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Anticoagulant effects of an antidiabetic drug on monocytes in vitro
Thrombosis Research 128 (2011) e100–e106
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Anticoagulant effects of an antidiabetic drug on monocytes in vitro C.E. Henriksson a,⁎, M. Hellum a, K.B.F. Haug a, H.C. Aass a, G.B. Joø a, R. Øvstebø a, A.M. Trøseid a, O. Klingenberg b, P. Kierulf a a b
Blood cell research group, Section for research, Department of Medical Biochemistry, Oslo University Hospital, Ullevaal, Oslo, Norway Department of Medical Biochemistry, Oslo University Hospital, Rikshospitalet, Oslo, Norway
a r t i c l e
i n f o
Article history: Received 28 May 2010 Received in revised form 1 July 2011 Accepted 7 July 2011 Available online 20 August 2011 Keywords: Tissue Factor Alternatively spliced Tissue Factor Microparticle Glibenclamide Monocyte
a b s t r a c t Introduction: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. Methods: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RTqPCR, western blot) and on cell viability and MP release (flow cytometry). Results: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. Conclusions: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated. © 2011 Elsevier Ltd. All rights reserved.
Introduction Diabetes leads to a hypercoagulable state. Patients with diabetes type 2 demonstrate increased tissue factor (TF) expression and activity [1–4], increased PS expression [5] and elevated levels of microparticles (MPs) in the circulation [6,7]. Sulfonylureas, such as glibenclamide, stimulate insulin secretion from pancreatic β-cells and are widely used to treat diabetes type 2 [8]. Glibenclamide is a general ATP-binding cassette (ABC) transporter inhibitor [9,10]. ABC transporters constitute a large family of proteins translocating various substances across biological membranes. Several ABC transporters are involved in disease pathogenesis. Among them are sulfonylurea receptors (SUR), ABCA1 (Tangier disease protein), multidrug resistance protein (MDR) and cystic fibrosis transmembrane conductance regulator (CFTR) [9]. To what extent inhibition of ABC-transporters may affect the hypercoagulable state in diabetes, remains unsettled.
Abbreviations: MP, Microparticle; TF, Tissue Factor; asTF, alternatively spliced Tissue Factor; LPS, Lipopolysacharride; PS, Phosphatidylserine; RBC, Red blood cells; ABC, ATP-binding cassette; SUR, Sulfonylurea receptor; mAb, monoclonal antibody; Ann V, Annexin V; 7-AAD, 7-amino-actinomycin D; PE, Phycoerythrin; FITC, Fluorescein isothiocyanate; TFPI, Tissue Factor Pathway Inhibitor. ⁎ Corresponding author. Tel.: + 47 22 11 83 56; fax: + 47 22 11 81 89. E-mail address: [email protected] (C.E. Henriksson). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.07.007
TF is the main initiator of the coagulation cascade [11]. Cell surface TF/Factor VIIa (FVIIa) initiates blood coagulation by limited proteolysis and activation of factor IX (FIX) and factor X (FX) (extrinsic tenase activity), which subsequently leads to generation of thrombin [12]. Despite being available at the cell surface, TF may not display full procoagulant activity. This phenomenon is termed encryption [13]. The encrypted form of TF is inactive, while the decrypted form confers the active procoagulant configuration [13]. The molecular mechanism of TF decryption is still obscure [14,15], but it has been associated with loss of cell membrane phospholipid asymmetry [16]. PS is normally sequestered at the inner plasma membrane leaflet, and cell surface relocalisation of PS is associated with TF decryption as well as with release of procoagulant MPs, which are defined as 0.1-1.0 μM shedded membrane vesicles [17]. Furthermore, the protein product of an alternatively spliced form of TF (asTF) is reported to circulate in a soluble form [18]. The asTF protein lacks both the transmembrane and the intracellular TF domains and instead contains a unique C-terminus. It has been suggested that this asTF exhibits phospholipid-dependent procoagulant activity [18,19]. It has previously been shown that lipopolysaccharide (LPS)stimulation of monocytes leads to synthesis of both TF and asTF, and to increased cell surface TF expression [20–22]. Endotoxin also enhances PS exposure at the cell surface [22] and results in release of procoagulant MPs [23]. Glibenclamide has previously been shown to reduce TF activity in an LPS-stimulated monocytic cell line (THP-1
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cells) [24], but the effects of glibenclamide on primary LPS-stimulated monocytes and their released MPs are not known. Using LPSstimulated TF-bearing human monocytes, we investigated the effects of glibenclamide on cell- and supernatant-associated procoagulant activity and related it to TF and asTF expression, cell viability and MP release. Our results showed that there was a concentration-dependent decrease in procoagulant activity in cells and supernatants up to 100 μmol/L glibenclamide. The decrease in procoagulant activity was associated with reduced expression of TF and asTF, and possibly also with a reduced number of MPs. Materials and methods Materials Roswell Park Memorial Institute culture medium (RPMI) 1640 (Gibco, Paisley, Scotland). Fetal calf serum (FCS) [25], heat and acid treated before use, was from BioWhittaker Inc. (Walkersville, MD). These reagents had endotoxin levels b0.25 EU/mL, as measured by a Limulus amoebocyte lysate assay. LPS from Escherichia coli (055:B5) was from BioWhittaker Inc. (Walkersville, MD). In all experiments, LPS was diluted in RPMI 1640 with 5% (v/v) FCS containing 2% (v/v) of penicillin and streptomycin (cat. no. P0906) from Sigma (St. Louis, MO). Glibenclamide (Glyburide) was from Sigma-Aldrich (St. Louis, MO). In all experiments, glibenclamide was diluted in Dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany). Tris buffered saline: 20 mmol/L Tris, 0.15 mol/L NaCl, pH 7.4. Factor X (FX) and Factor Xa (FXa) were from Enzyme Research Laboratories (South Bend, IN). S-2756 was from Chromogenix (Milano, Italy). Factor VIIa (FVIIa) was a kind gift from Professor Ulla Hedner at Novo Nordisk (Bagsvaerd, Denmark). Anti-TF monoclonal antibodies (mAbs) (TF9-5B7 and TF8-5G9, IgG1) were kind gifts from Professor James Morrissey at University of Illinois College of Medicine (Urbana, IL). Anti-asTF polyclonal antibodies and recombinant asTF were generously provided by Professor Vladimir Bogdanov at Mount Sinai school of Medicine (New York, NY) [18]. Anti-asTF polyclonal antibodies were kindly provided by Professor Ursula Rauch at Charite´- Universitätsmedizin (Berlin, Germany) [19]. Phycoerythrin (PE)-conjugated F(ab´)2 Fragment of Rabbit Anti-Mouse immunoglobulins was from DAKO (Glostrup, Denmark). Fluorescein isothiocyanate (FITC)-labeled Annexin V (Ann V) and 7-amino-actinomycin D (7-AAD) were from BD Biosciences Pharmingen (Hamburg, Germany). Monocyte isolation and thawing Full blood cell counting (Sysmex 2100XE) was performed on anonymous samples from healthy blood donors (upon written consent) at the Blood Bank, Oslo University Hospital, Ullevaal, Norway, and peripheral blood mononuclear cells (PBMC) were isolated from EDTA whole blood (450 mL) by density gradient centrifugation. Monocytes were purified from PBMC by elutriation centrifugation to a purity of N90% (flow cytometry), and cryopreserved in RPMI 1640 with 25% (v/v) FCS and 10% (v/v) DMSO at −150 °C essentially as previously described by Lund et al [26]. In all experiments, monocytes were thawed, washed once in RPMI 1640 with 20% (v/v) FCS, resuspended and cultivated in RPMI 1640 with 5% (v/v) FCS containing 2% (v/v) of penicillin and streptomycin. Monocytes used in the experiments came from different donors (I-XI). FXa-generating activity (extrinsic tenase activity) of cells Monocytes (4× 105/well) from three different donors were incubated (30 minutes, 37 °C) in 24-well plates (Ultra Low Attachment plates, Costar 3473, Corning) with 0, 5, 10, 25, 50, 75 or 100 μmol/L glibenclamide before LPS (1 μg/mL) was added for 4 h. Then, monocytes
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were centrifuged (300 g, 7 min, 4 °C), supernatants discarded, and the monocytes were resuspended in 1000 μL Tris buffered saline. Again, monocytes were centrifuged (300 g, 7 min, 4 °C), supernatants discarded, and the monocytes resuspended in 80 μL Tris-buffered saline with 0.5% (w/v) BSA (A-4503, Sigma). Recovery of monocytes was similar at all glibenclamide concentrations (microscopy counting). The ability of 1.5 × 105 resuspended cells/well (duplicates) to promote activation of FX to FXa, detected by a chromogenic FXa substrate (FXageneration) was assayed as previously described [22,23]. In control experiments, cells or LPS were omitted. Clot forming activity of cells and supernatants in citrated plasma Monocytes (1 × 10 6 cells/well) from three different donors were incubated with glibenclamide and LPS as above in a total volume of 1 mL. Next, monocytes were centrifuged (4300 g, 5 min, 20 °C), supernatants (950 μL of each well) collected and the monocytes resuspended in 950 μL RPMI 1640 with 5% (v/v) FCS containing 2% (v/ v) of penicillin and streptomycin. In control experiments, MPs but not contaminating cells were detected in the supernatants. Subsequently, clot forming ability of cells and supernatants (in duplicates) were determined in 96-well-plates (Cell culture cluster, Costar 3595, Corning) by adding pooled platelet poor citrated plasma (75 μL, 4 min, 37 °C), and clotting reaction initiated by CaCl2 (50 μL, 10 mmol/ L final concentration) [27]. Optical density changes (340 nm) were recorded kinetically (every 9th s for 20 min) (Versamax, Molecular Devices). Cell- and supernatant-associated thromboplastin activity was read from a standard curve (1–1000 mU/mL) of human brain thromboplastin (Dade Behring, Liederbach, Germany). Flow cytometry of cells Monocytes (4 × 10 5/well) were incubated with or without glibenclamide in the presence or absence of LPS (1 μg/mL) as above. After 4 h, monocytes were harvested by centrifugation as described for the FXa-generation assay. The supernatants were discarded, and the cells were resuspended in ice cold sterile filtered (0.2 μm) Annexin V Binding buffer (BD Pharmingen) containing 0.5% (w/v) human serum albumin (HSA) (ORHA 21, Dade Behring). In control experiments, the recovered monocytes were counted in a cell-counter (Advia, Bayer) and the number found to be similar at all glibenclamide concentrations. Monocytes (2 × 10 5/well) were labeled with anti-TF mAbs, FITC-labeled Ann V, and 7-AAD and analyzed as previously described [28].
Western blotting Monocytes (7.5 × 10 5/well) from three different donors were incubated with 0, 25 or 100 μmol/L glibenclamide, and then further cultured with or without LPS (1 μg/mL) for 4 h. Next, cells and supernatants were separated by centrifugation as described for the clot forming activity assay. The cells were washed in phosphate buffered saline (PBS) and lysed in Laemmli sample buffer (BIORAD) under reducing conditions. Proteins were analyzed by SDS-PAGE (10% TrisHCl ReadyGel, BIORAD) and electroblotted onto PVDF membranes. The membranes were blocked with 5% (w/v) skimmed milk powder in TBS with 0.05% (v/v) Tween 20 at 4 °C overnight and probed with a mixture of anti-TF primary mAbs TF9-5B7 (2.7 μg/mL) and TF8-5G9 (2.3 μg/mL) and a horseradish peroxidase (HRP)-labeled secondary antibody (Immun-star™ Goat Anti-Mouse (GAM)-HRP conjugate, BIORAD) and finally visualized with enhanced chemiluminescence (Immun-Star™ WesternC™ Chemiluminescent Kit, BIORAD) in a Kodak image station 440. Protein transfer was evaluated by Ponceau staining.
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RT-qPCR
Statistics
Monocytes (1.5 × 10 6 cells/well) from three different donors were incubated with 0, 5, 25 or 100 μmol/L glibenclamide as above in the presence and absence of LPS. After incubation, cells and supernatants were separated by centrifugation (200 g, 10 min, 20 °C), cells lysed in lysis/binding buffer (MagnaPure LC RNA isolation kit High Performance) and stored at − 80 °C until analysis (within one week). Total RNA was extracted (MagNa Pure LC RNA robot, Roche, UK) according to the manufacturer's instructions, quantified on a Nano Drop® spectrophotometer and analyzed on an Agilent BioAnalyzer 2100 System (RNA 6000 Pico assay). RNA integrity number (RIN) and ratio values (28S/18S) were obtained from 16 out of 18 samples. RIN values ranged from 7.1 to 9.4. Fifty ng of total RNA of each sample was reverse transcribed using Omniscript TM (Qiagen Ltd., Crawley, UK). qPCR was performed in triplets (or duplicates in some cases) using the LightCycler (Roche, UK). Primer sequences for each of the three transcripts were: asTF forward primer 5´-CAAGTTCAGGAAAGAAATATTCTACATCAT-3´, asTF reverse primer 5´-AGCTCCAACAGTGCTTCC3´, TF forward primer 5'-GACCGTAGAAGATGAACGGACT-3', TF reverse primer 5'-GGAGGGAATCACTGCTTGAA-3', TNF-α forward primer 5'CCCCAGGGACCTCTCTCTAA-3', TNF-α reverse primer 5'-GAGGTA CAGGCCCTCTGATG-3', Tissue factor pathway inhibitor (TFPI) forward primer 5´-ACTCGACAGTGCGAAGAA-3´, TFPI reverse primer 5´GGCATCCACCATACTTGA-3´. RT-PCR-reactions were controlled for efficiency and DNA contamination (−RT) and the PCR products were sequenced. The relative change in gene expression in glibenclamide-exposed compared to non-glibenclamide exposed monocytes was estimated using the comparative crossing threshold (Ct) method of relative quantification (ΔΔ Ct method) [29]. Gene expression levels for TF, asTF, TNF-α and TFPI in LPS-stimulated monocytes were normalized to endogenous reference genes (mean of GAPDH and β-actin mRNA). In a preliminary time course study TF, asTF and TNF-α mRNA levels were at the maximum level at 4 h, which is in accordance with previous reports [20]. ABCA1 mRNA was measured in LPS-stimulated monocytes pretreated with glibenclamide in increasing concentrations using the ABI 7900 HT (Applied biosystem).
Statistical analysis was performed with SPSS 16.0 for Windows and the MS-Excel add-in Analyse-IT. Univariate analysis of variance (ANOVA) with glibenclamide concentration as covariate and Kruskall Wallis test with pairwise comparisons and Bonferroni correction as well as t-test were performed. P-values less than 0.05 were regarded as statistically significant.
Flow cytometry of microparticles Monocytes (1 × 10 6/well) from three different donors were incubated with 0, 10, 25 or 100 μmol/L glibenclamide and 1 μg/mL LPS as above. Next, monocytes and supernatants were separated by centrifugation as described for the clot forming activity assay. After removal of cells, 250 μL of supernatant was centrifuged (17000 g, 30 min, 20 °C). Subsequently, 225 μL of supernatant was removed, and the remaining 25 μL (MP-enriched pellet) was resuspended in 225 μL of Annexin V Binding buffer and stored at 4 °C over night. A suspension of MPs (100 μL) was incubated 60 min on ice with 10 μL FITC-labeled Ann V, and then 200 μL Annexin V Binding buffer was added before analysis. The samples were processed on a BD FACS Aria cell sorter and analyzed with the BD FACSDiva Software, version 5.0.2 (BD, San Jose, CA). Forward scatter (FSC) and side scatter (SSC) were set at logarithmic gain and 100 000 events were recorded in most cases. Polystyrene microspheres (FluoSpheres, Molecular Probes, Invitrogen) were run to define MPs as particles between 0.1 μM and 1 μM. For each run, MPs were distinguished from instrumental noise by running sterile filtered buffer without MPs. In addition to scatter, MPs were identified by binding of Ann V-FITC. For quantification of MPs, a known number of fluorescent beads (BD Trucount™ Tubes, cat. no. 340334, BD Biosciences, CA) were added to the sample of MPs before analysis. The number of MPs in 1 μL was calculated according to the following formula: (MP events/bead events) × (beads per tube/ sample volume).
Results Glibenclamide reduced procoagulant activity of LPS-stimulated monocytes and their supernatants In accordance with our previous study [22], monocytes incubated in the absence of LPS for 4 h exhibited very low FXa-generating activity, whereas LPS stimulation increased FXa-generating activity between 200 and 700 times. FXa-generating activity of LPS-stimulated monocytes was completely blocked by mAbs against TF (TF9-5B7 and TF8-5G9, IgG1) [22]. The presence of glibenclamide reduced FXagenerating activity of LPS-stimulated monocytes in a concentrationdependent manner up to 100 umol/L (Fig. 1, panel a). Glibenclamide also reduced cell-associated (Fig. 1, panel b) and supernatant-associated clot forming activity (Fig. 1, panel c) in a concentration-dependent manner. In the absence of glibenclamide, we obtained approximately three times more procoagulant activity from the LPS-stimulated cells than from their corresponding supernatants. Cells incubated without LPS did not show any clot forming activity in platelet poor citrated plasma (data not shown). Glibenclamide reduced expression of TF protein of LPS-stimulated monocytes By flow cytometry we found that, upon LPS treatment, approximately half of the monocytes expressed TF (Fig. 2b, upper quadrants), and a small fraction was late apoptotic/necrotic (Ann V positive and 7-AAD positive) (Fig. 2b, red spots). We observed a dose-dependent decrease in TF expression by glibenclamide. At 50, 75 (not shown) and 100 μmol/L of glibenclamide, the fraction of TFpositive cells (upper quadrants) was significantly reduced (p b 0.05, Kruskall Wallis test with pairwise comparisons and Bonferroni correction for multiple comparisons). Glibenclamide did not significantly change the number of Ann V and 7-AAD positive (late apoptotic/necrotic) monocytes (Fig. 2, red spots). There was a trend towards increased PS positivity by glibenclamide above 50 μmol/L, but this increase was not statistically significant (Fig. 2, right quadrants). Interestingly, the mean number of cells positive for both TF and PS (upper right quadrants) was reduced to 49% already at 25 μmol/L glibenclamide. By western blot analysis, we could not detect TF protein in cells after 4 h of incubation in the absence of LPS (Fig. 3, lane 5). LPSstimulation resulted in strong TF protein expression (lane 4), which was not visibly reduced by 25 μmol/L glibenclamide (lane 3). This was in accordance with flow cytometry analysis which showed only 22% reduction in the mean number of TF-positive cells at 25 μmol/L glibenclamide (Fig. 2, compare panel b and panel c, upper quadrants). At 100 μmol/L glibenclamide (Fig. 3, lane 2) there was strong reduction of TF expression. In these experiments, we could not detect asTF in cell lysates using a mix of anti-TF mAbs (TF9-5B7 and TF8-5G9, IgG1). Furthermore, we could not visualize asTF protein using two different polyclonal antibodies directed against the unique C-terminus of asTF (cfr. materials) in pilot experiments (data not shown). However, in those experiments we could visualize recombinant asTF using the same two asTF-specific
a
Monocyte-associated Tenase activity (ng FXa/ 150 000 monocytes)
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Glibenclamide reduced expression of mRNAs for TF, asTF and TNF-α in LPS-stimulated monocytes
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Monocytes incubated for 4 h, in the absence of LPS and glibenclamide, have been shown to express small amounts of mRNA for TNF-α and TF [22], and even less asTF mRNA as quantified by RTqPCR. LPS-stimulation, in the absence of glibenclamide, increased the expression levels of mRNA approximately 270 times for TF, 440 times for asTF and 225 times for TNF-α (mean of three different donors). LPS-stimulation showed a coordinated induction of both full-length TF and asTF mRNA in all donors tested. Full-length TF was constantly expressed at a higher level than asTF (~ 100 fold) with a stable proportion between the two splice variants (data not shown). Glibenclamide dose-dependently decreased mRNA expression of all three transcripts in LPS-stimulated monocytes (Fig. 4, panel a). We also measured the expression of TFPI mRNA in the same donors as in Fig. 4. In accordance with previous reports, we found low expression of TFPI mRNA in unstimulated primary monocytes (0 hours) [30]. Even though TFPI was clearly upregulated by cultivation (with or without LPS for 4 hours), expression was still low. Glibenclamide (100 μmol/L) only slightly downregulated TFPI mRNA expression (2–4 fold) in LPS-stimulated monocytes (data not shown). Analysis of LPS-stimulated monocytes showed the presence of a low level ABCA1 mRNA. Increasing concentrations of glibenclamide appeared to downregulate ABCA1 in all three donors but considerable methodological variation was observed at this low level (data not shown). Glibenclamide may reduce the number of MPs released from LPS-stimulated monocytes
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A representative plot of Ann V-positive MPs released from LPSstimulated monocytes (without glibenclamide) is shown (Fig. 5, panel a). LPS stimulation of monocytes for 4 h resulted in approximately 70% PS positive and 30% PS negative MPs. The percentage of PS positive MPs did not change with glibenclamide treatment (data not shown). There was a trend that the total number of MPs (PS positive and PS negative) released from LPS-stimulated monocytes was reduced by glibenclamide (Fig. 5, panel b), but this change was not statistically significant.
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Glibenclamide (uM) Fig. 1. Effect of glibenclamide on procoagulant activity of LPS-stimulated cells and supernatants. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then LPS was added for 4 h. Next, cells and supernatants were separated by centrifugation and cell- (panels a and b) and supernatant-associated (panel c) procoagulant activity was measured as FXa-generating activity (panel a) or clot forming activity (panels b and c). In all panels single donor data are presented to display donor variation. Squares, circles, diamonds and triangles represent different experiments, using monocytes from six different donors. There was a statistically significant (p b 0.001, one-way anova) dose-dependent reduction of procoagulant activity in all three assays. # indicates p b 0.05 as compared to 0 μmol/L glibenclamide in post hoc analysis (Kruskall Wallis test, pairwise comparisons with Bonferroni correction for multiple comparisons).
antibodies. We also could not detect asTF in the supernatants by immunoprecipitation and western blotting.
The main findings in this work were that glibenclamide dosedependently decreased cell- and supernatant-associated procoagulant activity in LPS-stimulated monocytes, and that the reduction in procoagulant activity coincided with reduced cellular expression of TF and asTF. In addition, the number of released MPs was possibly decreased. Glibenclamide dose-dependently reduced TF activity of cells in two different functional coagulation assays, and this cell-associated reduction of TF activity was evident already at 25 μmol/L of glibenclamide. On the other hand, cell-associated TF antigen seemed to be strongly reduced only at higher concentrations of glibenclamide. Flow cytometry showed only 22% reduction in the mean number of TF-positive cells at 25 μmol/L glibenclamide. This reduction was too small to be visualized by western blotting. TF may be present on the cell surface without being fully functional (encrypted) [31], and one proposed explanation for the restrained TF activity is the asymetric distribution of cell surface PS in the plasma membrane. TF may need PS at the outer leaflet of the membrane to exhibit full activity. We have previously showed that after separation of LPS-stimulated human monocytes by sorting flow cytometry, PS positive monocytes were considerably more procoagulant than PS
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a) Glibenclamide 0 uM+LPS 0 ug/mL
d) Glibenclamide 50 uM+LPS 1 ug/mL
Quadrant distribution (%): Upper Left: TF+, PS-
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b) Glibenclamide 0 uM+LPS 1 ug/mL
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Quadrant distribution (%):
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Quadrant distribution (%):
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34.2 ± 7.2
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c) Glibenclamide 25 uM+LPS 1 ug/mL Upper Left: TF+, PS-
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Fig. 2. Flow cytometry analysis of TF and PS expression and of necrosis of LPS-stimulated monocytes pretreated with glibenclamide. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then further cultivated in the absence and presence of LPS for 4 h. Subsequently, cells were triple-labeled with anti-TF monoclonal antibodies (mAbs), Annexin V (Ann V) and 7-AAD and analyzed by flow cytometry. Dot plot from one representative experiment is shown (Donor V) on the left hand side. TF (PE-labeled anti-TF mAbs) is plotted on the Y-axis and PS (FITC-labeled Ann V) on the X-axis. TF positive cells were defined as events above the 95 percentile of monocytes incubated in the absence of LPS (panel a, upper quadrants). At the right side, corresponding quadrant distributions with percent positive cells in each quadrant are displayed as mean±standard deviation (SD) of five different experiments. Late apoptotic/necrotic cells (Ann V positive, 7-AAD positive) are represented by red spots. The data show a concentration-dependent reduction of TF expression (47.7% to 19.5% positive cells) by glibenclamide (from 0 to 100 μmol/L).
negative monocytes, even though the PS negative monocytes expressed more cell surface TF antigen than the PS positive monocytes [28]. In the present study, we observed by flow cytometry that glibenclamide dose-dependently decreased TF expression of both PS positive monocytes and PS negative monocytes. However, reduction of TF expression was evident at lower glibenclamide concentrations in PS positive than in PS negative cells. Thus, at 25 μmol/L glibenclamide mean reduction of cells positive for both TF and PS was 51%, while there was only a 19% reduction of cells positive for TF but PS negative. The procoagulant activity, therefore, seemed to relate closer to the number of double positive cells (TF and PS) than to the overall TF expression. This is in accord with our recent sorting flow cytometry data [28]. The data support a requirement for close physical proximity between TF and PS for full procoagulant activity. The apparently pivotal role for this small subset of double positive cells in inducing coagulation may also have been accentuated by our experimental setup using platelet-poor plasma. In contrast to Bogdanov et al., who subjected a multiple-tissue cDNA panel to quantitative PCR analysis and found that asTF mRNA was present in resting CD 14 positive monocytes [18], we did not detect asTF mRNA in primary monocytes immediately upon seeding
(“resting” monocytes). In agreement with Bajaj et al., we found that LPS upregulated cellular mRNA expression of both TF and asTF in monocytes [21]. In the present study, we also showed that glibenclamide, in a concentration dependent-manner, reduced expression of asTF mRNA in parallel with TF mRNA in LPS-stimulated monocytes. Recent work showed that asTF protein is present in monocyte-derived human dendritic cells [32]. In pilot experiments we did not detect asTF protein in human monocytes, using antibodies directed against its unique c-terminus. Furthermore, we neither detected asTF protein in the supernatant after immunoprecipitation under the above described conditions. However, further studies will be needed to address whether asTF protein is released from human monocytes, and if it contributes to the supernatant-associated procoagulant activity [33]. We were not able to unravel the molecular mechanisms for the effect of glibenclamide on TF expression in the present work. Glibenclamide may operate through the inhibition of several ABCtransporters. It has been shown to target the ATP-sensitive potassium channels (KATP) and to induce channel closure in pancreatic β-cells [8]. The KATP channel is a hetero-octameric complex with two different protein subunits: an inward rectifying K + channel, Kir6.x,
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Glibenclamide (μM) LPS (μg/mL) TF
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Fig. 3. Western blot of LPS- and glibenclamide-treated monocytes. Monocytes were preincubated with glibenclamide as indicated for 30 minutes and then further cultivated with or without LPS for 4 h. Subsequently, cell-associated TF was analyzed by western blotting. A representative experiment is shown. Human brain thromboplastin (TF) control (lane 1).
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and the sulfonylurea receptor, (SUR) [8]. SUR is a member of the ABC transporter family [8]. More than one isoform exists for both Kir6.x (Kir6.1, Kir6.2) and SUR (SUR1, SUR2a and SUR2b). In accordance with Schmid et al [34], we did not detect SUR 1 or SUR2 mRNA transcripts by RT-qPCR in monocytes incubated in the absence or presence of LPS. Furthermore, mRNA for the Kir6.1 isoform was not present, whereas low expression of Kir6.2 was detected. (data not shown). Schmid et al. showed that Kir subunits, in the absence of SUR subunits, were expressed and functional on human monocytes and suggested that they played an important role for stasis-induced thrombogenesis via the TF pathway [34]. Thus, glibenclamide may possibly reduce TF expression via the Kir6.2 pathway alone, but in our experiments we did not investigate this possibility further since we detected only very low levels of Kir6.2 mRNA. Alternatively, completely different mechanisms such as modulation of arachidonic acid metabolism [35,36], might be involved. It has been reported that MPs play an important role in type 2 diabetes [6,7] and other prothrombotic disorders [37,38]. Satta et al. showed that LPS-stimulation of human monocytes caused release of MPs with membrane associated procoagulant activity [23]. Glibenclamide may also inhibit the ABC-transporter ABCA1 [10], involved in MP shedding [39,40]. The present study showed that glibenclamide dose-dependently reduced procoagulant activity associated with the supernatant of LPS-stimulated monocytes. Furthermore, the reduction in supernatant-associated procoagulant activity seemed to be
Donor XI
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Glibenclamide (µM) Fig. 5. Flow cytometry analysis of microparticles released from LPS-stimulated monocytes. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then LPS was added for 4 h. Subsequently, MPs were isolated from the supernatants, labeled with Annexin V (Ann V) and analyzed by flow cytometry. PS (FITC labeled Ann V) is on the X-axis and Side scatter (SSC) on the Y-axis. The limit between PS positive and PS negative MPs was set between the two populations in a density plot. Panel a shows 70% PS positive MPs in a representative experiment of LPS-stimulated monocytes (without glibenclamide). Panel b shows the effect of glibenclamide on the total number of MPs from LPS-stimulated monocytes. Single donor data are presented to display donor variation. Statistical analysis (1-way Anova) for the effect of glibenclamide concentration: p = 0.3.
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Glibenclamide (μM) Fig. 4. Effect of glibenclamide on TF, asTF, TNF-α mRNA expression in LPS-stimulated monocytes. Monocytes were preincubated with different concentrations of glibenclamide, and then LPS was present for 4 h. Subsequently, cells were harvested, RNA was isolated and mRNAs for TF, asTF, TNF-α, β-actin and GAPDH were quantified by RTqPCR. The relative changes of each transcript were calculated using the comparative crossing threshold (Ct) method of relative quantification (ΔΔ Ct method) [28]. Data from three different donors are presented, using two to three parallels for each concentration. Data are presented as percent expression of non-glibenclamide exposed monocytes (cultured with LPS for 4 hours). Statistical analysis (1-way Anova) for the effect of glibenclamide concentration: TF; p = 0.0003, asTF; p = 0.0015, TNF-α; p = 0.0016. # indicates p b 0.05 as compared to 0 μM glibenclamide in post hoc analysis (t-test).
associated with a decrease in the number of MPs released into the medium. The mechanism for reduced MP-formation under our conditions might be through inhibition of ABCA1, but this needs to be further addressed. Analysis of LPS-stimulated monocytes showed the presence of ABCA1 mRNA transcripts, but at a low level. Increasing concentrations of glibenclamide seemed to downregulate ABCA1 in all three donors tested, but considerable methodological variation was observed at this low level. To our knowledge, the effect of glibenclamide on MP formation in vitro has not yet been studied, and as of today we have found no reported studies on changes in MP number or MP-associated procoagulant activity upon glibenclamide treatment in diabetic patients. In summary, we have demonstrated that glibenclamide decreased monocyte- and supernatant-associated procoagulant activity in a concentration-dependent manner. The decrease in procoagulant activity was associated with reduced expression of TF and asTF, and the number of MPs appeared to be decreased. Future clinical studies are needed to evaluate if glibenclamide also has a favourable effect on the hypercoagulable state in patients with diabetes.
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Conflict of Interest Statement None. References [1] Boden G, Vaidyula VR, Homko C, Cheung P, Rao AK. Circulating tissue factor procoagulant activity and thrombin generation in patients with type 2 diabetes: effects of insulin and glucose. J Clin Endocrinol Metab 2007;92:4352–8. [2] El-Ghoroury EA, El-Din HG. bdel-Kader M, Ragab S. Study of factor VII, tissue factor pathway inhibitor and monocyte tissue factor in noninsulin-dependent diabetes mellitus. Blood Coagul Fibrinolysis 2008;19:7–13. [3] Sambola A, Osende J, Hathcock J, Degen M, Nemerson Y, Fuster V, et al. Role of risk factors in the modulation of tissue factor activity and blood thrombogenicity. Circulation 2003;107:973–7. [4] Bogdanov VY, Osterud B. Cardiovascular complications of diabetes mellitus: The Tissue Factor perspective. Thromb Res 2010;125:112–8. [5] Wahid ST, Marshall SM, Thomas TH. Increased platelet and erythrocyte external cell membrane phosphatidylserine in type 1 diabetes and microalbuminuria. Diabetes Care 2001;24:2001–3. [6] Tan KT, Tayebjee MH, Lim HS, Lip GY. Clinically apparent atherosclerotic disease in diabetes is associated with an increase in platelet microparticle levels. Diabet Med 2005;22:1657–62. [7] Omoto S, Nomura S, Shouzu A, Nishikawa M, Fukuhara S, Iwasaka T. Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus. Diabetologia 2002;45:550–5. [8] Proks P, Reimann F, Green N, Gribble F, Ashcroft F. Sulfonylurea stimulation of insulin secretion. Diabetes 2002;51(Suppl 3):S368–76. [9] Hasko G, Deitch EA, Nemeth ZH, Kuhel DG, Szabo C. Inhibitors of ATP-binding cassette transporters suppress interleukin-12 p40 production and major histocompatibility complex II up-regulation in macrophages. J Pharmacol Exp Ther 2002;301:103–10. [10] Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G. Interleukin-1beta secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1. Blood 1997;90:2911–5. [11] Rapaport SI, Rao LV. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscler Thromb 1992;12:1111–21. [12] Roberts HR, Lozier JN. New perspectives on the coagulation cascade. Hosp Pract (Off Ed) 1992;27:97-12. [13] Monroe DM, Key NS. The tissue factor-factor VIIa complex: procoagulant activity, regulation, and multitasking. J Thromb Haemost 2007;5:1097–105. [14] Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol 2006;26:456–61. [15] Chen VM, Hogg PJ. Allosteric disulfide bonds in thrombosis and thrombolysis. J Thromb Haemost 2006;4:2533–41. [16] Pendurthi UR, Ghosh S, Mandal SK, Rao LV. Tissue factor activation: is disulfide bond switching a regulatory mechanism? Blood 2007;110:3900–8. [17] Freyssinet JM. Cellular microparticles: what are they bad or good for? J Thromb Haemost 2003;1:1655–62. [18] Bogdanov VY, Balasubramanian V, Hathcock J, Vele O, Lieb M, Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat Med 2003;9:458–62. [19] Szotowski B, Antoniak S, Poller W, Schultheiss HP, Rauch U. Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines. Circ Res 2005;96:1233–9. [20] Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989;9:2752–5.
[21] Bajaj MS, Ghosh M, Bajaj SP. Fibronectin-adherent monocytes express tissue factor and tissue factor pathway inhibitor whereas endotoxin-stimulated monocytes primarily express tissue factor: physiologic and pathologic implications. J Thromb Haemost 2007;5:1493–9. [22] Henriksson CE, Klingenberg O, Ovstebo R, Joo GB, Westvik AB, Kierulf P. Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis. Thromb Haemost 2005;94:1236–44. [23] Satta N, Toti F, Feugeas O, Bohbot A, Dachary-Prigent J, Eschwege V, et al. Monocyte vesiculation is a possible mechanism for dissemination of membraneassociated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 1994;153:3245–55. [24] Crutchley DJ, Conanan LB, Que BG. K + channel blockers inhibit tissue factor expression by human monocytic cells. Circ Res 1995;76:16–20. [25] Wharton SA, Riley PA. The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells. Cell Biochem Funct 1986;4:189–95. [26] Lund PK, Joo GB, Westvik AB, Ovstebo R, Kierulf P. Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience. Scand J Clin Lab Invest 2000;60:357–65. [27] Jungi TW. A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity. J Immunol Methods 1990;133: 21–9. [28] Henriksson CE, Hellum M, Landsverk KS, Klingenberg O, Joo GB, Kierulf P. Flow cytometry-sorted non-viable endotoxin-treated human monocytes are strongly procoagulant. Thromb Haemost 2006;96:29–37. [29] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) Method. Methods 2001;25:402–8. [30] van der Logt CP, Dirven RJ, Reitsma PH, Bertina RM. Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacterial lipopolysaccharide and phorbolester. Blood Coagul Fibrinolysis 1994;5:211–20. [31] Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol 2006;26:456–61. [32] Baroni M, Pizzirani C, Pinotti M, Ferrari D, Adinolfi E, Calzavarini S, et al. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J 2007;21:1926–33. [33] Mackman N. Alternatively spliced tissue factor - one cut too many? Thromb Haemost 2007;97:5–8. [34] Schmid D, Staudacher DL, Bueno R, Spieckermann PG, Moeslinger T. ATP-sensitive potassium channels expressed by human monocytes play a role in stasis-induced thrombogenesis via tissue factor pathway. Life Sci 2007;80:989–98. [35] Ozaki Y, Yatomi Y, Kume S. Effects of oral hypoglycaemic agents on platelet functions. Biochem Pharmacol 1992;44:687–91. [36] Satoh K, Ozaki Y, Yatomi Y, Kume S. Effects of sulfonylurea agents on platelet arachidonic acid metabolism; study on platelet homogenates. Biochem Pharmacol 1994;48:1053–5. [37] Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet JM, Tedgui A. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation 1999;99:348–53. [38] Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freyssinet JM, et al. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 2000;101:841–3. [39] Hamon Y, Broccardo C, Chambenoit O, Luciani MF, Toti F, Chaslin S, et al. ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol 2000;2:399–406. [40] Grau GE, Chimini G. Immunopathological consequences of the loss of engulfment genes: the case of ABCA1. J Soc Biol 2005;199:199–206.
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Anticoagulant effects of an antidiabetic drug on monocytes in vitro C.E. Henriksson a,⁎, M. Hellum a, K.B.F. Haug a, H.C. Aass a, G.B. Joø a, R. Øvstebø a, A.M. Trøseid a, O. Klingenberg b, P. Kierulf a a b
Blood cell research group, Section for research, Department of Medical Biochemistry, Oslo University Hospital, Ullevaal, Oslo, Norway Department of Medical Biochemistry, Oslo University Hospital, Rikshospitalet, Oslo, Norway
a r t i c l e
i n f o
Article history: Received 28 May 2010 Received in revised form 1 July 2011 Accepted 7 July 2011 Available online 20 August 2011 Keywords: Tissue Factor Alternatively spliced Tissue Factor Microparticle Glibenclamide Monocyte
a b s t r a c t Introduction: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. Methods: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RTqPCR, western blot) and on cell viability and MP release (flow cytometry). Results: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. Conclusions: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated. © 2011 Elsevier Ltd. All rights reserved.
Introduction Diabetes leads to a hypercoagulable state. Patients with diabetes type 2 demonstrate increased tissue factor (TF) expression and activity [1–4], increased PS expression [5] and elevated levels of microparticles (MPs) in the circulation [6,7]. Sulfonylureas, such as glibenclamide, stimulate insulin secretion from pancreatic β-cells and are widely used to treat diabetes type 2 [8]. Glibenclamide is a general ATP-binding cassette (ABC) transporter inhibitor [9,10]. ABC transporters constitute a large family of proteins translocating various substances across biological membranes. Several ABC transporters are involved in disease pathogenesis. Among them are sulfonylurea receptors (SUR), ABCA1 (Tangier disease protein), multidrug resistance protein (MDR) and cystic fibrosis transmembrane conductance regulator (CFTR) [9]. To what extent inhibition of ABC-transporters may affect the hypercoagulable state in diabetes, remains unsettled.
Abbreviations: MP, Microparticle; TF, Tissue Factor; asTF, alternatively spliced Tissue Factor; LPS, Lipopolysacharride; PS, Phosphatidylserine; RBC, Red blood cells; ABC, ATP-binding cassette; SUR, Sulfonylurea receptor; mAb, monoclonal antibody; Ann V, Annexin V; 7-AAD, 7-amino-actinomycin D; PE, Phycoerythrin; FITC, Fluorescein isothiocyanate; TFPI, Tissue Factor Pathway Inhibitor. ⁎ Corresponding author. Tel.: + 47 22 11 83 56; fax: + 47 22 11 81 89. E-mail address: [email protected] (C.E. Henriksson). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.07.007
TF is the main initiator of the coagulation cascade [11]. Cell surface TF/Factor VIIa (FVIIa) initiates blood coagulation by limited proteolysis and activation of factor IX (FIX) and factor X (FX) (extrinsic tenase activity), which subsequently leads to generation of thrombin [12]. Despite being available at the cell surface, TF may not display full procoagulant activity. This phenomenon is termed encryption [13]. The encrypted form of TF is inactive, while the decrypted form confers the active procoagulant configuration [13]. The molecular mechanism of TF decryption is still obscure [14,15], but it has been associated with loss of cell membrane phospholipid asymmetry [16]. PS is normally sequestered at the inner plasma membrane leaflet, and cell surface relocalisation of PS is associated with TF decryption as well as with release of procoagulant MPs, which are defined as 0.1-1.0 μM shedded membrane vesicles [17]. Furthermore, the protein product of an alternatively spliced form of TF (asTF) is reported to circulate in a soluble form [18]. The asTF protein lacks both the transmembrane and the intracellular TF domains and instead contains a unique C-terminus. It has been suggested that this asTF exhibits phospholipid-dependent procoagulant activity [18,19]. It has previously been shown that lipopolysaccharide (LPS)stimulation of monocytes leads to synthesis of both TF and asTF, and to increased cell surface TF expression [20–22]. Endotoxin also enhances PS exposure at the cell surface [22] and results in release of procoagulant MPs [23]. Glibenclamide has previously been shown to reduce TF activity in an LPS-stimulated monocytic cell line (THP-1
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cells) [24], but the effects of glibenclamide on primary LPS-stimulated monocytes and their released MPs are not known. Using LPSstimulated TF-bearing human monocytes, we investigated the effects of glibenclamide on cell- and supernatant-associated procoagulant activity and related it to TF and asTF expression, cell viability and MP release. Our results showed that there was a concentration-dependent decrease in procoagulant activity in cells and supernatants up to 100 μmol/L glibenclamide. The decrease in procoagulant activity was associated with reduced expression of TF and asTF, and possibly also with a reduced number of MPs. Materials and methods Materials Roswell Park Memorial Institute culture medium (RPMI) 1640 (Gibco, Paisley, Scotland). Fetal calf serum (FCS) [25], heat and acid treated before use, was from BioWhittaker Inc. (Walkersville, MD). These reagents had endotoxin levels b0.25 EU/mL, as measured by a Limulus amoebocyte lysate assay. LPS from Escherichia coli (055:B5) was from BioWhittaker Inc. (Walkersville, MD). In all experiments, LPS was diluted in RPMI 1640 with 5% (v/v) FCS containing 2% (v/v) of penicillin and streptomycin (cat. no. P0906) from Sigma (St. Louis, MO). Glibenclamide (Glyburide) was from Sigma-Aldrich (St. Louis, MO). In all experiments, glibenclamide was diluted in Dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany). Tris buffered saline: 20 mmol/L Tris, 0.15 mol/L NaCl, pH 7.4. Factor X (FX) and Factor Xa (FXa) were from Enzyme Research Laboratories (South Bend, IN). S-2756 was from Chromogenix (Milano, Italy). Factor VIIa (FVIIa) was a kind gift from Professor Ulla Hedner at Novo Nordisk (Bagsvaerd, Denmark). Anti-TF monoclonal antibodies (mAbs) (TF9-5B7 and TF8-5G9, IgG1) were kind gifts from Professor James Morrissey at University of Illinois College of Medicine (Urbana, IL). Anti-asTF polyclonal antibodies and recombinant asTF were generously provided by Professor Vladimir Bogdanov at Mount Sinai school of Medicine (New York, NY) [18]. Anti-asTF polyclonal antibodies were kindly provided by Professor Ursula Rauch at Charite´- Universitätsmedizin (Berlin, Germany) [19]. Phycoerythrin (PE)-conjugated F(ab´)2 Fragment of Rabbit Anti-Mouse immunoglobulins was from DAKO (Glostrup, Denmark). Fluorescein isothiocyanate (FITC)-labeled Annexin V (Ann V) and 7-amino-actinomycin D (7-AAD) were from BD Biosciences Pharmingen (Hamburg, Germany). Monocyte isolation and thawing Full blood cell counting (Sysmex 2100XE) was performed on anonymous samples from healthy blood donors (upon written consent) at the Blood Bank, Oslo University Hospital, Ullevaal, Norway, and peripheral blood mononuclear cells (PBMC) were isolated from EDTA whole blood (450 mL) by density gradient centrifugation. Monocytes were purified from PBMC by elutriation centrifugation to a purity of N90% (flow cytometry), and cryopreserved in RPMI 1640 with 25% (v/v) FCS and 10% (v/v) DMSO at −150 °C essentially as previously described by Lund et al [26]. In all experiments, monocytes were thawed, washed once in RPMI 1640 with 20% (v/v) FCS, resuspended and cultivated in RPMI 1640 with 5% (v/v) FCS containing 2% (v/v) of penicillin and streptomycin. Monocytes used in the experiments came from different donors (I-XI). FXa-generating activity (extrinsic tenase activity) of cells Monocytes (4× 105/well) from three different donors were incubated (30 minutes, 37 °C) in 24-well plates (Ultra Low Attachment plates, Costar 3473, Corning) with 0, 5, 10, 25, 50, 75 or 100 μmol/L glibenclamide before LPS (1 μg/mL) was added for 4 h. Then, monocytes
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were centrifuged (300 g, 7 min, 4 °C), supernatants discarded, and the monocytes were resuspended in 1000 μL Tris buffered saline. Again, monocytes were centrifuged (300 g, 7 min, 4 °C), supernatants discarded, and the monocytes resuspended in 80 μL Tris-buffered saline with 0.5% (w/v) BSA (A-4503, Sigma). Recovery of monocytes was similar at all glibenclamide concentrations (microscopy counting). The ability of 1.5 × 105 resuspended cells/well (duplicates) to promote activation of FX to FXa, detected by a chromogenic FXa substrate (FXageneration) was assayed as previously described [22,23]. In control experiments, cells or LPS were omitted. Clot forming activity of cells and supernatants in citrated plasma Monocytes (1 × 10 6 cells/well) from three different donors were incubated with glibenclamide and LPS as above in a total volume of 1 mL. Next, monocytes were centrifuged (4300 g, 5 min, 20 °C), supernatants (950 μL of each well) collected and the monocytes resuspended in 950 μL RPMI 1640 with 5% (v/v) FCS containing 2% (v/ v) of penicillin and streptomycin. In control experiments, MPs but not contaminating cells were detected in the supernatants. Subsequently, clot forming ability of cells and supernatants (in duplicates) were determined in 96-well-plates (Cell culture cluster, Costar 3595, Corning) by adding pooled platelet poor citrated plasma (75 μL, 4 min, 37 °C), and clotting reaction initiated by CaCl2 (50 μL, 10 mmol/ L final concentration) [27]. Optical density changes (340 nm) were recorded kinetically (every 9th s for 20 min) (Versamax, Molecular Devices). Cell- and supernatant-associated thromboplastin activity was read from a standard curve (1–1000 mU/mL) of human brain thromboplastin (Dade Behring, Liederbach, Germany). Flow cytometry of cells Monocytes (4 × 10 5/well) were incubated with or without glibenclamide in the presence or absence of LPS (1 μg/mL) as above. After 4 h, monocytes were harvested by centrifugation as described for the FXa-generation assay. The supernatants were discarded, and the cells were resuspended in ice cold sterile filtered (0.2 μm) Annexin V Binding buffer (BD Pharmingen) containing 0.5% (w/v) human serum albumin (HSA) (ORHA 21, Dade Behring). In control experiments, the recovered monocytes were counted in a cell-counter (Advia, Bayer) and the number found to be similar at all glibenclamide concentrations. Monocytes (2 × 10 5/well) were labeled with anti-TF mAbs, FITC-labeled Ann V, and 7-AAD and analyzed as previously described [28].
Western blotting Monocytes (7.5 × 10 5/well) from three different donors were incubated with 0, 25 or 100 μmol/L glibenclamide, and then further cultured with or without LPS (1 μg/mL) for 4 h. Next, cells and supernatants were separated by centrifugation as described for the clot forming activity assay. The cells were washed in phosphate buffered saline (PBS) and lysed in Laemmli sample buffer (BIORAD) under reducing conditions. Proteins were analyzed by SDS-PAGE (10% TrisHCl ReadyGel, BIORAD) and electroblotted onto PVDF membranes. The membranes were blocked with 5% (w/v) skimmed milk powder in TBS with 0.05% (v/v) Tween 20 at 4 °C overnight and probed with a mixture of anti-TF primary mAbs TF9-5B7 (2.7 μg/mL) and TF8-5G9 (2.3 μg/mL) and a horseradish peroxidase (HRP)-labeled secondary antibody (Immun-star™ Goat Anti-Mouse (GAM)-HRP conjugate, BIORAD) and finally visualized with enhanced chemiluminescence (Immun-Star™ WesternC™ Chemiluminescent Kit, BIORAD) in a Kodak image station 440. Protein transfer was evaluated by Ponceau staining.
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RT-qPCR
Statistics
Monocytes (1.5 × 10 6 cells/well) from three different donors were incubated with 0, 5, 25 or 100 μmol/L glibenclamide as above in the presence and absence of LPS. After incubation, cells and supernatants were separated by centrifugation (200 g, 10 min, 20 °C), cells lysed in lysis/binding buffer (MagnaPure LC RNA isolation kit High Performance) and stored at − 80 °C until analysis (within one week). Total RNA was extracted (MagNa Pure LC RNA robot, Roche, UK) according to the manufacturer's instructions, quantified on a Nano Drop® spectrophotometer and analyzed on an Agilent BioAnalyzer 2100 System (RNA 6000 Pico assay). RNA integrity number (RIN) and ratio values (28S/18S) were obtained from 16 out of 18 samples. RIN values ranged from 7.1 to 9.4. Fifty ng of total RNA of each sample was reverse transcribed using Omniscript TM (Qiagen Ltd., Crawley, UK). qPCR was performed in triplets (or duplicates in some cases) using the LightCycler (Roche, UK). Primer sequences for each of the three transcripts were: asTF forward primer 5´-CAAGTTCAGGAAAGAAATATTCTACATCAT-3´, asTF reverse primer 5´-AGCTCCAACAGTGCTTCC3´, TF forward primer 5'-GACCGTAGAAGATGAACGGACT-3', TF reverse primer 5'-GGAGGGAATCACTGCTTGAA-3', TNF-α forward primer 5'CCCCAGGGACCTCTCTCTAA-3', TNF-α reverse primer 5'-GAGGTA CAGGCCCTCTGATG-3', Tissue factor pathway inhibitor (TFPI) forward primer 5´-ACTCGACAGTGCGAAGAA-3´, TFPI reverse primer 5´GGCATCCACCATACTTGA-3´. RT-PCR-reactions were controlled for efficiency and DNA contamination (−RT) and the PCR products were sequenced. The relative change in gene expression in glibenclamide-exposed compared to non-glibenclamide exposed monocytes was estimated using the comparative crossing threshold (Ct) method of relative quantification (ΔΔ Ct method) [29]. Gene expression levels for TF, asTF, TNF-α and TFPI in LPS-stimulated monocytes were normalized to endogenous reference genes (mean of GAPDH and β-actin mRNA). In a preliminary time course study TF, asTF and TNF-α mRNA levels were at the maximum level at 4 h, which is in accordance with previous reports [20]. ABCA1 mRNA was measured in LPS-stimulated monocytes pretreated with glibenclamide in increasing concentrations using the ABI 7900 HT (Applied biosystem).
Statistical analysis was performed with SPSS 16.0 for Windows and the MS-Excel add-in Analyse-IT. Univariate analysis of variance (ANOVA) with glibenclamide concentration as covariate and Kruskall Wallis test with pairwise comparisons and Bonferroni correction as well as t-test were performed. P-values less than 0.05 were regarded as statistically significant.
Flow cytometry of microparticles Monocytes (1 × 10 6/well) from three different donors were incubated with 0, 10, 25 or 100 μmol/L glibenclamide and 1 μg/mL LPS as above. Next, monocytes and supernatants were separated by centrifugation as described for the clot forming activity assay. After removal of cells, 250 μL of supernatant was centrifuged (17000 g, 30 min, 20 °C). Subsequently, 225 μL of supernatant was removed, and the remaining 25 μL (MP-enriched pellet) was resuspended in 225 μL of Annexin V Binding buffer and stored at 4 °C over night. A suspension of MPs (100 μL) was incubated 60 min on ice with 10 μL FITC-labeled Ann V, and then 200 μL Annexin V Binding buffer was added before analysis. The samples were processed on a BD FACS Aria cell sorter and analyzed with the BD FACSDiva Software, version 5.0.2 (BD, San Jose, CA). Forward scatter (FSC) and side scatter (SSC) were set at logarithmic gain and 100 000 events were recorded in most cases. Polystyrene microspheres (FluoSpheres, Molecular Probes, Invitrogen) were run to define MPs as particles between 0.1 μM and 1 μM. For each run, MPs were distinguished from instrumental noise by running sterile filtered buffer without MPs. In addition to scatter, MPs were identified by binding of Ann V-FITC. For quantification of MPs, a known number of fluorescent beads (BD Trucount™ Tubes, cat. no. 340334, BD Biosciences, CA) were added to the sample of MPs before analysis. The number of MPs in 1 μL was calculated according to the following formula: (MP events/bead events) × (beads per tube/ sample volume).
Results Glibenclamide reduced procoagulant activity of LPS-stimulated monocytes and their supernatants In accordance with our previous study [22], monocytes incubated in the absence of LPS for 4 h exhibited very low FXa-generating activity, whereas LPS stimulation increased FXa-generating activity between 200 and 700 times. FXa-generating activity of LPS-stimulated monocytes was completely blocked by mAbs against TF (TF9-5B7 and TF8-5G9, IgG1) [22]. The presence of glibenclamide reduced FXagenerating activity of LPS-stimulated monocytes in a concentrationdependent manner up to 100 umol/L (Fig. 1, panel a). Glibenclamide also reduced cell-associated (Fig. 1, panel b) and supernatant-associated clot forming activity (Fig. 1, panel c) in a concentration-dependent manner. In the absence of glibenclamide, we obtained approximately three times more procoagulant activity from the LPS-stimulated cells than from their corresponding supernatants. Cells incubated without LPS did not show any clot forming activity in platelet poor citrated plasma (data not shown). Glibenclamide reduced expression of TF protein of LPS-stimulated monocytes By flow cytometry we found that, upon LPS treatment, approximately half of the monocytes expressed TF (Fig. 2b, upper quadrants), and a small fraction was late apoptotic/necrotic (Ann V positive and 7-AAD positive) (Fig. 2b, red spots). We observed a dose-dependent decrease in TF expression by glibenclamide. At 50, 75 (not shown) and 100 μmol/L of glibenclamide, the fraction of TFpositive cells (upper quadrants) was significantly reduced (p b 0.05, Kruskall Wallis test with pairwise comparisons and Bonferroni correction for multiple comparisons). Glibenclamide did not significantly change the number of Ann V and 7-AAD positive (late apoptotic/necrotic) monocytes (Fig. 2, red spots). There was a trend towards increased PS positivity by glibenclamide above 50 μmol/L, but this increase was not statistically significant (Fig. 2, right quadrants). Interestingly, the mean number of cells positive for both TF and PS (upper right quadrants) was reduced to 49% already at 25 μmol/L glibenclamide. By western blot analysis, we could not detect TF protein in cells after 4 h of incubation in the absence of LPS (Fig. 3, lane 5). LPSstimulation resulted in strong TF protein expression (lane 4), which was not visibly reduced by 25 μmol/L glibenclamide (lane 3). This was in accordance with flow cytometry analysis which showed only 22% reduction in the mean number of TF-positive cells at 25 μmol/L glibenclamide (Fig. 2, compare panel b and panel c, upper quadrants). At 100 μmol/L glibenclamide (Fig. 3, lane 2) there was strong reduction of TF expression. In these experiments, we could not detect asTF in cell lysates using a mix of anti-TF mAbs (TF9-5B7 and TF8-5G9, IgG1). Furthermore, we could not visualize asTF protein using two different polyclonal antibodies directed against the unique C-terminus of asTF (cfr. materials) in pilot experiments (data not shown). However, in those experiments we could visualize recombinant asTF using the same two asTF-specific
a
Monocyte-associated Tenase activity (ng FXa/ 150 000 monocytes)
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Glibenclamide reduced expression of mRNAs for TF, asTF and TNF-α in LPS-stimulated monocytes
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Monocytes incubated for 4 h, in the absence of LPS and glibenclamide, have been shown to express small amounts of mRNA for TNF-α and TF [22], and even less asTF mRNA as quantified by RTqPCR. LPS-stimulation, in the absence of glibenclamide, increased the expression levels of mRNA approximately 270 times for TF, 440 times for asTF and 225 times for TNF-α (mean of three different donors). LPS-stimulation showed a coordinated induction of both full-length TF and asTF mRNA in all donors tested. Full-length TF was constantly expressed at a higher level than asTF (~ 100 fold) with a stable proportion between the two splice variants (data not shown). Glibenclamide dose-dependently decreased mRNA expression of all three transcripts in LPS-stimulated monocytes (Fig. 4, panel a). We also measured the expression of TFPI mRNA in the same donors as in Fig. 4. In accordance with previous reports, we found low expression of TFPI mRNA in unstimulated primary monocytes (0 hours) [30]. Even though TFPI was clearly upregulated by cultivation (with or without LPS for 4 hours), expression was still low. Glibenclamide (100 μmol/L) only slightly downregulated TFPI mRNA expression (2–4 fold) in LPS-stimulated monocytes (data not shown). Analysis of LPS-stimulated monocytes showed the presence of a low level ABCA1 mRNA. Increasing concentrations of glibenclamide appeared to downregulate ABCA1 in all three donors but considerable methodological variation was observed at this low level (data not shown). Glibenclamide may reduce the number of MPs released from LPS-stimulated monocytes
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A representative plot of Ann V-positive MPs released from LPSstimulated monocytes (without glibenclamide) is shown (Fig. 5, panel a). LPS stimulation of monocytes for 4 h resulted in approximately 70% PS positive and 30% PS negative MPs. The percentage of PS positive MPs did not change with glibenclamide treatment (data not shown). There was a trend that the total number of MPs (PS positive and PS negative) released from LPS-stimulated monocytes was reduced by glibenclamide (Fig. 5, panel b), but this change was not statistically significant.
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Glibenclamide (uM) Fig. 1. Effect of glibenclamide on procoagulant activity of LPS-stimulated cells and supernatants. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then LPS was added for 4 h. Next, cells and supernatants were separated by centrifugation and cell- (panels a and b) and supernatant-associated (panel c) procoagulant activity was measured as FXa-generating activity (panel a) or clot forming activity (panels b and c). In all panels single donor data are presented to display donor variation. Squares, circles, diamonds and triangles represent different experiments, using monocytes from six different donors. There was a statistically significant (p b 0.001, one-way anova) dose-dependent reduction of procoagulant activity in all three assays. # indicates p b 0.05 as compared to 0 μmol/L glibenclamide in post hoc analysis (Kruskall Wallis test, pairwise comparisons with Bonferroni correction for multiple comparisons).
antibodies. We also could not detect asTF in the supernatants by immunoprecipitation and western blotting.
The main findings in this work were that glibenclamide dosedependently decreased cell- and supernatant-associated procoagulant activity in LPS-stimulated monocytes, and that the reduction in procoagulant activity coincided with reduced cellular expression of TF and asTF. In addition, the number of released MPs was possibly decreased. Glibenclamide dose-dependently reduced TF activity of cells in two different functional coagulation assays, and this cell-associated reduction of TF activity was evident already at 25 μmol/L of glibenclamide. On the other hand, cell-associated TF antigen seemed to be strongly reduced only at higher concentrations of glibenclamide. Flow cytometry showed only 22% reduction in the mean number of TF-positive cells at 25 μmol/L glibenclamide. This reduction was too small to be visualized by western blotting. TF may be present on the cell surface without being fully functional (encrypted) [31], and one proposed explanation for the restrained TF activity is the asymetric distribution of cell surface PS in the plasma membrane. TF may need PS at the outer leaflet of the membrane to exhibit full activity. We have previously showed that after separation of LPS-stimulated human monocytes by sorting flow cytometry, PS positive monocytes were considerably more procoagulant than PS
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a) Glibenclamide 0 uM+LPS 0 ug/mL
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Fig. 2. Flow cytometry analysis of TF and PS expression and of necrosis of LPS-stimulated monocytes pretreated with glibenclamide. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then further cultivated in the absence and presence of LPS for 4 h. Subsequently, cells were triple-labeled with anti-TF monoclonal antibodies (mAbs), Annexin V (Ann V) and 7-AAD and analyzed by flow cytometry. Dot plot from one representative experiment is shown (Donor V) on the left hand side. TF (PE-labeled anti-TF mAbs) is plotted on the Y-axis and PS (FITC-labeled Ann V) on the X-axis. TF positive cells were defined as events above the 95 percentile of monocytes incubated in the absence of LPS (panel a, upper quadrants). At the right side, corresponding quadrant distributions with percent positive cells in each quadrant are displayed as mean±standard deviation (SD) of five different experiments. Late apoptotic/necrotic cells (Ann V positive, 7-AAD positive) are represented by red spots. The data show a concentration-dependent reduction of TF expression (47.7% to 19.5% positive cells) by glibenclamide (from 0 to 100 μmol/L).
negative monocytes, even though the PS negative monocytes expressed more cell surface TF antigen than the PS positive monocytes [28]. In the present study, we observed by flow cytometry that glibenclamide dose-dependently decreased TF expression of both PS positive monocytes and PS negative monocytes. However, reduction of TF expression was evident at lower glibenclamide concentrations in PS positive than in PS negative cells. Thus, at 25 μmol/L glibenclamide mean reduction of cells positive for both TF and PS was 51%, while there was only a 19% reduction of cells positive for TF but PS negative. The procoagulant activity, therefore, seemed to relate closer to the number of double positive cells (TF and PS) than to the overall TF expression. This is in accord with our recent sorting flow cytometry data [28]. The data support a requirement for close physical proximity between TF and PS for full procoagulant activity. The apparently pivotal role for this small subset of double positive cells in inducing coagulation may also have been accentuated by our experimental setup using platelet-poor plasma. In contrast to Bogdanov et al., who subjected a multiple-tissue cDNA panel to quantitative PCR analysis and found that asTF mRNA was present in resting CD 14 positive monocytes [18], we did not detect asTF mRNA in primary monocytes immediately upon seeding
(“resting” monocytes). In agreement with Bajaj et al., we found that LPS upregulated cellular mRNA expression of both TF and asTF in monocytes [21]. In the present study, we also showed that glibenclamide, in a concentration dependent-manner, reduced expression of asTF mRNA in parallel with TF mRNA in LPS-stimulated monocytes. Recent work showed that asTF protein is present in monocyte-derived human dendritic cells [32]. In pilot experiments we did not detect asTF protein in human monocytes, using antibodies directed against its unique c-terminus. Furthermore, we neither detected asTF protein in the supernatant after immunoprecipitation under the above described conditions. However, further studies will be needed to address whether asTF protein is released from human monocytes, and if it contributes to the supernatant-associated procoagulant activity [33]. We were not able to unravel the molecular mechanisms for the effect of glibenclamide on TF expression in the present work. Glibenclamide may operate through the inhibition of several ABCtransporters. It has been shown to target the ATP-sensitive potassium channels (KATP) and to induce channel closure in pancreatic β-cells [8]. The KATP channel is a hetero-octameric complex with two different protein subunits: an inward rectifying K + channel, Kir6.x,
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Fig. 3. Western blot of LPS- and glibenclamide-treated monocytes. Monocytes were preincubated with glibenclamide as indicated for 30 minutes and then further cultivated with or without LPS for 4 h. Subsequently, cell-associated TF was analyzed by western blotting. A representative experiment is shown. Human brain thromboplastin (TF) control (lane 1).
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and the sulfonylurea receptor, (SUR) [8]. SUR is a member of the ABC transporter family [8]. More than one isoform exists for both Kir6.x (Kir6.1, Kir6.2) and SUR (SUR1, SUR2a and SUR2b). In accordance with Schmid et al [34], we did not detect SUR 1 or SUR2 mRNA transcripts by RT-qPCR in monocytes incubated in the absence or presence of LPS. Furthermore, mRNA for the Kir6.1 isoform was not present, whereas low expression of Kir6.2 was detected. (data not shown). Schmid et al. showed that Kir subunits, in the absence of SUR subunits, were expressed and functional on human monocytes and suggested that they played an important role for stasis-induced thrombogenesis via the TF pathway [34]. Thus, glibenclamide may possibly reduce TF expression via the Kir6.2 pathway alone, but in our experiments we did not investigate this possibility further since we detected only very low levels of Kir6.2 mRNA. Alternatively, completely different mechanisms such as modulation of arachidonic acid metabolism [35,36], might be involved. It has been reported that MPs play an important role in type 2 diabetes [6,7] and other prothrombotic disorders [37,38]. Satta et al. showed that LPS-stimulation of human monocytes caused release of MPs with membrane associated procoagulant activity [23]. Glibenclamide may also inhibit the ABC-transporter ABCA1 [10], involved in MP shedding [39,40]. The present study showed that glibenclamide dose-dependently reduced procoagulant activity associated with the supernatant of LPS-stimulated monocytes. Furthermore, the reduction in supernatant-associated procoagulant activity seemed to be
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Glibenclamide (µM) Fig. 5. Flow cytometry analysis of microparticles released from LPS-stimulated monocytes. Monocytes were preincubated with different concentrations of glibenclamide for 30 minutes and then LPS was added for 4 h. Subsequently, MPs were isolated from the supernatants, labeled with Annexin V (Ann V) and analyzed by flow cytometry. PS (FITC labeled Ann V) is on the X-axis and Side scatter (SSC) on the Y-axis. The limit between PS positive and PS negative MPs was set between the two populations in a density plot. Panel a shows 70% PS positive MPs in a representative experiment of LPS-stimulated monocytes (without glibenclamide). Panel b shows the effect of glibenclamide on the total number of MPs from LPS-stimulated monocytes. Single donor data are presented to display donor variation. Statistical analysis (1-way Anova) for the effect of glibenclamide concentration: p = 0.3.
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Glibenclamide (μM) Fig. 4. Effect of glibenclamide on TF, asTF, TNF-α mRNA expression in LPS-stimulated monocytes. Monocytes were preincubated with different concentrations of glibenclamide, and then LPS was present for 4 h. Subsequently, cells were harvested, RNA was isolated and mRNAs for TF, asTF, TNF-α, β-actin and GAPDH were quantified by RTqPCR. The relative changes of each transcript were calculated using the comparative crossing threshold (Ct) method of relative quantification (ΔΔ Ct method) [28]. Data from three different donors are presented, using two to three parallels for each concentration. Data are presented as percent expression of non-glibenclamide exposed monocytes (cultured with LPS for 4 hours). Statistical analysis (1-way Anova) for the effect of glibenclamide concentration: TF; p = 0.0003, asTF; p = 0.0015, TNF-α; p = 0.0016. # indicates p b 0.05 as compared to 0 μM glibenclamide in post hoc analysis (t-test).
associated with a decrease in the number of MPs released into the medium. The mechanism for reduced MP-formation under our conditions might be through inhibition of ABCA1, but this needs to be further addressed. Analysis of LPS-stimulated monocytes showed the presence of ABCA1 mRNA transcripts, but at a low level. Increasing concentrations of glibenclamide seemed to downregulate ABCA1 in all three donors tested, but considerable methodological variation was observed at this low level. To our knowledge, the effect of glibenclamide on MP formation in vitro has not yet been studied, and as of today we have found no reported studies on changes in MP number or MP-associated procoagulant activity upon glibenclamide treatment in diabetic patients. In summary, we have demonstrated that glibenclamide decreased monocyte- and supernatant-associated procoagulant activity in a concentration-dependent manner. The decrease in procoagulant activity was associated with reduced expression of TF and asTF, and the number of MPs appeared to be decreased. Future clinical studies are needed to evaluate if glibenclamide also has a favourable effect on the hypercoagulable state in patients with diabetes.
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