Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles.

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Crosstalk of human coronary perivascular adipose-derived stem cells with vascular cells: role of tissue factor.

The coronary perivascular adipose tissue (cPVAT) has been associated to the burden of cardiovascular risk factors and to the underlying vessel atherosclerotic plaque severity. Although the "outside to inside" hypothesis of PVAT-derived-adipokine regulation of vessel function is currently accepted, whether the resident mesenchymal stem cells (ASCs) in PVAT have a regulatory role on the underlying vascular arterial smooth muscle cells (VSMCs) is not known. Here, we investigated the interactions between resident PVAT-ASCs and VSMCs. ASCs were obtained from PVAT overlying the left anterior descending (LAD) coronary artery of hearts removed at heart transplant operations. PVAT was obtained both from patients with non-ischemic and ischemic heart disease as the cause of heart transplant. ASCs were isolated from PVAT, phenotypically characterized by flow cytometry, functionally tested for proliferation, and differentiation. Crosstalk between ASCs and VSMCs was investigated by co-culture studies. ASCs were detected in the adventitia of the LAD-PVAT showing differentiation capacity and angiogenic potential. ASCs obtained from PVAT of non-ischemic and ischemic hearts showed different tissue factor (TF) expression levels, different VSMCs recruitment capacity through the axis ERK1/2-ETS1 signaling and different angiogenic potential. Induced upregulation of TF in ASCs isolated from ischemic PVAT rescued their angiogenic capacity in subcutaneously implanted plugs in mice, whereas silencing TF in ASCs decreased the proangiogenic capacity of non-ischemic ASCs. The results indicate for the first time a novel mechanism of regulation of VSMCs by PVAT-ASCs in angiogenesis, mediated by TF expression in ASCs. Regulation of TF in ASCs may become a therapeutic intervention to increase cardiac protection.

TF-FVIIa PAR2-ß-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

BACKGROUND: Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. METHODS: Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. RESULTS: We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in ß-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of ß-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. CONCLUSIONS: These data provide insights into a TF-FVIIa signaling axis through PAR2-ß-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

BACKGROUND: Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. METHODS: The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity.  RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. CONCLUSIONS: The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. CLINICALTRIALS: gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

Hydroxytyrosol Alleviates Methotrexate-Induced Pulmonary Fibrosis in Rats: Involvement of TGF-ß1, Tissue Factor, and VEGF.

Methotrexate (MTX) is an indispensable drug used for the treatment of many autoimmune and cancerous diseases. However, its clinical use is associated with serious side effects, such as lung fibrosis. The main objective of this study is to test the hypothesis that hydroxytyrosol (HT) can mitigate MTX-induced lung fibrosis in rats while synergizing MTX anticancer effects. Pulmonary fibrosis was induced in the rats using MTX (14 mg/kg/week, per os (p.o.)). The rats were treated with or without HT (10, 20, and 40 mg/kg/d p.o.) or dexamethasone (DEX; 0.5 mg/kg/d, intraperitoneally (i.p.)) for two weeks concomitantly with MTX. Transforming growth factor beta 1 (TGF-ß1), interleukin-4 (IL-4), thromboxane A2 (TXA2), vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxy-guanosine (8-OHdG), tissue factor (TF) and fibrin were assessed using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and RT-PCR. Pulmonary fibrosis was manifested by an excessive extracellular matrix (ECM) deposition and a marked increase in TGF-ß1 and IL-4 in lung tissues. Furthermore, cotreatment with HT or dexamethasone (DEX) significantly attenuated MTX-induced ECM deposition, TGF-ß1, and IL-4 expression. Similarly, HT or DEX notably reduced hydroxyproline contents, TXA2, fibrin, and TF expression in lung tissues. Moreover, using HT or DEX downregulated the gene expression of TF. A significant decrease in lung contents of VEGF, IL-8, and 8-OHdG was also observed in HT + MTX- or DEX + MTX -treated animals in a dose-dependent manner. Collectively, the results of our study suggest that HT might represent a potential protective agent against MTX-induced pulmonary fibrosis.

Microparticle-associated tissue factor activity correlates with the inflammatory response in septic disseminated intravascular coagulation patients.

Background: Sepsis is often accompanied by the formation of disseminated intravascular coagulation (DIC). Microparticles can exert their procoagulant and proinflammatory properties in a variety of ways. The purpose of this study was to investigate the relationship between microparticle-associated tissue factor activity (TF+-MP activity) and the inflammatory response. Methods: Data from a total of 31 DIC patients with sepsis and 31 non-DIC patients with sepsis admitted to the ICU of the First Affiliated Hospital of Harbin Medical University from December 2017 to March 2019 were collected. Blood samples were collected and DIC scores were calculated on the day of enrollment. The hospital's clinical laboratory completed routine blood, procalcitonin, and C-reactive protein tests. TF+-MP activity was measured using a tissue factor-dependent FXa generation assay. Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) levels were determined using ELISA kits. Results: Compared with the non-DIC group, the DIC group had higher levels of leukocytes, neutrophils, procalcitonin, C-reactive protein, IL-1ß, and TNF-α, and more severe inflammatory reactions. TF+-MP activity in the DIC group was higher than that in the non-DIC group. In sepsis patients, TF+-MP activity was strongly correlated with inflammatory response indices and DIC scores. Conclusion: TF+-MP activity may play a major role in promoting inflammatory response in septic DIC.

Effect of factor XI inhibition on tumor cell-induced coagulation activation.

BACKGROUND: Cancer-associated thrombosis is a frequent complication in patients with malignancies. While factor XI (FXI)/FXIa inhibition is efficacious in preventing postoperative venous thromboembolism, its role in tumor cell-induced coagulation is less defined. OBJECTIVES: We thus aimed to provide mechanistic insights into FXI/FXIa inhibition in tumor cell-induced coagulation activation. METHODS: Procoagulant activity (PCA) of 4 different tissue factor (TF) expressing tumor cell lines was analyzed by single-stage clotting and thrombin generation assay in the presence of a FXIa inhibitor, BMS-262084 (BMS), an inhibitory FXI antibody (anti-FXI), or peak and trough concentrations of rivaroxaban or tinzaparin. Further, tumor cell-induced platelet aggregation was recorded. Recombinant human TF served as positive control. RESULTS: Although BMS and anti-FXI potently inhibited FXIa amidolytic activity, both inhibitors efficiently mitigated recombinant human TF- and tumor cell-induced fibrin clot formation and platelet aggregation only in the presence of low TF PCA. The anticoagulant effects showed an inverse correlation with the magnitude of cellular TF PCA expression. Similarly, BMS markedly interfered with tumor cell-induced thrombin generation, with the most prominent effects on peak and total thrombin. In addition, anticoagulant effects of FXIa inhibition by 10 µM BMS were in a similar range to those obtained by 600 nM rivaroxaban and 1.6 µM tinzaparin at low TF PCA levels. However, rivaroxaban and tinzaparin also exerted marked anticoagulant activity at high TF PCA levels. CONCLUSION: Our findings indicate that FXI/FXIa inhibition interferes with tumor cell-induced coagulation activation only at low TF PCA expression levels, a finding with potential implications for future in vivo studies.

Human Genetic Variation in F3 and Its Impact on Tissue Factor-Dependent Disease.

Tissue factor (TF) is the primary initiator of blood coagulation in humans. As improper intravascular TF expression and procoagulant activity underlie numerous thrombotic disorders, there has been longstanding interest in the contribution of heritable genetic variation in F3, the gene encoding TF, to human disease. This review seeks to comprehensively and critically synthesize small case-control studies focused on candidate single nucleotide polymorphisms (SNPs), as well as modern genome-wide association studies (GWAS) seeking to discover novel associations between variants and clinical phenotypes. Where possible, correlative laboratory studies, expression quantitative trait loci, and protein quantitative trait loci are evaluated to glean potential mechanistic insights. Most disease associations implicated in historical case-control studies have proven difficult to replicate in large GWAS. Nevertheless, SNPs linked to F3, such as rs2022030, are associated with increased F3 mRNA expression, monocyte TF expression after endotoxin exposure, and circulating levels of the prothrombotic biomarker D-dimer, consistent with the central role of TF in the initiation of blood coagulation.

Cancer-associated thrombosis: What about microRNAs targeting the tissue factor coagulation pathway?

Cancer patients are often diagnosed with venous thromboembolism (VTE), a cardiovascular disease that substantially decreases their quality of life and survival rate. Haemostasis in these patients is deregulated, which is reflected in the common presentation of a blood hypercoagulation state. Despite the inconsistent results, existing evidence suggests that the expression of microRNAs (miRNAs) is deregulated in the context of venous thrombogenesis in the general population. However, few miRNAs are known to be linked to cancer-associated VTE due to the lack of studies with oncological patients. Parallelly, coagulation factor III, also known as tissue factor (TF), tissue factor pathway inhibitor 1 (TFPI1) and tissue factor pathway inhibitor 2 (TFPI2) have been proposed to have a central role in cancer-associated VTE and tumour progression. Yet, contrary to what was expected, the role of miRNAs targeting the TF coagulation pathway (or extrinsic coagulation pathway) is poorly explored in cancer-induced thrombogenesis. In this review, in addition to miRNAs implicated in VTE, TF and TFPI1/2-targeting miRNAs were revised. Future studies should clarify the implications of these non-coding RNAs in tumour coagulome.

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development.

ABSTRACT: Antiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface-expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)-dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL-induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain-dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Expression of Tissue Factor and Platelet/Leukocyte Markers on Extracellular Vesicles Reflect Platelet-Leukocyte Interaction in Severe COVID-19.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet-leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP), and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples was associated with CRP, HMGB-1, PF4, and TF as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41+ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41+CD45+) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation.

INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1ß stimulation.

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) - a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1ß. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1ß alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1ß on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1ß, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1ß intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1ß-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1ß was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Coagulation factor VIIa enhances programmed death-ligand 1 expression and its stability in breast cancer cells to promote breast cancer immune evasion.

BACKGROUND: Immunotherapy for breast cancer has not gained significant success. Coagulation factor VIIa (FVIIa)-tissue factor (TF) mediated activation of protease-activated receptor 2 (PAR2) is shown to promote metastasis and secretion of the immune-modulatory cytokines but the role of FVIIa in cancer immunology is still not well understood. OBJECTIVES: Here, we aim to investigate whether FVIIa protects breast cancer cells from CD8 T-cell-mediated killing. METHODS: Peripheral blood mononuclear cell-derived CD8 T cells were cocultured with vehicle or FVIIa pretreated MDAMB468 cells. The proliferation and activity of CD8 T cells were measured by flow cytometry and ELISA. An allograft model, using wild-type or TF/PAR2-deleted 4T1 cells, was employed to determine the effect of FVIIa on breast cancer immune evasion in vivo. RESULTS: Here, we demonstrate that TF-FVIIa induces programmed death-ligand 1 (PD-L1) in breast cancer cells by activating PAR2. PAR2 activation triggers large tumor suppressor kinase 1 (LATS1) inactivation leading to loss of yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) phosphorylation and subsequent nuclear localization of YAP/TAZ. YAP/TAZ inhibition reduces PD-L1 expression and increases CD8 T-cell activity. We further demonstrate that, apart from transcriptional induction of PD-L1, PAR2 activation also increases PD-L1 stability by enhancing its glycosylation through N-glycosyltransferases STT3A and STT3B. CONCLUSION: In a mouse model of breast cancer, tumor cell-specific PAR2 depletion leads to PD-L1 downregulation and increases anti-PD-1 immunotherapy efficacy. In conclusion, we showed that FVIIa-mediated signaling cascade in cancer cells serves as a tumor intrinsic mechanism of immunosuppression to promote cancer immune evasion.

The unfolded protein response links ER stress to cancer-associated thrombosis.

Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non-small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.