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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the ...TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte–derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2–SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2–SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2–SP-PV–induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2–SP-PV–induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration–approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.
•SARS-CoV-2 spike protein pseudovirus infection enhances TF procoagulant activity and releases TF+ EVs via activation of ASMase.•FDA-approved tricyclic antidepressants that act as functional inhibitors of ASMase attenuate the pseudovirus-induced enhanced TF activity.
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Endothelial cell protein C receptor (EPCR) was first identified and isolated as a cellular receptor for protein C on endothelial cells. EPCR plays a crucial role in the protein C anticoagulant ...pathway by promoting protein C activation. In the last decade, EPCR has received wide attention after it was discovered to play a key role in mediating activated protein C (APC)-induced cytoprotective effects, including antiapoptotic, anti-inflammatory, and barrier stabilization. APC elicits cytoprotective signaling through activation of protease activated receptor-1 (PAR1). Understanding how EPCR-APC induces cytoprotective effects through activation of PAR1, whose activation by thrombin is known to induce a proinflammatory response, has become a major research focus in the field. Recent studies also discovered additional ligands for EPCR, which include factor VIIa, Plasmodium falciparum erythrocyte membrane protein, and a specific variant of the T-cell receptor. These observations open unsuspected new roles for EPCR in hemostasis, malaria pathogenesis, innate immunity, and cancer. Future research on these new discoveries will undoubtedly expand our understanding of the role of EPCR in normal physiology and disease, as well as provide novel insights into mechanisms for EPCR multifunctionality. Comprehensive understanding of EPCR may lead to development of novel therapeutic modalities in treating hemophilia, inflammation, cerebral malaria, and cancer.
Tissue factor (TF) is a cofactor for factor VIIa and the primary cellular initiator of coagulation. Typically, most TF on cell surfaces exists in a cryptic coagulant-inactive state but are ...transformed to a procoagulant form (decryption) following cell activation. Our recent studies in cell model systems showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells, and the hydrolysis of SM leads to decryption of TF. The present study was carried out to investigate the relevance of this novel mechanism in the regulation of TF procoagulant activity in pathophysiology. As observed in cell systems, administration of adenosine triphosphate (ATP) to mice enhanced lipopolysaccharide (LPS)-induced TF procoagulant activity in monocytes. Treatment of mice with pharmacological inhibitors of acid sphingomyelinase (ASMase), desipramine and imipramine, attenuated ATP-induced TF decryption. Interestingly, ASMase inhibitors also blocked LPS-induced TF procoagulant activity without affecting the LPS-induced de novo synthesis of TF protein. Additional studies showed that LPS induced translocation of ASMase to the outer leaflet of the plasma membrane and reduced SM levels in monocytes. Studies using human monocyte-derived macrophages and endothelial cells further confirmed the role of ASMase in LPS- and cytokine-induced TF procoagulant activity. Overall, our data indicate that LPS- or cytokine-induced TF procoagulant activity requires the decryption of newly synthesized TF protein by ASMase-mediated hydrolysis of SM. The observation that ASMase inhibitors attenuate TF-induced coagulation raises the possibility of their therapeutic use in treating thrombotic disorders associated with aberrant expression of TF.
•LPS-induced TF procoagulant activity in monocytes in vivo is dependent on LPS-induced activation of ASMase.•ASMase inhibitors attenuate LPS- and cytokine-induced TF procoagulant activity without decreasing the de novo synthesis of TF protein.
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Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon ...activation of inflammasome and secretion of interleukin 1β (IL-1β) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1β and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 CARD recruited membrane-associated guanylate kinase protein 3–BCL-10 B-cell lymphoma 10–MALT1 mucosa-associated lymphoid tissue lymphoma translocation protein 1) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1β–induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1β–induced NF-κB–dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2–MALT1 axis in thromboinflammation. Targeting the Gab2–MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.
•Our findings identify a previously unrecognized role of the Gab2–MALT1 axis in IL-1β-induced thromboinflammation.•Pharmacological inhibition of MALT1 attenuates venous thrombosis induced by flow restriction.
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Abstract
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.
Coagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated ...receptor-1 (PAR1)–mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB–mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.
•FVIIa induces the expression of anti-inflammatory miR10a by endothelial cells, and FVIIa-released EEVs carry miR10a in high abundance.•FVIIa-released EEVs suppress inflammation and protect barrier integrity in vitro and in vivo via the transfer of miR10a to target cells.
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Factor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent ...cytoprotective responses.
To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs.
Activation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs.
FVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage.
Our data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.
•Factor VIIa-released endothelial extracellular vesicles were enriched with microRNA-10a.•It is unknown whether the enrichment of microRNA-10a in factor VIIa-released extracellular vesicles come from increased cellular expression of microRNA-10a in FVIIa-treated endothelial cells or selective sorting of microRNA-10a into factor VIIa-released extracellular vesicles.•Factor VIIa induces microRNA-10a expression in endothelial cells via EPCR-PAR1–dependent intracellular activation of the ERK1/2-Elk-1-TWIST1 signaling axis.•Factor VIIa–induced acid sphingomyelinase activation leads to La protein expression, and La protein facilitates the sorting of microRNA-10a into the extracellular vesicles.
Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) ...and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia.
To investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS.
FVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase
mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model.
FVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase
mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase
mice.
Our study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.
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