© 1995 American Heart Association, Inc.
Articles |
K+ Channel Blockers Inhibit Tissue Factor Expression by Human Monocytic Cells
From the Miami Heart Research Institute, Miami Beach, Fla.
Correspondence to David J. Crutchley, PhD, Miami Heart Research Institute, 4701 Meridian Ave, Miami Beach, FL 33140.
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Abstract |
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Abstract Human monocytes express the important procoagulant protein, tissue factor (TF), after stimulation by a variety of agents, including bacterial lipopolysaccharide (LPS). Monocyte TF expression may contribute to intravascular coagulation in a number of disease states. The present studies show that monocytic cell TF expression can be inhibited by several agents known to block cellular K+ channels. Exposure of human peripheral blood to 100 ng/mL LPS for 2 hours led to pronounced TF procoagulant activity associated with the mononuclear cell fraction. This was inhibited by 4-aminopyridine (2 mmol/L), tetraethylammonium chloride (10 mmol/L), and apamin (1 µmol/L). In contrast, charybdotoxin (100 nmol/L) was inactive. More detailed studies were carried out in cultured human monocytic tumor THP-1 cells. These cells exhibited low but detectable levels of TF mRNA, measured by reverse transcription and polymerase chain reaction; cell surface procoagulant activity, measured by a plasma clotting assay; and cell homogenate TF antigen, measured by immunoassay. Exposure of THP-1 cells to 1 µg/mL LPS led to threefold to fivefold increases in all three parameters. Basal and LPS-induced levels of all three parameters were reduced in a dose-dependent manner by 4-aminopyridine (I50, 1 mmol/L) and tetraethylammonium chloride (I50, 20 mmol/L) but not by apamin or charybdotoxin. Expression of TF activity was also inhibited by glibenclamide, an inhibitor of ATP-dependent K+ channels (I50, 25 µmol/L). These results suggest that facilitation of TF synthesis may be an important role for K+ channels in monocytes.
Key Words: thromboplastin • blood coagulation • ion channels
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exposure of human monocytes to agents such as bacterial lipopolysaccharide (LPS), complement components, immune complexes, and various cytokines results in the expression of tissue factor (TF), a membrane-associated glycoprotein that binds with high affinity to factor VII/VIIa. The resulting TF/factor VIIa complex activates factors IX and X, leading to thrombin generation and blood clot formation (reviewed in Reference 11 ). Increased monocyte TF expression has been reported in a wide variety of disorders, including carcinoma,2 3 allograft rejection,4 crescentic glomerular nephritis,5 bacterial infection,6 and postoperative thrombosis7 8 9 and may contribute to the intravascular coagulation that is frequently observed with these conditions.
TF expression by monocytes is thought to be the result of gene transcription and/or mRNA stabilization,10 11 leading to protein synthesis and insertion of the mature protein into the plasma membrane. The signal transduction mechanisms and ionic requirements for these processes, however, are currently unclear. In the present study, we provide evidence that TF expression in human monocytic cells is inhibited by agents that block cellular K+ channels, suggesting that maintenance of these ion channels in a functional state may be a prerequisite for monocyte TF synthesis to occur.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Human monocytic tumor THP-1 cells were obtained from the American Type Culture Collection. Antibiotics and materials for the preparation of cell culture media were obtained from GIBCO. Fetal bovine serum was obtained from Hyclone. Tetraethylammonium chloride (TEA) and 4-aminopyridine were obtained from Aldrich. Apamin and glibenclamide were obtained from Sigma Chemical Co. Charybdotoxin was obtained from Bachem Bioscience. Ficoll-Hypaque was obtained from Pharmacia LKB. Bacterial endotoxin (lipopolysaccharide B, Escherichia coli 0111:B4) was obtained from Difco. DNA polymerase from Thermus aquaticus (Taq polymerase) was obtained from Perkin Elmer Cetus. Primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were obtained from Clontech. TF primers were prepared by Oligos, Etc. Recombinant Moloney murine leukemia virus reverse transcriptase was obtained from GIBCO BRL. Rabbit brain thromboplastin was obtained from Ortho. Enzyme immunoassay kits for human TF and human plasma deficient in factor VII were obtained from American Diagnostica.
Cell Treatment
For whole-blood studies, blood was obtained from three male and
two female volunteers who denied taking medication for at least 2 weeks
before donation. Informed consent was obtained in accordance with
Institutional Review Board requirements. Blood was drawn by forearm
venipuncture into sterile evacuated tubes containing lithium heparin.
The contents of the tubes contained <10 pg/mL LPS, as determined by a
sensitive chromogenic assay (Walker Bioproducts). Samples of blood (2
mL) were incubated for 15 minutes with K+ channel blockers
or saline vehicle and then incubated further for 2 hours with 100 ng/mL
LPS. Incubations were carried out at 37°C in sealed tubes, with
gentle mixing. Blood was then layered over 2 mL of Ficoll-Hypaque and
centrifuged at 250 g for 30 minutes. The mononuclear cell
band, consisting of 
45% monocytes and 55% lymphocytes, was
harvested, and cells were washed with Puck's saline A solution,
resuspended in Tris-saline buffer (100 mmol/L NaCl and 50 mmol/L
Tris [pH 7.4]), and assayed immediately for procoagulant activity.
For THP-1 cell studies, cells were grown in suspension culture by using RPMI 1640 medium supplemented with 100 µg/mL streptomycin, 100 U/mL penicillin, 10% heat-inactivated fetal bovine serum, and 10 mmol/L HEPES, pH 7.4. Cells (106/mL) were incubated for 15 minutes with growth medium containing K+ channel blockers or saline vehicle. Dimethyl sulfoxide (0.1% [wt/vol]) was the vehicle for glibenclamide. LPS was then added to a final concentration of 1 µg/mL, and cells were further incubated for 2 hours (mRNA measurement) or 4 hours (antigen and activity measurement). After incubation, cells were harvested by centrifugation at 250g for 10 minutes and washed with Puck's saline A solution. Cells were then resuspended in Tris-saline buffer and assayed immediately for procoagulant activity or resuspended in denaturing solution D12 for RNA extraction.
TF Assays
Procoagulant activity on the surface of intact cells was
determined by a single-stage clotting assay. Samples of cell suspension
were mixed with 25 mmol/L CaCl2, and clotting was initiated
by the addition of citrated normal human plasma. The time for clotting
to occur at 37°C was recorded by using a fibrometer, and procoagulant
activity was quantified by reference to a rabbit brain thromboplastin
standard. Procoagulant activity of both peripheral blood mononuclear
cells and THP-1 cells was due to TF, since it was (1) specifically
inhibited by HTF1-7B8, a blocking monoclonal antibody to human
TF,13 and (2) not expressed in plasma deficient in factor
VII. None of the K+ channel blockers affected the
procoagulant activity of cell suspensions or standard thromboplastin
when added at the assay stage.
For TF antigen assays, THP-1 cells were disrupted by brief sonication on ice and extracted with buffer (0.1 mol/L NaCl and 50 mmol/L Tris [pH 7.4]) containing 0.1% Triton X-100 for 18 hours at 4°C. Cell debris was pelleted by brief centrifugation in a microfuge, and TF in extracts was measured by using a commercial enzyme immunoassay kit (Imubind, American Diagnostica) according to the manufacturer's instructions. This kit uses a murine monoclonal antibody for antigen capture and a biotinylated rabbit polyclonal antibody for detection. None of the K+ channel blockers directly affected the immunoassay.
Reverse Transcription/Polymerase Chain Reaction of mRNA
Total RNA was prepared from THP-1 cells by the method of
Chomczynski and Sacchi.12 One to 10 µg of RNA was
reverse-transcribed with recombinant Moloney murine leukemia virus
reverse transcriptase in a reaction mixture containing 20 mmol/L Tris
(pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl, 100 µg/mL
bovine serum albumin, 0.5 mmol/L deoxyribonucleoside triphosphates, and
10 U placental RNase inhibitor. Reaction mixtures were incubated for 90
minutes at 42°C, heated to 95°C for 5 minutes, and then quickly
chilled on ice. One tenth of the reaction mixture was used for
polymerase chain reaction amplification14 15 by using the
following primers specific for TF: 5'-end primer,
5'-CTCGGACAGCCAACAATTCAGAGT-3'; 3'-end primer,
5'-TGTTCGGGAGGGAATCACTGCTTGAACACT-3'. For control purposes, primers for
the "housekeeping gene" G3PDH (5'-end primer,
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; 3'-end primer, 5'-CATG-
TGGGCCATGAGGTCCACCAC-3') were used in a side-by-side amplification with
the target gene for TF. Polymerase chain reaction was performed at a
final concentration of 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 50
µmol/L deoxyribonucleoside triphosphates, 0.1 µmol/L each of 5' and
3' primer, and 1 U of Taq polymerase in a total volume of 50
µL. The mixture was overlayered with mineral oil and then amplified
with the Perkin-Elmer Cetus thermal cycler. The amplification profile
consisted of denaturation at 95°C for 1 minute, primer-annealing at
58°C for 1 minute, and primer extension at 72°C for 2 minutes in a
20- to 50-cycle reaction. Five to 10 µL of each reaction mixture was
electrophoresed in 1.5% agarose gel or 8% polyacrylamide gels in
Tris/borate/EDTA buffer. Gels were stained with 0.5 µg/mL ethidium
bromide and photographed. Negative films were scanned with a laser
densitometer (Ultroscan XL, equipped with GelScan software, Pharmacia).
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Results |
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We first explored the effects of K+ channel blockers on TF expression by human peripheral blood monocytes. Experiments were performed in whole blood, rather than with purified monocytes, to approximate conditions in vivo and avoid inadvertent cell activation during purification. Mononuclear cells isolated from blood in the absence of LPS expressed negligible procoagulant activity (2±1 U/106 cells, mean±SEM of results from five donors), and this was unaffected by any of the agents tested. In contrast, cells isolated from blood incubated with LPS showed marked TF activity (55±7 U/106 cells), and this was strongly inhibited when a number of agents known to block K+ channels were added to the blood. These included 4-aminopyridine (2 mmol/L), TEA (10 mmol/L), and apamin (1 µmol/L), the bee venom peptide. In contrast, charybdotoxin (100 nmol/L), the scorpion venom peptide, was inactive (Table
). None of the agents appeared to affect cell
viability at the concentrations used, nor did they directly affect the
procoagulant activity of cells or standard thromboplastin when added at
the assay stage.
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TF activity associated with the mononuclear cell preparation can reasonably be attributed to monocytes, since they are the only blood cell type thought to express such activity.16 However, monocyte TF expression can be amplified by at least two other cell types present in blood: T lymphocytes17 18 19 20 and platelets.21 22 To investigate whether the K+ channel blockers affected monocytes directly or indirectly via inhibition of cellular cooperation, experiments were repeated with human monocytic THP-1 cells.23
Exposure of these cells to 1 µg/mL LPS for 4 hours increased cell
surface–associated TF activity to levels three to five times higher
than those observed with untreated cells. 4-Aminopyridine and TEA
inhibited both LPS-induced and basal TF activity in a dose-dependent
manner; I50 values were 1 mmol/L for 4-aminopyridine and
20 mmol/L for TEA (Fig 1). In addition, TF expression
was inhibited in a dose-dependent manner by glibenclamide
(I50, 25 µmol/L), a sulfonylurea thought to selectively
inhibit ATP-dependent K+ channels.24 In
contrast to results obtained with the whole-blood experiments, apamin
(10 nmol/L to 1 µmol/L) had no effect on TF activity in THP-1 cells,
and charybdotoxin (100 nmol/L) was again inactive (not shown). None of
these agents impaired the ability of THP-1 cells to exclude trypan
blue, suggesting that they did not exert their effects via general
cytotoxicity.
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In an attempt to define the mechanism of action of K+
channel blockers, we compared changes in cellular procoagulant activity
with those in TF antigen and mRNA. The LPS-induced increases in
procoagulant activity, measured on the surface of intact cells, were
accompanied by comparable increases in TF antigen, measured in cellular
homogenates, and both were inhibited to the same extent by
4-aminopyridine and TEA (Fig 2). Thus, the inhibition of
TF activity could be entirely accounted for by a reduction in TF
protein.
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Steady state levels of TF mRNA were measured by means of reverse
transcription/polymerase chain reaction.14 15 Primers were
selected according to nucleotides 8496 (exon 4) through 9372 (exon 5)
of the TF gene sequenced by Mackman et al25 to exclude
contamination with genomic DNA. The use of these primers resulted in a
270-bp fragment, corresponding to amino acids 140 to 229 in the mature
TF protein. As shown in Fig 3, exposure of THP-1 cells
to 1 µg/mL LPS for 2 hours led to an approximately threefold increase
in the 270-bp fragment corresponding to TF mRNA, with no appreciable
changes in levels of a 983-bp fragment corresponding to mRNA for the
"housekeeping gene" G3PDH. Both basal and LPS-stimulated levels
of TF mRNA were clearly diminished by 4-aminopyridine and TEA at the
same concentrations required to inhibit TF activity and antigen
expression. The effects of the K+ channel blockers appeared
to be specific, since G3PDH mRNA levels were unchanged.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies show for the first time that TF expression in human peripheral blood mononuclear cells and monocytic THP-1 cells can be inhibited by agents known to block K+ channels. The agents did not appear to specifically target the induction of TF by LPS, since basal TF expression in THP-1 cells was also inhibited. Basal TF activity in peripheral blood monocytes was too low to permit the effects of K+ channel blockers to be accurately assessed. In the more detailed studies conducted on THP-1 cells, the decreased procoagulant activity observed after treatment with K+ channel antagonists was accompanied by comparable declines in the levels of TF mRNA and protein. The K+ channel antagonists therefore appear to act at an early stage in TF synthesis by inhibiting gene transcription and/or by destabilizing mRNA. Alternatively, they may act to inhibit the activation of an as-yet-unidentified second-messenger system. A possible candidate is intracellular Ca2+; recent studies have suggested that Ca2+ may mediate TF expression in rat smooth muscle cells,26 and activation of K+ channels, leading to membrane hyperpolarization, has been suggested to facilitate Ca2+ mobilization.27 28 A possible interaction between K+ and Ca2+ in relation to TF expression would seem to warrant investigation.
K+ channels appear to be ubiquitous in eukaryotic cells and may be divided into several distinct classes on the basis of gating mechanism, conductance, specificity, and sensitivity to blocking agents (for review, see Reference 2929 ). In addition, a given cell type may possess several types of K+ channels. Human peripheral blood monocytes, monocyte-derived macrophages, and monocytic tumor U937 cells appear to possess at least four types of K+ channels, including a voltage-dependent inwardly rectifying channel, two types of Ca2+-activated channels, and at least one inactivating outward channel (reviewed in Reference 3030 ). The latter channel is thought to be absent in freshly isolated peripheral blood monocytes but may be induced by exposure to LPS.27 All four classes of channels show varying sensitivity to blockade by TEA and 4-aminopyridine at the concentrations used in the present study,31 and the Ca2+-activated channels may in addition be susceptible to block by the specific venom peptides, charybdotoxin and apamin.31 32 Although the precise nature of the K+ channel(s) involved in TF expression by THP-1 cells is not known, the ineffectiveness of charybdotoxin would seem to argue against a role for Ca2+-activated channels, whereas the activity of glibenclamide might suggest that ATP-dependent K+ channels are involved.24 Such channels have been well characterized in cardiac cells, pancreatic ß cells, skeletal muscle, and smooth muscle, where they are readily blocked by micromolar concentrations of glibenclamide.
The anomalous results obtained with apamin deserve further comment. Thus, apamin at 1 µmol/L effectively suppressed monocyte TF expression in the whole-blood preparation but was inactive against THP-1 cells. In contrast, 4-aminopyridine and TEA inhibited monocyte TF expression with equal effectiveness in whole blood and THP-1 cells. Apamin, an octapeptide found in bee venom, has been shown to specifically block low-conductance Ca2+-activated K+ channels in human T and B lymphocytes.30 32 Therefore, it is possible that apamin inhibition of TF expression in the whole-blood preparation may have been exerted indirectly, via inhibition of lymphocyte cooperation. Alternatively, the data may simply indicate that freshly harvested peripheral blood monocytes possess apamin-sensitive K+ channels, whereas the transformed THP-1 cells do not.
Although the electrophysiology of K+ channels has been
studied in great detail, less is known of their function, especially in
cells of monocyte-macrophage lineage.30 In human
monocytes, K+ channels are thought to contribute to the
maintenance of resting membrane potential and the hyperpolarization
response.33 34 Changes in K+ channel
expression during monocyte adherence, maturation, or activation have
also been reported,27 35 36 but their significance is
unclear. In mouse macrophages, K+ channel antagonists
inhibit LPS-induced secretion of the cytokine, tumor necrosis
factor-
.37 Our results provide the first pharmacologic
evidence that facilitation of TF synthesis may also be an important
function of these ion channels in monocytic cells.
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Acknowledgments |
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This study was supported in part by grants from the Walter G. Ross Foundation and Arthur F. Adams Foundation. We wish to thank Andy Toledo and Franco Arias for their excellent technical assistance. We would also like to thank Dr Steven Carson of the University of Nebraska Medical Center, Omaha, for his gift of monoclonal antibody to human TF.
Received August 5, 1994; accepted September 16, 1994.
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