Clinical Chemistry 48, No. 10, 2002
diagnostic tool for MI, thus allowing its implementation
in routine clinical practice where cardiac troponin tests
are used for both purposes.
This work was partially supported by First Medical
(Mountain View, CA). The study design and data analysis
were not influenced by First Medical. Drs. Apple and
Collinson have served as consultants to First Medical.
References
1. Ottani F, Galvani M, Nicolini FA, Ferrini D, Pozzati A, Di Pasquele G, et al.
Elevated cardiac troponin levels predict the risk of adverse outcome in
patients with acute coronary syndromes. Am Heart J 2000;140:917–27.
2. Heidenreich PA, Alloggiamento T, Melsop K, McDonald KM, Go AS, Tllatky
MA. The prognostic value of troponin in patients with non-ST elevation acute
coronary syndromes: a meta-analysis. J Am Coll Cardiol 2001;38:478 – 85.
3. Lindahl B, Diderholm E, Lagerqvist B, Venge P, Wallentin L. Mechanisms
behind the prognostic value of troponin T in unstable coronary artery
disease: a FRISC II substudy. J Am Coll Cardiol 2001;38:979 – 86.
4. Antman EM. Troponin measurements in ischemic heart disease: more than
just a black and white picture. J Am Coll Cardiol 2001;38:1497– 8.
5. Apple FS, Wu AHB. Myocardial infarction redefined: role of cardiac troponin
testing. Clin Chem 2001;47:377–9.
6. Apple FS. Cardiac troponin assays: analytical issues and clinical reference
range cutpoints. Cardiovasc Toxicol 2001;1:93– 8.
7. Apple FS, Wu AHB, Jaffe AS. European Society of Cardiology and American
College of Cardiology guidelines for redefinition of myocardial infarction: how
to use existing assays clinically and for clinical trials. Am Heart J 2002;in
press.
8. Jaffe AS, Ravkilde J, Roberts R, Naslund V, Apple FS, Galvani M, et al. It’s
time for a change to a troponin standard. Circulation 2000;102:1216 –20.
9. Alpert JS, Thygesen K. Myocardial infarction redefined—a consensus document of the Joint European Society of Cardiology/American College of
Cardiology Committee for the redefinition of myocardial infarction. J Am Coll
Cardiol 2000;21:959 – 69.
10. Wu AMB, Apple FS, Gibler WB, Jesse RL, Warshaw MM, Valdes R Jr. National
Academy of Clinical Biochemistry standards of laboratory practice: recommendations for the use of cardiac markers in coronary artery disease. Clin
Chem 1999;45:1104 –21.
11. Hamm CW, Goldmann BU, Heeschen C, Kregmann G, Berger J, Meinertz T.
Emergency room triage of patients with acute chest pain by means of a rapid
testing for cardiac troponin T or troponin I. N Engl J Med 1997;337:1648 –
53.
12. Apple FS, Anderson FP, Collinson P, Jesse RL, Kontos MC, Levitt MA, et al.
Clinical evaluation of the First Medical whole blood, point of cure testing
device for detection of myocardial infarction. Clin Chem 2000;46:1604 –9.
13. National Committee for Clinical Laboratory Standards. How to define and
determine reference intervals in the clinical laboratory; approved guideline—
2nd edition, Vol. 20, No. 13. Protocol C29 –A2. Wayne, PA: NCCLS, 2000.
14. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457– 81.
15. Newby LK, Christenson RH, Ohman EM, Armstrong PW, Thompson TD, Lee
KL, et al. Value of serial troponin T measures for early and late risk
stratification in patients with acute coronary syndromes. Circulation 1998;
98:1853–9.
16. Newby LK, Storrow AB, Gibler WB, Garvey JL, Tucker JF, Kaplan AL, et al.
Bedside multimarker testing for risk stratification in chest pain units: the
chest pain evaluation by creatine kinase-MB, myoglobin, and troponin I
(CHECKMATE) study. Circulation 2001;103:1832–7.
17. Heeschen C, Deu A, Langenbrink L, Goldmann BU, Hamm CW. Analytical and
diagnostic performance of troponin assays in patients suspicious acute
coronary syndromes. Clin Biochem 2000;33:359 – 68.
18. Venge P, Lagerqvist B, Diderholm E, Lindahl B, Wallentin L. Clinical
performance of three cardiac troponin assays in patients with unstable
coronary artery disease (a FRISC II Substudy). Am J Cardiol 2002;89:1035–
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19. Morrow DA, Cannon CP, Rifai N, Frey MJ, Vican R, Lakkis N, et al. Ability
of minor elevations of troponins I and T to predict benefit from an early
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myocardial infarction: results from a randomized trial. JAMA 2001;286:
2405–12.
1787
Validation of a Recombinant DNA Construct (␮LCR
and Full-Length ␤-Globin Gene) for Quantification of
Human ␤-Globin Expression: Application to Mutations
in the Promoter, Intronic, and 5ⴕ- and 3ⴕ-Untranslated
Regions of the Human ␤-Globin Gene, Leonid M. Irenge,1
Michel Heusterspreute,1 Marianne Philippe,2 Isabelle Derclaye,1 Annie Robert,3 and Jean-Luc Gala1,4* (1 Applied Molecular Technologies, Center for Human Genetics, Université Catholique de Louvain, Clos-Chapelle-aux-Champs,
30-UCL/30.46, B-1200 Bruxelles, Belgium; 2 Department
of Biochemistry, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Avenue Hippocrate, 30,
B-1200 Bruxelles, Belgium; 3 Biostatistics and Epidemiology, Clos-Chapelle-aux-Champs, 30-UCL/30.34, Université Catholique de Louvain, B-1200 Bruxelles, Belgium;
4
Applied Molecular Technologies, Queen Astrid Military
Hospital, Rue Bruyn, 2, B-1120 Bruxelles, Belgium; * address correspondence to this author at: Applied Molecular
Technologies, Center for Human Genetics, Clos-Chapelleaux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium;
fax 32-2-764-3959, e-mail gala@lbcm.ucl.ac.be)
␤-Thalassemia is characterized by the reduced production
of ␤-globin chains as a result of mutations in the ␤-globin
gene (1 ). This reduction is predictable when mutations
occur in the coding sequence, but not when they occur in
the 5⬘- and 3⬘-untranslated regions (UTRs), the locus
control region (LCR), the promoter, or the introns.
Whether such mutations are involved in the reduction of
the ␤-globin chain production or are simple polymorphisms cannot always be inferred from clinical data.
Transient transfection studies with a ␤-globin promoter
and an heterologous reporter gene have shown that
promoter mutations can decrease transcription (2 ) and are
then associated with the ␤-thalassemia phenotype, as
illustrated by the ⫺30T3 A mutation (3 ). However, such
studies have often failed to provide clear-cut data regarding the transcriptional effect of a mutation or a deletion
occurring in a noncoding sequence (4 ), and quantitative
data are lacking.
To bypass these limitations and to mimic as closely as
possible the regulatory mechanisms of ␤-human globin
gene expression in vivo, we created a construct (pBLG), in
which the entire human ␤-globin gene was cloned behind
the ␤-␮LCR. Whereas previous assays used constructs
bearing HS2 as a single LCR enhancer element (5, 6 ), we
used the entire ␤-␮LCR because it has been shown that
the other three HS elements play also a key role in
␤-globin transcription (7–11 ).
Nucleotides changes in various untranscribed or untranslated parts of the ␤-globin gene representing thalassemic mutations or deletions were introduced in the
construct. All the mutations assessed in our study were
found in members of proband families presenting with
␤-thalassemia or were created by directed mutagenesis. In
addition to the wild type, variant pBLG constructs carrying the following mutations were generated: ⫺101C3 T,
⫹20C3 T, IVS-I-108T3 C, and IVS-I-110G3 A mutations
(12–16 ); ⫹10 (-T), ⫹40343, and ⫹156531577 deletions
1788
Technical Briefs
(17–19 ); and two novel mutations (⫺223T3 C, and
⫺42C3 G). The ⫺30T3 A thalassemic mutation (3 ) was
included as a control. The constructs were expressed in
stably transfected mouse erythroleukemia (MEL) cells,
and the amount of human ␤-globin mRNA was measured
in total RNA extracted from transfected MEL cells grown
for 72 h in the presence of 5 mmol/L hexamethylene
bisacetamide, a chemical inducer of erythroid differentiation (20 ).
Quantification was performed by competitive reverse
transcription-PCR, using synthetic calibrator obtained by
directed mutagenesis and in vitro transcription. Reverse
transcription and coamplification of mRNA extracted
from transfected MEL cells and calibrator RNA were
performed in the same tubes with various ratios of target
to calibrator templates during cDNA synthesis and increasing numbers of PCR cycles during the exponential
phase of amplification. After separation on an agarose gel,
both DNA bands were photographed and quantified by
image analysis software. Target mRNA copy number was
calculated based on the number of copies generated in the
exponential phase of the PCR.
Data collected were analyzed with the Generalized
Linear Model program of SPSS Win 10.0TM (SPSS Inc.).
For the wild-type and mutated or deleted constructs, the
copy number of human ␤-globin mRNA per nanogram of
total RNA from MEL cells was submitted to ANOVA with
three trial factors (21 ) to assess the variability across PCR
cycles (the first trial factor with three PCR cycles as
levels), across the amount of calibrator RNA (the second
trial factor with six levels), and across transfections (the
third trial factor with three experiments as levels).
Results are reported as the grand mean ⫾ SD across the
levels of each trial factor, with the P value of the F-test.
The CV (%) was used to express the variability within
each trial factor of each construct. The reliability coefficient of the transfection factor is reported as the overall
interassay reproducibility. Contrasts between mean values of ␤-globin mRNA copy numbers were performed
with the Scheffé method (21 ). (Details on the materials
and methods used are available in a supplemental file
accompanying the online version of this Technical Brief at
http://www.clinchem.org/content/vol48/issue10/).
Human ␤-globin cDNA was consistently amplified
from the wild-type construct (Fig. 1), as confirmed by
sequence analysis the amplicon. Murine hemoglobin in
different MEL cells cultured with hexamethylene bisacetamide as well as quantification of murine glyceraldehyde
3-phosphate dehydrogenase in total RNA were highly
reproducible (see supplemental file for additional results).
Quantitative data obtained with different constructs are
shown in Table 1. Although expression of the ⫺223T3 C
construct did not differ from that of the wild type, mild to
markedly reduced expression was observed with the
other constructs. Accordingly, the novel ⫺223T3 C mutation is a polymorphism as also confirmed by normal
biological data in a single heterozygous patient. Despite
“silent” phenotypic features (12, 22 ) and previous in vitro
data suggesting a lack of binding activity (23, 24 ), the
⫺101C3 T mutation in the distal CACCC box has drastically reduced expression, like the known ⫺30T3 A mutation in the TATA box.
These apparently discrepant conclusions lead to several
comments: (a) a previous functional assay (12 ) produced
in vitro results similar to ours; (b) the human cellular line
used to show the lack of binding activity also lacks
␤-globin expression (25, 26 ), suggesting a lack of transcriptional factors essential for ␤-globin expression; (c)
several of these factors, such as the erythroid Krüppel-like
factor, bind to CACCC and are active at a level that can
not be detected by binding assays (27–29 ); (d) whereas
mutations in the proximal CACCC box are considered to
be more severe, some, like ⫺92C3 T, can also be silent
(30 ). Altogether, these data support our in vitro observations in favor of a transcriptional activity of the distal
CACCC box rather than the alleged lack of such activity.
The current discrepancy between in vitro and in vivo
observations could rather be the consequence of a cosegregate mutation located in a negative transcriptional
regulator, as suggested by other reports (31, 32 ).
The novel ⫺42C3 G mutation has a very mild transcriptional effect. This mutation is located in the ␤-globin
direct-repeat element, a highly conserved element found
in mammalian ␤-globin promoters (33 ). This is the first
report of a mutation in the human ␤-globin direct-repeat
element. The observed mild negative transcriptional effect
in vitro correlates closely with previous experiments on
single mutations in the mouse ␤-globin direct-repeat
element (33 ). In our patient, the phenotype observed with
⫺42C3 G/IVS-I-(⫺1)G3 C is comparable to the phenotype reported with ⫹33C3 G/codon 39C3 T mutations
(28 ), whereas the IVS-I-(⫺1)G3 C or codon 39C3 T mutations are ␤0-thalassemia mutations (34 ).
Several mutations or deletions found within the 5⬘-UTR
are associated with ␤-thalassemia (18 ), but are not always
confirmed by transient transfection studies (4 ). Assessment of 5⬘-UTR mutations with this assay brought further
insight in understanding of the 5⬘-UTR function and
mechanisms of disease. We tested three 5⬘-UTR mutations. In vitro data with ⫹10(-T) pinpoint the lack of
transcriptional defect associated with this mutation. This
reproduces previous observations and supports the hypothesis that a translational defect may reduce globin
chain synthesis in ⫹10(-T) heterozygotes (6 ). The
⫹20C3 T mutation and ⫹40343 deletion showed a twofold decrease in residual activity compared with the
wild-type construct. The 5⬘-UTR ⫹20C3 T mutation has
been observed only in cis with the IVS-II-745(C3 G)
mutation (5 ), as hypothesized several years ago (13 ).
Likewise and to the same extent, the 5⬘-UTR deletion
⫹40343 has also been shown to alter the ␤-globin transcription, despite a lack of evidence from previous transient transfection assays (4 ).
In addition to those quantitative data, our cell culture
expression system was also used to assess the alleged
abnormal mRNA splicing of the new IVS-I-108T3 C mutation (15 ). Discrepant conclusions have indeed been
drawn regarding the role of the IVS-I-108T3 C mutation
1789
Clinical Chemistry 48, No. 10, 2002
Fig. 1. Amplification of human ␤-globin cDNA and murine ␤-actin performed with primers specific for each transcript.
A 375-bp human ␤-globin transcript was consistently amplified from human reticulocytes used as a positive control (huRet; lane 1) and from various cDNA samples
from transfected MEL cells (lanes 3–9). No amplification was seen in untransfected MEL cells used as a negative control (Co; lane 2). A 645-bp fragment from murine
␤-actin was amplified from the same cDNA samples (lanes 2–9), but not from human reticulocytes cDNA (lane 2). Perfect identity between the 375-bp amplicons and
human ␤-globin transcripts was confirmed by sequence analysis (not shown).
(14, 15 ). We found no splicing abnormality in our study,
whereas the well-known IVS-I-110G3 A mutation, used
as a control, displayed abnormal splicing (16 ) as well as
an unexpected retention of IVS-I (see supplemental file for
additional results). Sequence analysis of both PCR bands
showed either the insertion of 19 intronic nucleotides, as
described previously (16 ), or full IVS-I retention. Quantitative data obtained with IVS-I-108T3 C showed de-
Table 1. Human ␤-globin mRNA copy number in the MEL cell line stably transfected with various ␤-globin variants: means
and CVs per trial factors.
Mutation
Mean (SD) copy number
of target RNA/ng
total RNA, ⴛ 105
Wild type
5.89 ⫾ 0.07
⫺223T3C
5.90 ⫾ 0.10
⫺42C3G
4.04 ⫾ 0.05
⫹20C3T
2.97 ⫾ 0.04
⫺30T3A
0.662 ⫾ 0.014
⫺101C3T
0.622 ⫾ 0.010
Del ⫹40343
3.14 ⫾ 0.07
Del ⫹10 (⫺T)
5.62 ⫾ 0.08
IVS-I-108T3C
2.78 ⫾ 0.04
Del ⫹156531577
2.10 ⫾ 0.02
Source of variability
(3 trial factors)
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
Cycle PCR
Calibrator RNA
Transfection
SD, ⴛ 105
CV, %
F-test
P
0.10
0.08
0.08
0.15
0.28
0.03
0.09
0.41
0.12
0.12
0.07
0.07
0.04
0.10
0.04
0.02
0.09
0.01
0.06
0.23
0.32
0.04
0.28
0.08
0.04
0.13
0.14
0.01
0.04
0.11
1.6
1.3
1.4
2.5
4.7
0.56
2.3
10
2.9
4.0
2.4
2.5
6.3
15
5.4
3.0
14
1.6
1.8
7.3
10
0.80
5.2
1.3
1.4
4.9
5.1
0.20
1.9
5.4
0.39
0.95
0.52
0.38
0.25
0.75
0.16
⬍0.001
0.05
0.01
0.60
0.16
0.02
⬍0.001
0.05
0.17
0.001
0.60
0.28
⬍0.001
⬍0.001
0.051
⬍0.001
0.001
0.33
⬍0.001
⬍0.001
0.99
0.94
0.02
1790
Technical Briefs
creased ␤-globin expression, which is consistent with the
observed thalassemic syndrome and pinpoints the potential role of a currently unknown cis-acting regulatory
element in this intron. Current in vitro and phenotypic
data highlight the role of IVS-I in ␤-globin gene regulation
and open the way to the characterization of as yet
uncharacterized cis-acting regulatory elements in this
region.
Functional effects of 3⬘-UTR mutations were also addressed with our assay. The 3⬘-UTR 13-bp deletion
⫹156531557, previously identified in the single heterozygous mother of a thalassemic Turkish child carrying a
compound mutation (19 ), was assessed. The mother presented with a typical thalassemic trait, but no study was
performed to assess the impact of this mutation on
␤-globin transcription. In our assay, the deletion showed
a strong negative transcriptional effect, consistent with
the biological observation and confirming the importance
of the 3⬘-UTR in the transcriptional regulation of the
␤-globin gene.
In conclusion, the combination of a cell culture expression system and competitive reverse transcription-PCR, as
described here, enables one to assess and quantify the
expression of wild-type and mutated human ␤-globin
genes. A full range of mutations can be introduced within
the human ␤-globin gene. Taking advantage of unique or
double restriction sites to introduce the mutation allows
answers to whether a particular sequence change is a
mutation or a silent polymorphism. Accurately measuring
the quantitative effect of single nucleotide changes or
deletions on ␤-globin gene expression should therefore
help to unravel the complex genotype–phenotype relationships in ␤-thalassemia, especially in complex cases of
thalassemia intermedia.
MEL cell lines and the pBluescript containing the human
␤-globin ␮LCR were kindly provided by Dr. F. Galacteros
(Hôpital Henri Mondor, Créteil, France). We thank F.
Lemaigre, G. Rousseau (Institute of Cellular Pathology,
Brussels, Belgium), B. Lethé (Ludwig Institute for Cancer
Research, Brussels, Belgium), and S. Loric (Hôpital St
Antoine, Paris, France) for reading the manuscript and for
helpful comments. We thank Dr. J. Billiet (AZ Brugge,
Brugge, Belgium) for providing some clinical data.
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Augusta, GA: The Sickle Cell Anemia Foundation, 1997:29pp.
Influence of Lepirudin, Argatroban, and Melagatran on
Prothrombin Time and Additional Effect of Oral Anticoagulation, Tivadar Fenyvesi, Ingrid Joerg, and Job Harenberg* (Fourth Department of Medicine, University Hospital Mannheim, Ruprecht-Karls-University Heidelberg,
Theodor-Kutzer-Ufer 1, 68167 Mannheim, Germany; * author for correspondence: fax 49-621-383-3308, e-mail
j-harenberg@t-online.de)
Oral anticoagulation (OAC) with coumarin derivatives
decreases the activities of coagulation factors II, VII, IX,
and X by inhibiting their vitamin K-dependent carboxylation (1 ). The effectiveness and safety of therapy with
oral anticoagulants for primary or secondary prophylaxis
of thromboembolism are usually monitored by the prothrombin time (PT), expressed as international normalized ratio (INR), prothrombin ratio, or percentage of
normal (2, 3 ). INR was standardized through large international reference studies (4 –7 ).
The direct thrombin inhibitors (DTIs) lepirudin and
argatroban are used to achieve effective anticoagulation in
patients with heparin-induced thrombocytopenia with or
without thrombosis (type II) (8 –10 ). Melagatran is currently under investigation in clinical trials (11–14 ). DTIs
prolong clotting times in PT assays and therefore interfere
with oral anticoagulants (15–18 ). During treatment of
deep venous thrombosis, heparins or DTIs are switched to
oral anticoagulants. During treatment for invasive diagnostics or surgery, patients on oral anticoagulant therapy
may temporarily be switched to a DTI. Decreased thrombin activity in the plasma of these patients leads to
prolongations of the PT (15, 16, 18 ). These additive effects
make it difficult to adjust dosage of either of the drugs
during concomitant use. In the case of heparins, additive
effects are antagonized by addition of protamine or heparinase to PT reagents (19 ). For DTIs, such antagonists are
missing. Antibodies against hirudin have been unsuitable
for neutralizing the drug’s anti-factor IIa effects because
of polyclonality, producing neutralizing or enhancing
antibodies, depending on the individual (20, 21 ). Argatroban and melagatran are small molecules and have not
been reported to be antigenic. Without the ability to
eliminate the additive effects of DTIs and oral anticoagulants on PT, it is important to address the issue of
handling these interactions in clinical practice and to
individually investigate them for each drug in vitro and
ex vivo.
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Here we describe the additive and synergistic actions of
the DTIs lepirudin, argatroban, and melagatran on the
effects of the oral anticoagulant phenprocoumon on PT.
These synergisms interfere with the analysis and dose
adjustment of oral anticoagulants during concomitant
therapy periods. Data were derived from plasma from
healthy volunteers and from patients treated with the
vitamin K antagonist phenprocoumon.
Blood from 6 healthy volunteers and 10 patients undergoing treatment with the vitamin K antagonist phenprocoumon (Hoffmann-La Roche) was collected by clean
cubital vein puncture into plastic vials containing 38
mL/L sodium citrate (9 mL of plasma in 1 mL of citrate).
All donors (volunteers and patients) gave informed consent in accordance with the current revision of the Helsinki Declaration. After centrifugation (1800g for 10 min),
plasma samples were shock frozen in liquid nitrogen,
stored at ⫺80 °C, and analyzed within 4 weeks. After
thawing, plasma samples were supplemented with lepirudin (molecular mass ⬃6500 Da; obtained from Aventis)
and argatroban (molecular mass 526.7 Da; kindly provided by Mitsubishi Chemical Corp., Tokyo, Japan) in
concentrations ranging from 300 to 3000 ␮g/L. Melagatran (molecular mass 473.6 Da; courtesy of Astra Zeneca,
Mölndal, Sweden) was added at lower concentrations,
between 30 and 1000 ␮g/L, because of its higher gravimetric potency observed in preliminary experiments.
Clotting time measurements were carried out in a KC
10a microdevice (22 ) from Amelung Co. PT was determined with a recombinant thromboplastin reagent (Aventis Behring; lot number 526935; international sensitivity
index ⫽ 1.09). With this thromboplastin reagent, clotting
times can be determined up to ⬃600 s with this device.
Interassay CVs were 10%, 8.7%, and 9.9% at clotting times
of 10, 50, and 300 s, respectively (n ⫽ 12). To start the
clotting time assay, 50 ␮L of plasma was incubated at
37 °C for 3 min (micromethod). To initiate clot formation,
100 ␮L of PT reagent (dissolved according to the manufacturer’s instructions) was added. The clotting times (PT)
were transformed into INRs with an equation appropriate
for the KC 10a device, obtained from the manufacturer of
the thromboplastin reagent: PT (in seconds) ⫻ 0.09 ⫹
0.2 ⫽ INR. The mean value obtained for the healthy
volunteers was 10.2 ⫾ 0.3 s, corresponding to an INR
range of 1.1 ⫾ 0.03.
All data are presented as the mean ⫾ SD. Calculation
factors (ng ⫻ factor ⫽ nmol/L) for transformation of the
data from gravimetric scaling to an equimolar scale were
0.154 for lepirudin, 1.901 for argatroban, and 2.11 for
melagatran, respectively. For reverse transformation, the
factors were 6.5 for lepirudin, 0.526 for argatroban, and
0.474 for melagatran.
The PT was prolonged in OAC plasma (OACP; samples
from patients undergoing a stable phase of OAC therapy)
to 26.0 ⫾ 5.0 s. All DTIs prolonged PT values in a
concentration-dependent manner in plasma from the controls and patients receiving OAC therapy. The results are
displayed in Fig. 1 in double scaling (nmol/L and ␮g/L).
In Fig. 1, the upper therapeutic gravimetric concentrations