The Role of the Tissue Factor Pathway in the Hypercoagulable State in Patients with the Antiphospholipid Syndrome

1998 ◽  
Vol 79 (02) ◽  
pp. 276-281 ◽  
Author(s):  
Olga Amengual ◽  
Tatsuya Atsumi ◽  
Graham Hughes ◽  
Munther Khamashta
Keyword(s):  
Tissue Factor ◽  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Umbilical Vein ◽  
Antigen Expression ◽  
Peripheral Blood Mononuclear ◽  
Tissue Factor Pathway

SummaryThe antiphospholipid syndrome (APS) is characterised by both arterial and venous thrombosis, recurrent pregnancy loss and thrombocytopaenia in association with antiphospholipid antibodies (aPL). To explore further the pathogenesis of thrombosis in APS, we evaluated the behaviour of tissue factor (TF) pathway in patients with APS. Plasma antigen levels of soluble TF and tissue factor pathway inhibitor (TFPI), a physiological regulator of TF dependent coagulation activation, were measured in 57 APS patients (36 primary and 21 secondary to systemic lupus erythematosus). Significantly elevated levels of both TF and TFPI were found in APS patients compared with 25 healthy controls (279 ± 15 vs. 217 ± 17 pg/ml, p = 0.01; 56.24 ± 2.00 vs. 47.92 ± 2.22 ng/ml, p = 0.01, respectively), suggesting in vivo upregulation of TF pathway in patients with APS. By flow-cytometry, monocytes from a healthy donor displayed higher TF antigen expression when incubated in the presence of APS plasmas than in control plasmas (24.23 ± 3.11 vs. 12.78 ± 1.57%, p = 0.002). Peripheral blood mononuclear cells (PBMC) also expressed more procoagulant activity (PCA) when incubated in the presence of APS plasmas than in control plasmas (1.80 ± 0.12 vs. 1.35 ± 0.054, p = 0.001) implying that TF up-regulation in APS was reproducible in vitro. Human monoclonal anticardiolipin antibodies induced PCA on PBMC and also TF mRNA on both PBMC and human umbilical vein endothelial cells shown by reverse-transcription polymerase chain reaction. These data strongly suggest that the TF pathway is implicated in the pathogenesis of aPL related thrombosis.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

1997 ◽  
Vol 78 (02) ◽  
pp. 864-870 ◽  
Author(s):  
Hideki Nagase ◽  
Kei-ichi Enjyoji ◽  
Yu-ichi Kamikubo ◽  
Keiko T Kitazato ◽  
Kenji Kitazato ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Free Form ◽  
Initial Reaction ◽  
Extrinsic Pathway ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Preliminary Report of In vitro and In vivo Effectiveness of Dornase Alfa on SARS-CoV-2 Infection (Preprint)

2020 ◽  
Author(s):  
Hacer Kuzu Okur ◽  
Koray Yalcin ◽  
Cihan Tastan ◽  
Sevda Demir ◽  
Bulut Yurtsever ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Tract ◽  
Mononuclear Cells ◽  
Multiple Organ ◽  
Peripheral Blood Mononuclear ◽  
Dornase Alfa ◽  
Tract Infections ◽  
Adult Peripheral Blood

UNSTRUCTURED Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). Coronavirus disease 2019 (COVID-19) pandemic affected more than two million people over the world, resulting in unprecedented health, social and economic crises. The COVID-19, viral pneumonia that progresses to ARDS and even multiple organ failure, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and predict severe respiratory diseases. A similar mucus structure is detected in COVID-19 patients due to the accumulation of excessive NET in the lungs. Here, we show our preliminary results with dornase alfa that may have an in-vitro anti-viral effect against SARS-CoV-2 infection in a bovine kidney cell line, MDBK without drug toxicity on healthy adult peripheral blood mononuclear cells. In this preliminary study, we also showed that dornase alfa can promote clearance of NET formation in both an in-vitro and three COVID-19 cases who showed clinical improvement in radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea and coughing.

scholarly journals A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rachel Tanner ◽  
Andrew D. White ◽  
Charelle Boot ◽  
Claudia C. Sombroek ◽  
Matthew K. O’Shea ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mononuclear Cells ◽  
Functional Assay ◽  
Intermediate Precision ◽  
Peripheral Blood Mononuclear ◽  
Growth Inhibition Assay ◽  
Early Evaluation ◽  
Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

scholarly journals The effect of serum on monocyte tissue factor generation

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 707-714 ◽  
Author(s):  
RL Edwards ◽  
D Perla
Keyword(s):  
T Cells ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
High Serum ◽  
Peripheral Blood Mononuclear ◽  
Serum Factors ◽  
Normal Peripheral Blood ◽  
Stimulatory Activity ◽  
Normal Hemostasis

Abstract Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

Circulating and inducible IL-32α in chronic hepatitis C virus infection

2019 ◽  
Vol 2 (1) ◽  
pp. 23-30
Author(s):  
Mark Collister ◽  
Julia Rempel ◽  
Jiaqi Yang ◽  
Kelly Kaita ◽  
Zach Raizman ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Chronic Hepatitis C Virus ◽  
Chronic Hcv

Background: Interleukin 32 (IL-32) is a recently described pro-inflammatory cytokine implicated in chronic hepatitis C virus (HCV)-related inflammation and fibrosis. IL-32α is the most abundant IL-32 isoform. Methods: Circulating IL-32α levels were documented in patients with chronic HCV infections ( n = 31) and compared with individuals who spontaneously resolved HCV infection ( n = 14) and HCV-naive controls ( n = 20). In addition, peripheral blood mononuclear cells (PBMC) from the chronic HCV ( n = 12) and HCV-naive ( n = 9) cohorts were investigated for responses to HCV core and non-structural (NS)3 protein induced IL-32α production. Finally, correlations between IL-32α levels, hepatic fibrosis and subsequent responses to interferon-based therapy were documented in patients with chronic HCV. Results: Circulating IL-32α levels in patients with chronic HCV were similar to those of spontaneously resolved and HCV-naive controls. HCV protein induced IL-32α responses were similar in chronic HCV patients and HCV-naive controls. In patients with chronic HCV, serum IL-32α levels correlated with worsening METAVIR fibrosis (F) scores from F0 to F3 ( r = 0.596, P < 0.001) as did NS3 induced IL-32α responses ( r = 0.837, P < 0.05). However, these correlations were not sustained with the inclusion of IL-32α levels at F4 scores, suggesting events at F4 interfere with IL-32α synthesis or release. In chronic HCV patients who underwent treatment ( n = 28), baseline in vivo and in vitro induced IL-32α concentrations were not predictive of therapeutic outcomes. Conclusions: IL-32α activity is associated with worsening fibrosis scores in non-cirrhotic, chronic HCV patients.

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