scholarly journals Canine Neutrophil Extracellular Traps Enhance Clot Formation and Delay Lysis

2017 ◽  
Vol 55 (1) ◽  
pp. 116-123 ◽  
Author(s):  
Unity Jeffery ◽  
Dana N. LeVine
Keyword(s):  
Autoimmune Diseases ◽  
Neutrophil Extracellular Traps ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Clot Lysis ◽  
Elastase Activity ◽  
Platelet Poor Plasma ◽  
Extracellular Traps ◽  
Tissue Plasminogen

Autoimmune diseases increase the risk of thrombosis. Neutrophil extracellular traps (NETs) are webs of DNA and protein that may mediate thrombosis in autoimmune diseases. Human and murine studies show NET-releasing neutrophils within a thrombus promote its growth, but it is unclear to what extent NET fragments released into circulation during inflammation are prothrombotic. This study hypothesized that canine NETs promote clot formation and impair lysis even in the absence of neutrophils. NETs were prepared from PMA-stimulated neutrophils and added to fibrinogen and thrombin or to recalcified pooled canine platelet-poor plasma, tissue factor, and tissue plasminogen activator. Clot formation and lysis were measured spectrophotometrically. NETs did not alter fibrin clot formation, but NETs increased maximum clot formation velocity ( P = .001) and delayed lysis ( P = .009) of plasma clots compared with supernatants from nonstimulated neutrophils. DNase digestion of NETs reduced their effect on clot lysis but not maximum clot formation velocity. This suggested impaired lysis was principally mediated by DNA within NETs but that NET proteins were principally responsible for increased speed of clot formation. Previous reports suggested elastase or histones might be responsible for the effect of NETs on clot formation. Elastase activity was greatly reduced by plasma, and addition of histones to plasma did not increase formation velocity, suggesting these proteins were not responsible for increasing maximum formation velocity. This study showed that NETs enhanced clot formation and impaired clot lysis in canine platelet-poor plasma. These in vitro findings suggest both NET proteins and DNA may contribute to thrombosis in inflammatory disease.

Enhanced Fibrin Clot Formation and Stability with a Superactive Analog of Factor VIIa in an In Vitro Model of Hemophilia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4056-4056
Author(s):  
Alisa S. Wolberg ◽  
Mirella Ezban ◽  
Ulla Hedner ◽  
Egon Persson
Keyword(s):  
Factor Viia ◽  
Fibrin Clot ◽  
Model System ◽  
Clot Formation ◽  
In Vitro Assays ◽  
Clot Lysis ◽  
High Dose ◽  
Total Blood ◽  
Subsequent Decrease

Abstract Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrated its clinical efficacy in treating bleeding in hemophilia patients with inhibitors. Using in vitro assays, we and others have previously shown that high dose rFVIIa shortens the onset of fibrin clot formation and normalizes the fibrin structure and porosity of clots formed under hemophilic conditions (absence of factors VIII and/or IX (Wolberg et al. (2001) 98(11): 826a). Recently, several superactive analogs of rFVIIa have been described, demonstrating increased thrombin-generating activity on platelets (Persson et al. (2001) 98(24): 13583–8), and increased capability to reduce the tail bleeding time and total blood loss in a mouse model of hemophilia A (Tranholm et al. (2003) 102(10): 3615–20). In the current study, we have used a cell-based reconstituted model system of coagulation to compare the abilities of rFVIIa and one of the superactive analogues, NN1731, to modulate the onset and rate of fibrin clot formation, as well as the ability of these molecules to increase the stability of fibrin clots formed in the presence of a fibrinolytic challenge. The model system consists of tissue factor-bearing monocytes, purified pro- and anti-coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), freshly-isolated, unactivated platelets and fibrinogen. This system permits the inclusion of protein and cell concentrations at their physiologic levels. “Coagulation” is initiated by combining the components and following clot formation by optical density. “Hemophilia” is simulated by omitting factors IX and VIII. We observed that NN1731 shortened the clot time of hemophilic conditions to normal and did so at lower doses than were required of rFVIIa. Further, NN1731 increased the rate of clot formation to normal. To test the ability of clots to form in a fibrinolytic environment, we performed a Plasmin Challenge Assay, in which clot formation is initiated in the presence of a low concentration of plasmin. In this assay, clot formation is indicated as an increase in turbidity, and clot lysis is seen as a subsequent decrease in turbidity. There is no turbidity development (no clot formation) under hemophilic conditions. We observed that NN1731 increased the rate of clot formation and the length of time that fibrin was present under hemophilic conditions, and did so at lower concentrations than were required for rFVIIa. Moreover, in all of these assays, NN1731 normalized hemophilic conditions. Our results suggest that NN1731 promotes clot formation and stability to a greater degree than equal doses of rFVIIa and may possess clinical utility in patients that are refractory to current doses of rFVIIa.

In Vitro Studies on the Fibrinolytic, Thrombolytic and Fibrinogenolytic Properties of a Tissue Plasminogen Activator from Guinea Pig Keratocytes

1985 ◽  
Vol 53 (02) ◽  
pp. 200-203 ◽  
Author(s):  
A Electricwala ◽  
R J Ling ◽  
P M Sutton ◽  
B Griffiths ◽  
P A Riley ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Thrombolytic Activity ◽  
Tissue Plasminogen ◽  
In Vitro Systems

SummaryThe fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37° C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (<50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3–6 hr with the concentration of GPK activator in the range of 1–5 μg/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

scholarly journals Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 915 ◽  
Author(s):  
Esther Fousert ◽  
René Toes ◽  
Jyaysi Desai
Keyword(s):  
Autoimmune Diseases ◽  
Lupus Erythematosus ◽  
Current Knowledge ◽  
Neutrophil Extracellular Traps ◽  
Clinical Diagnostics ◽  
Extracellular Traps ◽  
Self Tolerance ◽  
Antigen Presenting

Following fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in vitro, in vivo and clinical diagnostics suggest that NETs may play a crucial role in inflammation and autoimmunity in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Most likely, NETs contribute to breaking self-tolerance in autoimmune diseases in several ways. During this review, we discuss the current knowledge on how NETs could drive autoimmune responses. NETs can break self-tolerance by being a source of autoantigens for autoantibodies found in autoimmune diseases, such as anti-citrullinated protein antibodies (ACPAs) in RA, anti-dsDNA in SLE and anti-myeloperoxidase and anti-protein 3 in AAV. Moreover, NET components could accelerate the inflammatory response by mediating complement activation, acting as danger-associated molecular patterns (DAMPs) and inflammasome activators, for example. NETs also can activate other immune cells, such as B cells, antigen-presenting cells and T cells. Additionally, impaired clearance of NETs in autoimmune diseases prolongs the presence of active NETs and their components and, in this way, accelerate immune responses. NETs have not only been implicated as drivers of inflammation, but also are linked to resolution of inflammation. Therefore, NETs may be central regulators of inflammation and autoimmunity, serve as biomarkers, as well as promising targets for future therapeutics of inflammatory autoimmune diseases.

The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

1993 ◽  
Vol 70 (02) ◽  
pp. 301-306 ◽  
Author(s):  
Linda A Robbie ◽  
Nuala A Booth ◽  
Alison M Croll ◽  
Bruce Bennett
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Dependent Inhibition ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

scholarly journals Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Leela Goel ◽  
Huaiyu Wu ◽  
Bohua Zhang ◽  
Jinwook Kim ◽  
Paul A. Dayton ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Negative Pressure ◽  
Boiling Point ◽  
Clot Lysis ◽  
Control Group ◽  
Peak Negative Pressure ◽  
Blood Clots ◽  
Tissue Plasminogen ◽  
First Time

AbstractOne major challenge in current microbubble (MB) and tissue plasminogen activator (tPA)-mediated sonothrombolysis techniques is effectively treating retracted blood clots, owing to the high density and low porosity of retracted clots. Nanodroplets (NDs) have the potential to enhance retracted clot lysis owing to their small size and ability to penetrate into retracted clots to enhance drug delivery. For the first time, we demonstrate that a sub-megahertz, forward-viewing intravascular (FVI) transducer can be used for ND-mediated sonothrombolysis, in vitro. In this study, we determined the minimum peak negative pressure to induce cavitation with low-boiling point phase change nanodroplets and clot lysis. We then compared nanodroplet mediated sonothrombolysis to MB and tPA mediate techniques. The clot lysis as a percent mass decrease in retracted clots was 9 ± 8%, 9 ± 5%, 16 ± 5%, 14 ± 9%, 17 ± 9%, 30 ± 8%, and 40 ± 9% for the control group, tPA alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups, respectively. In retracted blood clots, combined ND- and tPA-mediated sonothrombolysis was able to significantly enhance retracted clot lysis compared with traditional MB and tPA-mediated sonothrombolysis techniques. Combined nanodroplet with tPA-mediated sonothrombolysis may provide a feasible strategy for safely treating retracted clots.

scholarly journals Incorporation of α2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 347
Author(s):  
Zsuzsa Bagoly ◽  
Barbara Baráth ◽  
Rita Orbán-Kálmándi ◽  
István Szegedi ◽  
Réka Bogáti ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Intracranial Hemorrhage ◽  
Intravenous Thrombolysis ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Stroke Patients ◽  
Plasmin Inhibitor ◽  
Fibrin Clots

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

scholarly journals Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Ondracek ◽  
T.M Hofbauer ◽  
A Mangold ◽  
T Scherz ◽  
V Seidl ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Endothelial Cells ◽  
Plaque Rupture ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Neutrophil Extracellular Traps ◽  
Funding Source ◽  
Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

755 CXCR1 and CXCR2 chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A803-A803 ◽  
Author(s):  
Alvaro Teijeira ◽  
Saray Garasa ◽  
Itziar Migueliz ◽  
Assunta Cirella ◽  
Ignacio Melero
Keyword(s):  
Cancer Patients ◽  
Tumor Cells ◽  
Mouse Models ◽  
Chemokine Receptor ◽  
Suppressor Cells ◽  
Neutrophil Extracellular Traps ◽  
Myeloid Derived Suppressor Cells ◽  
Extracellular Traps ◽  
Tumor Associated Neutrophils

BackgroundNeutrophils are expanded and abundant in an important fraction (up to 35% of patients) in cancer-bearing hosts. When neutrophils are expanded, they usually promote exert immunomodulatory functions promoting tumor progression and the generation of metastases. Neutrophils can undergo a specialized form of cell death called NETosis that is characterized by the extrusion of their DNA to contain infections. In cancer NETs have been described to promote metastases in mouse models. IL-8, a CXCR1/2 ligand clinically targeted by blocking antibodies, has been described to induce NETosis and is upregulated in many cancer patients. Our hypothesis is that chemokines secreted by cancer cells can mediate NETosis in tumor associated neutrophils and that NETs can be one of the immunomodulatory mechanisms provided by tumor associated neutrophils.MethodsNETosis induction of peripheral neutrophils and granulocytic myeloid derived suppressor cells by different chemotactic stimuli, tumor cell supernatants and cocultures upon CXCR1/2 blockade. NET immunodetection in mouse models and xenograft tumors upon CXCR1/2 blockade. In vitro tumor cytotoxicity assays in the presence/absence of NETs, and videomicroscopy studies in vitro and by intravital imaging to test NETs inhibition of immune cytotoxicity by immune-cell/target-cell inhibition. Tumor growth studies and metastases models in the presence of NETosis inhibitors and in combination with checkpoint blockade in mouse cancer models.ResultsUnder the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.ConclusionsCXCR1 and 2 are the main receptors mediating NETosis of tumor associated neutrophils in our in-vitro and in vivo systems expressing high levels of CXCR1 and 2 ligands. NETs limit cancer cell cytotoxicity by impeding contacts with cancer cells.

scholarly journals The mechanism of in vitro clot lysis induced by vascular plasminogen activator

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1331-1337 ◽  
Author(s):  
C Tran-Thang ◽  
EK Kruithof ◽  
F Bachmann
Keyword(s):  
Plasminogen Activator ◽  
Whole Blood ◽  
Apparent Molecular Weight ◽  
Clot Lysis ◽  
Initial Concentration ◽  
Low Concentrations ◽  
Sds Page ◽  
Tissue Plasminogen ◽  
Low Rate

Abstract The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.

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