy ... MDA-231t(+CV) or MDA-231t(+EPCR) cells were injected into the mammary fat pad (m.f.p) of nude mice, and the growth of tumor in m.f.p. was monitored for 2 months. As shown in Fig. 1D (in set), tumor growth rate is statistically significantly higher in mice injected with MDA-231t(+EPCR) cells compared to MDA-231t(+CV) cells until 40 days following tumor cell implantation. However, in the last two weeks, tumors derived from MDA231t(+EPCR) cells grew less rapidly than tumors originating from MDA-231t(+CV) cells. At the end of 60 days, the tumor volume of MDA-231t(+EPCR) cell-derived tumors was about 30% lower than that of MDA-231t(+CV) cell-derived tumors (Fig. 1D). Although this difference did not reach statistical significance, it was substantial and consistent. At the time of euthanasia (day 60), the mice bearing MDA-231t(+CV) cell-derived tumors appeared to be lethargic, and developed swollen lymph nodes (Fig. 1E and ​and1F),1F), whereas mice bearing MDA-231t(+EPCR) cell-derived tumors exhibited no outward sickness and did not develop any swollen lymph nodes (Fig. 1F). Histological examination of lymph node sections showed extensive infiltration of cells into this region in mice injected with MDA-231t(+CV) cells and not in mice injected with MDA-231(+EPCR) cells (Fig. 1G). The skin over the tumors of the mice injected with MDA-231t(+CV) cells turned blood red and looked different from that of the tumors generated by MDA-231(+EPCR) cells, starting around 30 to 35 days following tumor cell inoculation. At the time of sacrifice (60 days), all tumors developed in mice injected with MDA-231t(+CV) cells were highly inflamed and necrotic, most of which developed hematogenous ulcers at the top skin of tumors (Fig. 1H). Some necrotic tumors collapsed and had leaky liquid centers. None of the tumors in mice bearing MDA-231t(+EPCR) cells showed necrotic ulcerations. Interestingly, analysis of tumor tissue sections for EPCR and TF expression showed that a majority of tumor cells stained negative for EPCR irrespective of whether MDA-231t(+CV) or MDA-231t(+EPCR) cells were used for implantation (Fig. 1I). In both cases, tumor cells stained intensively positive for TF. Analysis of tumor tissue sections for macrophage infiltration and angiogenesis by staining them for F4/80 antigen and CD31, respectively, showed significant reduction in macrophage infiltration (Fig. 1J) and microvessel density (Fig. 1K) in tumors derived from MDA-231t(+EPCR) cells compared to tumors derived from MDA-231t(+CV) cells. It may be pertinent to note here that tissue sections analyzed for tumors derived from both MDA-231t(+CV) and MDA-231t(+EPCR) cells represent the actively growing regions of the tumor. During the preparation of this manuscript, Schaffner et al. [6] reported that EPCR-expressing cells, selected from expansion of EPCR+ cancer stem cell-like population from MDA-MB-231 mfp cells, had markedly increased tumor cell-initiating activity compared to EPCR− cells. Although the experimental approach and MDA-MB-231 cells used for implantation in the present study and the recently published study [6] differed, the results obtained from both the studies are similar to some extent. Schaffner et al. [6] observed that MDA-MB-231 mfp cells exhibit two distinct populations, EPCR+ cells with moderate levels of TF and EPCR negative cells with high levels of TF expression, and cell sorting was used to select EPCR+ and EPCR− cells for their experiments. Although varied levels of EPCR and TF expression were also found in our MDA-MB-231t cell population, we did not find two clearly distinctive populations of cells (see Fig. 1C). Most of the cells expressed very low levels of EPCR and high levels of TF. Therefore, we genetically engineered them to express EPCR to obtain EPCR+ cells. As reported by Schaffner et al. [6], EPCR expression in breast cancer cells increased the tumor cell growth potential, although not drastically, but in a statistically significant fashion. However, we found that in the later stages of tumor progression, the differences in tumor growth between tumors derived from EPCR+ or EPCRlow cells vanished. In fact, at the end of experimental period, tumor volume in mice injected with EPCR+ cells was 30% lower than in mice injected with EPCRlow cells. It is difficult to predict whether the earlier study [6] could have found similar results if their observation was not terminated when the tumor size reached less than 1 cm3. It is interesting to note that, as found in the earlier study [6], irrespective of the EPCR status in tumor cells that were inoculated, the majority of outgrown tumor cells were EPCR negative, which indicates a conversion from EPCR+ cells to EPCR− cells in the tumor microenvironment. Here, it is important to point out that our observation on loss of EPCR expression in tumor tissues derived from EPCR+ tumor cells is not due to a lack of sensitivity to detect EPCR in tumor tissues by immunohistochemistry method. Loss of EPCR in tumors derived from EPCR+ cells is also confirmed by immunoblot analysis of tumor tissue extracts (data not shown). At present, it is unknown at which stage of tumor growth the EPCR expression was lost and the underlying mechanism for it. A notable finding of the present study is that while we observed solid and liquefaction necrosis in tumors originated from EPCRlow cells, no necrosis was found in tumors originated from EPCR+ cells. Necrosis is a common feature of aggressive breast cancer and has been associated with a poor prognosis [7]. Tumor necrosis is the direct result of chronic ischemia caused by vascular collapse when the rate of tumor cell growth exceeds that of angiogenesis [8]. Necrotic liquefaction occurs when the cellular structures are broken down by proteolytic enzymes released from ruptured lysosomes of tumor cells and/or similar enzymes released by infiltrating inflammatory cells [9]. Although it appears to be counterintuitive at first, necrosis resulting from chronic ischemia is associated with increased angiogenesis [8]. Prolonged hypoxic conditions were known to increase tumor progression and angiogenesis, and to promote metastatic potential [10]. Therefore, EPCR expression in breast cancer cells, despite having initial growth advantage, may limit cancer progression at an advanced stage.

Glycosylation of tissue factor is not essential for its transport or functions.

See also Morrissey JH. Low‐carb tissue factor? This issue, pp 1508–10.DOI: 10.1111/j.1538‐7836.2011.04332.x.