ABSTRACT:Repeated bleeding into joints in hemophilia leads to chronic inflammation that plays a central role in the pathogenesis of hemophilic arthropathy (HA). Our recent studies revealed that factor VIIa (FVIIa) treatment releases extracellular vesicles from endothelial cells (eEVs) and FVIIa-released eEVs exhibit anti-inflammatory and barrier-protective functions. The present study was undertaken to investigate the effect of FVIIa-released eEVs on HA and the mechanism of their protective effect. Joint bleeding in hemophilia (F8-/-) mice was induced by a needle puncture injury. Injured mice were treated with saline, control eEVs, or FVIIa-released eEVs, and the changes in the knee joints were analyzed by gross examination of knees as well as histological and immunohistochemical analysis. Joint tissues were examined for evidence of synovial hyperplasia, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. The data showed that treatment of mice with control eEVs had no significant effect on the development of HA, whereas treatment with FVIIa-released eEVs markedly reduced all pathological features of joint bleed-induced HA. Incorporation of microRNA10a (miR10a) inhibitor into FVIIa-released eEVs abrogated the protective effect of FVIIa-released eEVs on HA. More importantly, loading miR10a mimic into control eEVs conferred a protective effect. Administration of miR10a-containing FVIIa-released eEVs or control eEVs loaded with miR10a mimic was found to abrogate joint bleed-induced interleukin-6 production in the synovium. miR10a in eEVs had no effect on hemostasis. Cumulatively, our data indicate that EVs containing miR10a that effectively suppress synovial inflammation would have immense therapeutic value in treating HA.

Background: 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. Objectives: To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. Methods: Activation of Elk -1 and TWIST1 expression was analyzed by immunofluo- rescence 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. Results: 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. Conclusion: 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.

Background 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. Objective To investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS. Methods 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. Results 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. Conclusion Our study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.

Supplementary File PDF 277K, Supplementary Methods: Includes additional details of reagents and methodology, and references. Supplementary Figure 1: Effect of FVIIa and thrombin on cell proliferation of wild-type REN, TF- or PAR1- knocked-down REN MPM cells. Supplementary Figure 2: TF- or PAR1 knock-down in REN MPM cells suppresses in vivo tumor cell proliferation. Supplementary Figure 3: TF- or PAR1 knock-down in REN MPM cells increases tumor cell apoptosis in vivo. Supplementary Figure 4: Over expression EPCR did not alter in vitro growth potential of REN MPM cells. Supplementary Figure 5: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) protein levels in parental MS-1 and M9K MPM cells and their variants as analyzed by western blot analysis. Supplementary Figure 6: EPCR knock-down did not alter cell proliferation of MS-1 or M9K MPM cells in vitro

Recurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)(-/-) mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII-/- mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII-/- mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed-induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.

Recombinant FVIIa (rFVIIa) is used as a therapeutic agent to treat bleeding disorders in hemophilia patients with inhibitors and other congenital and acquired bleeding disorders.1.Hedner U. Factor VIIa and its potential therapeutic use in bleeding‐associated pathologies.Thromb Haemost. 2008; 100: 557-562Crossref PubMed Scopus (67) Google Scholar Most of the experimental evidence indicates that FVIIa‐induced hemostatic response in hemophilia therapy is dependent on platelets.2.Hoffman M. Monroe D.M. Platelet binding and activity of recombinant factor VIIa.Thromb Res. 2010; 125: S16-S18https://doi.org/10.1016/j.thromres.2010.01.025Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 3.Giansily‐Blaizot M. Schved J.F. Recombinant human factor VIIa (rFVIIa) in hemophilia: mode of action and evidence to date.Ther Adv Hematol. 2017; 8: 345-352https://doi.org/10.1177/2040620717737701Crossref PubMed Google Scholar However, patients with congenital and acquired platelet disorders have also been treated successfully with rFVIIa.4.Goodnough L.T. Experiences with recombinant human factor VIIa in patients with thrombocytopenia.Semin Hematol. 2004; 41: 25-29https://doi.org/10.1053/j.seminhematol.2003.11.006Crossref PubMed Scopus (35) Google Scholar, 5.Poon M.C. The use of recombinant activated factor VII in patients with Glanzmann's thrombasthenia.Thromb Haemost. 2021; 121: 332-340https://doi.org/10.1055/s-0040-1718373Crossref PubMed Scopus (8) Google Scholar Our recent study indicated that ⁓30% of thrombin generated by FVIIa at the site of injury in FVIII−/− mice might come from a platelet‐independent mechanism.6.Keshava S. Pendurthi U.R. Esmon C.T. Mohan Rao L.V. Therapeutic doses of recombinant factor VIIa in hemophilia generates thrombin in platelet‐dependent and ‐independent mechanisms.J Thromb Haemost. 2020; 18: 1911-1921https://doi.org/10.1111/jth.14881Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Our recent studies revealed that FVIIa binding to endothelial cell protein C receptor (EPCR) induces the release of extracellular vesicles (EVs) from the endothelium via activation of protease‐activated receptor 1, and these EVs are capable of correcting bleeding in platelet‐depleted or hemophilia mice without causing systemic activation of coagulation.7.Das K. Keshava S. Ansari S.A. et al.Factor VIIa induces extracellular vesicles from the endothelium; a potential mechanism for its hemostatic effect.Blood. 2021; 137: 3428-3442Crossref PubMed Scopus (7) Google Scholar The follow‐up studies showed that FVIIa‐released endothelial EVs (EEVs) are enriched with microRNA (miR) 10a, and FVIIa‐released EEVs confer anti‐inflammatory and barrier protective responses to monocytes and vascular endothelium, respectively, through the transfer of miR10a to target cell types.8.Das K. Keshava S. Pendurthi U.R. Rao L.V.M. Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a.Blood. 2022; 139: 118-133https://doi.org/10.1182/blood.2021012358Crossref PubMed Scopus (5) Google Scholar To determine the relevance of FVIIa‐released EVs in the treatment of hemophilia and pathophysiology, it is important to obtain proof that FVIIa treatment increases circulating EVs in human subjects. Ideally, it is desirable to perform a well‐designed and controlled clinical study to obtain the above evidence in humans. In the absence of extensive resources, secured funding, and involvement of multiple hemophilia treatment centers, such studies are not feasible. However, analysis of EVs in existing plasma samples from patients treated with rFVIIa may provide the necessary evidence to the above proof of concept. After several months of exploration, we identified plasma samples from 10 patients with severe hemophilia (nine FVIII deficient and one FIX deficient) treated with rFVIIa (90 μg/kg) with matching pre‐infusion plasma, 30 min, and 6 h post‐infusion plasma samples (from Erika J. Martin and Donald F. Brophy at Virginia Commonwealth University's Coagulation Advancement Laboratory). The same group of investigators also provided nine plasma samples from patients with severe FVIII deficiency (no inhibitor) with matching baseline/pre‐infusion and 4 h post‐infusion of rFVIII (the mean FVIII dose of 32 IU/kg). These samples were used in earlier studies.9.Brophy D.F. Martin E.J. Christian Barrett J. et al.Monitoring rFVIIa 90 mug kg(−)(1) dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.Haemophilia. 2011; 17: e949-e957https://doi.org/10.1111/j.1365-2516.2011.02492.xCrossref PubMed Scopus (15) Google Scholar, 10.Agerso H. Brophy D.F. Pelzer H. et al.Recombinant human factor VIIa (rFVIIa) cleared principally by antithrombin following intravenous administration in hemophilia patients.J Thromb Haemost. 2011; 9: 330-338Crossref Scopus (39) Google Scholar, 11.Al Hawaj M.A. Martin E.J. Venitz J. et al.Monitoring rFVIII prophylaxis dosing using global haemostasis assays.Haemophilia. 2013; 19: 409-414https://doi.org/10.1111/hae.12110Crossref PubMed Scopus (28) Google Scholar In addition, plasma samples from three patients with severe hemophilia treated with rFVIIa (between 72 and 123 μg/kg) were obtained from Marilyn Manco‐Johnson, Hemophilia and Thrombosis Center, School of Medicine, University of Colorado. Quantification of EVs isolated from plasma of 10 patients with hemophilia treated with rFVIIa by nanoparticle tracking analysis in a blinded fashion showed that the EV number was markedly higher in the plasma of FVIIa‐treated patients compared with pre‐infusion plasma samples or plasma samples from healthy volunteers (Figure 1A). Although the EV number was significantly higher even at 0.5 h post rFVIIa infusion, the number was much higher at 6 h post rFVIIa infusion (Figure 1A). Similar results were obtained with the analysis of EVs in the plasma of three patients with hemophilia A treated with rFVIIa, where the plasma was collected 1 h post rFVIIa infusion (data not shown). In contrast to the above data, we found no significant increase in the EV number in the plasma of patients with hemophilia A treated with FVIII (Figure 1B). Differences in the collection time of post‐treatment samples (4 h post for FVIII infusion vs. 0.5 h, 1 h, or 6 h post rFVIIa infusion) could not be a reason for the observed differences in EVs in FVIIa‐treated and FVIII‐treated patients as we found a significant increase in circulating EVs in all post rFVIIa plasma samples. When isolated EVs were probed for an endothelial marker, VE‐cadherin, we observed a significant increase in VE‐cadherin in EVs isolated from post rFVIIa samples compared with pre‐infusion samples (Figure 1C), indicating that FVIIa‐released EVs were derived from endothelial cells. We found no significant differences in VE‐cadherin antigen levels between EVs isolated from post FVIII treatment compared with pre‐infusion plasma samples (Figure 1C). Analysis of an equal number of EVs isolated from pre‐ and post‐infusion plasma samples for their ability to support FVIIa‐catalyzed activation of FX showed that EVs from 6 h post rFVIIa infusion plasma samples markedly increased the rate of FX activation (Figure 1D). Pre‐incubation of EVs with annexin V abolished the enhancing effect of EVs on FX activation, indicating that the increased FX activation observed in EVs from the 6 h post rFVIIa infusion plasma samples stem from the increased phosphatidylserine (PS) levels in the outer membrane of these EVs. Here, it is pertinent to note that FVIIa‐released EEVs do not contain tissue factor.7.Das K. Keshava S. Ansari S.A. et al.Factor VIIa induces extracellular vesicles from the endothelium; a potential mechanism for its hemostatic effect.Blood. 2021; 137: 3428-3442Crossref PubMed Scopus (7) Google Scholar Interestingly, we found no increase in the hemostatic potential in EVs isolated from 30 min post rFVIIa infusion plasma samples. These data are consistent with our recent observations that showed increased PS levels in FVIIa‐released EEVs coming from the externalization of PS on endothelial cells in response to FVIIa, and the process of FVIIa‐induced PS externalization in endothelial cells requires 2 h or longer.7.Das K. Keshava S. Ansari S.A. et al.Factor VIIa induces extracellular vesicles from the endothelium; a potential mechanism for its hemostatic effect.Blood. 2021; 137: 3428-3442Crossref PubMed Scopus (7) Google Scholar miR10a levels were markedly higher (about 10‐fold) in EVs from 6 h post rFVIIa samples compared with EVs from pre‐infusion plasma or plasma from healthy volunteers (Figure 2A). No significant increase in miR10a levels was found in FVIIa‐released EVs isolated from 0.5 h post‐rFVIIa samples. These data are consistent with our earlier observations that showed FVIIa‐induced miR10a expression in endothelial cells, and its sorting to EVs would require more than 2 h.8.Das K. Keshava S. Pendurthi U.R. Rao L.V.M. Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a.Blood. 2022; 139: 118-133https://doi.org/10.1182/blood.2021012358Crossref PubMed Scopus (5) Google Scholar No significant differences were found in miR10a levels between pre‐infusion and post FVIII infusion samples (Figure 2A). Although EVs isolated from both FVIIa‐treated and control samples were readily taken up by human monocyte‐derived macrophages (Figure 2B), only FVIIa‐released EVs conferred anti‐inflammatory phenotype to macrophages as they significantly reduced lipopolysaccharide (LPS)‐induced expression of proinflammatory cytokines tumor necrosis factor α (TNFα) (Figure 2C), interleukin (IL)‐1β (Figure 2D), and IL‐6 (Figure 2E). If EVs were incorporated into recipient macrophages that were pre‐transfected anti‐miR10a, the anti‐inflammatory effect of FVIIa‐released EVs is markedly diminished (Figures 2C–E). As observed with macrophages, EVs isolated from either pre‐infusion or 6 h post rFVIIa plasma samples were taken up by the endothelial cells (Figure 2F). Incorporating FVIIa‐released EVs and not constitutively circulating EVs from pre‐infusion plasma samples significantly reduced LPS‐induced endothelial barrier disruption (Figure 2G). If endothelial cells were transfected with anti‐miR10a prior to incorporating EVs, the protective effect of FVIIa‐released EVs significantly reduced the LPS‐induced endothelial barrier disruption. Taken together, the data shown in Figure 2 strongly indicate that FVIIa‐released EVs in hemophilia patients could confer anti‐inflammatory and barrier protective effects in target cells. Our current observations that FVIIa treatment increases circulating EVs in hemophilia patients and FVIIa‐released EVs promote FX activation in ex vivo experiments raise the possibility that FVIIa‐released EVs in patients may contribute to the hemostatic effect of rFVIIa. However, it is difficult to determine the extent of the contribution of FVIIa‐released EVs to hemostasis, among other potential mechanisms. Our data may also provide potential clues on why rFVIIa treatment is ineffective in a small fraction of patients.12.Hedner U. Lee C.A. First 20 years with recombinant FVIIa (NovoSeven).Haemophilia. 2011; 17: e172-e182https://doi.org/10.1111/j.1365-2516.2010.02352.xCrossref PubMed Scopus (68) Google Scholar, 13.Young G. Cooper D.L. Gut R.Z. Investigators H. Dosing and effectiveness of recombinant activated factor VII (rFVIIA) in congenital haemophilia with inhibitors by bleed type and location: the experience of the haemophilia and thrombosis research society (HTRS) registry (2004‐2008).Haemophilia. 2012; 18: 990-996https://doi.org/10.1111/j.1365-2516.2012.02864.xCrossref PubMed Scopus (26) Google Scholar It is possible that patients having a lower number of EVs released in response to FVIIa treatment may not respond to FVIIa treatment adequately. Our data may also have implications for FVIIa treatment modalities. If FVIIa‐released EVs contribute to the hemostatic effect in therapeutic settings, the pharmacodynamics of EVs may influence the dosing schedule. The observation that FVIIa‐released EVs exert anti‐inflammatory and barrier protective effects may have a greater implication on rFVIIa prophylaxis to prevent joint bleeding‐induced inflammation and arthropathy. We recognize the limitations in our study in working with patients' archived plasma samples. Biofluid storage methods can impact EV yield, composition, and function,14.Jeyaram A. Jay S.M. Preservation and storage stability of extracellular vesicles for therapeutic applications.AAPS J. 2017; 20: 1https://doi.org/10.1208/s12248-017-0160-yCrossref PubMed Scopus (192) Google Scholar, 15.Gelibter S. Marostica G. Mandelli A. et al.The impact of storage on extracellular vesicles: a systematic study.J Extracell Vesicles. 2022; 11: e12162https://doi.org/10.1002/jev2.12162Crossref PubMed Scopus (34) Google Scholar and processing EVs from fresh samples is preferable.15.Gelibter S. Marostica G. Mandelli A. et al.The impact of storage on extracellular vesicles: a systematic study.J Extracell Vesicles. 2022; 11: e12162https://doi.org/10.1002/jev2.12162Crossref PubMed Scopus (34) Google Scholar However, EVs in plasma seem relatively stable during storage, particularly when the plasma was stored −80°C.14.Jeyaram A. Jay S.M. Preservation and storage stability of extracellular vesicles for therapeutic applications.AAPS J. 2017; 20: 1https://doi.org/10.1208/s12248-017-0160-yCrossref PubMed Scopus (192) Google Scholar, 16.Coumans F.A.W. Brisson A.R. Buzas E.I. et al.Methodological guidelines to study extracellular vesicles.Circ Res. 2017; 120: 1632-1648https://doi.org/10.1161/CIRCRESAHA.117.309417Crossref PubMed Scopus (565) Google Scholar, 17.Yuan F. Li Y.M. Wang Z. Preserving extracellular vesicles for biomedical applications: consideration of storage stability before and after isolation.Drug Deliv. 2021; 28: 1501-1509https://doi.org/10.1080/10717544.2021.1951896Crossref PubMed Scopus (36) Google Scholar The plasma of patients used in the study were stored at −80°C. Moreover, the comparison between pre‐ and post‐treatment samples collected within a few hours and stored for the same length of time in identical settings should minimize any concerns about how the storage of our patients' samples would impact our data or interpretation. Limited availability of patients' plasma samples precludes extensive characterization of FVIIa‐released EVs. Despite these limitations, our study provides strong proof of concept evidence that FVIIa administration to patients releases pro‐hemostatic and cytoprotective EVs from endothelial cells, very similar to those observed in cell and murine models.7.Das K. Keshava S. Ansari S.A. et al.Factor VIIa induces extracellular vesicles from the endothelium; a potential mechanism for its hemostatic effect.Blood. 2021; 137: 3428-3442Crossref PubMed Scopus (7) Google Scholar, 8.Das K. Keshava S. Pendurthi U.R. Rao L.V.M. Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a.Blood. 2022; 139: 118-133https://doi.org/10.1182/blood.2021012358Crossref PubMed Scopus (5) Google Scholar The present information may stimulate further research on the analysis of FVIIa‐released EVs in patients and their potential significance in the treatment of bleeding disorders of hemophilia patients and other groups, and off‐label use of rFVIIa. KD participated in the study design, performed experiments, analyzed data, and wrote the first draft of the manuscript. URP contributed to the study design and provided technical advice in performing the study. MM‐J, EJM, and DFB provided patient plasma samples. LVMR conceived and designed the research, analyzed data, and wrote the manuscript. All authors read and reviewed the manuscript and contributed to preparing the final version of the manuscript. The authors report no conflict of interests. Currently, D.F. Brophy is an employee of SanofiGenzyme. The Institutional Review Board at the University of Texas Health Science Center at Tyler approved the protocol for obtaining blood from healthy volunteers. A written informed consent was obtained prior to blood collection. Pre‐existing hemophilia patients' plasma samples were obtained under an exempt protocol. The study was conducted in compliance with the Declaration of Helsinki. National Heart, Lung, and Blood InstituteHL124055National Hemophilia Foundation

Background:Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. Methods:Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. Results:Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase(-)(/-) mice, while their levels were increased in SMS2(-/-) mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. Conclusions:Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

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.

Objective: In response to inflammatory insult, endothelial cells express cell adhesion molecules and TF (tissue factor), leading to increased adhesion of leukocytes to the endothelium and activation of coagulation. Enhanced coagulation could further exacerbate inflammation. Identifying key signaling molecule(s) that drive both inflammation and coagulation may help devise effective therapeutic strategies to treat inflammatory and thrombotic disorders. The aim of the current study is to determine the role of Gab2 (Grb2-associated binder2), which is known to play a crucial role in the signaling evoked by growth factors and antigen receptors, in inflammatory signaling pathways and its contribution to vascular dysfunction. Approach and Results: WT (wild type) and Gab2-silenced endothelial cells were treated with TNFα (tumor necrosis factor alpha), IL (interleukin)-1β, or lipopolysaccharide (LPS). Activation of key signaling proteins in the inflammatory signaling pathways and expression of cell adhesion molecules, TF, and inflammatory cytokines were analyzed. Gab2 −/ − and WT littermate mice were challenged with LPS or S pneumoniae (Streptococcus pneumoniae ), and parameters of inflammation and activation of coagulation were assessed. Gab2 silencing in endothelial cells markedly attenuated TNFα-induced, IL-1β–induced, and LPS-induced expression of TF, cell adhesion molecules, and inflammatory cytokines/chemokines. Gab2 silencing suppressed TNFα-induced, IL-1β–induced, and LPS-induced phosphorylation and ubiquitination of TAK1 (transforming growth factor beta-activated kinase 1) and activation of MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa B). Immunoprecipitation studies revealed that the Src kinase Fyn phosphorylates Gab2. Gab2 −/− mice are protected from LPS or S pneumoniae –induced vascular permeability, neutrophil infiltration, thrombin generation, NET formation, cytokine production, and lung injury. Conclusions: Our studies identify, for the first time, that Gab2 integrates signaling from multiple inflammatory receptors and regulates vascular inflammation and thrombosis.

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.

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

Recombinant factor FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patientswith 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 PAR1R46Q-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 plateletdepleted 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.

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.

In hemophilia bypass therapy, a platelet-dependent mechanism is believed to be primarily responsible for recombinant factor VIIa (rFVIIa)'s hemostatic effect. rFVIIa may also possibly interact with other cells through its binding to endothelial cell protein C receptor (EPCR) or cell surface phospholipids.We aim to investigate the relative contribution of platelet-dependent and platelet-independent mechanisms in rFVIIa-mediated thrombin generation in hemophilic conditions at the injury site.Platelets were depleted in acquired and genetic hemophilia mice using anti-platelet antibodies. The mice were subjected to the saphenous vein injury, and the hemostatic effect of pharmacological concentrations of rFVIIa was evaluated by measuring thrombin generation at the injury site.Administration of anti-mouse CD42 antibodies to mice depleted platelets by more than 95%. As expected, hemophilia mice, compared with wild-type mice, generated only a small fraction of thrombin at the injury site. The depletion of platelets in hemophilia mice further reduced thrombin generation. However, when pharmacological doses of rFVIIa were administered to hemophilia mice, substantial amounts of thrombin were generated even in the platelet-depleted hemophilia mice. No differences in thrombin generation were detected among FVIII-/- , EPCR-deficient FVIII-/- , and EPCR-overexpressing FVIII-/- mice depleted of platelets or not. Evaluation of platelets by flow cytometry as well as immunoblot analysis showed no detectable expression of EPCR.Our data suggest that pharmacological concentrations of rFVIIa generate thrombin in hemophilia in both platelet-dependent and platelet-independent mechanisms.

We recently showed that clotting factor VIIa (FVIIa) binding to endothelial cell protein C receptor (EPCR) induces anti-inflammatory signaling and protects vascular barrier integrity. Inflammation and vascular permeability are thought to be major contributors to the development of hemophilic arthropathy following hemarthrosis. The present study was designed to investigate the potential influence of FVIIa interaction with EPCR in the pathogenesis of hemophilic arthropathy and its treatment with recombinant FVIIa (rFVIIa). For this, we first generated hemophilia A (FVIII-/-) mice lacking EPCR (EPCR-/-FVIII-/-) or overexpressing EPCR (EPCR++ FVIII-/-). Joint bleeding was induced in FVIII-/-, EPCR-/- FVIII-/-, and EPCR++FVIII-/- mice by needle puncture injury. Hemophilic synovitis was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections. EPCR deficiency in FVIII-/- mice significantly reduced the severity of hemophilic synovitis. EPCR deficiency attenuated the elaboration of interleukin6, infiltration of macrophages, and neoangiogenesis in the synovium following hemarthrosis. A single dose of rFVIIa was sufficient to fully prevent the development of milder hemophilic synovitis in EPCR-/-FVIII-/- mice. The development of hemophilic arthropathy in EPCR-overexpressing FVIII-/- mice did not significantly differ from that of FVIII-/- mice, and 3 doses of rFVIIa partly protected against hemophilic synovitis in these mice. Consistent with the data that EPCR deficiency protects against developing hemophilic arthropathy, administration of a single dose of EPCR-blocking monoclonal antibodies markedly reduced hemophilic synovitis in FVIII-/- mice subjected to joint bleeding. The present data indicate that EPCR could be an attractive new target to prevent joint damage in hemophilia patients.

Crohn’s disease and ulcerative colitis are the two forms of disorders of the human inflammatory bowel disease with unknown etiologies. Endothelial cell protein C receptor (EPCR) is a multifunctional and multiligand receptor, which is expressed on the endothelium and other cell types, including epithelial cells. Here, we report that EPCR is expressed in the colon epithelial cells, CD11c+, and CD21+/CD35+ myeloid cells surrounding the crypts in the colon mucosa. EPCR expression was markedly decreased in the colon mucosa during colitis. The loss of EPCR appeared to associate with increased disease index of the experimental colitis in mice. EPCR−/− mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, manifested by increased weight loss, macrophage infiltration, and inflammatory cytokines in the colon tissue. DSS treatment of EPCR−/− mice resulted in increased bleeding, bodyweight loss, anemia, fibrin deposition, and loss of colon epithelial and goblet cells. Administration of coagulant factor VIIa significantly attenuated the DSS-induced colon length shortening, rectal bleeding, bodyweight loss, and disease activity index in the wild-type mice but not EPCR−/− mice. In summary, our data provide direct evidence that EPCR plays a crucial role in regulating the inflammation in the colon during colitis.

Objective: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1–mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. Conclusions: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.

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.

Recent studies from our laboratory showed that clotting factor VIIa (FVIIa), whose primary function is to initiate the coagulation cascade following its binding to procoagulant cofactor tissue factor (TF), also binds anticoagulant cofactor, endothelial cell protein C receptor (EPCR). EPCR plays a key role in the activated protein C (APC)-mediated anticoagulant pathway by promoting the activation of protein C bound to it. Pharmacological concentrations of rFVIIa could compete with protein C for the EPCR and downregulate APC generation by preventing protein C binding to the EPCR. Our recent studies showed that FVIIa binding to EPCR also induces barrier-protective and anti-inflammatory effects both in vitro and in vivo model systems. Bio-distribution of rFVIIa in mice revealed that a small fraction of rFVIIa administed to mice i.v. accumulates in knee joints and retained there for three to seven days. The present study was conducted to investigate the relevance of FVIIa binding to EPCR in pharmacological FVIIa treatment of joint disease in hemophilia. For this, we first generated hemophilia A mice lacking EPCR or overexpressing EPCR. To generate EPCR deficient FVIII-/- mice, Procrflox/flox and Procr+/floxMeox2+/cre mice were first backcrossed with FVIII-/- mice to generate Procrflox/flox/FVIII-/- and Meox2+/cre/FVIII-/- mice. EPCR deficient FVIII-/- mice (Procr-/-/FVIII-/-) were generated by breeding female Procrflox/flox/FVIII-/- with male Procr+/floxMeox2+/cre /FVIII-/- mice. EPCR- overexpressing FVIII-/- were generated by crossing Tie2-EPCR mice with FVIII-/- mice. We induced joint bleeding in FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice by needle puncture injury. Right knee joint intra articular space was pierced with 30G needle to induce joint bleeding; left knee joint served as an uninjured control. Injured mice were treated with a single dose of rFVIIa (1 mg/kg) at 20 min following the injury or three doses of rFVIIa (1 mg/Kg) at 20 min, 24 h and 72 h following the injury. In controls, saline was administered in place of rFVIIa. The knee injury was evaluated by measuring knee joint diameter and visual bleeding score. At the end of 14 days post injury, mice were euthanized, and the knee joints were excised and fixed for immunohistochemical analysis. Hemophilic arthropathy was evaluated by scoring a set of pathological parameters (e.g., synovial hyperplasia, neoangiogenesis, presence of blood, iron score, villus formation, and cartilage degeneration). A subset of mice was used to collect synovial fluid at 7 days post injury. Assessment of joint bleeding at 5 h following the needle injury, by extracting and measuring hemoglobin levels in joint tissues and monitoring hematocrit, showed an equal amount of bleeding in all three genotypes of mice (FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice). Administration of three doses of FVIIa was effective in preventing hemophilic arthropathy in FVIII-/- and EPCR-overexpressing FVIII-/- mice, whereas a single dose of rFVIIa had a minimal effect in reducing hemophilic arthropathy. Interestingly, a single dose of FVIIa fully corrected the needle injury-induced hemophilic arthropathy in EPCR-deficient FVIII-/- mice. More importantly, EPCR-deficient FVIII-/- mice failed to develop full-blown hemophiic arthropathy even in the absence of any treatment. Measurement of inflammatory cytokines in the synovial fluid showed a robust increase in IL-6 levels in FVIII-/- mice and a single dose of FVIIa substantially reduced IL-6 levels. Interestingly, IL-6 levels in the synovial fluid of injured EPCR-overexpressing FVIII-/- mice were markedly higher compared to injured FVIII-/- mice, three doses of rFVIIa was required to attain a significant reduction in IL-6 levels in these mice. In contrast to these data, we found very little IL-6 levels in the synovial fluids of injured EPCR-deficient FVIII-/- mice. Overall, these data indicate that EPCR levels profoundly influence hemophilic arthorpathy. EPCR-deficiency protects from development hemophilic arthropathy because down-regulation of APC generation in EPCR deficiency could allow sufficient thrombin generation in hemophilia to prevent joint bleeding and bleeding-associated inflammation. Disclosures Rao: Takeda: Research Funding.

Tissue factor (TF), an integral membrane glycoprotein, is a cofactor for coagulation factor VIIa (FVIIa) and primary cellular initiator of the coagulation. Upon vascular injury or in disease conditions, blood comes in contact with TF, and the formation of TF-FVIIa complex initiates activation of the coagulation cascade. While TF is critical for the maintenance of hemostasis, aberrant expression of TF activity could lead to thrombotic disorders. Typically, most of TF on cell surfaces exist in a cryptic, coagulant inactive state, and an "activation" step (decryption) is essential for the transformation of cryptic TF to prothrombotic TF. Our recent studies showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells. The hydrolysis of SM, by either bacterial sphingomyelinase (bSMase) or acid-sphingomyelinase (ASMase) translocated from lysosomes to the outer leaflet in response to ATP, LPS or cytokine stimulation, increased TF activity on intact cells without altering TF protein levels. SM hydrolysis also led to the release of TF+ microvesicles (MVs). Inhibition of ASMase by functional inhibitors blocked LPS-induced TF procoagulant activity without impairing LPS-induced TF antigen levels in both in vitro and in vivo model systems. SM levels in the plasma membrane are regulated primarily by SM synthesizing enzymes, such as sphingomyelin synthases (SMS) 1 and 2 or SM hydrolyzing enzymes, such as ASMase and neutral SMases (nSMase). Many disease conditions, including diabetes, ischemia/hypoxia, and cancer, alter SM metabolism by altering the activities of the above enzymes. These diseases are also known to have increased thrombotic risk. To investigate the importance of SM metabolism in regulating TF procoagulant activity through TF encryption and decryption, we either overexpressed or silenced the enzymes involved in SM metabolism and determined their effect on TF procoagulant activity on intact cells and the release of TF+ MVs. Human monocyte-derived macrophages (MDMs) or human embilical vein endothelial cells (HUVEC) were chosen as cell model systems. In the first set of experiments, MDMs were transfected with adenovirus encoding SMS1, SMS2, or both to overexpress SMS. Analysis of SM levels in the outer leaflet by confocal microscopy and flow cytometry using SM specific binding protein (lysenin) revealed that overexpression of SMS1 or SMS2 increased SM levels in the outer leaflet. Measurement of TF activity on intact cells showed that overexpression of either SMS1 or SMS2 reduced both basal TF activity and the extent of increased TF activity following ATP or bSMase treatment. Overexpression of SMS1 or SMS2 also decreased the release of TF+ MVs. Overexpression of SMS1 or SMS2 had no significant effect on TF antigen levels. In the next set of experiments, MDMS were transfected with control scrambled RNA (scRNA) or siRNA specific for ASMase, nSMase1, nSMase2, or nSMase3. As expected from our earlier studies, ASMase silencing attenuated both basal and ATP-induced increased TF activity in MDMs. In case of nSMases, the knock-down of nSMase2 or nSMase3, but not nSMase1, reduced basal TF activity as well as ATP-induced TF decryption in MDMs. Analysis of SM levels in the outer leaflet showed that silencing of ASMase, nSMase2, or nSMase3 enhanced the SM content. The knock-down of either ASMase or nSMases did not affect TF antigen levels. In additional studies, HUVECs were transfected with control scRNA or siRNA specific for nSMase1, nSMAse2, or nSMase3. Forty eight hour post-transfection, HUVECs were stimulated with TNFα (10 ng/ml) plus IL-1β (10 ng/ml) for 6 h to induce TF expression. Analysis of cell surface TF activity showed that silencing nSMase2 or nSMase3, but not nSMase1, attenuated TNFα+IL-1β-induced TF procoagulant activity without decreasing TNFα+IL-1β-induced TF antigen levels. Overall, our data support the hypothesis that alterations in SM metabolism regulate TF procoagulant activity through encryption and decryption. Disclosures Rao: Takeda: Research Funding.