Monocyte Tissue Factor Expression Research Articles

Reduced Monocyte and Neutrophil Infiltration and Activation by P-Selectin/CD62P Inhibition Enhances Thrombus Resolution in Mice.

Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions. Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed. Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus, and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin. Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis.

Abstract 458: A Study Of Short-term Low-saturated Fat Versus High-saturated Fat Diet In Atherothrombosis Risk Factors In Subjects With Hypertriglyceridemia

Introduction: Dietary fat and composition may impact the risk of atherosclerotic cardiovascular disease (ASCVD) in subjects with hypertriglyceridemia (HTG). Hypothesis: We hypothesized that a low-saturated fat diet (LSFD), in contrast to a high-saturated fat diet (HSFD), improves blood lipid levels and reduces atherothrombosis risk factors in subjects with HTG. Method: Subjects with HTG and metabolic syndrome (n=19) were randomly placed on an LSFD (~25% of calories from fat, 5% from saturated fat) or an HSFD (~52% of calories from fat, 25% from saturated fat) for 4 days (day 1-4) and a breakfast on day 5 and then switched to the other diet after a 4- to 6-week washout period. Blood was collected on day 1 fasting before the start of the diet and on day 5 fasting and at 4 and 6 hours after breakfast. Lipid profiles and flow cytometry to measure monocyte intracellular lipid accumulation, monocyte expression of tissue factor (TF), and monocyte-platelet aggregates (MPAs) were performed. Results: On day 5, LSFD, compared to HSFD, induced lower plasma levels of postprandial total triglyceride and LDL-triglyceride and fasting and postprandial total cholesterol, non-HDL-cholesterol, LDL-cholesterol, and small dense LDL-cholesterol. Classical and intermediate monocytes with LSFD, in contrast to HSFD, exhibited reduced intracellular lipid accumulation indicated by decreases in cellular granularity and nile red staining examined by flow cytometry. LSFD vs. HSFD also reduced classical monocyte uptake of oxidized LDL ex vivo at 4 hours postprandially on day 5. Surface level of TF was higher on classical and intermediate monocytes than on nonclassical monocytes assessed by mean fluorescence intensity. Classical and intermediate monocytes, compared to nonclassical monocytes, were also more prone to form aggregates with platelets. However, there was no significant difference between LSFD and HSFD in TF levels on any monocyte subsets and in MPAs of any monocyte subsets at any time points recorded. Conclusion: In subjects with HTG and metabolic syndrome, short-term LSFD, in contrast to HSFD, reduced monocyte lipid accumulation and oxidized LDL uptake but did not induce significant differences in monocyte surface levels of TF and MPA formation.

11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability.

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. The findings of the present study have further clarified the procoagulant properties of CNT.

P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

VALPROIC ACID INHIBITS CLASSICAL MONOCYTE-DERIVED TISSUE FACTOR AND ALLEVIATES HEMORRHAGIC SHOCK-INDUCED ACUTE LUNG INJURY IN RATS.

Background: Monocytes and monocyte-derived tissue factor (TF) promote the development of sepsis-induced acute lung injury (ALI). Classical monocytes (C-Mcs) can be induced to express TF. Valproic acid (VPA) alleviates hemorrhagic shock (HS)-induced ALI (HS/ALI) and inhibits TF expression in monocytes. We hypothesized that C-Mcs and C-Mc-derived TF promoted HS/ALI and that VPA could inhibit C-Mc-derived TF expression and attenuate HS/ALI. Methods: Wistar rats and THP-1 cells were used to evaluate our hypothesis. Monocyte subtypes were analyzed by flow cytometry; mRNA expression was measured by fluorescence quantitative polymerase chain reaction; protein expression was measured by Western blotting, immunofluorescence, or immunohistology; inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay; and ALI scores were used to determine the degree of ALI. Results: The blood %C-Mcs and C-Mcs/non-C-Mcs ratios, monocyte TF levels, serum and/or lung inflammatory cytokine levels, and ALI scores of HS rats were significantly increased ( P < 0.05). After monocyte depletion and thrombin inhibition, the inflammatory cytokine levels and ALI scores were significantly decreased ( P < 0.05). VPA reduced the %C-Mcs and C-Mc/non-C-Mc ratios, TF expression, inflammatory cytokine levels, and ALI scores during HS ( P < 0.05) and inhibited HS-induced monocyte Egr-1 and p-ERK1/2 expression ( P < 0.05). VPA inhibited hypoxia-induced TF expression in THP-1 cells by regulating the p-ERK1/2-Egr-1 axis. Conclusion: C-Mcs and C-Mc-derived TF accelerate the development of HS/ALI by increasing thrombin production. VPA inhibits HS-induced C-Mc production of TF by regulating the p-ERK1/2-Egr-1 axis and alleviates HS/ALI.

Pathophysiological mechanisms of thrombosis in acute and long COVID-19.

COVID-19 patients have a high incidence of thrombosis, and thromboembolic complications are associated with severe COVID-19 and high mortality. COVID-19 disease is associated with a hyper-inflammatory response (cytokine storm) mediated by the immune system. However, the role of the inflammatory response in thrombosis remains incompletely understood. In this review, we investigate the crosstalk between inflammation and thrombosis in the context of COVID-19, focusing on the contributions of inflammation to the pathogenesis of thrombosis, and propose combined use of anti-inflammatory and anticoagulant therapeutics. Under inflammatory conditions, the interactions between neutrophils and platelets, platelet activation, monocyte tissue factor expression, microparticle release, and phosphatidylserine (PS) externalization as well as complement activation are collectively involved in immune-thrombosis. Inflammation results in the activation and apoptosis of blood cells, leading to microparticle release and PS externalization on blood cells and microparticles, which significantly enhances the catalytic efficiency of the tenase and prothrombinase complexes, and promotes thrombin-mediated fibrin generation and local blood clot formation. Given the risk of thrombosis in the COVID-19, the importance of antithrombotic therapies has been generally recognized, but certain deficiencies and treatment gaps in remain. Antiplatelet drugs are not in combination with anticoagulant treatments, thus fail to dampen platelet procoagulant activity. Current treatments also do not propose an optimal time for anticoagulation. The efficacy of anticoagulant treatments depends on the time of therapy initiation. The best time for antithrombotic therapy is as early as possible after diagnosis, ideally in the early stage of the disease. We also elaborate on the possible mechanisms of long COVID thromboembolic complications, including persistent inflammation, endothelial injury and dysfunction, and coagulation abnormalities. The above-mentioned contents provide therapeutic strategies for COVID-19 patients and further improve patient outcomes.

Complement Shields Fc Regions on HIT Immune Complexes and Prevents Recognition By Cellular Fcγ Receptors

Heparin-induced thrombocytopenia (HIT) is a life-threatening immune-mediated thrombotic disorder caused by immune complexes (ICs) containing IgG antibodies to platelet factor 4 (PF4)/heparin complexes. In previous studies, we established that complement opsonization of HIT ICs is essential for cellular binding of ICs and subsequent engagement of Fcγ receptors (FcγRs). We additionally showed that complement inhibition alone prevented FcγR-mediated effector functions, such as neutrophil degranulation, monocyte tissue factor expression, and procoagulant activity (Khandelwal, Blood 2021). As KKO and HIT ICs can also bind FcγRs in media without complement, we undertook these studies to examine the direct effects of complement on IC-FcγR interactions. For these studies, we used THP-1 cells or U937 cells, monocytic cell lines expressing FcγRI and FcγRII, but not complement receptors (CRs). Cells were incubated with AF-647 labeled KKO (50 mg/ml), a HIT-like monoclonal antibody, either alone or with PF4 (25 mg/ml) and heparin (1U/ml) to form in situ KKO ICs. Labeled KKO or KKO ICs were added to RPMI-containing media (lacking complement) or undiluted citrated plasma (source of complement) in the presence or absence of anti-FcγRIIA. Binding of labeled IgG or anti-C3c was examined by flow cytometry. Consistent with prior observations, THP-1 cells incubated in media show robust binding of KKO ICs in a FcγRII dependent manner (70% reduction in binding with anti-FcγRII; Figure 1A). Surprisingly, in undiluted plasma, there was minimal KKO IC binding to THP-1 cells, despite adequate complement generation (Figure 1B). Similar effects of plasma vs. media were seen in confocal experiments using KKO ICs with THP-1 cells (Figure 1C & 1D), HIT ICs incubated with THP-1 cells (containing HIT patient IgG, PF4/heparin; data not shown), or KKO ICs incubated with U937 cells (data not shown). In other studies, we note that binding of KKO ICs was dependent on the source of complement, with undiluted human plasma showing maximal inhibitory effects (100%), while undiluted normal human serum (NHS) and fetal bovine serum (FBS) reduced KKO IC binding by 70% and 50%, respectively (data not shown). To demonstrate that the inhibitory effects of undiluted plasma on KKO IC binding was complement-mediated, THP-1 cells were incubated in plasma containing the complement inhibitors BBK32 (C1r inhibitor) or CP40 (C3 inhibitor) prior to addition of labeled KKO ICs. In other experiments, THP-1 cells were incubated with labeled KKO ICs in media containing human complement proteins (C1+C3+C4). As shown, complement inhibition in plasma restored KKO IC binding to cellular FcγR (Figure 2A). Conversely, addition of purified complement components to media resulted in reduced KKO IC binding (Figure 2B). Similar effects of complement proteins and inhibitors were noted in ELISA-based assays using immobilized FcγRIIA incubated with KKO ICs in plasma or media (data not shown). These studies demonstrate striking inhibitory effects of complement on IC-FcγR interactions. Specifically, complement shields Fc regions on ICs from recognition and binding to cellular FcγRs and this masking effect is reversed by complement inhibition. We additionally show that these masking effects of complement are less apparent in media containing FBS, the most commonly used source of complement for cell-based studies. Taken together with our recent observations showing requirements for complement receptors in binding complement-opsonized KKO ICs, these studies suggest that the masking effects of complement on ICs prevent FcγR activation on cells lacking CRs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes.

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.

Does hsa-miR-223-3p from platelet-derived extracellular vesicles regulate tissue factor expression in monocytic cells?

Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation. Abbreviations: Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT)

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

Interferon-γ Produced by EBV-Positive Neoplastic NK-Cells Induces Differentiation into Macrophages and Procoagulant Activity of Monocytes, Which Leads to HLH.

Simple SummaryEpstein–Barr virus (EBV), a common virus all over the world, infects not only B-cells but also T- and NK-cells. Once infected with EBV, human beings remain infected for life, and EBV renders infected B-cells immortal. EBV-positive NK-cell neoplasms, such as extranodal NK/T-cell lymphoma of nasal type, aggressive NK-cell leukemia, and chronic active EBV infection, are relatively rare but lethal disorders. They show systemic inflammation and progress to hemophagocytic lymphohistiocytosis (HLH), a life-threatening state of immune hyperactivation. The suppression and prevention of HLH are important to treat the neoplasms. Revealing the mechanism will pave a new path for treatment. We show herein that IFN-γ produced by EBV-positive neoplastic NK-cell is responsible for inducing the differentiation and the activation of M1-like macrophages. Suppressing IFN-γ may regulate HLH in EBV-positive NK-cell neoplasms.Epstein–Barr virus (EBV)-positive T- or NK-cell neoplasms show progressive systemic inflammation and abnormal blood coagulation causing hemophagocytic lymphohistiocytosis (HLH). It was reported that inflammatory cytokines were produced and secreted by EBV-positive neoplastic T- or NK-cells. These cytokines can induce the differentiation of monocytes into macrophages leading to HLH. To clarify which products of EBV-positive neoplastic T- or NK-cells have effects on monocytes, we performed a co-culture assay of monocytes with the supernatants of EBV-positive T- or NK-cell lines. The expression of differentiation markers, the phagocytosis ability, and the mRNA expression of the inflammatory cytokines of THP-1, a monocytic cell line, clearly increased after culturing with the supernatants from EBV-NK-cell lines. Co-culturing with the supernatants promoted the expression of CD80 and CD206 as well as M1 and M2 macrophage markers in human monocytes. Co-culturing with the supernatants of EBV-NK-cell lines significantly enhanced the procoagulant activity and the tissue factor expression of monocytes. Interferon (IFN)-γ was elevated extremely not only in the supernatant of EBV-NK-cell lines but also in the plasma of EBV-positive NK-cell neoplasms patients accompanying HLH. Finally, we confirmed that IFN-γ directly enhanced the differentiation into M1-like macrophages and the procoagulant activity of monocytes. Our findings suggest that IFN-γ may potentially serve as a therapeutic target to regulate HLH in EBV-positive NK-cell neoplasms.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p&amp;lt;0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p&amp;lt;0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p&amp;lt;0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p&amp;lt;0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p&amp;lt;0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p&amp;lt;0.01; CLI-095: −100±6,6%, n=6, p&amp;lt;0.01; BAY-11-7082: −100±2,1%, n=6, p&amp;lt;0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. Funding Acknowledgement Type of funding sources: None.

The role of oxidized phospholipids in COVID-19-associated hypercoagulopathy.

There is more pronounced hypercoagulation in COVID-19 infection than in other viral lung infections. Oxidized phospholipids (OxPLs) appear in COVID-19-infected lungs due to oxidative stress, after which they promote the induction of tissue factor (TF) expression and inflammatory programmers in monocytes, as well as activate endothelial cells to recruit and bind to monocytes. Therefore, we aimed to demonstrate the role of OxPLs in inflammatory and procoagulant responses in COVID-19 infection. Patients with a positive SARS-CoV-2 polymerase chain reaction test and ten healthy donors were included in the study. Peripheral blood was drawn at inclusion for OxPAPC, IFN-γ, and CCL2 serum level measurements. Clinical data were collected from electronic patient medical files. The serum levels of OxPAPC, IFNγ, and CCL2 were measured by immune assays. Seventy-two patients were included in the study. OxPAPC and CCL2 were higher in the patients than in the controls (<0.003 and 0.011, respectively). INF-γ did not significantly differ between groups. There was no difference between the patients with lung involvement and those without CCL2, INF-γ, and OxPAPC. D-dimer, CRP, and ferritin were higher in the patients with lung involvement. Serum levels of INF-γ and CCL2 were positively correlated with each other (r:0.757, p<0.0001), but no correlation was detected between OxPAPC and INF-γ or CCL2. There was no correlation between OxPAPC and hematologic or biochemical parameters. OxPAPC, which is thought to contribute to hypercoagulability, was found to be high in the patients with Covid-19 infection. The role of OxPLs in COVID-19-associated hypercoagulopathy should be investigated further in experimental models and in larger patient groups.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex–mediated thrombotic disorders.

Association of Soluble Suppression of Tumorigenesis 2 (sST2) With Platelet Activation, Monocyte Tissue Factor and Ischemic Outcomes Following Angioplasty and Stenting

Background: Peripheral artery disease (PAD) patients undergoing infrainguinal angioplasty with stenting suffer high rates of target lesion restenosis and ischemic events. Blood-based prognostic markers in these patients are currently limited. The IL-33/ST2-system is involved in atherothrombosis. Soluble ST2 has been proposed as a biomarker in patients with cardiovascular disease.Aim: To investigate the association of sST2 with platelet activation and monocyte tissue factor (TF) in 316 patients undergoing elective angioplasty and stenting for cardiovascular disease, and its predictive value for ischemic outcomes following infrainguinal angioplasty with stent implantation in 104 PAD patients within this cohort.Methods and Results: Circulating levels of sST2, platelet surface P-selectin, monocyte TF expression as well as soluble P-selectin were determined in 316 consecutive patients on dual antiplatelet therapy following angioplasty and stenting. sST2 was independently associated with soluble P-selectin (B = 6.4, 95% CI 2.0–10.7, p = 0.004) and TF expression (B = 0.56, 95% CI 0.02–1.1, p = 0.041) but not with platelet surface P-selectin (B = 0.1, 95% CI −0.1–0.3, p = 0.307) after adjustment for age, sex, clinical risk factors and inflammatory parameters. During the follow-up of 24 months, the primary endpoint occurred in 41 of 104 PAD patients (39.4%). However, circulating levels of sST2 did not predict the primary endpoint in PAD patients (HR 1.1, 95% CI 0.76–1.71, p = 0.527).Conclusion: sST2 is associated with soluble P-selectin and monocyte TF expression in atherosclerosis but not with ischemic outcomes following infrainguinal angioplasty with stent implantation for PAD.

Complement Regulates the Procoagulant Effects of HIT Immune Complexes

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p &amp;lt;0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p&amp;lt;0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p&amp;lt;0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents &amp; Royalties; Apotex: Consultancy, Research Funding.

Role of toll-like receptor 4 on tissue factor regulation in human monocytes

Abstract Background Inflammation and coagulation play a pivotal role in the pathogenesis of acute coronary events and an extensive cross-talk links the two systems, whereby inflammation activates coagulation and coagulation affects inflammatory activity. Infact, pro-inflammatory stimuli can induce tissue factor (TF) expression, the principal initiator of the clotting cascade, in circulating monocytes and activate pathways leading to thrombin generation.In turn, TF may bind cellular receptors which may affect the production and release of inflammatory mediators. According to our recent results, proprotein convertase subtilisin/kexin9 (PCSK9) and Gamma-Glutamyltransferase (GGT), two molecules involved in the pathogenesis of cardiovascular disease, are able to up-regulate TF expression in monocytes by activating NFkB pathway but the mechanism and the receptor involved in this biological response is unknown. One plausible possibility is that both molecules bind a Toll-like Receptor (TLR)4 located on membranes of human and cultured monocytes,activating the TLR4/MyD88-NFkB pathway and eventually leading to stimulation of TF expression. Aim To assess whether both molecules are able to bind to TLR4 located on the surface of human monocytes and whether this specific binding involves the TLR4/MyD88-NFkB pathway on TF modulation. Methods THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, was used as in vitro model. The cells were stimulated with human (h) recombinant (r) PCSK9 (5 μg/ml) and hrGGT (1 μg/ml) or pre-incubated with BAY-117082 (BAY, 10–5M) a NFκB inhibitor, CLI-095 (3x10–6M), a highly Myd88/TLR4 signaling specific inhibitor and LPS-RS (1 μg/ml) a TLR4 antagonist. TF procoagulant activity (PCA), was assessed by 1-stage clotting assay and the results expressed by ρg/mL of active protein. Experimental series were carried out in endotoxin-free conditions, in order to exclude lipopolysaccharide (LPS)-dependent immune responses. Results hrPCSK9 and hrGGT stimulated TF expression (PCA: from 50±20 to 120±20, n=10, p&amp;lt;0.01) and (PCA: from 190±140 to 460±360, n=15, p&amp;lt;0.001) respectively, an effect down-regulated by BAY,a NFκB inhibitor (PCA by hrPCSK9: −71±23%, n=5, p&amp;lt;0.01; PCA by hrGGT: −90±21%, n=7, p&amp;lt;0.001). CLI-095, a TLR4 inhibitor (PCA by hrPCSK9: −86±26%, n=3, p&amp;lt;0.05; and PCA by GGT: −89±10%, n=5, p&amp;lt;0.001).LPS-RS, a TLR4 antagonist, (PCA by hrPCSK9: −74±25%, n=3, p&amp;lt;0.05; PCA by hrGGT: −70±17%, n=5, p&amp;lt;0.001) abolished both PCSK9 and GGT-induced TF expression. Conclusions These data are the first demonstration of a direct role of PCSK9 and GGT as active mediators of inflammatory-based thrombotic diseases. The possible mechanism of action involves recognition of two proteins by TLR4 on monocytes membrane surface, lead to activation of the transcription factor NFκB. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind TLR4 modulation and TF expression. Funding Acknowledgement Type of funding source: None

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/β3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

Clinical Evaluation for the Role of High-Sensitivity C-Reactive Protein in Combination with D-Dimer and Wells Score Probability Test to Predict the Incidence of Deep Vein Thrombosis Among Cancer Patients.

BackgroundDeep vein thrombosis (DVT) is a frequent complication in cancer patients and is the second leading cause of death. The high level of C-reactive protein (CRP) is an acute phase reactant that induces tissue factor (TF) expression in monocytes, smooth muscle cells, and endothelial cells. The CRP level positively correlates with the incidence, extension, and volume of thrombus. TF expression triggers the coagulation system including the formation of thrombin and circulating fibrin such as prothrombin fragment 1+2 (F1 + 2) and D-dimer.ObjectiveTo determine the diagnostic value of high-sensitivity (hs)-CRP, D-dimer, and Wells score combination to predict the incidence of DVT on clinically suspected DVT (Wells score ≥2) cancer patients.Subjects and MethodsThis study was a cross-sectional study on a diagnostic test to determine the diagnostic value of hs-CRP and D-dimer for early detection of DVT on clinically suspected DVT (Wells score ≥2) cancer patients. It was conducted in Dr. Kariadi Hospital, Semarang Indonesia on 35 subjects. The diagnosis of DVT was confirmed by color duplex sonography. The diagnostic accuracy of combination of hs-CRP, D-dimer, and Wells score was analyzed by logistic regression.ResultsDVT was confirmed in 10 subjects (28,6%). The cut-off point of hs-CRP levels for probable DVT was ≥51.05 mg/L and for D-dimer was ≥5030 µg/L. The median levels of both variables were higher in the subjects with DVT compared with the subjects without DVT, but it was not statistically significant. The combination of hs-CRP (≥51.05 mg/L), D-dimer (≥5030 µg/L), and Wells score ≥3 had the high accuracy (94.1%) to predict the incidence of DVT compared with hs-CRP (65.0%), D-dimer (54.7%), and combination of hs-CRP and D-dimer (71.0%).ConclusionThe combination of hs-CRP (≥51.05 mg/L), D-dimer (≥5030 µg/L), and Wells score ≥3 can predict the incidence of DVT in cancer.