scholarly journals Neutrophils: back in the thrombosis spotlight

Blood ◽  
2019 ◽  
Vol 133 (20) ◽  
pp. 2186-2197 ◽  
Author(s):  
Denis F. Noubouossie ◽  
Brandi N. Reeves ◽  
Brian D. Strahl ◽  
Nigel S. Key
Keyword(s):  
Animal Models ◽  
Tissue Factor ◽  
Contact System ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Extrinsic Pathway ◽  
Vessel Occlusion ◽  
Activated Neutrophils

Abstract Reactive and clonal neutrophil expansion has been associated with thrombosis, suggesting that neutrophils play a role in this process. However, although there is no doubt that activated monocytes trigger coagulation in a tissue factor-dependent manner, it remains uncertain whether stimulated neutrophils can also directly activate coagulation. After more than a decade of debate, it is now largely accepted that normal human neutrophils do not synthetize tissue factor, the initiator of the extrinsic pathway of coagulation. However, neutrophils may passively acquire tissue factor from monocytes. Recently, the contact system, which initiates coagulation via the intrinsic pathway, has been implicated in the pathogenesis of thrombosis. After the recent description of neutrophil extracellular trap (NET) release by activated neutrophils, some animal models of thrombosis have demonstrated that coagulation may be enhanced by direct NET-dependent activation of the contact system. However, there is currently no consensus on how to assess or quantify NETosis in vivo, and other experimental animal models have failed to demonstrate a role for neutrophils in thrombogenesis. Nevertheless, it is likely that NETs can serve to localize other circulating coagulation components and can also promote vessel occlusion independent of fibrin formation. This article provides a critical appraisal of the possible roles of neutrophils in thrombosis and highlights some existing knowledge gaps regarding the procoagulant activities of neutrophil-derived extracellular chromatin and its molecular components. A better understanding of these mechanisms could guide future approaches to prevent and/or treat thrombosis.

scholarly journals Mast Cell Tryptase Potentiates Neutrophil Extracellular Trap Formation

2021 ◽  
pp. 1-14
Author(s):  
Gunnar Pejler ◽  
Sultan Alanazi ◽  
Mirjana Grujic ◽  
Jeremy Adler ◽  
Anna-Karin Olsson ◽  
...  
Keyword(s):  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Functional Communication ◽  
Extracellular Milieu ◽  
Extracellular Traps ◽  
Inflammatory Conditions ◽  
Histone 3 ◽  
Activated Neutrophils ◽  
Core Histones

Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.

scholarly journals Histone Deacetylase Inhibitors Dose-Dependently Switch Neutrophil Death from NETosis to Apoptosis

Biomolecules ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 184 ◽  
Author(s):  
Hussein J. Hamam ◽  
Nades Palaniyar
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Histone Deacetylase Inhibitors ◽  
Significant Inhibition ◽  
The Body ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Post Translational Modification

Acetylation is an important post translational modification of histone that plays a role in regulation of physiological and pathological process in the body. We have recently shown that the inhibition of histone deacetylases (HDAC) by low concentrations of HDAC inhibitors (HDACis), belinostat (up to 0.25 µM) and panobinostat (up to 0.04 µM) promote histone acetylation (e.g., AcH4) and neutrophil extracellular trap formation (NETosis). Clinical use of belinostat and panobinostat often leads to neutropenia and the in vivo concentrations vary with time and tissue locations. However, the effects of different concentrations of these HDACis on neutrophil death are not fully understood. We considered that increasing concentrations of belinostat and panobinostat could alter the type of neutrophil death. To test this hypothesis, we treated human neutrophils with belinostat and panobinostat in the presence or absence of agonists that promote NOX-dependent NETosis (phorbol myristate acetate or lipopolysaccharide from Escherichia coli 0128) and NOX-independent NETosis (calcium ionophores A23187 or ionomycin from Streptomyces conglobatus). Increasing concentrations of HDACis induced histone acetylation in a dose-dependent manner. ROS analyses showed that increasing concentrations of HDACis, increased the degree of NOX-derived ROS production. Higher levels (>1 µM belinostat and >0.2 µM panobinostat) of AcH4 resulted in a significant inhibition of spontaneous as well as the NOX-dependent and -independent NETosis. By contrast, the degree of neutrophil apoptosis significantly increased, particularly in non-activated cells. Collectively, this study establishes that increasing concentrations of belinostat and panobinostat initially increases NETosis but subsequently reduces NETosis or switches the form of cell death to apoptosis. This new information indicates that belinostat and panobinostat can induce different types of neutrophil death and may induce neutropenia and regulate inflammation at different concentrations.

P6396LDL cholesterol promotes neutrophil extracellular trap formation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A S Ondracek ◽  
T M Hofbauer ◽  
P P Wadowski ◽  
A Mangold ◽  
T Gremmel ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Troponin T ◽  
Ex Vivo ◽  
Plasma Concentrations ◽  
Density Lipoprotein ◽  
Neutrophil Activation ◽  
Neutrophil Extracellular Trap ◽  
Dependent Manner ◽  
Activated Neutrophils

Abstract Background Pro-protein convertase subtilisin/kexin 9 (PCSK9) is a regulator of low density lipoprotein (LDL) receptor (LDLR) expression and has gained attention in the treatment of hyperlipidemia. Serum levels of LDL are correlated with numbers of activated neutrophils in the circulation of hyperlipidemic patients. Activated neutrophils can form neutrophil extracellular traps (NETs) by expelling their chromatin, and NETs have been recognized as important risk factors for acute myocardial infarction (AMI) and stroke. Purpose We analyzed the influence of serum LDL levels on neutrophil activation, their propensity to form NETs, and the correlation of NET surrogate markers with systemic inflammatory responses in AMI. Methods We recruited 249 consecutive patients with AMI (mean age 58 years, 20% females) and assessed laboratory parameters, inflammatory markers, and serum lipid profiles 72 h after primary percutaneous coronary intervention. Double-stranded DNA (dsDNA) and citrullinated histone H3 were determined from plasma as markers of in vivo NET formation. In a subset of patients (n=25), ex vivo NET formation in response to PCSK9 and ionomycin was analyzed, neutrophils were stained for CD11b, LDLR, and lectin-like oxidized LDLR (LOX-1) using flow cytometry at baseline and after activation with phorbol myristate acetate. PCSK9 levels were measured by ELISA. Results Patients with serum LDL levels above the median [median (IQR) LDL 111 mg/dl (87–141), mean ± SD LDL 115±41 mg/dl] had significantly higher concentrations of circulating dsDNA. Levels of dsDNA [median 121 ng/ml, IQR 101–156] were linked with levels of the specific NET marker citH3 [median (IQR) 252 ng/ml (655–1600), rs=0.157]. Plasma concentrations of dsDNA and citH3 correlated significantly with CRP (dsDNA rs=0.328; citH3 rs=0.209), proBNP (dsDNA rs=0.286; citH3 rs=0.192), troponin T (dsDNA rs=0.282; citH3 rs=0.197), and IL-6 (dsDNA rs=0.242; citH3 rs=0.216). In the subset of patients in whom neutrophils were characterized, pre-treatment of neutrophils with PCSK9 could significantly decrease ionomycin induced NET release in a dose-dependent manner. Levels of serum LDL were associated with spontaneous NET formation (r=0.504) ex vivo and activated CD11b on neutrophils (r=0.495). LOX-1 expression correlated significantly with LDLR expression (rs=0.670) and PCSK9 levels (r=0.520). Patient neutrophil stimulation in whole blood led to a significant decrease of the LDLR positive neutrophil population. This effect was greater when PCSK9 levels were higher. Conclusion Our data indicate a role for LDL in boosting neutrophil activation depending on PCSK9.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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.

scholarly journals Caveolin-1 Enhances Tissue Factor Pathway Inhibitor Exposure and Function on the Cell Surface

2005 ◽  
Vol 280 (23) ◽  
pp. 22308-22317 ◽  
Author(s):  
Cristina Lupu ◽  
Xiaohong Hu ◽  
Florea Lupu
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Protease Production ◽  
Extrinsic Pathway ◽  
Caveolin 1 ◽  
Tissue Factor Pathway ◽  
Quaternary Complex ◽  
And Function

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.

Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 1973-1978 ◽  
Author(s):  
Guyu Ho ◽  
Masaaki Narita ◽  
George J. Broze ◽  
Alan L. Schwartz
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Feedback Inhibition ◽  
Factor Xa ◽  
Full Length ◽  
Pharmacokinetic Studies ◽  
Dependent Manner ◽  
Carboxy Terminus ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissuefactor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPIC127), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPIC127) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor–related protein. Pharmacokinetic studies of TFPIC127 in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI.

Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

2002 ◽  
Vol 88 (07) ◽  
pp. 123-130 ◽  
Author(s):  
Matthieu Broussas ◽  
Pascale Cornillet-Lefèbvre ◽  
Gérard Potron ◽  
Philippe Nguyên
Keyword(s):  
Tissue Factor ◽  
Rank Order ◽  
Mrna Level ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Extrinsic Pathway ◽  
A3 Adenosine Receptor ◽  
Factor Expression ◽  
Cardioprotective Effects ◽  
Dose Dependent

SummaryTissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5’-N’-methyluronamide (IB-MECA) inhibited LPSinduced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

scholarly journals Leukocyte proteases cleave von Willebrand factor at or near the ADAMTS13 cleavage site

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1666-1674 ◽  
Author(s):  
Thomas J. Raife ◽  
Wenjing Cao ◽  
Bonnie S. Atkinson ◽  
Bruce Bedell ◽  
Robert R. Montgomery ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Hot Spot ◽  
Peptide Bond ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Activity Levels ◽  
Activated Neutrophils ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The function of von Willebrand factor (VWF) is regulated by proteolysis, which limits its multimeric size and ability to tether platelets. The importance of ADAMTS13 metalloprotease in VWF regulation is demonstrated by the association between severe deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura (TTP). However, ADAMTS13 activity levels do not always correlate with the clinical course of TTP, suggesting that other proteases could be important in regulating VWF. We identified 4 leukocyte proteases that cleave the synthetic VWF substrate FRETS-VWF73 and multimeric VWF. Elastase and proteinase 3 (PR3) cleave multimeric VWF and FRETS-VWF73 at the V1607-T1608 peptide bond; cathepsin G and matrix metalloprotease 9 cleave VWF substrates at the Y1605-M1606 and M1606-V1607 bonds, respectively. Isolated intact human neutrophils cleave FRETS-VWF73 at the V1607-T1608 peptide bond, suggesting that elastase or PR3 expressed on leukocyte surfaces might cleave VWF. In the presence of normal or ADAMTS13-deficient plasma, cleavage of FRETS-VWF73 by resting neutrophils is abolished. However, activated neutrophils retain proteolytic activity toward FRETS-VWF73 in the presence of plasma. Although the in vivo relevance remains to be established, these studies suggest the existence of a “hot spot” of VWF proteolysis in the VWF A2 domain, and support the possibility that activated leukocytes may participate in the proteolytic regulation of VWF.

scholarly journals Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1069-1073 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Activate Factor ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Hemophilic Patient ◽  
Factor X Activation ◽  
Rate Of Activation

Abstract Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

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