Increased Tissue Factor Activity Research Articles

Characterization of extracellular vesicles from patients with severe aortic stenosis undergoing transcatheter aortic valve implantation and assessment of their coagulation potential

Abstract Funding Acknowledgements None. Background The pathogenesis of aortic stenosis (AS) includes processes of inflammation and calcification. Patients with aortic stenosis have increased risk of stroke. The amount of circulating extracellular vesicles (EVs) is elevated in patients with severe AS and decreases following valve intervention. The function of these EVs in the clinical setting of severe AS is not fully known. Aim With flow cytometry characterize EVs in patients with severe AS before and after Transcatheter Aortic Valve Implantation (TAVI). To investigate whether these EVs have a procoagulant effects by themselves or when incubated with human aortic endothelial cells (hAoECs), human cardiac microvascular endothelial cells (hCMVEC), human cardiac myocytes (hCM) and spheroids of hCMVEC and hCM. Methods 25 patients with severe AS were included in the study and blood samples collected before and 2 months after TAVI. The CytoFLEX flow cytometer (Beckman Coulter) was used for characterization of surface antigens on purified EVs. 2D-cultures of hAoECs, hCMVEC and hCM were incubated with EVs (100 mg/mL) for 6 hours (h) and spheroids of hCM and hCMVEC with EVs for 48h. An inhouse assay determining Tissue Factor (TF) activity by means of FXa generation in cell lysates were utilized. TF activity in purified EVs alone was determined using Tissue Factor Activity Assay kit (Human, colorimetric) from abcam. Results We found an increased level of TF positive EVs, phosphatidylserine (PS) positive EVs as well as double positive PS / TF expressing EVs in AS patients before TAVI compared to healthy controls and reduction of PS+ and TF+/PS+ expressing EVs after TAVI (Fig 1). When analyzing TF activity by means of FXa generation we found no procoagulant effect in the purified EVs alone nor when incubating EVs with hAoEC or hCM. In contrast, we found an increased TF activity in hCMVEC and spheroids of hCM and hCMVEC after incubation with EVs from AS patients compared to healthy EVs. Conclusion Patients with AS have increased amount of EVs expressing typically procoagulant surface antigens. The EVs by themselves have no procoagulant activity. However, upon interaction with hCMVEC and spheroids, consisting of hCMVEC and hCM, EVs appear to induce procoagulant activity in the recipient cells thus suggesting a possible link to thromboembolic events in coronary arteries. Additional studies are warranted to elaborate the mechanism and function of these TF, PS and TF / PS positive EVs.

CA19-9-Positive Extracellular Vesicle Is a Risk Factor for Cancer-Associated Thrombosis in Pancreatic Cancer

Cancer-associated venous thromboembolism (VTE) is a frequent complication associated with high mortality in patients with cancer, particularly pancreatic cancer. While biological factors such as coagulation factors released from cancer cells may underlie the mechanisms of cancer-associated VTE, the detailed mechanisms have not been determined. Here, we aimed to determine whether extracellular vesicles carrying a glycan sialyl-Lewisa, known as carbohydrate antigen 19-9 (CA19-9), which is a clinically used serum tumor marker and selectin ligand, are a significant cause of cancer-associated VTE. Risk factors for cancer-associated VTE were determined using clinical data. EVs derived from CA19-9-deficient or overexpressing pancreatic cancer cells were characterized. The protein levels of coagulation factors on the surface of the EVs were quantified using our newly developed sensitive method. Higher CA19-9 levels in the sera of patients were significantly associated with the occurrence of VTE. Using CA19-9-negative or overexpressing pancreatic cancer cells, we found that EVs derived from these cells interacted with E-selectin of endothelial cells in a CA19-9-dependent manner in cell-based assays and invitro blood vessel models. EVs derived from cancer cells have higher tissue factor levels on their surfaces, and increased tissue factor activity is induced locally, where CA19-9-positive EVs bind to activated endothelial cells. These results suggest that the binding between CA19-9-positive EVs released fromcancer cells and endothelial cell E-selectin explains theincreased frequency of VTE in patients with pancreatic cancer.

Sars-Cov-2 Spike Protein Triggers Inflammasome-Mediated Tissue Factor Activity

Introduction: An exaggerated inflammatory response both locally and systemically mediated by innate immune signaling is a near universal feature of severe COVID-19 illness and has been associated with multi-organ damage and thrombosis. As part of an antiviral response, activation and formation of the intracellular NLRP3 inflammasome leads to expression of proinflammatory IL-1 family cytokines. In macrophages, the NLRP3 inflammasome is linked to the release of tissue factor (TF) bearing microvesicles. Multiple studies provide evidence of NLRP3 inflammasome activation in COVID-19 including increased IL-1β in monocytes, sera, and bronchoalveolar lavage fluid from COVID-19 patients. Patient monocytes bear features of inflammasome activation, which correlate with disease severity (Potere et al. Cardiovascular Research 2023). Given the central role of thromboinflammation in COVID-19, we explored the ability of various coronavirus spike proteins to increase TF activity in a monocytic cell line. Mechanistically, we sought to explore the role of the NLRP3 inflammasome in SARS-CoV-2 spike protein triggered tissue factor generation. Methods: The SARS-CoV-2 α variant S1 subunit (S1α) and Omicron variant BA.1.1.529 S1 (S1O) recombinant proteins were obtained from Raybiotech and OC43, a non-COVID respiratory human coronavirus, recombinant spike protein (OC43 S) from Sino Biological. THP-1 cells at a concentration of 2x10 6/mL were incubated with spike proteins at 37°C in RPMI, supplemented with 10% fetal bovine serum. A specific NLRP3 inflammasome inhibitor (MCC950) was incubated with select samples for 1 hour following addition of the spike protein. The cells were harvested, subjected to RNA extraction and cDNA synthesis by reverse transcription. TF, NLRP3, caspase (CASP)-1, and IL-1β relative mRNA expression was quantified using RT-PCR and normalized to GADPH expression. For quantifying microparticle TF activity, cells were incubated with the spike proteins for 6 hours then microparticles were isolated from the supernatant after centrifugation at 20,000 g for 30 minutes at 4°C, washed and TF activity was measured using a two-stage chromogenic assay kit (Abcam). A monoclonal anti-TF antibody (Santa Cruz) was used to confirm specificity for TF generation. Experiments were performed in triplicate. Data is presented as mean ± standard deviation. The student t test was used to assess the difference between unpaired groups with a value of p <.05 considered statistically significant. Results: Our previous data showed that S1α, but not S1O or OC43 S, leads to increased TF mRNA expression in THP-1 cells in a dose dependent manner. Here we show TF microparticle activity was increased with S1α (20 µg/mL) as compared to incubation of THP-1 cells with S1O or OC43 spike proteins (20 µg/mL), which were comparable to the activity of cells alone ( Figure 1). After treatment with S1α (20 µg/mL) for 1 hour, we found increased expression of TF (10-fold), NLRP3 (2-fold), CASP-1 (3-fold), and IL-1β (3.5-fold). Treatment with S1O and OC43 S did not lead to a significant change in relative mRNA expression of TF, NLRP3, CASP-1, and IL-1β. Following incubation with the S1α spike protein for 1 hour, cells were re-plated in fresh media containing 10 µM MCC950. Treatment with MCC950 significantly reduced expression of TF, NLRP3, CASP-1, and IL-1β ( Figure 2). Conclusion: We confirmed that TF expression induced by the S1α spike protein leads to an increase in TF activity, as assessed by microparticle generation. The NLRP3 inflammasome is a mediator of coagulopathy induced by the spike protein, and TF, CASP-1, and IL-1β expression are abrogated after specific NLRP3 inflammasome inhibition. TF activity and inflammation induced by the coronavirus spike proteins in vitro correlates with the respective clinical phenotype and disease severity of the variants. This suggests that the effects of SARS-CoV-2 on TF and inflammasome activation derive in part from the spike protein, and these assays may be useful in predicting the effects of spike protein variants.

Bystander effect of SARS-CoV-2 spike protein on human monocytic THP-1 cell activation and initiation of prothrombogenic stimulus representing severe COVID-19

BackgroundHypercoagulable state and thromboembolic complications are potential life-threatening events in COVID-19 patients. Our previous studies demonstrated that SARS-CoV-2 infection as well as viral spike protein expressed epithelial cells exhibit senescence with the release of inflammatory molecules, including alarmins.FindingsWe observed extracellular alarmins present in the culture media of SARS-CoV-2 spike expressing cells activate human THP-1 monocytes to secrete pro-inflammatory cytokines to a significant level. The release of THP-1 derived pro-inflammatory cytokine signature correlated with the serum of acute COVID-19 patient, but not in post-COVID-19 state. Our study suggested that the alarmins secreted by spike expressing cells, initiated phagocytosis property of THP-1 cells. The phagocytic monocytes secreted complement component C5a and generated an autocrine signal via C5aR1 receptor. The C5a-C5aR1 signal induced formation of monocyte mediated extracellular trap resulted in the generation of a prothrombogenic stimulus with activating platelets and increased tissue factor activity. We also observed an enhanced C5a level, platelet activating factor, and high tissue factor activity in the serum of acute COVID-19 patients, but not in recovered patients.ConclusionOur present study demonstrated that SARS-CoV-2 spike protein modulates monocyte responses in a paracrine manner for prothrombogenic stimulus by the generation of C5a complement component.

The role of coagulation hemostasis for development of early-onset preeclampsia in low-risk patients

Aim: to determine a role of specific parameters of coagulation hemostasis for development of severe early-onset preeclampsia (еPE) in low-risk patients.Materials and Methods. A single-center prospective study included 40 patients with a physiological course of pregnancy and term delivery (control group) as well as 41 pregnant women with developed severe еPE (study group). Patients at the gestational age of 19–21 and 27–28 weeks in the comparison groups were examined for serum tissue factor (TF) activity; tissue factor pathway inhibitor (TFPI) and potential to generate thrombin in calibrated thrombography test.Results. Patients with developed ePE vs. control group were found to have significantly increased TF activity at both time points: 49.1 pmol/ml vs. 5.3 pmol/ml (p < 0.0001) and 56.7 pmol/ml vs. 6.6 pmol/ml (p < 0.0001); respectively. At gestational age of 19–21 weeks; patients in the study vs. control group had serum TFPI level significantly lower (1.1 U/ml vs. 1.6 U/ml; p < 0.0001) that was significant elevated up to 2.1 U/ml by 82 % at 27–28 weeks of pregnancy. At both time points; a significantly increased thrombin generation was detected in women with developed ePE.Conclusion. The development of ePE is characterized by increased potential to generate thrombin. TF has been identified as a more sensitive marker of activated coagulation hemostasis; and its significantly increased activity was recorded at gestational age of 19–21 weeks that allows us to consider TF as a prognostic biomarker.

Coagulation activation during extracorporeal membrane oxygenation (ECMO)

IntroductionExtracorporeal membrane oxygenation (ECMO) can be life-saving, but suffers from thrombus formation in the circuit with associated risks of oxygenator occlusion, hemolysis and arterial embolism. The formation of thrombin is the key step to thrombus formation and two factors are needed for sustained thrombin generation, a coagulation activator to initiate the process and a procoagulant phospholipid surface for the coagulation system to assemble on. Materials and methodsThe purpose of this study was to use thrombin generation potential (TGP) and other assays to determine the specific coagulation activators and sources of procoagulant phospholipid that are present in ECMO patient plasma. Samples were collected from 60 patients on ECMO (age 1d-19y) followed evaluation of native and stimulated TGP, measurement of factor II levels and determination of procoagulant extracellular vesicle levels by flow cytometry. ResultsDuring ECMO, native (unstimulated) TGP was increased, followed by a decrease back towards normal after ECMO ended. The main activator of TGP in ECMO plasma was increased FXIa (100% of samples tested), while increased tissue factor activity was present in 7%. Procoagulant phospholipids were present in plasma from ECMO patients in the form of circulating platelet and red cell extracellular vesicles, which were increased 2 to 7-fold compared to normal. Procoagulant extracellular vesicle levels correlated with increased plasma native TGP activity. ConclusionsECMO activates coagulation in plasma primarily through activation of the contact system and formation of activated factor XIa and generation of circulating procoagulant extracellular vesicles through platelet and red cell activation.

Bile acid-induced tissue factor activity in hepatocytes correlates with activation of farnesoid X receptor

Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.The potential of various bile acids to induce procoagulant tissue factor (TF) activity in viable HepG2 cells and primary human hepatocytes was investigated. Increased TF activity correlated with the molecules' ability to enter the cells and activate the farnesoid X receptor (FXR) suggesting a crucial role of FXR in bile acid-mediated TF activity within the liver parenchyma.

Defining the Balance between the Expression of Procoagulant, Anticoagulant and Angiogenic Molecules in Myeloma Plasma Cells

Introduction: Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and other tissues. This disease significantly increases the risk of venous thromboembolic disorders. The interplay between Myeloma Plasma Cells (MPCs) with coagulation and angiogenesis is currently under investigation. It has been shown that MPCs promotes angiogenesis through VEGF expression. Whether MPCs express procoagulant and/or anticoagulant proteins remains to be shown.Aim: To determine the expression of tissue factor (TF), tissue factor pathway inhibitor 1 (TFPI 1), tissue factor pathway inhibitor 2 (TFPI 2), vascular endothelial growth factor (VEGF-A) and thrombomodulin (TM) by MPCs.Methods: Human MPCs (RPMI 8226) and human umbilical vein endothelial cells (HUVECs) that were used as control were cultured according to standard procedures. Total RNA was extracted and reversely transcribed as previously described. Briefly, 1 μg cDNA was PCR amplified using the Go TAq® qPCRMaster mix (Promega) and the Mx3000P qPCR system according to the manufacturer's protocol. Quantitative RT-PCR was used to measure the relative mRNA levels using TaqMan assays for VEGF-A, TFPI 1, TFPI 2, TF or TM (Sigma Aldrich). Ct values were normalized against the endogenous control B-Actine (from Sigma Aldrich).Relative mRNA expression was calculated using the comparative threshold method (1/PUISSANCE (2 (Ct (gene)-Ct (endogenous control))).Results: MPCs exhibited 4-fold higher TF expression compared to HUVECs but significantly lower expression of TFPI1 and TFPI2. For both MPCs and HUVECs, the expression of TFPI 1 was more pronounced compared to the expression of TF gene. Interestingly, MPCs showed lower expression of TFPI 2 compared to TF. Therefore, the TF/TFPI 1 ratio was higher in MPCs, reflecting a potentially increased TF activity compared to HUVECs. MPCs and HUVECs expressed comparable levels of TM. Finally, VEGF expression was significantly higher in the MPCs as compared to HUVECs. Results are summarized in Table 1.Conclusions. Plasmocytes from human multiple myeloma cells are characterized by a marked proangiogenic potential driven by high gene expression of VEGF which is accompanied by increased expression of TF. However, TF expression seems to be at least partially compensated by increased expression of TFPIs leading to the assumption that the TF-dependent procoagulant properties of plasmocytes are restricted by a balanced increase in TFPI. These data indicate that TF expression by myeloma cells may be associated with cell proliferation rather than a procoagulant [Display omitted] DisclosuresGarderet:Amgen: Honoraria; Takeda: Honoraria.

SARS-CoV-2 infection induces the activation of tissue factor–mediated coagulation via activation of acid sphingomyelinase

AbstractSevere 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.

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

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. HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Increased tissue factor activity promotes thrombin generation at type 1 diabetes onset in children

ObjectiveIn type 1 diabetes (T1D), a prothrombotic status due to elevated coagulation factors coincides with metabolic derailment. In a previous study, we discovered altered thrombin generation profiles in children with T1D. These alterations are potentially most pronounced at T1D onset and ameliorated after insulin treatment. We tested this hypothesis in a longitudinal study, measuring thrombin generation together with coagulation parameters in children at T1D onset and during follow‐up.Materials and methodsTwenty‐three children (12 female, age: 9.4 [2.7‐17.3] years; median [range]) were tested at T1D onset and after long‐term insulin treatment. Thrombin generation was measured using calibrated automated thrombography. Tissue factor (TF) activity and tissue factor pathway inhibitor (TFPI) activity were measured using enzyme‐linked immunosorbent assay (ELISA).ResultsA procoagulant shift was observed in thrombin generation traces at T1D onset compared to follow‐up (time to peak: 5.67 [4.11‐7.67] min vs 6.39 [4.89‐10.44] min, P < .001). These alterations at T1D onset coincided with increased TF activity (5.18 [0.01‐12.97] pmol/L vs 2.67 [0.04‐10.41] pmol/L, P < .05) and increased TFPI activity (0.051 [0.038‐0.074] U/mL vs 0.035 [0.026‐0.056] U/mL, P < .05).ConclusionThe procoagulant shift in thrombin generation at T1D onset is a result of increased TF activity, but this effect is partially counterbalanced by increased TFPI levels. Elevated TF and TFPI levels hint to a fragile hemostatic balance at the endothelial lining of blood vessels. Additional prothrombotic stimuli may tip over this balance explaining the increased thrombotic risk of children with T1D.

Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells.

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.

Alterations in Sphingomyelin Metabolism Influences Tissue Factor Procoagulant Activity and the Release of TF-Positive Microvesicles

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.

A Lipid Peroxidation Product, 4-Hydroxy-2-Nonenal, Triggers Intravascular Coagulation and Inflammation through Generation of Tissue Factor-Positive Microvesicles

Tissue factor (TF) is a cellular receptor for the clotting protease factor VIIa (FVIIa), and TF-FVIIa complex initiates activation of the coagulation cascade. TF-FVIIa also influences non-hemostatic functions via direct or indirect cell signaling. Constitutive expression of TF is primarily restricted to the surface of perivascular cells, sequestered from circulating blood clotting factors, to prevent aberrant intravascular clotting. It has been suggested that TF is also present in the blood associated with microvesicles (MVs). These circulating TF, however, do not induce any thrombotic complications as they are either cleared from the system before they could accumulate to a pathological level or lack procoagulant activity. However, various pathological conditions such as sepsis, diabetes, bacterial infection, atherosclerosis, and cancer can increase the levels of circulating TF+ MVs, which can then trigger intravascular coagulation and inflammation. We hypothesized that oxidative stress, a pathological condition, central to most of the diseases mentioned above, plays a role in the generation of TF+ MVs in vivo, which could contribute to intravascular coagulation and inflammation. We recently showed that 4-hydroxy-2-nonenal (HNE), the most abundant and stable unsaturated aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids, enhances TF activity in monocytic cells and macrophages. However, the role of HNE in activation of intravascular coagulation is unknown. To investigate this, C57Bl/6J wild-type mice were administered with HNE intraperitoneally (10 mg/kg body weight) and generation of TF+ MVs, activation of intravascular coagulation, and inflammation was assessed. HNE administration markedly increased TF activity associated with MVs. Nanoparticle tracking analysis (NTA) showed a 3-fold increase in the number of MVs in the plasma of HNE-treated mice. Measurement of thrombin-antithrombin (TAT) complexes revealed a significant increase in TAT levels in HNE treated mice. Pre-treatment of the mice with anti-murine TF antibody attenuated the HNE-induced increase in TAT levels. Measurement of plasma clotting times showed a significant reduction in HNE treated mice (118.3 ± 10.42 sec) compared to saline treated control mice (&gt; 300 sec). Incubation of plasma with anti-TF antibody reversed the reduction observed in the plasma clotting time of HNE treated mice. Assessment of clotting in the saphenous vein injury bleeding model showed an increased number of clots, and reduced blood loss in HNE-administered mice compared to saline treated mice. Blockade of TF with 1H1 anti-TF antibody negated the HNE-mediated reduction in the clotting time. Next, we investigated whether HNE administration also induces a proinflammatory response in mice. Blood cells analysis showed approximately a 4-fold increase in circulating neutrophils in HNE-treated mice at 6 h following HNE treatment. Immunohistochemistry studies showed a robust infiltration of neutrophils into the lungs. Cytokine analysis of plasma showed a significant increase in IL-6 and CXCL1 levels in HNE-treated mice. Blockade of TF using 1H1 anti-TF antibody significantly reduced the HNE-mediated neutrophil infiltration into the lungs and completely abrogated the increase in plasma IL-6 levels. In additional studies, we attempted to identify the source of TF+ MVs. It is unlikely that they are originated from monocytes or endothelial cells as HNE did not induce TF expression in these cell types. LPS, used as a positive control, showed significant induction of TF on both PBMCs and the endothelium Immunohistochemistry revealed that HNE disrupted endothelial barrier integrity of vessel wall, raising a possibility that TF+ MVs could have come from perivascular cells. In summary, our data indicate that oxidative stress could induce the release of TF+ MVs, and this would contribute to intravascular coagulation and inflammation. Our studies also suggest that TF-positive microvesicles can be a useful therapeutic target in the treatment of thromboinflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Vincristine induces procoagulant activity of the human lymphoblastic leukemia cell line Jurkat through the release of extracellular vesicles.

Thromboembolic events are frequent and serious complications of acute lymphoblastic leukaemia treatment. The importance of chemotherapy in the pathogenesis of this increased risk is enhanced by the fact that thrombosis rarely occurs at diagnosis. Our study aims at investigating the effect of chemotherapy on pro-coagulant activity (PCA), phosphatidylserine (PS) exposure, tissue factor (TF) activity and derived extracellular vesicles (EV) of Jurkat cells. Jurkat cells were treated with two commonly used chemotherapeutics: Vincristine (VCR) or Daunorubicin (DNR), at relevant concentrations. PCA of cells and derived EV were evaluated using Thrombin generation Assay (TGA). Cells or EV were incubated with annexin V or anti TF antibodies to assess the respective contribution of TF and PS. PS exposure on cells was analysed by flow cytometry. Derived EV were evaluated in fluorescence microscopy and flow cytometry. Untreated Jurkat cells and EV support thrombin generation. Thrombin generation was abolished when PS activity was inhibited by annexin V. VCR treatment resulted in a time dependent increase of thrombin generation. After VCR exposure, TF activity increased as well as PS exposure increased on the cell surface. The increase in TF activity was abolished by annexin V indicating that PS was required. A spontaneous release of EV from Jurkat cells was observed and VCR treatment increased the number of generated EV. Our results indicate that VCR increased the PCA of Jurkat cells predominantly through PS exposure and increased EV generation. Lymphoid blasts derived EV could be biomarkers to determine high thrombotic risk ALL patients.

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles

ABSTRACTAirway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

Investigating a novel recombinant antibody to attenuate prostate cancer progression by targeting cell surface GRP78.

206 Background: Prostate cancer (PCa) is characterized by increased activation of the procoagulant protein, tissue factor (TF) that drive tumour progression. We now show that this process is modulated by GRP78 on the cell surface (cs). In PCa GRP78 is a endoplasmic reticulum-resident chaperone that localizes to the cell surface where it functions as a signaling molecule with antigenic properties. In response to csGRP78 presentation, PCa patients produce autoantibodies (AutoAbs) against the N-terminus of GRP78. AutoAbs:csGRP78 complex acts as a potent driver of tumor growth via upregulation of the unfolded protein response (UPR) and TF activity. We hypothesize that inhibiting the binding of anti-GRP78 AutoAbs to csGRP78 will supress UPR and TF activity. Here we describe a recombinant anti-GRP78 antibody (AEP8587) that competes with the binding of AutoAbs to csGRP78 and may act as a novel therapeutic antibody with antitumor activity. Methods: Changes in TF activity or UPR markers were evaluated in vitro in the PCa cell line DU145 following treatment with anti-GRP78 AutoAbs or co-treatment with either enoxaparin, a low molecular weight heparin (LMWH), or AEP8587. Protein expression of TF and UPR markers was determined using western blotting and qRT-PCR. TF activity was determined using a real-time continuous assay. AutoAbs were purified PCa patients (St. Joseph’s Healthcare Hamilton). Results: Pre-prostatectomy PCa patients display high levels of anti-GRP78 AutoAbs (~60µg/ml), compared to healthy controls (~5µg/ml). Here, we show that anti-GRP78 AutoAb increases TF activation in vitro and leads to increased tumor progression in a DU145 xenograft model. In contrast, we show a co-treatment of anti-GRP78 AutoAb with either enoxaparin or AEP8587 completely abolishes the AutoAb-mediated increase in TF activity in vitro. Enoxaparin or AEP8587 co-treatment reversed the AutoAb effect on increased UPR markers. Conclusions: We have identified anti-GRP78 AutoAb as a driver of PCa progression. Our results indicate that a recombinant antibody, AEP8587, can bind to csGRP78 and prevent the binding of anti-GRP78 AutoAbs. This represents a potential novel means to manage PCa progression.

Trimethylamine N-oxide promotes tissue factor expression and activity in vascular endothelial cells: A new link between trimethylamine N-oxide and atherosclerotic thrombosis

Trimethylamine-N-oxide (TMAO), one of the products in choline metabolite, is recently reported to be associated with cardiovascular diseases (CVD) that mainly attribute to atherothrombosis. However, the mechanisms how TMAO functions in the pathogenesis of CVD and atherothrombosis remain elusive. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. In the present study, we demonstrated that TMAO promoted TF (but not TF pathway inhibitor) expression via activation of NF-κB signaling pathway in primary human coronary artery endothelial cells (HCAECs). TMAO strongly increased TF activity and thrombin production. Further, a small dose of TMAO significantly increased TF expression and nuclear translocation of NF-κB with the synergistic action of low-dose of pro-atherosclerotic factors, such as TNF-α and HMGB1. Importantly, plasma TMAO level was positively correlated with TF activity in patients with ST-elevation myocardial infarction (STEMI). Altogether, our data revealed that TMAO promoted thrombosis through increasing TF expression and activity. The understanding of the new link between TMAO and atherothrombosis may facilitate therapeutic strategy in the prevention and treatment of atherothrombosis.

Melatonin improves hyperglycemia induced damages in rat brain.

Diabetes mellitus is an endocrine disorder which is characterized by the development of resistance to the cellular activity of insulin or inadequate insulin production. It leads to hyperglycemia, prolonged inflammation, and oxidative stress. Oxidative stress is assumed to play an important role in the development of diabetic complications. Melatonin is the hormone that interacts with insulin in diabetes. Therefore, in this study, the effects of melatonin treatment with or without insulin were examined in diabetic rat brain. Rats were divided into five groups as control, diabetes, diabetes+insulin, diabetes+melatonin, and diabetes+melatonin+insulin. Experimental diabetes was induced by streptozotocin (60mg/kg, i.p.). Twelveweeks after diabetes induction, rats were decapitated. Malondialdehyde, glutathione, sialic acid and nitric oxide levels, superoxide dismutase, catalase, glutathione-S-transferase, myeloperoxidase, and tissue factor activities were determined in brain tissue. Melatonin alone showed its antioxidant effect by increasing brain glutathione level, superoxide dismutase, catalase, and glutathione-S-transferase activities and decreasing malondialdehyde level in experimental diabetes. Although insulin did not have a significant effect on glutathione and glutathione-S-transferase, its effects on lipid peroxidation, superoxide dismutase, and catalase were similar to melatonin; insulin also decreased myolopeoxidase activity and increased tissue factor activity. Combined melatonin and insulin treatment mimicked the effects of insulin. Addition of melatonin to the insulin treatment did not change the effects of insulin, but the detailed role of melatonin alone in the treatment of diabetes merits further experimental and clinical investigation.

4‐Hydroxy‐2‐Nonenal (HNE), a product of Lipid Peroxidation, Induces Tissue Factor Decryption by modulating Thioredoxin system and mitochondrial ROS generation independently

Oxidative stress plays a major role in the pathogenesis of various diseases such as atherosclerosis. Aberrant expression of a coagulation cofactor tissue factor (TF) is thought to contribute to the pathogenesis of atherosclerosis. Recently, we found that 4‐hydroxy‐2‐nonenal (HNE), a major aldehyde produced by oxidation of ω‐6 polyunsaturated fatty acids, increases the procoagulant activity of cell surface tissue factor (TF). These studies revealed that HNE generates ROS and decrypts TF through p38 MAPK‐dependent externalization of phosphatidylserine (PS). The present study is carried out to elucidate the detailed molecular mechanism involved in HNE‐induced TF decryption. Colocalization of ROS generation and mitochondrial staining indicated that mitochondria are the major source of HNE‐induced ROS generation. Inhibitors of the mitochondrial electron transport chain (ETC) complexes III and V significantly inhibited the HNE‐induced ROS generation. These inhibitors also attenuated the HNE‐induced PS externalization and TF activation, indicating that HNE‐induced ROS generation contributes to PS exposure critical for TF decryption. Interestingly, inhibition of ROS by these inhibitors did not block the HNE‐induced p38 MAPK activation. Additional studies showed that HNE inhibits thioredoxin (Trx) and thioredoxin reductase (TrxR) activities in a dose‐ and time‐dependent manner. Antioxidant N‐acetylcysteine (NAC) prevented the HNE‐induced inhibition of TrxR but the inhibitors of mitochondrial ETC that prevented HNE‐induced ROS generation failed to block the HNE‐induced TrxR inhibition. To investigate whether HNE‐induced inhibition of Trx or TrxR is responsible for HNE‐induced TF decryption, we determined whether inhibition of the activity of Trx or TrxR by their pharmacological inhibitors would also activate TF. Treatment of the cells with either curcumin, Auranofin or PMX‐464 (TrxR inhibitors) or PX‐12 (Trx inhibitor) markedly increased TF activity in a PS‐dependent manner. Inhibition of the TrxR/Trx also activated MKK3/6 and p38 MAPK as observed with HNE. Active Trx binds ASK1 and inactivation of Trx disrupts the complex, enabling ASK1 activation that leads to p38 MAPK activation. Therefore, we investigated whether HNE‐induced inactivation of Trx leads to ASK1 activation that could lead to p38 MAPK activation. Our data show HNE neither induces ASK1 activation nor ASK1 inhibitors block HNE‐induced TF decryption, suggesting that p38 MAPK activation is independent of ASK1. In additional experiments, we found that blockade of thiol groups by phenylarsine oxide (PAO) attenuated HNE‐induced PS exposure and TF decryption. In summary, our present data suggest that HNE induces TF decryption by two separate pathways – one is ROS‐dependent but independent of p38 MAPK activation and the second is via Trx‐TrxR‐ and p38 MAPK‐dependent. Both the mechanisms result in the exposure of PS at the cell surface that contributes to TF decryption. Blockade of thiol groups by PAO appears to inhibit HNE‐induced TF decryption by blocking the upstream signaling molecules that regulate HNE‐induced PS exposure.