Unlike thrombin and trypsin, VIIa has to bind its cellular receptor, TF, to activate PARs. Therefore, TF-VIIa could only activate PARs that are in the close vicinity of TF. Unless TF and PAR2 are present in high density, it is unlikely that TF-VIIa encounters PAR2 on the cell surface. This raises an important question on how TF-VIIa could activate PAR2-mediated cell signaling. In this study, we show that a fraction of both TF and PAR2 are compartmentalized in cholesterol-rich specialized plasma membrane domains, lipid rafts/caveolae, in breast carcinoma cells (MDA-MB-231), a cell system in which TF-VIIa was shown to induce robust PAR2-mediated cell signaling.6
Depletion or sequestration of plasma membrane cholesterol impaired the TF-VIIa—induced cell signaling in these cells. Silencing of caveloin-1, an integral protein of caveolae, diminished the TF-VIIa—induced cell signaling without affecting the TF-VIIa coagulant activity. Overall, the data presented in this report suggest that sequestration of TF and PAR2 in lipid rafts/caveolae, probably coupled with differential segregation of heterotrimeric G-proteins into these microdomains, facilitates an effective environment for TF-VIIa—induced cell signaling.
Cholesterol plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin-1, form caveolae, flask-shaped invaginations in the plasma membrane. Because these microdomains have been shown to be enriched with a variety of signaling molecules,13,16,22–24
it is believed that they play a role in compartmentalizing signaling molecules at the cell surface and modulating signaling functions.9,10,18,21,25
Although certain G-protein-coupled receptors and G-proteins are known to be segregated into lipid rafts and caveolae,16–18
we are not aware of any report on whether PARs are segregated into lipid rafts. Consistent with our recent observations in fibroblasts,15
TF in MDA-MB-231 tumor cells is also localized in lipid rafts/caveolae. Further, our studies also show that a fraction of PAR2 is colocalized with TF presumably in lipid rafts/caveolae. Segregation of a fraction of PAR2 with TF into the lipid rafts may facilitate PAR2 access to VIIa bound to the TF. TF-VIIa cleavage of PAR2 in these microdomains could activate the signaling pathways as these microdomains were also shown to be enriched with certain heterotrimeric G-proteins16,21
and a number of other signaling molecules.10–13
The integrity of cholesterol-rich membrane microdomains appears to be critical for the TF-VIIa—induced PAR2-mediated cell signaling. Depletion of membrane cholesterol, which leads to the loss of the caveolar structure as observed by transmission electron microscopy, reduced TF and PAR2 association with low-density membrane microdomains/caveolae, and impaired the TF-VIIa—induced PI hydrolysis and IL-8 gene expression. Although the depletion of membrane cholesterol also impairs the assembly of TF-VIIa complex at the cell surface,15
this alone cannot explain the complete loss of TF-VIIa—induced cell signaling in the cholesterol-depleted cells. In this context, it is pertinent to point out that cholesterol depletion by 10 mmol/L mβ
CD treatment reduced the TF-VIIa activation of factor X by ≈50%, whereas it completely attenuated the TF-VIIa—induced cell signaling. The importance of the integrity of cholesterol-rich membrane microdomains in the TF-VIIa—induced cell signaling is better illustrated in cells where membrane cholesterol is sequestered rather than depleted. Filipin, which sequesters the membrane cholesterol by forming cholesterol-filipin complexes,20
impaired the signaling function of TF-VIIa despite the increased TF-VIIa coagulant activity in these cells. The increased TF-VIIa coagulant activity observed in filipin-treated cells could have been the result of increased concentration of cholesterol in membranes patches because filipin treatment is shown to result in cholesterol aggregation in the membrane20
or movement of TF from inactive glycosphingolipid-rich microdomains to active anionic phospholipid region of the membrane.
Although exact underlying mechanisms are not fully known, it is well-established that most of the TF activity at the cell surface is encrypted.26,27
Ultrastructural localization of TF in smooth muscle cells,28
activated endothelial cells,29
showed that a fraction of TF in these cells is associated with caveolae. Based on the increased TF activity and the enlargement of caveolar structures in smooth muscle cells after their detachment, Mulder et al28
speculated that caveolae-associated TF might function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost. Recently, Lupu et al30
reported that caveolae may regulate TF activity indirectly through regulation of tissue factor pathway inhibitor activity. These investigators showed caveolin-1 silencing decreased the tissue factor pathway inhibitor activity on activated endothelial cells and thereby increases TF activity by several fold. The data presented in this report show that caveloin-1 silencing had no significant effect on TF coagulant activity at the surface of tumor cells. Similar results were also obtained in lung fibroblasts (data not shown). These data strongly suggest that the caveolae are not negative regulators of TF procoagulant activity, as previously thought. In this context, it may be pertinent to note that, unlike endothelial cells, other cell types synthesize little or no tissue factor pathway inhibitor and most of it was secreted and not associated with the cell surface.31,32
Similarly, MDA-MB-231 tumor cells, the cell model system used in the present study, do not produce tissue factor pathway inhibitor (unpublished data of the authors). Thus, it is unlikely that tissue factor pathway inhibitor plays a role in localizing TF into caveolae or modulating TF-VIIa activity or signaling in our model system.
In contrast to its lack of effect on TF coagulant activity, caveloin-1 silencing significantly reduced the TF-VIIa—induced cell signaling in tumor cells. It is likely that the loss of the structural integrity of caveolae might have caused the relocalization of TF and PAR2 at the cell surface without affecting their functions per se. Such reorganization could have placed the PAR2 beyond the physical reach of TF-VIIa, thus impairing the ability of TF-VIIa to activate PAR2. This hypothesis is supported by our observation that showed lipid raft/caveolar disruption by cholesterol binding drugs or caveolin-1 silencing impaired TF-VIIa cleavage of PAR2 but not trypsin cleavage of PAR2.
A number of G-protein-coupled receptors and their interacting proteins are known to be localized within lipid rafts and caveolin-enriched microdomains.16,21,33,34
Further, caveolins may act as scaffolding proteins to cluster and regulate signaling molecules targeted to the caveolae, such as Src-family tyrosine kinase, epidermal growth factor receptor, and G-protein α
Therefore, disruption of lipid rafts or caveolae may also cause uncoupling of G-proteins and other signaling proteins with their membrane receptor. This could explain why lipid raft disruption by cholesterol binding drugs had no effect on trypsin activation of PAR2 at the cell surface but impaired trypsin-induced PAR2-mediated cell signaling.
In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa—induced cell signaling. The cholesterol content in the plasma membrane and not the structural integrity of the rafts/caveolae appear to influence the TF procoagulant activity, whereas the structural integrity of cholesterol-rich, caveolin-1— enriched microdomains plays a greater role in regulating the TF-VIIa signaling. Our data also suggest that localization of TF and PAR2 in membrane microdomains is critical for TF-VIIa to trigger PAR2-mediated cell signaling, whereas PAR2 localization is less critical for PAR2 peptide-mediated cell signaling. These findings may have several biological implications. This could explain why some cells that express both TF and functional PAR2 may fail to respond to FVIIa.35
Further, compartmentalization of TF-VIIa signaling to the microdomains may facilitate the cells to respond to other proteases in tandem or subsequent to FVIIa exposure because these proteases (or peptide agonists) can access the PAR2 that is not readily accessible to TF-VIIa. Because the cholesterol is one of the major components of the rafts/caveolae, their structure is sensitive to the amount of cholesterol in the membrane, and therefore the concentration of cholesterol in the membrane not only regulates the TF-VIIa coagulant function but also the TF-VIIa—induced cell signaling. Because recent studies suggest that TF-VIIa, in addition to triggering blood coagulation, plays a role in many pathophysiological processes,1,2
cholesterol lowering could provide additional health benefits, in addition to reducing atherosclerosis.