Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

2005 ◽  
Vol 94 (12) ◽  
pp. 1236-1244 ◽  
Author(s):  
Olav Klingenberg ◽  
Reidun Øvstebø ◽  
Gun-Britt Joø ◽  
Åse-Brit Westvik ◽  
Peter Kierulf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Procoagulant Activity ◽  
Clinical Samples ◽  
Meningococcal Infection ◽  
High Concentrations ◽  
Apoptosis And Necrosis ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryTissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.

scholarly journals The Effect of Oleic and Palmitic Acid on Induction of Steatosis and Cytotoxicity on Rat Hepatocytes in Primary Culture

2015 ◽  
pp. S627-S636 ◽  
Author(s):  
A. MORAVCOVÁ ◽  
Z. ČERVINKOVÁ ◽  
O. KUČERA ◽  
V. MEZERA ◽  
D. RYCHTRMOC ◽  
...  
Keyword(s):  
Palmitic Acid ◽  
Functional Capacity ◽  
Rat Hepatocytes ◽  
In Vitro Models ◽  
Albumin Production ◽  
Rat Primary Hepatocytes ◽  
Apoptosis And Necrosis ◽  
Dose Dependent Increase ◽  
Dose Dependent

In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone.

scholarly journals The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

2007 ◽  
Vol 54 (3) ◽  
pp. 625-639 ◽  
Author(s):  
Marian Tomasiak ◽  
Halina Stelmach ◽  
Tomasz Rusak ◽  
Michał Ciborowski ◽  
Piotr Radziwon
Keyword(s):  
Morphological Changes ◽  
Membrane Curvature ◽  
Procoagulant Activity ◽  
Calcium Signal ◽  
Left Shift ◽  
Cell Sizing ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
In Vitro Treatment

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.

Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor–dependent procoagulant activity

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3962-3970 ◽  
Author(s):  
Florence Sabatier ◽  
Veronique Roux ◽  
Francine Anfosso ◽  
Laurence Camoin ◽  
José Sampol ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Cellular Responses ◽  
Procoagulant Activity ◽  
Immunoperoxidase Staining ◽  
Monocytic Cells ◽  
Endothelial Microparticles ◽  
Blocking Antibodies ◽  
Β2 Integrins

In the present study we investigated whether endothelial microparticles (EMPs) can bind to monocytic THP-1 cells and modulate their procoagulant properties. Using flow cytometry, we demonstrated that EMPs express adhesive receptors similar to those expressed by activated endothelial cells. Expression of endothelial antigens by THP-1 cells incubated with EMP was shown by immunoperoxidase staining and flow cytometry using antibodies directed against E-selectin, VCAM-1, and endoglin. EMP binding to THP-1 cells was time- and concentration- dependent, reached a plateau at 15 minutes, and had an EMP-to-monocyte ratio of 50:1. EMP binding was not affected by low temperature and was not followed by the restoration of phosphatidylserine asymmetry, suggesting that adhesion was not followed by fusion. A 4-hour incubation of THP-1 cells with EMP led to an increase in procoagulant activity as measured by clotting assay. Concomitantly, THP-1 exhibited increased levels of tissue factor (TF) antigen and TF mRNA compared to control cells. The ability of EMP to induce THP-1 procoagulant activity was significantly reduced when THP-1 cells were incubated with EMP in the presence of blocking antibodies against ICAM-1 and β2 integrins. These results demonstrate that EMPs interact with THP-1 cells in vitro and stimulate TF-mediated procoagulant activity that is partially dependent on the interaction of ICAM-1 on EMP and its counterreceptor, β2 integrins, on THP-1 cells. Induction of procoagulant activity was also demonstrated using human monocytes, suggesting a novel mechanism by which EMP may participate in the dissemination and amplification of procoagulant cellular responses.

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.

scholarly journals Applications of Flow Cytometry to Clinical Microbiology

2000 ◽  
Vol 13 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Alberto Álvarez-Barrientos ◽  
Javier Arroyo ◽  
Rafael Cantón ◽  
César Nombela ◽  
Miguel Sánchez-Pérez
Keyword(s):  
Flow Cytometry ◽  
Clinical Microbiology ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Commercial Kits ◽  
Antimicrobial Treatments ◽  
Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

scholarly journals The stable prostacyclin analog, iloprost, and prostaglandin E1 inhibit monocyte procoagulant activity in vitro

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 382-386 ◽  
Author(s):  
DJ Crutchley ◽  
MJ Hirsh
Keyword(s):  
Tissue Factor ◽  
Prostaglandin E1 ◽  
Human Peripheral Blood ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Dependent Inhibition ◽  
Clinical Potential ◽  
Prostacyclin Analog

Abstract Exposure of human peripheral blood to 100 ng/mL of bacterial endotoxin for 2 hours resulted in a 20-fold increase in monocyte procoagulant activity. The activity was functionally identified as tissue factor, because it was not expressed in plasma deficient in factor VII and was specifically inhibited by a monoclonal antibody directed against human tissue factor. When the stable prostacyclin analog, iloprost, was added to blood 30 minutes before endotoxin, a dose-dependent inhibition of monocyte procoagulant activity occurred, with an I50 of 20 nmol/L. Prostaglandin E1 (PGE1) produced similar effects, with an I50 of 150 nmol/L. Exposure of THP-1 monocytic cells to 100 ng/mL endotoxin resulted in a threefold increase in procoagulant activity after 2 hours and a 20-fold increase after 6 hours. A 30-minute pretreatment with iloprost or PGE1 again inhibited development of procoagulant activity, with I50 values of 5 nmol/L and 150 nmol/L, respectively. Treatment of THP-1 cells with iloprost 2 hours after exposure to endotoxin significantly inhibited further increases in procoagulant activity. Iloprost was less potent under these conditions, 30% inhibition being obtained at 100 nmol/L and 70% at 1 mumol/L. These results suggest that prostacyclin may be a physiologic modulator of monocyte tissue factor expression; in addition, its stable analog, iloprost, may have clinical potential for the treatment of thrombotic disorders in which elevated monocyte procoagulant activity plays a role.

scholarly journals Increased in vitro and in vivo generation of procoagulant activity (tissue factor) by mononuclear phagocytes after intralipid infusion in rabbits

Blood ◽  
1985 ◽  
Vol 65 (6) ◽  
pp. 1391-1395 ◽  
Author(s):  
P Montemurro ◽  
A Lattanzio ◽  
G Chetta ◽  
L Lupo ◽  
L Caputi-Iambrenghi ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Mononuclear Phagocyte ◽  
Mononuclear Phagocytes ◽  
Procoagulant Activity ◽  
Sterile Saline ◽  
Functional Changes ◽  
Before And After

Abstract Intralipid, a fat emulsion widely used in parenteral nutrition, can produce marked functional changes of the mononuclear phagocyte system. We investigated the effect of Intralipid administration on the generation of procoagulant activity by rabbit mononuclear phagocytes. Two groups of ten rabbits given either a single infusion of Intralipid 10% or a similar volume of sterile saline were studied before and after infusion. Procoagulant activity was measured on isolated blood mononuclear cells after incubation with and without endotoxin, using a one-stage clotting assay. Cells from animals infused with Intralipid produced significantly more procoagulant activity than controls (P less than .01). Results were similar when freshly collected whole blood was incubated with and without endotoxin, and procoagulant activity was measured on subsequently isolated mononuclear cells (P less than .01). In addition, when rabbits were given a single injection of endotoxin, blood and spleen mononuclear cells harvested 50 to 60 minutes after the injection from animals pretreated with Intralipid expressed five to seven times more procoagulant activity than did cells from animals pretreated with saline. In all instances, procoagulant activity was identified as tissue factor. These findings suggest that Intralipid may cause functional changes in mononuclear phagocytes, resulting in increased production of tissue factor on incubation in short-term culture in vitro and in response to endotoxin in vivo.

scholarly journals Immunologic Identification of Tissue Factor (Thromboplastin) Synthesized by Cultured Fibroblasts

Blood ◽  
1973 ◽  
Vol 41 (5) ◽  
pp. 671-678 ◽  
Author(s):  
Leo R. Zacharski ◽  
Leon W. Hoyer ◽  
O. Ross McIntyre
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Coagulation Factors ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Cultured Fibroblasts ◽  
Procoagulant Effect ◽  
Tissue Thromboplastin ◽  
Thromboplastin Factor

Abstract Immunologic methods were employed in an attempt to identify a potent procoagulant present in homogenates of human skin fibroblasts cultured in vitro. The activity of this procoagulant was restricted to the early stages of coagulation and was heretofore considered to be due to tissue factor (tissue thromboplastin, factor III) either alone or in combination with one or more of the first-stage coagulation factors (VIII, IX, XI, XII). The present studies demonstrated that procoagulant activity was not diminished by incubation with anti-VIII or anti-IX antibodies of human origin or with anti-VIII antibody of rabbit origin. Furthermore, cell culture homogenates failed to bind antifactor VIII antibody and did not contain an inhibitor of the reaction between factor VIII and its antibody. By contrast, procoagulant activity was obliterated by an antibody to tissue factor protein regardless of whether plasmas deficient in factor VIII, IX, XI, or XII were used in the assay system. The antitissue factor antibody failed to block the procoagulant effect after tissue factor had complexed factor VII. The procoagulant, therefore, consisted entirely of tissue factor.

Stimulation of HCO3- transport in isolated proximal bullfrog duodenum by prostaglandins

1980 ◽  
Vol 239 (3) ◽  
pp. G198-G203 ◽  
Author(s):  
G. Flemstrom
Keyword(s):  
Electrical Potential ◽  
Potential Difference ◽  
Electrical Potential Difference ◽  
Morphological Examination ◽  
Minimal Concentration ◽  
Dependent Transport ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Stimulation Of

An in vitro preparation of proximal duodenum from the bullfrog transported alkali into the luminal solution (approximately 1 mueq x h-1 x cm-2) and generated a transepithelial electrical potential difference (5-10 mV, lumen negative). Transport was inhibited by 2,4-dinitrophenol (10(-5) M), CN- (5 X 10(-3) M), indomethacin (5 X 10(-5) M), and acetazolamide (5 X 10(-3) M) indicating that metabolism is required. Both alkali transport and the electrical potential difference showed a dose-dependent increase on administration of the prostaglandins E2, 16,16-dimethyl E2, and F2 alpha. The minimal concentration stimulating transport was lower with the E-type prostaglandins (10(-8) M than with F2 alpha (10(-6) M), and the former also produced greater maximal responses. In addition to metabolic-dependent transport of alkali, there was passive transmucosal migration of HCO3-, amounting to approximately 40% of basal (unstimulated) transport and sensitive to variation of the transmucosal hydrostatic pressure. Morphological examination showed that the preparation is devoid of Brunner glands. Stimulation of duodenal epithelial HCO3- transport by prostaglandins may contribute to their previously demonstrated ability to prevent duodenal ulceration.

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