Tissue Factor Activity Research Articles

Evaluation of four commercial ELISAs to measure tissue factor in human plasma

BackgroundUnder pathological conditions, tissue factor (TF)-positive extracellular vesicles (EVs) are released into the circulation and activate coagulation. Therefore, it is important to identify methods that accurately quantitate levels of TF in plasma. Enzyme-linked immunosorbent assays (ELISAs) are a fast and simple method to quantitate levels of proteins. However, there are several specific challenges with measuring TF antigen in plasma including its low concentration and the complexity of plasma. ObjectivesWe aimed to evaluate the ability of 4 commercial ELISAs to measure TF in human plasma. MethodsWe determined the ability of 4 commercial ELISAs (Imubind, Quantikine, Human SimpleStep, and CD142 Human) to detect recombinant human TF (Innovin) (12.5-100 pg/mL), TF-positive EVs isolated from the culture supernatant from a human pancreatic cancer cell line (57 pg/mL), TF in plasma containing low levels of EV TF activity (1.2-2.6 pg/mL) from lipopolysaccharide-stimulated whole blood, and plasma containing high levels of EV TF activity (151-696 pg/mL) from patients with acute leukemia. ResultsThe CD142 Human ELISA could not detect recombinant TF. Imubind and Quantikine but not Human SimpleStep detected recombinant TF spiked into plasma and TF-positive EVs isolated from the culture supernatant of a human pancreatic cancer cell line. Quantikine and Imubind could not detect low levels of TF in plasma from lipopolysaccharide-stimulated whole blood. However, Quantikine but not Imubind detected TF in plasma from acute leukemia patients with high levels of EV TF activity. ConclusionOur results indicate that commercial ELISAs have different abilities to detect TF. Quantikine and Imubind could not detect low levels of TF in plasma, but Quantikine detected TF in plasma with high levels of TF.

Testosterone therapy increases the anticoagulant potential in men with opioid-induced hypogonadism: a randomized, placebo-controlled study.

Hypogonadism is prevalent during opioid treatment, and low testosterone concentrations are associated with cardiovascular disease. The effect of testosterone replacement therapy (TRT) on the coagulation system in men with hypogonadism is not clarified. We investigate the effects of TRT on the tissue factor (TF) and contact activation pathways of coagulation in opioid-treated men. This was a double-blinded, placebo-controlled study in 37 men with total testosterone < 12 nmol/L randomized to 24 weeks of testosterone injections (n = 17) or placebo (n = 20). Variables of the coagulation system were analysed at baseline and after 24 weeks. Measurements included the TF pathway (endogenous thrombin potential (ETP) and peak thrombin), the contact activation pathway (endogenous kallikrein potential (EKP) and peak kallikrein), coagulation factors (FVII, FX, prothrombin, and FXII), and inhibitors (tissue factor pathway inhibitor (TFPI), protein C, protein S, antithrombin, and C1 esterase inhibitor (C1inh)). Between-group differences at 24 weeks were determined with analysis of covariance. Within-group changes in TRT and placebo were analysed with paired t-test. Between-group differences at 24 weeks were observed for ETP (P = 0.036), FVII (P = 0.044), FX (P = 0.015), prothrombin (P = 0.003), protein C (P = 0.004), and protein S (P = 0.038). Within the TRT group, ETP, peak thrombin, FVII, FX, prothrombin, TFPI, protein C, FXII, and C1inh decreased and protein S increased (all P < 0.05). Within the placebo group, coagulation outcomes were unchanged. TRT affects the coagulation system in an anticoagulant direction through suppressed TF pathway in men with opioid-induced hypogonadism.

Mild Acquired von Willebrand Syndrome and Cholestasis in Pediatric and Adult Patients with Fontan Circulation.

Background: Hemodynamic alterations in Fontan patients (FP) are associated with hemostatic dysbalance and Fontan-associated liver disease. Studies of other hepatopathologies indicate an interplay between cholestasis, tissue factor (TF), and von Willebrand factor (VWF). Hence, we hypothesized a relationship between the accumulation of bile acids (BA) and these hemostatic factors in FP. Methods: We included 34 FP (Phenprocoumon n = 15, acetylsalicylic acid (ASA) n = 16). BA were assessed by mass spectrometry. TF activity and VWF antigen (VWF:Ag) were determined by chromogenic assays. VWF collagen-binding activity (VWF:CB) was assessed via ELISA. Results: Cholestasis was observed in 6/34 FP (total BA ≥ 10 µM). BA levels and TF activity did not correlate (p = 0.724). Cholestatic FP had lower platelet counts (p = 0.013) from which 5/6 FP were not treated with ASA. VWF:Ag levels were increased in 9/34 FP and significantly lower in FP receiving ASA (p = 0.044). Acquired von Willebrand syndrome (AVWS) was observed in 10/34-FP, with a higher incidence in cholestatic FP (4/6) (p = 0.048). Conclusions: Cholestasis is unexpectedly infrequent in FP and seems to be less frequent under ASA therapy. Therefore, ASA may reduce the risk of advanced liver fibrosis. FP should be screened for AVWS to avoid bleeding events, especially in cholestatic states.

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions

BackgroundVascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. ObjectivesWe sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. MethodsWe assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. ResultsWe found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. ConclusionsBy using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte–endothelial adhesion.

Fibroblast growth factor 2 (FGF2) ameliorates the coagulation abnormalities in sepsis

BackgroundSepsis is defined as a life-threatening organ dysfunction caused by dysregulation of the host response to infection. There is still a lack of specific treatment for sepsis. Here, we report that Fibroblast growth factor-2 (FGF2) can reduce the mortality of sepsis by ameliorating the coagulation abnormalities. MethodsFGF2 was intraperitoneally injected into septic mice induced by lipopolysaccharide (LPS) and then assessed for coagulation response, organ damage and survival. RAW264.7 cells with or without FGF2 pretreating were exposed to LPS, and then changes in coagulation related factors expression and signaling were tested. ResultsThe findings showed that intraperitoneal injection of FGF2 inhibited coagulation activity, reduced lung and liver damage, and increased survival in septic mice. In RAW264.7 cells, LPS upregulated the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); however, pretreatment with FGF2 prevented this upregulation, while FGF2 knockdown exacerbated TF upregulation. Moreover, FGF2 suppressing the AKT/mTOR/S6K1 signaling pathway in septic mice and RAW264.7 cells stimulated by LPS. ConclusionsThis study revealed a therapeutic role of FGF2 in ameliorating the coagulation abnormalities during sepsis.

Severe COVID-19 classified by SCRI is associated with higher levels of AOPP and 8- OHDG.

SARS-CoV-2 has become one of the most important and challenging medical research topics in recent years. The presence of endothelial dysfunction, immune thrombosis, and oxidative stress contributes to complications and requires more extended hospitalisation of patients. In this article, we focused on analysing the impact of oxidative stress on the severity of COVID-19 infection. The study group consisted of 72 patients with laboratory-confirmed SARS-CoV enrolled. The patients were divided into moderate and severe diseases according to the SCRI (Simple Covid Risk Index, including lymphocyte/D-dimer ratio). Using the ELISA kit, we determined the level of AOPP and 8-OHdG. Patients with severe COVID-19 had higher levels of both AOPP (P<0.05) and 8-OHdG (P<0.05) compared to patients with moderate disease. Albumin levels were significantly lower (P<0.001), although fibrinogen (P<0.01), D-dimer (P<0.001), and TF (P<0.05) levels were higher in severe patients than in moderate course. AOPP/Alb was also higher among severe patients (P<0.05). Our data suggest a potential role for AOPP and 8-OHdG in predicting the outcome of SARS-CoV-2 patients. Elevated AOPP levels were associated with increased Dimer-D, TF, and vWF activity levels.

Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice.

Introduction Certain low-molecular-weight heparins have been reported to reduce tumor growth and metastasis in tumor cell-inoculated mouse models and cancer patients. Recently, direct oral anticoagulants (DOACs) have been widely used in patients with thromboembolism. This study was aimed at investigating the effect of DOACs, which target thrombin or factor Xa, on tumor growth in a syngeneic mouse model comprising BALB/c mice inoculated with colon cancer Colon26 cells. Materials and Methods DOACs targeting thrombin (dabigatran etexilate [DABE]) or factor Xa (rivaroxaban [RVX] and edoxaban [EDX]) were orally administered daily to male BALB/c mice inoculated with Colon26 cells, followed by analyses of tumor growth and plasma levels of coagulation- and tumor-related factors such as tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), and matrix metalloproteinase-2 (MMP-2). Results Colon26 cells expressed significant amounts of functionally active TF. Tumor growth in Colon26-inoculated mice was significantly suppressed in DABE- or RVX-treated mice ( p <0.05) and was suppressed more significantly in EDX-treated mice ( p <0.01). Therefore, the antitumor mechanism of action of EDX was investigated next. Plasma levels of TF, PAI-1, IL-6, and MMP-2 were elevated in Colon26-inoculated mice but were significantly reduced in EDX-treated mice ( p <0.01). The expression of protease-activated receptor (PAR)1, PAR2, signal transducer and activator of transcription-3 (STAT3), cyclin D1, and Ki67 was increased in tumor tissue of Colon26-inoculated mice but (except for PAR1) was significantly decreased in tumor tissues of EDX-treated mice ( p <0.01). In addition, apoptotic cells and p53 protein levels were significantly increased in tumor tissues of EDX-treated mice. Conclusion The data suggest that among the tested DOACs, EDX significantly suppresses tumor cell proliferation via the factor Xa-PAR2 pathway, which is activated by coagulation and inflammation in Colon26-inoculated mice and induces tumor cell apoptosis.

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.

Cytotoxic CD8+ T Cells Are Involved in the Thrombo-Inflammatory Response during First-Diagnosed Atrial Fibrillation.

Atrial myopathy and atrial fibrillation (AF) accompany thrombo-inflammation. This facilitates disease progression and promotes major adverse cardiovascular events (MACEs). Thrombin receptor (protease-activated receptor 1, PAR1) signalling is central in mediating thrombo-inflammation. We hypothesised that PAR1 signalling links coagulation and inflammation through cytotoxic CD8+ T lymphocytes in patients presenting with first-diagnosed AF (FDAF). A total of 210 patients were studied. We included data and blood samples from patients presenting with FDAF (n = 160), cardiac tissue from patients with paroxysmal AF (n = 32) and 20 controls. During early AF, a pro-inflammatory and cytotoxic subset of T lymphocytes (CD8+) circulated more frequently when compared to patients with chronic cardiovascular disease but without AF, accompanied by elevated plasma levels of CD8+ effector molecules, which corresponded to biomarkers of adverse cardiac remodelling and atrial dysfunction. Activation of tissue factor (TF) and PAR1 was associated with pro-inflammatory and cytotoxic effector functions. PAR1-related CD8+ cell activation was more frequent in FDAF patients that experienced a MACE. In patients with FDAF, the TF-factor Xa-factor IIa-axis contributes to thrombo-inflammation via PAR1 in CD8+ T cells. Intervening in this cascade might be a promising synergistic approach to reducing disease progression and the vascular complications of AF.

Peripheral blood mononuclear cell tissue factor (F3 gene) transcript levels and circulating extracellular vesicles are elevated in severe coronavirus 2019 (COVID-19) disease

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with excessive coagulation, thrombosis, and mortality. ObjectiveTo provide insight into mechanisms that contribute to excessive coagulation in coronavirus 2019 (COVID-19) disease. Patients/MethodsBlood from COVID-19 patients was investigated for coagulation–related gene expression and functional activities. ResultsSingle-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from severe COVID-19 patients revealed a 5.2-fold increase in tissue factor (TF [F3 gene]) transcript expression levels (P < .05), the trigger of extrinsic coagulation; a 7.7-fold increase in C1-inhibitor (SERPING1 gene; P < .01) transcript expression levels, an inhibitor of intrinsic coagulation; and a 4.4-fold increase in anticoagulant thrombomodulin (TM [THBD gene]) transcript expression levels (P < .001). Bulk RNA-seq analysis of sorted CD14+ monocytes on an independent cohort of COVID-19 patients confirmed these findings (P < .05). Indicative of excessive coagulation, 41% of COVID-19 patients’ plasma samples contained high D-dimer levels (P < .0001); of these, 19% demonstrated extracellular vesicle TF activity (P = .109). COVID-19 patients’ ex vivo plasma–based thrombin generation correlated positively with D-dimer levels (P < .01). Plasma procoagulant extracellular vesicles were elevated ∼9-fold in COVID-19 patients (P < .01). Public scRNA-seq data sets from bronchoalveolar lung fluid and our peripheral blood mononuclear cell scRNA-seq data show CD14+ monocytes/macrophages TF transcript expression levels are elevated in severe but not mild or moderate COVID-19 patients. ConclusionsBeyond local lung injury, SARS-CoV-2 infection increases systemic TF (F3) transcript levels and elevates circulating extracellular vesicles that likely contribute to disease-associated coagulation, thrombosis, and related mortality.

Sex-dependent balance between thrombin and plasmin generation in the presence of thrombomodulin.

Assessing simultaneous generation of thrombin (TG) and plasmin (PG) is an approach to evaluate the balance between coagulation and fibrinolysis with sensitivity to predict endogenous thrombin and plasmin generation. The addition of thrombomodulin (TM), provides the essential component for thrombin activation of protein C and thrombin-activatable fibrinolysis inhibitor. However, the influence of sex on the balance between TG and PG with and without TM addition has not been investigated to date. To investigate the possible sex-based differences in TG and PG in the presence and absence of TM. Simultaneous TG and PG were measured in plasma samples obtained from 17 males and 17 females upon tissue factor and tissue plasminogen activator addition. Thrombin- and plasmin-specific fluorogenic substrates Z-Gly-Gly-Arg-AMC and Boc-Glu-Lys-Lys-AMC were used in the study. Thrombin and plasmin peak height (TPH and PPH) and production rate (TPR and PPR) values were determined. To evaluate the balance between TG and PG, the ratios between TPH and PPH (TPH/PPH) and TPR and PPR (TPR/PPR) were calculated. TPH between males and females demonstrated significant difference regardless of TM addition. TPR demonstrated differences between males and females only upon TM addition, while PG parameters was not dependent on the sex of the donor. TM significantly lowered TPH/PPH in males, and enhanced TPR/PPR in females. Thus, TPH/PPH and TPR/PPR significantly differed between men and women. Our results indicate that TM may act differently in males and females by shifting the underlying TG/PG balance to fibrinolysis in males and to coagulation in females.

Pathogenesis of abdominal aortic aneurysms from the vascular surgeon perspective – knowledge summary

&#x0D; Abdominal aortic aneurysms (AAA) affect 2.4% of the population, with men being five times more likely to be affected than women. The development of AAA is linked to changes in the elastin and vascular wall collagen. The enzymes that damage the cell wall are called metalloproteinases. AAA forms as a result of damage to elastic fibres and the loss of the property of reversible deformation of the aortic wall. The degradation of elastin and other stem proteins in the aortic wall is caused by metalloproteinases and serine proteases, accompanied by cysteine proteases and asparagine proteases. Increased calprotectin levels are observed in AAA patients in comparison to patients with a healthy aorta. A significant role in the pathogenesis of AAA and its rupture is played by inflammatory response cells; proteases of the tissue plasma coagulation and fibrinolysis. Plasminogen activator and plasmin accelerate the degradation of the aortic wall. Microbial involvement of C. pneumoniae, Helicobacter pylori, CMV, and HIV is considered in this inflammatory reaction. The local activation of platelets and the plasma coagulation system leads to the formation of a mural thrombus filling the lumen of the aneurysm. The mural thrombus shows a high tissue factor (TF) activity. The formation of AAA is conditioned by a combination of multiple factors. The factors impacting the formation of AAA discovered so far include genetic factors, sex, age, lifestyle (abuse of alcohol, tobacco misuse, obesity, stress), health conditions (hypertension, high cholesterol level, atherosclerosis), and infectious factors: bacteria, viruses, and other microorganisms.&#x0D;

The histone methyltransferase MLL1/KMT2A in monocytes drives coronavirus-associated coagulopathy and inflammation

AbstractCoronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, “coagulopathy-related factors”) in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA–mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation.

Dietary fat intake is associated with insulin resistance and an adverse vascular profile in patients with T1D: a pooled analysis

BackgroundInsulin resistance (IR) increases vascular risk in individuals with Type 1 Diabetes (T1D). We aimed to investigate the relationship between dietary intake and IR, as well as vascular biomarkers in T1D.MethodsBaseline data from three randomised controlled trials were pooled. Estimated glucose disposal rate (eGDR) was used as an IR marker. Employing multivariate nutrient density substitution models, we examined the association between macronutrient composition and IR/vascular biomarkers (tumour necrosis factor-α, fibrinogen, tissue factor activity, and plasminogen activator inhibitor-1).ResultsOf the 107 patients, 50.5% were male with mean age of 29 ± 6 years. Those with lower eGDR were older with a longer diabetes duration, higher insulin requirements, and an adverse vascular profile (p < 0.05). Patients with higher degrees of IR had higher total energy intake (3192 ± 566 vs. 2772 ± 268 vs. 2626 ± 395 kcal/d for eGDR < 5.1 vs. 5.1–8.6 vs. ≥ 8.7 mg/kg/min, p < 0.001) and consumed a higher absolute and proportional amount of fat (47.6 ± 18.6 vs. 30.4 ± 8.1 vs. 25.8 ± 10.4%, p < 0.001). After adjusting for total energy intake, age, sex, and diabetes duration, increased carbohydrate intake offset by an isoenergetic decrease in fat was associated with higher eGDR (β = 0.103, 95% CI 0.044–0.163). In contrast, increased dietary fat at the expense of dietary protein intake was associated with lower eGDR (β = − 0.119, 95% CI − 0.199 to − 0.040). Replacing fat with 5% isoenergetic amount of carbohydrate resulted in decreased vascular biomarkers (p < 0.05).ConclusionHigher fat, but not carbohydrate, intake is associated with increased IR and an adverse vascular profile in patients with T1D.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

Beyond uterine atony: characterizing postpartum hemorrhage coagulopathy

Postpartum hemorrhage is a leading cause of morbidity and mortality worldwide, yet the associated early coagulopathy is not well defined. We hypothesized that women who develop postpartum hemorrhage have a distinct derangement of thrombin generation and coagulation factors compared with postpartum women without postpartum hemorrhage. This prospective study of pregnant patients with postpartum hemorrhage was completed at a single urban hospital. Blood was drawn on postpartum hemorrhage diagnosis and 2 and 4 hours later. Assays of patients with postpartum hemorrhage included thrombelastography, whole blood thrombin generation, coagulation factor activity, tissue factor levels and activity, and tissue factor pathway inhibitor levels, which were compared with that of patients without postpartum hemorrhage. A total of 81 patients were included in this study. Of those patients, 66 had postpartum hemorrhage, and 15 served as controls. Compared with patients without PPH, patients with postpartum hemorrhage had lower fibrinogen levels (469.0 mg/dL vs 411.0 mg/dL; P=.02), increased tissue plasminogen activator resistance (fibrinolysis 30 minutes after maximal clot strength: 8.7% vs 4.2%; P=.02), decreased peak thrombin concentration (150.2 nM vs 40.7 nM; P=.01), and decreased maximal rate of thrombin generation (60.1 nM/minute vs 2.8 nM/minute; P=.02). Furthermore, compared with patients without postpartum hemorrhage, patients with postpartum hemorrhage had decreased tissue factor levels (444.3 pg/mL vs 267.1 pg/mL; P=.02) and increased tissue factor pathway inhibitor levels (0.6 U/mL vs 0.8 U/mL; P=.04), with decreased tissue factor pathway inhibitor ratios (624 vs 299; P=.01). PPH is not only an issue of uterine tone and mechanical bleeding but also a distinct coagulopathy that is characterized by decreased fibrinogen level, clot breakdown resistance, and markedly low thrombin generation. This pathology seemed to be driven by low tissue factor and high tissue factor pathway inhibitor levels.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

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

Endothelial SIRT6 deficiency promotes arterial thrombosis in mice

ObjectiveArterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD+-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. Approach and resultsUsing a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6−/− mice as compared to Sirt6fl/fl controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. ConclusionsEndothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.

Sex Hormone Levels, Biomarkers of Hypercoagulability and Endothelial Cell Activation Are Determinants of Severity of COVID-19 in Women and Men. the Roadmap-COVID-19 Prospective Study

Background: Patients with COVID 19 disease have haemostatic dysfunction and are at higher risk of thrombotic complications. Although age and sex are a major risk factor for outcome. Even though the incidence of COVID 19 seems to be similar in both genders, men are at a higher risk of a worse outcome, with the odds up to 2.8 times higher than those for women. It is not known whether sex hormones contribute to this preposition. The real impact of sex hormone levels on the severity of COVID 19 remains to be demonstrated. Objectives: We aimed to investigate the impact of variations in sex hormone levels on the hypercoagulability and severity of COVID 19 in women and men according to age. Methods: 186 patients with symptomatic newly diagnosed Covid 19 were enrolled in the study. Specially we included postmenaupausal women (>50 years) and age matched men. Participants were divided into two groups: moderate Covid-19 (ARDS) and severe Covid-19. The control group consisted of 60 healthy individuals, with the same age as patients. Biomarkers of hypercoagulability and endothelial cell activation: Procoagulant phospholipid (PPL) TFPI, D-Dimer, Soluble thrombomodulin , Tissue factor activity, Thrombin generation (TG) with the Calibrated Automated Thrombogram (CAT) were all measured with assays were from Diagnostica Stago, Asnières, France. Total testosterone, estradiol, and progesterone were measured by ELISA method (Abcam, France). IL-6, Complement C3a and C5a by Human IL-6 ELISA kit, ThermoFisher (Scientific Asnieres-sur-Seine, France). The Upper Normal Limit (UNL) and the Lower Normal Limit (LNL) for each studied biomarker were defined in the control group as follows: UNL= mean + 2 standard deviation (SD), and LNL: = mean - 2 SD. Results: The levels of biomarkers of hypercoagulability, endothelial cell activation and inflammation in patients and controls are reported in Table 1. Thrombogram was marked by significantly prolonged lag time, prolonged time to peak and decreased ETP compared to the control group. The comparison between females and males showed that the lag-time was over the UNL in 18% of men over 50 years versus 8% in men under 50 years, and in 10% and 0% of women respectively. ETP values were below the LNL in 82% of men over 50 years versus 63% for men under 50 years and in 54% and 92% for women respectively. In the same way, the peak of thrombin was over the UNL in 39% and 15% for men over 50 and men under 50 versus 12% and 0% for women respectively. The TFPI were over the UNL in 99% of men over 50 years and in 84% of men under 50 years versus 91% and 87% for women respectively. D-Di increased in 88% of patients under or over 50 years independently of the group. The PPL were shorter than the LNL in 90 % and 80% of men over and under 50 years respectively, and for women in 86% and 77% respectively. The levels of TM were significantly lower in women for the 2 groups in comparison with men over or under 50 years. The levels of C3a, were above the UNL in 86%, and 63% for men over and under 50 years respectively and in 55% and 87% of women over and under 50 years respectively. The testosterone levels in males both younger and older than 50 years were significatively below the lower limit of the normal range of each age group. The estradiol levels were in normal range for women but significantly increased men (Table2). Stratification by the severity of COVID-19 showed that the testosterone levels were significantly lower in men who developed ARDS or severe COVID-19 than in those who did not (p>0.05). In men, the estradiol levels were significantly increased but the difference was not significant between those with severe or mild COVID-19. Males who developed severe COVID-19 had a lower progesterone level. No statistically significant difference in testosterone or progesterone levels were found in the women compared to normal range, the estradiol level was lower in females with ARDS (Table 3.)Relationship analysis of sex hormones with markers of immune activation, testosterone levels and estradiol/testosterone were correlated with, IL-6, fibrinogen, and D-Di. Conclusion: Our study highlights that disturbance in circulating sex hormone levels is a hallmark of critical COVID-19 in males and reinforces the concept that determination of testosterone and E2/T ratio in association with biomarkers of hypercoagulability upon diagnosis might lead to an a accurate risk assessment tool for prompt identification of patients at high risk of COVID-19 worsening. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)

Introduction: Venous thromboembolism (VTE) is an important adverse event associated with immune checkpoint inhibitors (ICI) in cancer patients (12-15% annual rate). We have developed a murine model of venous thrombosis associated with ICI therapy (referred to as ICI-CAT). We found that mice bearing CT26 colon carcinoma treated with ICI developed larger thrombi following IVC occlusion compared with mice treated with IgG. ICI did not increase thrombus weight in mice without tumors. Tissue factor (TF) plays an important role in cancer-associated thrombosis (CAT) in patients treated with conventional therapies, and TF+ extracellular vesicles (EV) were associated with the development of VTE in pancreatic cancer. However, little is known about the impact of ICI on TF expression in patients treated with ICI. To address the underlying mechanism of ICI-associated thrombosis (IAT), we have examined the expression of TF in tumor and immune cells, as well as circulating EV, in our murine CT26 model. Methods: A syngeneic colon carcinoma model (CT26) in BALB/c mice was utilized to evaluate ICI-CAT following inferior vena cava ligation (stasis). Tumor tissue was analyzed for TF expression by immunoblotting using an anti-TF antibody (ab189483, Abcam). Tumor tissue was also dissociated, and TF-positive cells were assessed by flow cytometry analysis with CD45+ and CD45- gating using antibodies for CD11b, Ly6C, Ly6G (BioLegend), and TF (R&D). EV were isolated from platelet poor plasma from ICI or control IgG-treated mice by centrifugation (20,000 x g). Procoagulant activity of EV TF was measured using a chromogenic substrate to detect FXa generation following incubation of EV with FVIIa in the presence or absence of an anti-TF antibody. Results: Treatment of tumor-bearing mice with ICI markedly induced TF expression in tumors (Fig. A, Leftupper). Furthermore, levels of EV TF antigen (Fig. A, Left bottom) and EV TF activity (Fig. A, Right) were increased in mice treated with ICI. To explore the cellular origin of TF, we performed flow cytometry with dissociated tumor cells. A greater percentage of TF-positive cells in both the CD45+ (37.8%) and CD45- (74.6%) populations were detected in tumors from ICI treated mice compared to those treated with control IgG (27.9% and 49.8%, respectively), suggesting both tumor and tumor-infiltrating immune cells contribute to the larger thrombi observed in ICI-treated mice (Fig. B). Further analysis of CD45+ TF+ cells by CD11b gating (data not shown) revealed that a TF expression in a monocyte population (CD11b+ Ly6C+) was increased 3-fold by ICI (46.6%) compared to control IgG (14.2%) (Fig. C). We also found that TF expression was increased in cultured CT26 cells in response to interferon-g, but not in response to anti-PD-1 and anti-CTLA-4 antibodies, suggesting that ICIs do not directly upregulate TF and that more complex cellular interactions and inflammatory changes may be involved. Conclusions: ICI treatment of tumor-bearing mice results in increased tumor TF expression and EV TF expression. TF was also upregulated in immune cells in the tumor microenvironment after ICI treatment, including both tumor/stromal cells (CD45-), and tumor-associated monocytes and neutrophils (CD45+). These results suggest that multiple underlying mechanisms and diverse cellular expression underlie TF expression in this model, and that these processes many contribute to the larger thrombi observed in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal