Factor VIIa induces extracellular vesicles from the endothelium: a potential mechanism for its hemostatic effect.

Recombinant factor FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type (WT), EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice. In vivo studies revealed that administration of FVIIa to WT, EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice, increased the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

OBJECTIVE: TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. CONCLUSIONS: HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to ß-catenin accumulation via the AKT/GSK3ß pathway and contributes to breast cancer progression.

Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3ß inactivation is involved in these processes and that ß-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of ß-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. ß-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent ß-catenin accumulation may represent a potential therapeutic approach to control breast cancer.

Role of Cell Surface Lipids and Thiol-Disulphide Exchange Pathways in Regulating the Encryption and Decryption of Tissue Factor.

Tissue factor (TF), a transmembrane glycoprotein, is the cellular receptor of the coagulation factors VII (FVII) and VIIa (FVIIa). The formation of TF-FVIIa complex triggers the initiation of the blood coagulation pathway. TF plays an essential role in haemostasis, but an aberrant expression of TF activity contributes to thrombotic disorders. In health, TF pro-coagulant activity on cells is controlled tightly to allow sufficient coagulant activity to achieve haemostasis but not to cause thrombosis. It is achieved largely by selective localization of TF in the body and encryption of TF at the cell surface. A vast majority of TF on resting cells exists in an encrypted state with minimal pro-coagulant activity but becomes pro-thrombotic following cell injury or activation. At present, the mechanisms that are responsible for TF encryption and activation (decryption) are not entirely clear, but recent studies provide important mechanistic insights into these processes. To date, externalization of phosphatidylserine to the outer leaflet and thiol-disulphide exchange pathways that either turn on and off the allosteric disulphide bond in TF are shown to play a major role in regulating TF pro-coagulant activity on cell surfaces. Recent studies showed that sphingomyelin, a major phospholipid in the outer leaflet of plasma membrane, plays a critical role in the encryption of TF in resting cells. The present review provides an overview of recent literature on the above-described mechanisms of TF encryption and decryption with a particular emphasis on our recent findings.

The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system.

Many pathophysiologic agents transform cryptic tissue factor (TF) on cells to prothrombotic TF, and one such stimulus is 4-hydroxy-2-nonenal (HNE), the most abundant aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids. HNE was shown to induce reactive oxygen species (ROS) generation and p38 MAPK activation, but the link between them and their role in TF decryption are unclear. The present study was carried out to elucidate potential mechanisms involved in HNE-induced TF decryption in monocytic cells. The data presented herein show that mitochondria are the primary source for HNE-induced ROS generation. The inhibition of mitochondrial electron transport chain complex III and V blocked HNE-induced ROS generation, but not p38 MAPK activation. These inhibitors reduced phosphatidylserine (PS) externalization and TF decryption significantly, but not completely. HNE treatment inhibited the activities of thioredoxin reductase (TrxR) and thioredoxin (Trx), independent of ROS. Inhibition of the TrxR/Trx system by HNE or pharmacological inhibitors induced p38 MAPK activation, PS externalization, and TF decryption. Additional studies revealed that the inhibition of TrxR/Trx led to activation of apoptosis signal-regulating kinase (ASK-1) and mitogen-activated protein kinase kinase 3/6. Inhibition of ASK-1 expression by small interfering RNA or its activity by pharmacological inhibitors diminished HNE-induced TF decryption. Overall, our data suggest that HNE induces TF decryption by 2 distinctive pathways. One is ROS dependent but independent of p38 MAPK activation, and the other is via TrxR/Trx and is p38 MAPK activation dependent. However, both mechanisms result in the enhancement of PS at the outer leaflet that is responsible for TF decryption.

The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface.

Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF) decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF.