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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 569-576
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Investigative Science, National Heart and Lung
Institute, Division of Biomedical Sciences, Imperial College School of
Medicine, London and the Matilda and Terence Kennedy Institute of
Rheumatology, London.
Thrombomodulin is an endothelial cell receptor for thrombin. It
functions as a natural anticoagulant by greatly accelerating activation
of protein C by thrombin. Using a direct gene screening strategy we
identified a frameshift insertion mutation, insT 1689, in the
thrombomodulin gene of a patient with myocardial infarction. The
mutation predicts an elongated gene product because of substitution of
the 12 C-terminal amino acids by 61 abnormal residues. Pedigree analysis showed that the mutation was also likely to have been present
in a sibling who had had fatal myocardial infarction. Carriers of the
mutant allele express significantly lower amounts of thrombomodulin on
the surface of their monocytes detected by flow cytometry and have
lower levels of soluble thrombomodulin in plasma. Wild type and the
mutant thrombomodulin were expressed in COS-7 cells. Cellular
distribution of the expressed proteins was evaluated by
immunofluorescence microscopy, which showed reduced cell surface
expression and intense juxtanuclear localization of the abnormal
protein. This suggests impaired translocation through the endoplasmic
reticulum/Golgi apparatus. Cells expressing abnormal thrombomodulin had
reduced ability (~2.5-fold) to accelerate the thrombin mediated
activation of protein C. This is the first demonstration of reduced
expression arising from a natural thrombomodulin gene mutation. The
results provide support for the suggestion that gene mutation of
thrombomodulin may be important in the pathogenesis of some cases of
occlusive thrombotic disease.
(Blood. 2000;95:569-576)
The pathogenetic process leading to myocardial
infarction (MI) involves the formation of the atherosclerotic plaque
followed by its rupture, with subsequent thrombosis and coronary
occlusion. It is now considered that thrombin is involved both in the
atherosclerotic process and in arterial thrombus
formation.1 Thrombin has mitogenic effects on smooth muscle
cells2 and fibroblasts3 and facilitates leukocyte activation and adhesion.4 Thrombin is also the
key enzyme in blood coagulation. It promotes clot formation, platelet activation and, by activating factors V and VIII, the positive feed-back mechanism that amplifies its generation.5
Under physiologic conditions, procoagulant and natural anticoagulant
mechanisms ensure that the production and inhibition of thrombin are in
equilibrium. Two major anticoagulant mechanisms are involved in the
suppression of excessive thrombin generation.6 Antithrombin
forms an inactive complex with thrombin, which is then rapidly removed
from the circulation. Activated protein C (APC), together with its
cofactor protein S inactivates factors Va and VIIIa, thereby
suppressing the major positive feedback mechanism in coagulation.
Efficient protein C activation requires formation of a complex between
thrombin and its endothelial cell receptor thrombomodulin
(TM).7
TM is a transmembrane glycoprotein expressed mainly on the endothelial
surface of blood vessels, but also on several other cells such as
platelets,8 monocytes,9 and certain tumor cells.10 TM is a modular protein composed of 5 structural
domains.11 The 3 extracellular domains consist of a large
hydrophobic region, 6 EGF-like repeats and a Ser/Thr rich region. A
transmembrane region is followed by a short cytoplasmic domain. The
function of the last 3 EGF-like repeats and of the Ser/Thr rich domain is to mediate thrombin binding and protein C activation.12
On binding to TM, thrombin undergoes conformational change and loses its ability to activate platelets and to cleave
fibrinogen.13 The rate of protein C activation by the
thrombin-TM complex is a 20 000-fold increase compared with thrombin alone.
The TM-APC pathway appears to be the main mechanism by which excessive
thrombin formation is counteracted in the small vessels, including the
coronary arteries. Impaired activity of this natural anticoagulant
mechanism leads to an increased predisposition toward thrombosis. It
has been shown that genetic defects of protein C, protein S, factor V
(1691G to A), and prothrombin (20 210G to A) predispose to venous
thrombosis.14,15 There is increasing evidence that the
latter 2 defects can contribute to the development of arterial
thrombosis, although the relative risk associated with a mutation is
less than that in venous thrombosis.16-18
Inherited deficiency of TM in thrombotic disease has remained largely
unexplored because of the difficulty of examining the phenotype of this
transmembrane protein. Recently, 2 systematic programs of study
involving a direct gene screening strategy have been
conducted.19,20 These studies have provided highly
suggestive evidence for a role of TM mutations in both venous and
arterial thrombosis. To date, however, there has been no experimental
evidence defining the effects of naturally occurring TM mutations on TM expression and function in human disease. We report here the
identification and functional characterization of a TM gene mutation in
a kindred with a history of MI.
The propositus in the kindred under investigation was part of a
study of TM mutations in patients with MI. The investigation has been
subject of prior reports.20,21 Briefly, the patient group
consisted of 104 unselected patients admitted to a west London hospital
who had confirmed MI using WHO criteria. Controls matched on a 1:1
basis for sex, age, and race were patients attending the Outpatient
Department of Charing Cross Hospital, London, for blood tests. The only
criterion used for exclusion of controls was that they had had venous
or arterial thrombosis, which was ascertained by questionnaire.
Clinical data
Blood collection
DNA extraction
Single strand conformation polymorphism (SSCP) analysis The TM fragment spanning nucleotides 1577 (numbering from the translation start site) to 1760 was amplified by polymerase chain reaction (PCR) using primers 21A (sense): 5'-TGTGCCTGGTGGTGGCGCTT-3' and 21B (antisense): 5'-TGGACGGAGCCAGGCTCCT-3'. The fragment was denatured for 4 minutes at 96°C, strands were separated by electrophoresis for 650 Vhs on a 20% precast polyacrylamide gel and silver-stained using the Phast System (Pharmacia, Uppsala, Sweden).Direct sequencing A fragment spanning nucleotides 1376 to 1789 was amplified with primers 18A (sense): 5'-CGACGGTTTCATCTGCACGG-3' and 22B (antisense): 5'-CAAA GCTGGGGGTGAGG-3' and then both strands were sequenced with primers 18A and 22B, respectively. The sequencing was performed with ABI Prism Big Dye Terminator Cycle sequencing kit on an automated sequencer type 373 XL (Perkin-Elmer Applied Biosystems, Warrington, Cheshire, UK), using the services of the Advanced Biotechnology Centre, Charing Cross Hospital Site. The PCR product amplified from the genomic DNA of the propositus was cloned into the pCR® 2.1-TOPO vector (Invitrogen, Leek, The Netherlands). After propagation in Escherichia coli, 10 clones containing either of the amplified alleles were sequenced.Flow cytometry Packed cells were separated from heparinized whole blood by centrifugation at 300 × g for 8 minutes. They were then diluted in 15 mL of RPMI medium (Sigma-Aldrich, England) and layered above 5mL Lymphoprep (Nycomed Pharma, Oslo, Norway). After centrifugation at 500 × g for 25 minutes, the white cells were collected from the interphase and washed once with RPMI. The cells were fixed with 3% formaldehyde in phosphate buffered saline (2.7 mmol/L potassium chloride, 137 mmol/L sodium chloride, pH 7.4, PBS), washed, and stored in PBS with 0.2% sodium azide at 4°C until staining. Indirect fluorescent staining of the monocytes was performed in duplicate. Each sample was incubated on ice for 1 hour with 1.25 µg of mouse monoclonal antihuman TM antibody (Dako Corp, Carpinteria, CA), washed with PBS, then incubated on ice for 1 hour with 4 µg of FITC conjugated goat antimouse antibody [F (ab')2 specific, SIGMA-Aldrich Co, Ltd, England, UK], and washed once more to remove excess antibody. Cells were blocked for 30 minutes in 20% normal mouse serum and, after removal of the blocking solution, were incubated for 1 hour with CD33 PE antibody (Becton Dickinson, Oxford, UK) to tag the monocytes. This was followed by a final washing step with PBS. Unstained cells and cells stained with the FITC conjugated secondary antibody or with the CD33 PE antibody only, were included as reference for analysis. The samples were analyzed on a Becton Dickinson FACScan flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA). Data acquisition and analysis were carried out using CELLQuest software. Monocytes identified by positive staining with CD33PE were live gated at acquisition. Calculation was performed for each sample, by subtracting the nonspecific fluorescence (measured in the absence of the specific antibody) from the TM associated fluorescence (measured in the presence of the specific antibody).Mutagenesis The expression vector pRSVSVOTM22 was a kind gift from Prof. E. Sadler (Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO). This vector contains the full length TM cDNA and the SV40 origin, therefore is suitable for high-level transient TM expression in COS cells. It will be termed below as pRSVSVOTMwt. Plasmid pRSVSVOTMmut, which contained the identified mutations, 1686G to C and ins T 1689 was constructed by oligonucleotide-directed mutagenesis using the PCR strategy.23 The sequence of the in vitro amplified insert was directly confirmed.Cell culture COS-7 cells were purchased from the European Cell Culture Collection and propagated in DME containing 4.5 mg/mL glucose, 3.7 mg/mL sodium bicarbonate supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Gibco BRL, Glasgow, UK).Transfections COS-7 cells were transfected using 6 × 104 exponentially growing cells seeded in 35 mm well plates 24 hours before transfection. Cells (50% confluent) in each well were cotransfected by the standard calcium phosphate method24 with 2 µg of plasmid pSEAP2-Control (Clontech Laboratories, Inc, Palo Alto, CA) (for assessment of transfection efficiency) and 3 µg of either pRSVSVOTMwt or pRSVSVOTMmut. Transfections were performed in triplicate on 3 separate occasions. Transfection efficiency was determined by measuring the concentration of secreted alkaline phosphatase in the culture medium 56 hourss after transfection by means of chemiluminiscence assay (Great Escape SEAP Reporter System, Clontech Laboratories) with a Fluroscan FL plate luminometer (Labsystems, Finland).Indirect immunofluorescence staining For these studies, autoclaved thin coverslips were laid in the bottom of the cell culture wells before seeding. Fifty-six hours after transfection, cells were washed 3 times with PBS, fixed with ice cold 3.7% paraformaldehyde in PBS for 20 minutes at 4°C, washed with PBS, and permeabilized with 0.1% Triton-X in PBS for 10 minutes. After 3 further washes, nonspecific binding sites were blocked with 5% normal goat serum and 1% BSA in PBS for 1 hour at room temperature. After removal of the blocking solution, the cells were incubated with 2 µg of monoclonal anti-TM antibody per well for 1 hour at 37°C. The cells were washed in PBS 3 times, for 10 minutes, with gentle rocking to remove any unbound antibody and incubated for 1 hour at 37°C with 7 µg of FITC conjugated goat antimouse antibody per well. Cells were washed in PBS 3 times with rocking, the coverslips were removed from the well, drained, and mounted in Vectashield (Vector Labs Inc, Peterborough, UK) onto microscope slides. The slides were examined with epi-fluorescence optics on an Olympus PROVIS microscope (Olympus Optical Co, UK) and captured onto 35 mm transparency film. In addition, cells were imaged using a Color Coolview CCD camera (Photonic Science, Robertsbridge, UK) mounted onto a BioRad DVC250 (Bio-Rad Laboratories, UK) confocal microscope. In this case, single confocal images (1 µm in Z-axis) were captured through individual cells, at a level where the optical section cut through the maximum diameter of the nucleus.Western blotting Fifty-six hours after transfection, the cells transfected with pRSVSVOTM or pRSVSVOTMmut or vector without TM cDNA, were washed twice with ice cold PBS; 1 mL of lysis buffer containing 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP 40, 0.5% sodium deoxycholate, with a cocktail of protease inhibitors (Boehringer Mannheim, UK) was added. The lysing cells were scraped and collected into 1.5 mL tubes on ice. The lysate was centrifuged at maximum speed for 15 minutes at 4°C to remove debris; 10 µL of the cell lysate or 1 µL of protein molecular weight marker (ECL protein molecular weight markers code RPN 2107) or 250 ng recombinant truncated TM lacking the transmembrane and cytoplasmic domain (Recombinant TM, American Diagnostica, Greenwich, CT), respectively, were mixed each with 10 µL of sample buffer (50 mmol/L Tris-HCl, pH 6.8, 2% SDS, 0.1% BPB, 10% glycerol, 200 mmol/L DTT). For nonreducing conditions, the DTT was omitted from the sample buffer used for truncated TM and cell lysates. The mixtures were boiled for 3 minutes, and electrophoresed in SDS-polyacrylamide gel. Proteins were electroblotted onto Hybond P (PVDF) membrane (Amersham, Little Chalfont, UK). The membrane was incubated in blocking solution (5% BSA in TBS-T [100 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20]), overnight at 4°C. Immunodetection was performed by incubating the membrane for 1 hour at room temperature with a 50 ng/mL dilution of polyclonal sheep anti-TM antibody (American Diagnostica Inc, Greenwich, CT) in TBS-T, followed by a 1 hour incubation with a 1:1000 dilution of HRP conjugated antigoat antibody in TBS-T (Dako Corp, Carpinteria, CA). After electroblotting and blocking, the lane with the molecular weight marker was separately incubated with 1:1500 dilution of streptavidin-HRP and realigned for final detection. The final detection step was peformed using Enhanced Chemiluminiscence (ECL) Western blotting detection system and exposure of the membrane to Hyperfilm ECL (both Amersham, Little Chalfont, UK).Thrombomodulin cofactor activity assay Cell surface TM cofactor activity was assayed essentially as previously described.25 Transfected cells were washed 3 times with assay buffer (50 mmol/L Tris-HCl, pH 8.0, 2 mmol/L CaCl2, 100 mmol/L sodium chloride, 1% BSA), scraped into 1 mL of the same buffer and pelleted by centrifugation at 3000 × g for 3 minutes. The cells were gently resuspended in 180 µL of assay buffer and assayed in 2 dilutions, each at a final volume of 180 µL. The cells were incubated with 0.89 µmol human protein C and 14.9 nmol human thrombin for 30 minutes at 37°C. The reaction was stopped by addition of 0.4 µmol antithrombin and 13 IU /mL heparin. The amidolytic activity of APC was assayed with the chromogenic substrate S 2366 at a concentration of 200 µmol. A reference curve was constructed with known dilutions of purified APC. Cofactor activity is expressed in nanomoles (nmol) APC generated in 30 minutes per 106 transfected cells. Human thrombin, protein C, antithrombin, and APC were purchased from Enzyme Research Laboratories (Swansea, UK) and S 2366 was from Chromogenix (Molndal, Sweden). The cofactor activity was normalized for transfection efficiency. In negative controls, either protein C or thrombin were omitted.Measurement of TM antigen Plasma TM was measured in 10 family members, 5 carriers of the mutation and 5 noncarriers (see Table 1). For each of the carriers, plasmas from 2 nonfamily normal subjects matched for age and sex (n = 10 controls in all) were also assayed. Soluble TM in plasma and TM antigen in the cell lysates prepared as above from transfected cells, was quantified with a commercially available ELISA (Immunbind TM, American Diagnostica Inc, Greenwich, CT). For the latter, measurements were performed on triplicate samples from 2 separate transfections (n = 6).Quantitation of activation markers D-dimer in plasma was measured using the Asserachrom D-Di commercial ELISA from Diagnostica Stago (Asnieres, France). Measurement of F1 + 2 activation fragment was performed with the ELISA method previously developed in our laboratory.26 All the measurements were done using citrate anticoagulated plasma stored at 70°C.
Statistical methods Means, variance, and standard error were calculated for data sets and all results are expressed in the text as means ± SE (standard error). Significance was calculated after logarithmic transformation of the data, with the t test for sample means with unequal variance. All operations were performed in Microsoft Excel (Microsoft Corp, Redmond, WA).
The propositus who was identified by an abnormal SSCP pattern of TM
fragment 1577-1760, had suffered MI at the age of 52 years. Sequence
analysis identified a complex mutation consisting of a base
substitution, 1686G to C and a base insertion, ins T
1689. No other mutation was detected within the entire coding
sequence. Cloning and direct sequencing of both alleles identified 1 normal allele and 1 allele with both mutations (Figure
1), confirming that the 2 sequence
alterations are present on the same allele. The G to C substitution has
no effect on the predicted amino acid sequence of the protein, whereas
the insertion creates a shift in the reading frame. In the protein
predicted by the abnormal nucleotide sequence, the last 12 normal amino
acids of the cytoplasmic domain are replaced by 61 different residues.
There is no predicted change to the transmembrane domain. Figure
2 illustrates the effect of the nucleotide
insertion on the deduced amino acid sequence of the carboxy-terminal
region of TM. The propositus was normal at the factor V Leiden
polymorphic site (F V 1691) and the prothrombin 20 210 polymorphic
site.
Pedigree analysis As shown in Figure 3, the frameshift mutation identified by SSCP analysis was also confirmed by sequence analysis in 2 living brothers aged 63 years (II /4) and 60 years (II /5), in 2 daughters aged 34 years (III /1) and 27 years (III /3) and in the niece aged 30 years (III /5). Identification of the niece as a carrier of the mutation (III/5) provides indirect evidence that the deceased brother of the propositus (II/8), who had suffered fatal MI, had also been a carrier. The father of the propositus was reported by the family to have died of MI at 74 years; his carrier status was unknown. The propositus and his family members have known risk factors for MI, including smoking habit, increased blood pressure, elevated triglycerides, and elevated cholesterol, see Table 1.
TM expression on the monocyte surface TM antigen on the monocyte surface was detected by flow cytometry using anti-TM mouse monoclonal antibody and a secondary, FITC conjugated antimouse antibody. Ten blood samples were blind tested, 5 from carriers of the mutation ins T 1689 and 5 from normal family members. The results are shown in Table 2. Although fluorescence is low because of weak expression of TM on the surface of monocytes, the results show clearly a significantly lower cell surface expression of TM resulting from the mutation (carriers vs non carriers, 8.0 ± 1.1 vs 16.3 ± 2.2 arbitrary units of fluorecsence, P = .01). Representative histograms are shown in Figure 4.
Transient expression of wild type and mutant TM in COS-7 cells After transient expression with wild type and mutant expression vectors, Western blotting revealed bands of appropriate mobility (~100 kd, under reducing conditions), with the mutant TM having slightly lower mobility than the wild-type TM and the truncated TM, Figure 5, panel B. The molecular weight (mol wt) of all TM molecules (truncated, wild type, and mutant) appeared anomalous (low) under nonreducing conditions with respect to the mol wt standards used, but the differences between them were consistent (Figure 5). There is also evidence of higher molecular forms of TM, that may represent either membrane fragments or TM polymers. A lower mol wt band, 60 kd, is also present under reducing conditions. As it also appears in the lysates from mock transfected cells (Figure 5), we conclude that it represents nonspecific protein crossreacting with the polyclonal antibody. The discrete mobility difference between the wild type and the mutant bands presumably represents the predicted difference in mol wt caused by the C-terminal extension. This difference between wild type and mutant expressed proteins, was also observed by immunoprecipitation of labeled expressed proteins (results not illustrated).
Cellular distribution of the expressed proteins Visualization of TM expression by COS-7 cells was accomplished with confocal microscopy after indirect immunofluorescent staining with monoclonal anti-TM antibody and secondary FITC labeled antibody. Cells transfected with plasmid pRSVSVOTMwt, showed localization of TM in the plasma membrane (Figure 6A, B). In contrast, in the cells transfected with plasmid pRSVSVOTMmut, a much reduced membrane incorporation of the TM antigen and a high juxtanuclear staining was observed (Figure 6C, D, and E). Untransfected cells and cells transfected with plasmid without TM insert showed no staining.
Thrombomodulin cofactor activity To assess the functional impact of the lowered membrane incorporation of the mutant compared with the wild type TM, a 2-stage TM cofactor activity assay was performed on the surface of transfected cells. Protein C activation was significantly reduced on the surface of cells expressing mutant TM, 214 ± 25 nmol APC/106 transfected cells compared with cells expressing the normal TM, 553 ± 117 nmol APC/106 transfected cells, n = 9, P = .0001. Untransfected cells and cells transfected with the vector without TM insert had no detectable activity.Plasma levels of activation markers As shown in Table 2, a significantly lower concentration of soluble TM was found in the plasma from carriers of the mutation ins T 1689, 3.46 ± 0.08 versus 4.06 ± 0.15 ng/mL in normal individuals (P = .01). The difference was also significant when the levels in carriers were compared with those measured in the normal age and sex matched control group, 4.69 ± 0.52 versus 3.46 ± 0.08, P = .02. The concentrations of coagulation activation markers F1 + 2 and D-dimer were higher in the mutation carriers compared with noncarriers of the mutation, 43.3 ± 15.7 versus 26.6 ± 4.42 ng/mL and 419 ± 98 versus 293 ± 44.2 ng/mL, respectively, but the increases were not statistically significant (P = .56 and P = .31, respectively).
We report a mutation in the TM gene of a patient with MI, consisting of a silent base substitution, 1686G to C, and a base insertion, insT 1689, the latter predicted to cause a shift in the reading frame and an elongated gene product. The predicted mutant protein has normal sequences for its extracellular and transmembrane domains, but an elongated intracellular C terminal tail. The consequences of the mutation are decreased surface expression of TM on the monocytes of carriers and decreased levels of soluble TM in plasma.
We are grateful to Dr Philip Mason and Dr Blandine Mille
(Imperial College School of Medicine) for their help with mutagenesis.
Submitted January 22, 1999; accepted September 13, 1999.
Supported by grants from the British Heart Foundation (PG/98152 and
PG/96065), the Graham Dixon Trust, the Coronary Thrombosis Trust and
the Special Trustees of Charing Cross Hospital.
Reprints: Gabriella Kunz, Department of Hematology, Imperial
College School of Medicine, Charing Cross Campus, Hammersmith, London
W6 8RP, UK.
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
specific hemostasis and hypercoagulable states.
N Engl J Med.
1999;340:1555-1564
