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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2696-2706
RAPID COMMUNICATION
By
From the Division of Molecular and Genetic Medicine, University of
Sheffield, Royal Hallamshire Hospital, Sheffield, UK; and the
Department of Haematology, University of Cambridge, MRC Centre,
Cambridge, UK.
The inherent variability of conformational diseases is demonstrated
by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the
main © 1998 by The American Society of Hematology.
IT IS NOW BECOMING apparent that a
variety of diseases, such as the prion encephalopathies,1
the degenerative neuropathies,2 and Alzheimer's
disease,3 all arise from inappropriate changes in the
conformation of an underlying protein. The risk of such changes
particularly occurs with proteins whose physiological function involves
an inherent alteration in their conformation.4 A prototypic
example of this type of conformational change is provided by the serpin
family of proteins that includes the principal protease inhibitors of
human plasma such as The triggering of the conformational trapping mechanism of the serpins
is dependent on precise interactions that allow the opening of the
5-stranded sheet of the molecule by a sliding movement on the
structures that underlie it.9 Mutations of amino acids within these underlying structures can result in a premature triggering of the 5- to 6-stranded conformational change to give dysfunction and
other consequences that have been graphically described for the serpins
as a whole as the syndrome of the hypersensitive
mousetrap.4 Thus, the same homologous mutation in different
serpins can give10 with antithrombin, thrombosis; with
C1-inhibitor, angioedema; and with
We report here studies of two new variants of antithrombin associated
with premature thrombosis that illustrate the characteristic phenotypic
variability of conformational diseases. The two antithrombin variants,
descriptively named Wibble and Wobble, both have amino acid
substitutions of the same conserved residue, threonine-85, which
directly underlies the opening of the 5-stranded sheet of the molecule.
In antithrombin Wobble, threonine-85 is replaced by a positively
charged amino acid, causing a predictable gross decrease in stability
compatible with its presentation as an apparent type I antithrombin
deficiency. The other variant, antithrombin Wibble, is of particular
interest, as here the less severe substitution of the threonine, by a
bulky but non-charged aminoacid, results in apparently normal secretion
of the antithrombin. However, the variant is susceptible to spontaneous
transition from the normal 5-stranded to the abnormal 6-stranded form.
This is confirmed by the finding in the patient's plasma of monomeric
latent antithrombin.
A bonus from the thorough studies of mutants of proteins associated
with disease is the unexpected clues that these natural mutants provide
as to aspects of normal function. Recently, we have completed a series
of crystallographic structures showing in detail the changes that take
place to give the heparin activation of antithrombin.15
However, a new observation in our study here, that antithrombin Wibble
has a profound increase in heparin affinity, opens a unique and quite
unexpected insight into the mechanism by which antithrombin binds and
is activated by heparin.
Materials
Methods
Measurement of plasma antithrombin levels.
Plasma antithrombin antigen levels were measured by a sandwich
enzyme-linked immunosorbent assay (ELISA) using a polyclonal antiserum
to antithrombin (Dako, High Wycombe, UK) as a capture antibody and its
peroxidase conjugate for detection. Thrombin inhibitory activity was
measured in the presence of heparin using a
BioMèrieux (Basingstoke, UK) kit on an ACL analyzer.
Antithrombin gene analysis.
Standard procedures were used to isolate high molecular weight genomic
DNA from peripheral whole blood. Oligonucleotide primers, designed to
amplify all coding regions of the antithrombin gene (exons 2 to 6)
together with intron/exon boundaries,18 were synthesized on
an Applied Biosystems (Foster City, CA) automated DNA
synthesiser. With the exception of exons 3a and 3b, which were amplified as a single 1.3-kb fragment, all exons were amplified individually. Two hundred fifty micrograms of genomic DNA samples were
diluted in a final volume of 50 µL containing 30 pmol of each primer,
200 µmol/L each dNTP, and 1 U Biotaq (Bioline, London, UK) in 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1%
Triton X-100, 10 mmol/L Tris-HCl, pH 8.8. Samples were denatured by
heating for 7 minutes at 95°C and then subjected to 35 cycles of
denaturation at 94°C, annealing at different temperatures depending
on the exon, and extension at 72°C. All steps were for 1 minute,
except where exons 3a and 3b were being amplified, when extension was for 2 minutes. In all cases, the final extension step was increased by
10 minutes. Amplified polymerase chain reaction (PCR) product was
purified either directly using the Reagent Pack for PCR Product Pretreatment (Amersham, Little Chalfont, UK) or, after
electrophoresis in 1% agarose, using the Qiaex Gel Extraction kit
(Qiagen, Düsseldorf, Germany) and elution of the DNA into 40 µL
of H2O. One to 9 µL of the purified PCR product was then
sequenced using one of the amplification primers end-labeled with
32P and the Thermo Sequenase Cycle Sequencing kit from
Amersham.
Isolation of RNA and synthesis of cDNA.
Buffy coats were collected by centrifugation of peripheral blood for 10 minutes at 2,800g and total RNA then isolated using Trizol
reagent (GIBCO BRL, Paisley, UK) and the procedure
supplied by the manufacturer. To synthesize cDNA, 1 µg of RNA was
diluted in 19 µL water, denatured at 65°C for 5 minutes, and then
placed on ice. Three hundred units of Moloney murine leukaemia virus reverse transcriptase (GIBCO BRL) and 30 U of RNAsin (Promega, Southampton, UK), diluted in 21 µL of 153 mmol/L KCl,
6.1 mmol/L MgCl2, 2 mmol/L DTT, 2 mmol/L dNTPs, 0.214 mg/mL
random hexamers, 102 mmol/L Tris-HCl, pH 8.3, was added and the sample
was incubated at 37°C for 2 hours. The reverse transcriptase was
denatured by heating at 65°C for 10 minutes and the cDNA was stored
at Analysis of AT3 gene expression.
The two mutations identified at nucleotide 2718 create an additional
recognition site for the restriction enzyme Sty I within exon
2, providing a marker with which to examine expression of mRNA derived
from the normal and mutated alleles. Thus, the PCR was used to amplify
a 447-bp fragment spanning exons 2 and 3a of the antithrombin gene from
the randomly generated cDNA prepared as described above. PCRs were
performed in a 50 µL volume containing 4 µL random cDNA, 100 µmol
of each dNTP, 15 pmol of each of the primers AT24 and AT20, and 1 U
Biotaq (Bioline) diluted in 16 mmol/L
(NH4)2SO4, 0.01% Tween-20, 1.5 mmol/L MgCl2, 67 mmol/L Tris-HCl, pH 8.8. Samples were
denatured for 7 minutes at 95°C and then subjected to 35 cycles of
1-minute steps of denaturation at 94°C, annealing at 56°C, and
extension at 72°C. The final extension step was increased by 10 minutes. The amplified product was digested overnight at 37°C with
Sty I (New England Biolabs, Boston, MA) and then analyzed by
electrophoresis in 8% polyacrylamide. Two fragments of
251 and 196 bp were observed where C was present at nucleotide position
2718, as in the normal allele, whereas the presence of either A or T at
position 2718 gave rise to three fragments of 196, 155, and 96 bp.
Purification of antithrombin.
Variant antithrombin was purified from the patient plasma using
precipitation of plasma with dextran sulfate and calcium
chloride,19 after which the supernatant was diluted with an
equal volume of equilibration buffer (50 mmol/L Tris-HCl, 10 mmol/L
sodium citrate, 5 mmol/L sodium EDTA, 150 mmol/L NaCl, pH 7.4). This
mixture was applied to a heparin-Sepharose affinity chromatography
column previously equilibrated with the same buffer and the column was washed with equilibration buffer, followed by the same buffer containing 0.4 mol/L NaCl. The antithrombin was eluted using a gradient
from 0.4 to 2 mol/L NaCl in the equilibration buffer. Antithrombin
peaks were further purified by anion exchange chromatography on
Q-Sepharose fast flow, as described previously.13
Preparation of latent antithrombin and thermal stability assessment.
Latent antithrombin was prepared as described.20 Fifty
milligrams of pure Electrophoresis methods.
The purity of antithrombin preparations was analyzed by PAGE in a
Tris-Glycine gel system with a stacking gel pH of 6.8 and running gel
pH of 8.8.23 The analysis of the formation of polymers in
antithrombin preparations was performed using PAGE as
described,24 with a stacking gel pH of 6.9 and a running
gel pH of 8.9. Samples of plasma that were analyzed were applied to the
PAGE system, after which they were blotted to
nitrocellulose25 and probed using rabbit anti-antithrombin
antibodies (Dako, Ely, UK), followed by a goat antirabbit
IgG-horseradish peroxidase conjugate, with detection via an ECL kit
(Amersham). Transverse urea gradient (TUG) gels containing a gradient
of 0 to 8 mol/L urea were prepared in the PAGE system24
according to methods described.26 Rocket immunoelectrophoresis was performed according to the method of Laurell.27 N-terminal sequencing was performed on proteins
that had been electrophoresed in a Tris-Tricine gel
system28 and blotted to polyvinylidene difluoride
membrane.29
Determination of heparin binding affinity for antithrombin.
Equilibrium binding constants for the interaction between antithrombin
and heparin pentasaccharide or high-affinity heparin were determined
using titrations of the heparin species (0 to 500 µmol/L) into
antithrombin solutions (25 nmol/L) in which the increase of
fluorescence emission at 340 nm, with excitation at 280 nm, was
followed, as described.17 Buffers used were 20 mmol/L Tris-HCl, 0.1% PEG 8000, 0.1 mmol/L EDTA, pH 7.4, containing 100 mmol/L NaCl (I0.15) or 250 mmol/L NaCl (I0.3).
Rapid kinetics of heparin binding to antithrombin variants.
The kinetics of heparin pentasaccharide binding to antithrombin
variants were determined using stopped-flow spectrofluorimetry essentially as described.30 The antithrombin molecules
( Association rate constant with factor Xa.
The association rate constant for the interaction between antithrombin
and factor Xa was analyzed under pseudo-first order conditions using a
discontinuous assay system essentially as described.17
Variant Identification and Family Histories
Plasma Findings
Thermal Stability Tests
Latent Antithrombin
Latent Antithrombin in Normal Plasma To check the finding of latent antithrombin in the plasma of the affected individuals in family 1, pre-prepared latent antithrombin was added to a normal control plasma sample to show that it eluted in a peak position identical to that of peak II in Fig 2 at an NaCl concentration of 0.36 mol/L. This was confirmed, but in doing so it was noticed that a similar, although smaller shoulder at peak II was present with normal plasma even in the absence of added latent antithrombin. Recovery of the material from this peak from normal plasma showed it to have the characteristic stability of latent or cleaved antithrombin. SDS-PAGE electrophoresis and amino-terminal sequencing confirmed that the shoulder contained, as expected, some cleaved antithrombin, but also a greater amount of uncleaved, latent antithrombin.Heparin Affinity The heparin affinity-chromatography results indicated that antithrombin Wibble and Wobble both had an extraordinary and equivalent increase in heparin affinity. This was quantifiable for the isolated antithrombin Wibble as in Table 2, which compares the affinity constants (Kd) for native -antithrombin, the Wibble
variant, and another conformational variant Rouen-VI.13 The
increase in affinity for the core pentasaccharide is 25-fold with the
Wibble variant, versus threefold with the Rouen-VI variant.
Association Rate Constant With Factor Xa
We record here a unique experiment of nature in which separate mutations in two families provide both an archetypal example of a severe episodic conformational disease and also a matching forme fruste version that gives a model, effectively in slow motion, of the molecular mechanism involved. A particular advantage of this natural experiment is that it allows the study of the mutant antithrombin in its authentically glycosylated and functional form in a way that could not, at present, be reproduced in the laboratory by recombinant expression. Conformational Disease and Thrombosis The serpin protease inhibitors of human plasma provide the best characterised example of a newly recognized clinical entity the conformational diseases.4 The first example of a
conformational disease to be studied in the serpins was the common
genetic deficiency of 1-antitrypsin resulting from a
conformational change in a variant
1-antitrypsin35 at its site of synthesis in
the liver. A consequence of this conformational change is the
aggregation of polymers of the variant in the liver,11 with
resultant risk of cirrhosis and, because of the associated plasma
deficiency of the inhibitor, the slow onset of emphysematous lung
degeneration.
Molecular Lesions and Instability Interest in the detailed structural pathology of these two new variants was aroused by the recognition that the mutations had occurred at a site on the molecule known to have a critical function in controlling the conformational stability of antithrombin.9 A previous study of the structural pathology of the serpins10 had identified a vulnerable zone of the molecule that underlies the opening of its main -pleated sheet, the A-sheet (Fig 6A and B). In particular, the
studies of Yu et al36 had shown that even a minor steric
decrease at the site of the conserved threonine 85 (59 in
1-antitrypsin) resulted in a significant increase in the
stability of the molecule. They replaced this threonine by a smaller
alanine and showed that this resulted in a tightening of the closed
5-stranded A-sheet present in the active form of the molecule (Fig
6A-I). As compared with this stable recombinant mutant, the two
mutations identified in this study both involve the substitutions of
bulkier sidechains at position 85 (Fig 6B) and hence will predictably
result in a decreased conformational stability. As expected, the
replacement of threonine 85 by the large and highly polar lysine in
antithrombin Wobble gives an instability of such severity that the
variant is substantially (but not completely) lost from the plasma.
However, replacement of threonine 85 by the bulky but nonpolar
methionine in antithrombin Wibble results in a small, but demonstrable,
decrease in thermal stability, as shown in Fig 4. Strong evidence that
this instability results from a more ready opening of the A-sheet is
provided by the reversion of the variant to full stability (Fig 4) on
the bolstering of the 5-stranded conformation by the addition of the high-affinity heparin pentasaccharide (Fig 6C-II).
Latent Transition It was proposed12 in 1991 that the intact reactive center loop of the serpins could become incorporated as the middle strand of the A-sheet and that this would not only explain the observed properties of latent plasminogen activator inhibitor (PAI-1), but also be inducible in other plasma serpins, notably in antithrombin. This has indeed proved to be so, with, as well as PAI-1,37 the formation of the latent conformation being inducible in1-antitrypsin,21 1-antichymotrypsin,38 and, as has been
crystallographically confirmed,15,39 in antithrombin (Fig
6A-II). The occurrence of the latent transition of antithrombin has
been shown to have biological implications in that it is a complicating
product in the pasteurization of fractionated plasma
antithrombin14 and it has also been shown to be formed by
the in vitro incubation of the unstable Rouen-VI variant of
antithrombin.13 However, until the investigation of family
1 with antithrombin Wibble, there had been no evidence that the latent
transition could occur as a spontaneous in vivo phenomenon.
Heparin Affinity and Activation The bonus finding from the investigation of antithrombins Wibble and Wobble are the insights they provide as to the conformational contribution to the binding and activation of antithrombin by heparin. Both variant antithrombins showed an extraordinary increase in heparin affinity as evidenced in Fig 2, with the elution of antithrombin requiring 1.5 mol/L NaCl and that of the Wobble variant (not shown) requiring 1.6 mol/L NaCl. Quantitatively the Kd for binding of antithrombin Wibble and the heparin pentasaccharide (Table 2) is by far the highest affinity yet recorded for either natural or modified forms of antithrombin. Analysis of the kinetics of binding (Fig 5 and Table 3) shows that the increase primarily results from the greater stability of the complex formed between the pentasaccharide and heparin. The large increase in the constant defining the conformational change upon heparin pentasaccharide binding, k2, indicates that the rate of this change is increased significantly in antithrombin Wibble, both as compared with normal antithrombin and also with the already increased rate in antithrombin Rouen VI. The higher rate order of interaction of antithrombin Wibble with factor Xa (Table 2) is also an indication of an increased release of its reactive loop into the active inhibitory configuration. Both of these results fit with the predicted likelihood that the Wibble and Wobble variants will have an increased flexibility in the opening and shutting of the A-sheet, giving a more ready release of the reactive loop and also a more ready transition to the closed form of the sheet on the addition of heparin (Fig 6C).
Submitted May 21, 1998;
accepted August 5, 1998.
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© 1998 by the American Society of Hematology.
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A. Zhou, P. E. Stein, J. A. Huntington, and R. W. Carrell Serpin Polymerization Is Prevented by a Hydrogen Bond Network That Is Centered on His-334 and Stabilized by Glycerol J. Biol. Chem., April 18, 2003; 278(17): 15116 - 15122. [Abstract] [Full Text] [PDF]
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M. Makris, M. Leach, N. J. Beauchamp, M. E. Daly, P. C. Cooper, K. K. Hampton, P. Bayliss, I. R. Peake, G. J. Miller, and F. E. Preston Genetic analysis, phenotypic diagnosis, and risk of venous thrombosis in families with inherited deficiencies of protein S Blood, March 15, 2000; 95(6): 1935 - 1941. [Abstract] [Full Text] [PDF]
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A. E. Mast, J. E. Stadanlick, J. M. Lockett, and D. J. Dietzen Solvent/Detergent-Treated Plasma Has Decreased Antitrypsin Activity and Absent Antiplasmin Activity Blood, December 1, 1999; 94(11): 3922 - 3927. [Abstract] [Full Text] [PDF]
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A. Zhou, J. A. Huntington, and R. W. Carrell Formation of the Antithrombin Heterodimer In Vivo and the Onset of Thrombosis Blood, November 15, 1999; 94(10): 3388 - 3396. [Abstract] [Full Text] [PDF]
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A. Zhou, R. Faint, P. Charlton, T. R. Dafforn, R. W. Carrell, and D. A. Lomas Polymerization of Plasminogen Activator Inhibitor-1 J. Biol. Chem., March 16, 2001; 276(12): 9115 - 9122. [Abstract] [Full Text] [PDF]
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| Copyright © 1998 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
Abstract
20°C until required.
)
antithrombin Wibble, (
) antithrombin Rouen VI, and (
)
1 · s
Asp).
J Clin Invest
94:2265,
1994