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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 554-559
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratory for Experimental Internal Medicine and
Department of Intensive Care, Academic Medical Center, University of
Amsterdam, and Organon Teknika, Boxtel, The Netherlands.
Triggering of the tissue factor (TF)-dependent coagulation pathway
is considered to underlie the generation of a procoagulant state during
endotoxemia. To determine the in vivo pattern of monocytic TF messenger
RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were
injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected
before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS
administration. Total blood RNA was isolated and amplified by NASBA
(nucleic acid sequence-based amplification), followed by quantitation
of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the
pattern of coagulation activation with the kinetics of monocytic TF
mRNA expression, we measured plasma levels of markers of thrombin
generation, thrombin-antithrombin (TAT) complexes, and prothrombin
fragment 1 + 2 (F1 + 2). Baseline value (mean ± SEM) of the
number of TF mRNA molecules per monocytic cell was 0.08 ± 0.02. A
progressive and significant (P < .0001) increase in TF
expression was observed after LPS injection (+0.5 hour:
0.3 ± 0.1, +1 hour: 1.3 ± 0.9, +2 hours: 4.1 ± 0.9),
peaking at +3 hours (10 ± 1.9 TF mRNA molecules per monocyte). As
TF mRNA levels increased, thrombin generation was augmented. Peak
levels of TAT and F1 + 2 were reached later (at t +4 hours) than
those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to
baseline after 24 hours. In conclusion, we used a NASBA/ECL-based
technique to quantify TF mRNA in whole blood during human endotoxemia
and observed a 125-fold increase in TF mRNA levels. Our data
demonstrate a pivotal role for enhanced TF gene activity in the
activation of coagulation after LPS challenge.
(Blood. 2000;96:554-559)
Tissue factor (TF) is a membrane-bound glycoprotein
that is considered to be the main initiator of the coagulation cascade by acting as a cofactor of activated factor VII.1 In fact, factor VIIa itself bears limited procoagulant activity, but in complex
with TF, factor VIIa is capable of proteolytic activation of factors IX
and X, which leads to thrombin formation and conversion of fibrinogen
into fibrin. It is assumed that TF is not normally expressed on cells
in direct contact with blood, but TF expression may become expressed on
intravascular cells (mainly monocytes and endothelial cells) by the
action of inflammatory stimuli, including lipopolysaccharide (LPS,
endotoxin).1 This augmented TF expression is thought to be
responsible for the thrombotic manifestations of various inflammatory
states.1
Endotoxemia triggered by intravenous injection of Escherichia
coli LPS into humans is a powerful model to investigate the pattern
of inflammatory responses and hemostatic changes that occur during
gram-negative sepsis.2-6 Human endotoxemia is associated with a well-documented state of cytokine activation, transient activation of fibrinolysis, and sustained activation of coagulation, resulting in a net procoagulant state.5,6 There is good
evidence for a role of the extrinsic TF-dependent coagulation pathway
in eliciting the procoagulant state during endotoxemia and
gram-negative sepsis. In particular, the use of antibodies directed
against TF or factor VII/VIIa resulted in attenuation of the activation of coagulation in models of endotoxemia and septicemia in
primates.7-13
Based largely on in vitro data, both monocytes and endothelial cells
are assumed to be the sites of induced intravascular TF expression, but
direct experimental evidence for enhanced TF gene activity in these
cells in humans is lacking. Endothelial cells are not accessible for TF
quantitation. In addition, though some groups have reported increased
TF antigen expression in whole blood,14-17 flow cytometric
analysis of circulating leukocytes has been negative as reported by
others.18 The latter finding contrasts with the relative
ease with which it is possible to follow TF expression on human
endothelial cells and on leukocytes in ex vivo
experiments.19-27 The reason(s) for this discrepancy are
poorly understood, but may be related to plasmatic factors that obscure
TF epitopes in vivo. To help resolve this issue, we have looked for
alternative assessment of TF induction during endotoxemia. We used a
sensitive method to accurately quantify TF messenger RNA (mRNA)
expression by blood leukocytes, based on the amplification of RNA by a
nucleic acid sequence-based amplification (NASBA) system28
and precise mRNA quantitation by an electrochemiluminescence (ECL)
assay.29 We used this methodology to determine the kinetics of TF mRNA expression in healthy volunteers after exposure to intravenous endotoxin. Additionally, we measured plasma levels of
markers of thrombin generation to directly relate the pattern of
coagulation activation with the kinetics of monocytic TF mRNA expression during human endotoxemia.
Patients
Methods
Nucleic acid isolation.
Total nucleic acids were isolated from whole blood according
to a solid-phase extraction method described by Boom et
al.30 Briefly, 100 µL whole blood was mixed with 900 µL
lysis buffer (50 mmol/L Tris-HCl [pH 6.4], 20 mmol/L EDTA, 1.3%
[wt/vol] Triton X-100, 5.25 mol/L guanidine thiocyanate). For
quantification purposes, in vitro synthesized internal calibrator RNA
(Q-RNA) was designed to be identical to the wild-type with only a small
region of the sequence replaced with a sequence enabling specific
detection of this RNA. Twenty microliters of a Q-RNA solution (prepared by dissolving a freeze-dried Q-RNA sphere in 220 µL elution buffer) was added to each tube containing lysed whole blood. Specifically, 2 × 104 molecules of TF Q-RNA were present in the
20 µL solution that was added to the sample. Next, 50 µL of
activated silica suspension (1 g/mL) was added to the lysis mixture.
After washing and drying the silica, nucleic acid was eluted with 50 µL elution buffer and stored at Nucleic acid sequence-based amplification.
NASBA reactions were carried out according to Kievits et
al31 with modifications. Briefly, 5 µL of nucleic acid
solution was added to 10 µL NASBA mixture. The final concentrations
in 20 µL reaction mixture were 40 mmol/L Tris-HCl, pH 8.5, 12 mmol/L MgCl2, 70 mmol/L KCl, 15% vol/vol DMSO, 1 mmol/L each
dNTP, 2 mmol/L each NTP, 0.2 µmol/L of each TF primers (TF-1 and
TF-2), and 2 × 104 molecules of TF Q-RNA. TF
oligonucleotide primers were designed for specific amplification of a
213-nucleotide long fragment of TF mRNA. The TF primer sequences were
TF-1:
5'-AATTCTAATACGACTCACTATAGGGAGAGGGCTGTCTGTACTCTTCGGTTTA-3' (T7 promoter part underlined) and TF-2:
5'-GAAGGAACAACACTTTCCTA-3' (positions 788-765 [TF-1] and
575-594 [TF-2] of TF mRNA sequence, GenBank accession number M16553).
The reactions were incubated for 5 minutes at 65°C to destabilize
secondary structures in the RNA and then for 5 minutes at 41°C
(primer annealing temperature). Subsequently, 5 µL of the NASBA
enzyme solution (1.28 U/µL AMV-reverse transcriptase
[Seikagagu America, Ijamsville, MD], 0.016 U/µL RNAse H
[Pharmacia], 6.4 U/µL T7-RNA polymerase [Amersham Pharmacia Biotech, Rosendaal, Netherlands] and 0.43 g/L bovine serum albumin (BSA) [Roche Diagnostics, Almere, Netherlands]) was added to each reaction tube to initiate amplification, and reactions were incubated for 90 minutes at 41°C. Hence, isothermal nucleic acid
amplification was accomplished by the simultaneous activity of the 3 enzymes. To check for possible contamination, we included a tube
containing water instead of nucleic acid solution in each set of
amplification reactions. NASBA products were visualized on a 2%
electrophoresis agarose gel containing ethidium bromide. To exclude the
possibility of nonspecific amplification in the initial experiments,
separation of amplified RNA on an agarose gel, followed by blotting
onto a filter and subsequent hybridization with a labeled
oligonucleotide probe was also carried out. These experiments confirmed
the presence of specific amplification products for TF mRNA. NASBA
reactions were stored at Electrochemiluminescence assay.
The ECL method has been previously adapted for the detection of
amplified nucleic acids using ECL-labeled oligonucleotides in sandwich
hybridization assays.29,32 Amplified RNA was detected using
a 1-step probe hybridization method, followed by detection and
quantitation in an ECL reader, which automatically separates free from
bound label. The subsequent detection of bound probes uses ECL. The
computer linked to the instrument directly calculates results. In our
experiments, fresh separate mixtures of TF capture probe with TF
wild-type detection probe (1:1 ratio) and TF capture probe with TF
Q-ECL detection probe, specific for the internal calibrator RNA, (1:1
ratio) were prepared. Oligonucleotide sequences of the probes were TF
capture probe: 5'-[Biotine] GCCTCCGGGATGTTTTTGGCAAGGA-3' (positions 596-620 in mRNA sequence, GenBank accession number M16553),
TF wild-type detection probe: 5'-[ECL label]
GTTCAGGAAAGAAAACAGCCA-3' (positions 656-676 in mRNA sequence,
GenBank accession number M16553), and TF Q detection probe:
5'-[ECL label] AAGTAAAGTCGACAAGCACAG-3' (replaced
sequence at the position of the TF wild-type detection probe). Five
microliters NASBA reaction (1:5 diluted) was added to 20 µL probe
mixture (10 µL capture and 10 µL detection probe; the 2 probe
combinations in separate tubes) in ECL tubes. Samples were incubated 15 minutes at 60°C, and tubes were mixed by vortexing every 5 minutes.
Three hundred microliters assay buffer (100 mmol/L tripropylamine, pH
7.5, Organon Teknika, Boxtel, The Netherlands) was then added to tube
reactions and ECL counts were read in an Origen 1.5 ECL Analyzer
(Organon Teknika). During ECL detection, we included a water control
for each probe combination tested; these ECL signals were used as
background levels for the other reactions analyzed with the same probe
combinations. The quantitation of mRNA levels is based on the ECL
signals and the amount of Q-RNA spiked per reaction. Specific
activities of the wild-type (WT) and Q-ECL probes are known. WT ECL and
Q-ECL signals are measured over a range of WT RNA input at a fixed
Q-RNA concentration. Therefore, the ratios of WT and Q-NASBA
amplificates could be determined from the signal ratios of the
respective probes and the initial amount of WT RNA calculated. All ECL
signals were corrected for the background before performing the
quantitation. Final quantitation of TF mRNA was obtained according to
the following formula: log TF = 0.88 × (log TF WT ECL Markers of coagulation activation.
Blood was drawn from the antecubital vein and collected in tubes
containing buffered 3.2% citrate solution. Activation of coagulation
was assessed by plasma measurements of markers for thrombin formation,
TAT complexes (µg/L) and prothrombin F1 + 2 (nmol/L), with specific
enzyme-linked immunosorbent assays (ELISAs) (Behringwercke AG, Marburg, Germany).
Leukocyte responses.
Global and differential white cell counts were performed by flow
cytometry in blood samples collected in tubes containing EDTA. Monocyte
counts were used to correct the absolute log values of TF obtained in
the ECL assay (described below).
In vitro whole blood stimulation with lipopolysaccharide.
We performed in vitro assays involving whole blood stimulation with
different concentrations of LPS, followed by both measurement of TF
mRNA levels by NASBA/ECL technique and of TF antigen expression on
monocytes by flow cytometry (FACscan analysis, described later). Blood
samples obtained from several volunteers were collected into tubes
containing 100 µL (500 IE) endotoxin-free heparin per 10 mL whole
blood. LPS preparations were shaken continuously for 30 minutes before
the addition to whole blood samples. Samples were diluted 1:1 with
HBSS, and whole blood stimulation was performed in the absence or
presence of LPS at 37°C and 5% CO2 for 4 hours. The
following concentrations of LPS were added to whole blood obtained from
2 volunteers: 0, 0.01, 0.1, 1, 10, and 100 ng/mL.
FACScan analysis.
Fifty microliters cell suspension at a concentration of 107
cells/mL was incubated with 50 µL primary antibody (concentration 10 µg/mL) for 60 minutes at 4°C. The mouse monoclonal IgG1 antihuman TF antibodies used were 5G9 (kindly donated by Dr T. S. Edgington, The Scripps Research Institute, La Jolla, CA), 4509 (American Diagnostica Inc, Greenwich, CT), and TFE
(Kordia/Enzyme Research Laboratories Inc, Leiden, The Netherlands).
Thereafter, cells were washed with ice-cold PBS containing 0.1% BSA
(wt/vol). Subsequently, a secondary RPE-conjugated rabbit antimouse
antibody was diluted in PBS containing 1% BSA and the cells were
incubated for 60 minutes at 4°C. After washing, cells were
resuspended in PBS containing 1% BSA and analyzed using a FACscan
(Becton Dickinson, Mountain View, CA). The monocytes were gated by
their specific forward and side-scatter pattern. Furthermore, the
monocyte population was identified by high CD14 expression. A total of
20 000 events was recorded for each file. After subtracting control
IgG1 mean fluorescence, specific antibody binding was expressed as mean fluorescence intensity (MFI).
Tissue factor antigen levels.
Circulating TF antigen was measured using a commercially available
assay at time points Statistical analysis
TF mRNA (NASBA/ECL) and TF antigen (FACscan) analysis in LPS-stimulated whole blood cells In an initial in vitro experiment, we examined whether TF mRNA levels reflect TF protein expression on the cell membrane. This indeed appeared to be the case. LPS concentrations between 0.01 and 100 ng/mL were used. A dose-dependent increase, as shown in Figure 1, was found for both flow cytometric antigen levels (using 3 different antibodies) and TF mRNA levels. The increase is particularly impressive for the mRNA levels. Log TF levels (log molecules per milliliter) were 3.7 at baseline and were raised to 6.2, ie, 316-fold increased, after 4 hours incubation with 100 ng LPS. Figure 2 shows a linear plot of TF mRNA levels against MFI as detected with monoclonal antibody 4509. The relationship between antigen and mRNA levels appears to be linear up to a dose of 10 ng/mL LPS. This is well within the range of LPS levels attainable in human volunteer studies, in which 4 ng/kg is administered intravenously.
Clinical and hematologic parameters in human endotoxemia Administration of LPS was associated with a transient rise in body temperature, peaking after 3 hours (38.3 ± 0.5°C, P < .05). All subjects experienced flu-like symptoms, such as headache, nausea, and myalgia. LPS injection also induced a biphasic change in leukocyte counts, involving early neutropenia, followed by neutrophilia, monocytopenia, and lymphopenia (Table 1).
TF expression in human endotoxemia TF antigen levels on monocytes (measured by FACs analysis with the monoclonal antibody 4509) tended to increase in 8 of the 10 subjects (data not shown). However, in accordance with our previous findings,18 it was not possible to establish a significant increase of TF levels after LPS infusion, because of the high interindividual variation in TF surface antigen levels and the differences in time at which peak levels were reached.
Coagulation activation markers in human endotoxemia
We have successfully used a NASBA-based method for RNA
amplification, followed by an ECL-based detection system to investigate the kinetics of TF mRNA expression in vivo, in a model of human endotoxemia induced by IV injection of LPS. We observed a maximum 125-fold increase of TF mRNA levels in monocytes that was directly related to activation of the coagulation system. The combined NASBA/ECL
technology has been previously used for accurate quantitation of viral
copies in blood of HIV-infected patients.30,31 Our data
confirm the usefulness of these techniques for mRNA measurements and
extend their application to the field of hemostasis. Indeed, this
methodology may have numerous applications to accurately quantify TF
mRNA expression in clinical situations in which TF is known to play a
role, including gram-negative septicemia, atherosclerosis, autoimmune
diseases, adult respiratory distress syndrome, and cancer.
We are grateful to Angelique Groot for her help with TF antigen assays,
and to Michel de Baar and Suzanne Jurriaans (Department of Human
Retrovirology, Academic Medical Center, Amsterdam) for helpful
discussions regarding the ECL experiments.
Submitted August 16, 1999; accepted March 9, 2000.
Supported by a FAPESP Grant (98/02821) (R.F.F.); by the E. Dekker
program of the Dutch Heart Foundation (H.t.C. and C.A.S.); and by a
grant from the Dutch Organization for Scientific Research (J.J.T.).
T.v.d.P. is a fellow of the Royal Dutch Academy of Arts and Sciences.
Reprints: Pieter H. Reitsma, Laboratory for Experimental
Internal Medicine, G2-135, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; e-mail: p.h.reitsma{at}amc.uva.nl.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
70°C. This
material was used as input in the amplification reactions.
.05 was considered statistically significant.
