Extracellular Vesicles in the Pathogenesis, Clinical Characterization, and Management of Dermatomyositis: A Narrative Review.

Extracellular vesicles (EVs) are lipid-bilayer particles secreted from cells that primarily assist in cell-to-cell communication through the content of their cargo, such as proteins and RNA. EVs have been implicated in the pathogenesis of various autoimmune diseases, including dermatomyositis (DM), an inflammatory autoimmune disease characterized by distinct cutaneous manifestations, myopathy, and lung disease. We sought to review the role of EVs in DM and understand how they contribute to the pathogenesis and clinical characterization of the disease. We summarized the research progress on EVs in dermatomyositis based on recent publications. EV cargoes, such as double-stranded DNA, microRNA, and proteins, contribute to DM pathogenesis and mediate the proinflammatory response and cytokine release through signaling pathways such as the stimulator of interferon genes (STING) pathway. These nucleic acids and proteins have been proposed as disease-specific, stable biomarkers to monitor disease activity and responses to therapy. They also correlate with clinical parameters, inflammatory markers, and disease severity scores. Furthermore, some markers show an association with morbidities of DM, such as muscle weakness and interstitial lung disease. The continued study of EVs will help us to further elucidate our understanding of dermatomyositis.

Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly.

The nuclear lamina is essential for the structural integration of the nuclear envelope. Nuclear envelope rupture and chromatin externalization is a hallmark of the formation of neutrophil extracellular traps (NETs). NET release was described as a cellular lysis process; however, this notion has been questioned recently. Here, we report that during NET formation, nuclear lamin B is not fragmented by destructive proteolysis, but rather disassembled into intact full-length molecules. Furthermore, we demonstrate that nuclear translocation of PKCα, which serves as the kinase to induce lamin B phosphorylation and disassembly, results in nuclear envelope rupture. Decreasing lamin B phosphorylation by PKCα inhibition, genetic deletion, or by mutating the PKCα consensus sites on lamin B attenuates extracellular trap formation. In addition, strengthening the nuclear envelope by lamin B overexpression attenuates NET release in vivo and reduces levels of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Our findings advance the mechanistic understanding of NET formation by showing that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for chromatin release in neutrophils.

Resolving primary membranous glomerulopathy (MGN) reveals a dynamically metabolic pathway from sub-epithelium to glomerular basement membranes.

In idiopathic (primary) membranous glomerulopathy (MGN), there is a phenomenon of subepithelial deposits (stages 1 and 2) transitioned to intramembranous deposits, with lucent resolving features (stages 3 and 4). This phenomenon has not been described in other types of immune complex mediated glomerulonephritis with either subendothelial or mesangial deposits. The goal of this study was to evaluate what unique immunostaining pattern could occur in primary MGNs with intramembranous resolving features. PLA2R and IgG4 immunostains were performed in 50 primary MGNs, and 39 secondary MGNs after the clinical history was reviewed. Primary MGNs with resolving features were further evaluated in detail. A total of 84% (42/50) of primary MGN cases had diffuse positive immunostaining for IgG4 in the glomeruli, and most of them were also positive for PLA2R staining. Eight of the remaining primary MGN cases (8/50) with positive PLA2R but negative IgG4 staining in the glomeruli had diffuse resolving features as observed by electron microscopy. All secondary MGNs were stained negatively for both IgG4 and PLA2R except for one case with positive IgG4 staining but negative staining for PLA2R. Our data indicate that IgG4 staining on paraffin tissue is a very reliable screening tool to confirm the presence of primary MGN. Primary MGN with PLA2R+/IgG4- stains were seen in those with intramembranous resolving features. This finding is consistent with the known weak-binding capacity of IgG4 to the glomerular basement membranes. The transitional phenomenon from PLA2R+/IgG4+ subepithelial deposits to PLA2R+/IgG4- intramembranous resolving deposits in primary MGN implies that there may be a continuous metabolic activity from podocyte to glomerular basement membrane.

Extracellular vesicles mediate cellular interactions in renal diseases-Novel views of intercellular communications in the kidney.

The kidney is a complicated and important internal organ receiving approximately 20% of the cardiac output and mediates numerous pathophysiologic actions. These include selectively filtering macromolecules of the blood, exquisite reclaimation of electrolyctes, urine concentration via an elegant osmotic mechanism, and excretion of an acid load. In addition, the renal tubules carry out secretory functions and produce hormones and cytokines. The kidney receives innervation and hormonal regulation. Therefore, dysfunction of the kidney leads to retention of metabolic waste products, and/or significant proteinuria and hematuria. In the past several decades, the role of extracellular vesicles (EVs) in intercellular communications, and the uptake of EVs by recipient cells through phagocytosis and endocytosis have been elucidated. The new knowledge on EVs expands over the classical mechanisms of cellular interaction, and may change our way of thinking of renal pathophysiology in the subcellular scale. Based on some ultrastructural discoveries in the kidney, this review will focus on the role of EVs in intercellular communications, their internalization by recipient cells, and their relationship to renal pathology.

Extracellular vesicles and lupus nephritis - New insights into pathophysiology and clinical implications.

Lupus nephritis (LN) is a major cause for overall morbidity and mortality in patients with systemic lupus erythematosus (SLE), while its pathogenic mechanisms are still not well understood. Extracellular vesicles (EVs) are membrane vesicles that are released from almost all cell types. EVs can be subdivided into exosomes, microvesicles, and apoptotic bodies. Latest studies have shown that EVs can be released during several cellular events, including cell activation, autophagy, and several types of programed cell death, i.e. apoptosis, necroptosis, pyroptosis, and NETosis. Emerging evidence demonstrates that EVs harbor different bioactive molecules, including nucleic acids, proteins, lipids, cytokines, immune complexes (ICs), complements, and other molecules, some of which may contribute to pathogenesis of autoimmune diseases. EVs can serve as novel information shuttle to mediate local autocrine or paracrine signals to nearby cells, and distant endocrine signals to cells located far away. In LN, EVs may have pathogenic effects by transportation of autoantigens or complements, promotion of IC deposition or complement activation, and stimulation of inflammatory responses, renal tissue injury, or microthrombus formation. Additionally, EVs released from kidney cells may serve as specific biomarkers for diagnosis or monitoring of disease activity and therapeutic efficacy. In this review, we will summarize the latest progress about EV generation from basic research, their potential pathologic effects on LN, and their clinical implications. The cutting-edge knowledge about EV research provides insights into novel therapeutic strategy, new tools for diagnosis or prognosis, and evaluation approaches for treatment effectiveness in LN.

Spermine on Endothelial Extracellular Vesicles Mediates Smoking-Induced Pulmonary Hypertension Partially Through Calcium-Sensing Receptor.

Objective- This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results- Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine-a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N1-dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions- Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.

Pathogenic roles of microvesicles in diabetic retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes and has been recognized as the leading cause of blindness in adults. Several interrelated molecular pathways are involved in the development of DR. Microvesicles (MVs) are cell membrane vesicles, which carry many biologic molecules, such as mRNAs, microRNAs, transcription factors, membrane lipids, membrane receptors, and other proteins. They may be involved in intercellular communication that can promote inflammation, angiogenesis, and coagulation. Recent studies have indicated that changes in the number and composition of MVs may reflect the pathologic conditions of DR. At present, MVs are well recognized as being involved in the pathophysiological conditions of tumors and cardio-metabolic diseases. However, the roles of MVs in DR have yet to be investigated. In this review, we provide an overview of DR-induced microvascular injury that is caused by MVs derived from endothelial and circulating cells, and discuss the possible mechanisms by which MVs can lead to endothelial dysfunction, coagulation and inflammation. In addition, the protective effects of preconditioned MVs and stem cell-derived MVs are also described . Understanding the involvement of MVs in the pathophysiological conditions of DR may provide insight into the disease mechanisms and may suggest novel therapeutic strategies for DR in the future.

Translocation of Endogenous Danger Signal HMGB1 From Nucleus to Membrane Microvesicles in Macrophages.

High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

Novel proteolytic microvesicles released from human macrophages after exposure to tobacco smoke.

Cigarette smoking damages the extracellular matrix in a variety of locations, leading to atherosclerotic plaque instability and emphysematous lung destruction, but the underlying mechanisms remain poorly understood. Here, we sought to determine whether exposure of human macrophages, a key participant in extracellular matrix damage, to tobacco smoke extract (TSE) induces the release of microvesicles (MVs; or microparticles) with proteolytic activity; the major proteases involved; and the cellular mechanisms that might mediate their generation. We found that MVs released from TSE-exposed macrophages carry substantial gelatinolytic and collagenolytic activities that surprisingly can be predominantly attributed to a single transmembrane protease of the matrix metalloproteinase (MMP) superfamily (namely, MMP14). Flow cytometric counts revealed that exposure of human macrophages to TSE for 20 hours more than quadrupled their production of MMP14-positive MVs (control, 1112 ± 231; TSE-induced, 5823 ± 2192 MMP14-positive MVs/µL of conditioned medium; means ± SEM; n = 6; P < 0.01). Our results indicate that the production of these MVs by human macrophages relies on a series of regulated steps that include activation of two mitogen-activated protein kinases (MAPKs, i.e., the Jun N-terminal kinase and p38 MAPK), and then MAPK-dependent induction and maturation of cellular MMP14, a remarkable accumulation of MMP14 into nascent plasma membrane blebs, and finally caspase- and MAPK-dependent apoptosis and apoptotic microvesicle generation. Proteolytically active MVs induced by tobacco smoke may be novel mediators of clinical important matrix destruction in smokers.

Microvesicles and diabetic complications--novel mediators, potential biomarkers and therapeutic targets.

Microvesicles (MVs), also known as microparticles, are small membrane vesicles released from different cell types under different conditions. MVs have been detected in the circulation and in organs/tissues in various diseases, including diabetes. Patients with different types of diabetes and complications have different cellular MV patterns. Studies have shown that MVs may mediate vascular thrombosis, vascular inflammation, angiogenesis, and other pathological processes of the disease through their procoagulant, pro-inflammatory, pro-angiogenic, proteolytic, and other properties. Therefore, MVs contribute to the development of diabetic macrovascular and microvascular complications. In addition, clinical studies have indicated that changes in MV number and composition may reflect the pathophysiological conditions of disease, and therefore, may serve as potential biomarkers for diagnostic and prognostic use. Understanding MVs' involvement in the pathophysiological conditions may provide insight into disease mechanisms and would also be helpful for the development of novel therapeutic strategies in the future. Here, we review the latest publications from our group and other groups and focus on the involvement of MVs in diabetic complications.

Microparticles: new light shed on the understanding of venous thromboembolism.

Microparticles are small membrane fragments shed primarily from blood and endothelial cells during either activation or apoptosis. There is mounting evidence suggesting that microparticles perform a large array of biological functions and contribute to various diseases. Of these disease processes, a significant link has been established between microparticles and venous thromboembolism. Advances in research on the role of microparticles in thrombosis have yielded crucial insights into possible mechanisms, diagnoses and therapeutic targets of venous thromboembolism. In this review, we discuss the definition and properties of microparticles and venous thromboembolism, provide a synopsis of the evidence detailing the contributions of microparticles to venous thromboembolism, and propose potential mechanisms, by which venous thromboembolism occurs. Moreover, we illustrate a possible role of microparticles in cancer-related venous thromboembolism.

Cholesterol-induced membrane microvesicles as novel carriers of damage-associated molecular patterns: mechanisms of formation, action, and detoxification.

OBJECTIVE: Cholesterol enrichment occurs in vivo when phagocytes ingest retained and aggregated lipoproteins, damaged or senescent cells, and related debris. We previously reported that enrichment of human monocyte/macrophages with unesterified cholesterol (UC) triggers the release of highly procoagulant microvesicles ([MVs], also called microparticles) through induction of apoptosis. We determined whether UC-induced MVs (UCMVs) might transmit endogenous danger signals and, if so, what molecular processes might be responsible for their production, recognition, and detoxification. METHODS AND RESULTS: Injection of UCMVs into rats provoked extensive leukocyte rolling and adherence to postcapillary venules in vivo. Likewise, exposure of mouse aortic explants or cultured human endothelial cells to UCMVs augmented the adhesion of human monocytes by several fold and increased endothelial cell intercellular adhesion molecule-1 via nuclear factor-κB activation. To explore molecular mechanisms, we found that UC enrichment of human monocytes, in the absence of other stimuli, induced mitochondrial complex II-dependent accumulation of superoxide and peroxides. A subset of these moieties was exported on UCMVs and mediated endothelial activation. Strikingly, aortic explants from mice lacking lectin-like oxidized low-density lipoprotein receptor-1, a pattern-recognition receptor, were essentially unable to respond to UCMVs, whereas simultaneously treated explants from wild-type mice responded robustly by increasing monocyte recruitment. Moreover, high-density lipoprotein and its associated enzyme paraoxonase-1 exerted unexpected roles in the detoxification of UCMVs. CONCLUSIONS: Overall, our study implicates MVs from cholesterol-loaded human cells as novel carriers of danger signals. By promoting maladaptive immunologic and thrombotic responses, these particles may contribute to atherothrombosis and other conditions in vivo.

Neutrophil extracellular traps drive lupus flares with acute skin and kidney inflammation triggered by ultraviolet irradiation.

Sunlight triggers lupus flares causing both local skin and systemic inflammation, including lupus nephritis, through poorly understood mechanisms. To address this knowledge gap, we found that UVB irradiation of asymptomatic, young female lupus-prone mice induced skin and kidney inflammation with proteinuria, accompanied by neutrophil infiltration and neutrophil extracellular trap (NET) formation. Furthermore, UVB irradiation induced co-expression of CXCR4 and cytokines/C3 by neutrophils in vitro and in vivo, in the skin and kidneys of lupus-prone mice, indicating their transmigratory and pro-inflammatory potentials. A causality study demonstrated that inhibiting CXCR4 attenuated renal neutrophil infiltration, accumulation of NETs, NET-associated cytokines/C3, and proteinuria in UVB-irradiated lupus-prone mice. Remarkably, inhibiting NETosis through a novel strategy targeting nuclear envelope integrity reduced deposition of NET-associated cytokines/C3 in skin and kidneys, attenuating proteinuria in UVB-irradiated MRL/lpr·lmnB1 Tg mice. Our investigation unveils a new mechanism by which neutrophil NETs drive the early onset of lupus flares triggered by UVB-irradiation. Targeting neutrophil transmigration and NETosis could be promising therapeutic strategies.

Moonlighting chromatin: when DNA escapes nuclear control.

Extracellular chromatin, for example in the form of neutrophil extracellular traps (NETs), is an important element that propels the pathological progression of a plethora of diseases. DNA drives the interferon system, serves as autoantigen, and forms the extracellular scaffold for proteins of the innate immune system. An insufficient clearance of extruded chromatin after the release of DNA from the nucleus into the extracellular milieu can perform a secret task of moonlighting in immune-inflammatory and occlusive disorders. Here, we discuss (I) the cellular events involved in the extracellular release of chromatin and NET formation, (II) the devastating consequence of a dysregulated NET formation, and (III) the imbalance between NET formation and clearance. We include the role of NET formation in the occlusion of vessels and ducts, in lung disease, in autoimmune diseases, in chronic oral disorders, in cancer, in the formation of adhesions, and in traumatic spinal cord injury. To develop effective therapies, it is of utmost importance to target pathways that cause decondensation of chromatin during exaggerated NET formation and aggregation. Alternatively, therapies that support the clearance of extracellular chromatin are conceivable.

Recent progress in the mechanistic understanding of NET formation in neutrophils.

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation.

Objective: Ultraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKCα-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKCα nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation. Methods: We studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKCα nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation. Results: Our time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKCα in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKCα nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNFα, IFNγ, and IFNα in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency. Conclusions: ROCK regulated NETosis through modulation of PKCα nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB-induced skin inflammation by attenuation of NETosis and NET-associated cytokines.

Type 2 diabetes in mice induces hepatic overexpression of sulfatase 2, a novel factor that suppresses uptake of remnant lipoproteins.

UNLABELLED: Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial remnant lipoproteins. Our work and that of others have identified syndecan-1 heparan sulfate proteoglycans (HSPGs) as remnant lipoprotein receptors. Nevertheless, defects in the T2DM liver have not been molecularly characterized, and neither has the correction that occurs upon caloric restriction. We used microarrays to compare expression of proteoglycan-related genes in livers from control db/m mice; obese, T2DM db/db littermates fed ad libitum (AL); and db/db mice pair-fed to match the intake of db/m mice. Surprisingly, the arrays identified only one gene whose dysregulation by T2DM would disrupt HSPG structure: the heparan sulfate glucosamine-6-O-endosulfatase-2 (Sulf2). SULF2 degrades HSPGs by removing 6-O sulfate groups, but had no previously known role in diabetes or lipoprotein biology. Follow-up quantitative polymerase chain reaction assays revealed a striking 11-fold induction of Sulf2 messenger RNA in the livers of AL T2DM mice compared with controls. Immunoblots demonstrated induction of SULF2 in AL livers, with restoration toward normal in livers from pair-fed db/db mice. Knockdown of SULF2 in cultured hepatocytes doubled HSPG-mediated catabolism of model remnant lipoproteins. Notably, co-immunoprecipitations revealed a persistent physical association of SULF2 with syndecan-1. To identify mechanisms of SULF2 dysregulation in T2DM, we found that advanced glycosylation end products provoked a 10-fold induction in SULF2 expression by cultured hepatocytes and an approximately 50% impairment in their catabolism of remnants and very low-density lipoprotein, an effect that was entirely reversed by SULF2 knockdown. Adiponectin and insulin each suppressed SULF2 protein in cultured liver cells and in murine livers in vivo, consistent with a role in energy flux. Likewise, both hormones enhanced remnant lipoprotein catabolism in vitro. CONCLUSION: SULF2 is an unexpected suppressor of atherogenic lipoprotein clearance by hepatocytes and an attractive target for inhibition.

Tobacco smoke induces the generation of procoagulant microvesicles from human monocytes/macrophages.

OBJECTIVE: To investigate whether exposure of human monocytes/macrophages to tobacco smoke induces their release of membrane microvesicles (MVs) that carry tissue factor (TF) released from cells, whether smoke-induced MVs are procoagulant, and what cellular processes might be responsible for their production. METHODS AND RESULTS: We found that exposure of human THP-1 monocytes and primary human monocyte-derived macrophages to 3.75% tobacco smoke extract (TSE) significantly increased their total and TF-positive MV generation. More importantly, MVs released from TSE-treated human monocytes/macrophages exhibited 3 to 4 times the procoagulant activity of control MVs, as assessed by TF-dependent generation of factor Xa. Exposure to TSE increased TF mRNA and protein expression and cell surface TF display by both THP-1 monocytes and primary human monocyte-derived macrophages. In addition, TSE exposure caused activation of C-Jun-N-terminal kinase (JNK), p38, extracellular signal regulated kinase (ERK) mitogen-activated protein kinases (MAPK), and apoptosis (a major mechanism for MV generation). Treatment of THP-1 cells with inhibitors of ERK, MAP kinase kinase (MEK), Ras, or caspase 3, but not p38 or JNK, significantly blunted TSE-induced apoptosis and MV generation. Surprisingly, neither ERK nor caspase 3 inhibition altered the induction of cell surface TF display by TSE, indicating an effect solely on MV release. Inhibition of ERK or caspase 3 essentially abolished TSE-induced generation of procoagulant MVs from THP-1 monocytes. CONCLUSIONS: Tobacco smoke exposure of human monocytes/macrophages induces cell surface TF display, apoptosis, and ERK- and caspase 3-dependent generation of biologically active procoagulant MVs. These processes may be novel contributors to the pathological hypercoagulability of active and secondhand smokers.