Human megakaryocytes confer tissue factor to a subset of shed platelets to stimulate thrombin generation

SummaryTissue factor (TF), the main activator of the blood coagulation cascade, has been shown to be expressed by platelets. Despite the evidence that both megakaryocytes and platelets express TF mRNA, and that platelets can make de novo protein synthesis, the main mechanism thought to be responsible for the presence of TF within platelets is through the uptake of TF positive microparticles. In this study we assessed 1) whether human megakaryocytes synthesise TF and transfer it to platelets and 2) the contribution of platelet-TF to the platelet hemostatic capacity. In order to avoid the cross-talk with circulating microparticles, we took advantage from an in vitro cultured megakaryoblastic cell line (Meg-01) able to differentiate into megakaryocytes releasing platelet-like particles. We show that functionally active TF is expressed in human megakaryoblasts, increased in megakaryocytes, and is transferred to a subset of shed platelets where it contributes to clot formation. These data were all confirmed in human CD34pos- derived megakaryocytes and in their released platelets. The effect of TF silencing in Meg-megakaryoblasts resulted in a significant reduction of TF expression in these cells and also in Meg-megakaryocytes and Meg-platelets. Moreover, the contribution of platelet-TF to the platelet hemostatic capacity was highlighted by the significant delay in the kinetic of thrombin formation observed in platelets released by TF-silenced megakaryocytes. These findings provide evidences that TF is an endogenously synthesised protein that characterises megakaryocyte maturation and that it is transferred to a subset of newly-released platelets where it is functionally active and able to trigger thrombin generation.

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In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

Thrombosis and Haemostasis ◽

10.1160/th09-10-0690 ◽

2010 ◽

Vol 104 (09) ◽

pp. 514-522 ◽

Cited By ~ 3

Author(s):

Thomas Lecompte ◽

Agnès Tournier ◽

Lise Morlon ◽

Monique Marchand-Arvier ◽

Claude Vigneron ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Cathepsin G ◽

Coagulation Cascade ◽

Clotting Factor ◽

Factor V ◽

Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

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The Mycotoxin Deoxynivalenol Significantly Alters the Function and Metabolism of Bovine Kidney Epithelial Cells In Vitro

Toxins ◽

10.3390/toxins11100554 ◽

2019 ◽

Vol 11 (10) ◽

pp. 554

Author(s):

Bailey ◽

Breton ◽

Panic ◽

Cogan ◽

Bailey ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Synthesis ◽

Cellular Response ◽

De Novo ◽

Fumonisin B1 ◽

Economic Cost ◽

Bovine Kidney ◽

Low Concentrations ◽

De Novo Protein Synthesis

Bovine mycotoxicosis is a disorder caused by the ingestion of fungal toxins. It is associated with chronic signs, such as reduced growth rate and milk yield, and causes significant economic cost to the dairy industry. The mycotoxins deoxynivalenol (DON), zearalenone (ZEN), and fumonisin B1 (FB1) are commonly found in grain fed to cattle. Patulin (PA) is a common grass silage contaminant but is also found in grain. The effects of these mycotoxins on cellular function at low concentrations are not well understood. Using Madin–Darby bovine kidney cells we evaluated the cellular response to these mycotoxins, measuring cytotoxicity, de novo protein synthesis, cell proliferation, cell cycle analysis, and also metabolic profiling by 1H NMR spectroscopy. DON, ZEN, and PA induced cytotoxicity, and PA and FB1 induced a decrease in metabolic activity in surviving cells. DON was the only mycotoxin found to have a significant effect on the metabolic profile, with exposed cells showing increased cellular amino acids, lactate, 2-oxoglutarate, 3-hydroxybutyrate, and UDP-N-acetylglucosamine and decreased β-alanine, choline, creatine, taurine, and myo-inositol. Cells exposed to DON also showed reductions in protein synthesis. DON has previously been documented as being a ribotoxin; the results here suggest that exposure of bovine cells to DON causes a decrease in protein synthesis with corresponding cellular accumulation of precursors. Cell proliferation was also arrested without causing apoptosis. It is likely that exposure triggers hypoxic, hypertonic, and ribotoxic responses in bovine cells, and that these responses contribute to reduced productivity in exposed cattle.

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Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

International Journal of Toxicology ◽

10.1177/1091581820907324 ◽

2020 ◽

Vol 39 (3) ◽

pp. 207-217

Author(s):

F. Poitout-Belissent ◽

D. Culang ◽

D. Poulin ◽

R. Samadfan ◽

S. Cotton ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

In Vivo Studies ◽

Coagulation Cascade ◽

Dependent Manner ◽

Kinetic Assay ◽

Thrombin Generation Assay ◽

Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

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Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Scientific Reports ◽

10.1038/s41598-019-49635-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Ivelin I. Ivanov ◽

Bonita H. R. Apta ◽

Arkadiusz M. Bonna ◽

Matthew T. Harper

Keyword(s):

Protein Synthesis ◽

Tissue Factor ◽

Platelet Activation ◽

Whole Blood ◽

Vessel Wall ◽

De Novo ◽

Vascular Damage ◽

Coagulation Cascade ◽

Activated Platelets ◽

Necessary And Sufficient

Abstract Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation.

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Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line

Immunology Letters ◽

10.1016/s0165-2478(01)00213-9 ◽

2001 ◽

Vol 77 (3) ◽

pp. 127-131 ◽

Cited By ~ 3

Author(s):

Yong-Hoon Chung ◽

Jeehee Youn ◽

Yong Choi ◽

Doo Jin Paik ◽

Yang-Ja Cho

Keyword(s):

Protein Synthesis ◽

Cell Line ◽

De Novo ◽

Myeloma Cell ◽

Myeloma Cell Line ◽

Mouse Myeloma Cell ◽

Mouse Myeloma Cell Line ◽

Induced Apoptosis ◽

De Novo Protein Synthesis ◽

Mouse Myeloma

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Lectin Stimulation of Tissue Thromboplastin Activity in Human Monocytes in vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657060 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1574-1579 ◽

Cited By ~ 4

Author(s):

T Lyberg ◽

H Prydz

Keyword(s):

Protein Synthesis ◽

Concanavalin A ◽

Wheat Germ ◽

Actinomycin D ◽

De Novo ◽

Human Monocytes ◽

Tissue Thromboplastin ◽

De Novo Protein Synthesis ◽

Stimulation Of

SummaryLectins (phytohaemagglutinin, concanavalin A and wheat germ agglutinin) trigger an increase in tissue thromboplastin activity of human monocytes in vitro. The presence of serum was not necessary and did not enhance the activity. The increase was inhibited by cycloheximide and actinomycin D, suggesting that de novo protein synthesis is involved.

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Insulin-like growth factor I stimulates sodium-phosphate cotransport in the rat osteoblastic cell line UMR-106-01

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1992.262.4.e389 ◽

1992 ◽

Vol 262 (4) ◽

pp. E389-E393

Author(s):

P. I. Campbell ◽

D. K. Breedlove ◽

S. A. Kempson

Keyword(s):

Protein Synthesis ◽

Growth Factor ◽

Cell Line ◽

De Novo ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Umr 106 ◽

De Novo Protein Synthesis ◽

Stimulation Of

The effect of recombinant insulin-like growth factor I (IGF-I) on Pi uptake by a rat osteoblast-like cell line (UMR-106-01) in culture was investigated. IGF-I (10(-6)-10(-8) M) caused a dose-related stimulation of Na(+)-Pi cotransport. A 30-70% increase in Na(+)-dependent Pi uptake over control values was observed after 1- to 5-h exposure of these cells to 10(-7) M IGF-I. The increase was detected within 45 min, in contrast to the slower action of insulin. This effect of IGF-I was specific for Na(+)-Pi uptake, because Na(+)-independent Pi uptake and Na(+)-alanine cotransport were unaffected by IGF-I. A reversal of IGF-I induced stimulation of Na(+)-Pi cotransport was observed within 1 h of removal of the hormone. Kinetic analysis of the IGF-I effect indicates a significant change only in the apparent maximum velocity (Vmax) of Na(+)-Pi cotransport. The Vmax was 5.22 +/- 0.47 vs. 3.33 +/- 0.45 nmol Pi.mg protein-1.10 min-1 in confluent monolayers exposed to 10(-7) M IGF-I and vehicle alone, respectively, for 3 h (P less than 0.05, group t test). Blocking de novo protein synthesis with cycloheximide had no effect on this stimulatory effect of IGF-I. These observations indicate that IGF-I specifically stimulates Pi uptake in osteoblastic cells. The effect is characterized by an increase in Vmax and is not dependent on de novo protein synthesis. The mechanism remains to be determined.

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