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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3388-3396
Formation of the Antithrombin Heterodimer In Vivo and the Onset of
Thrombosis
By
Aiwu Zhou,
James A. Huntington, and
Robin W. Carrell
From the University of Cambridge Department of Haematology, Cambridge
Institute for Medical Research, Cambridge, UK.
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ABSTRACT |
Antithrombin is shown to undergo a slow spontaneous conversion to
its inactive latent conformation with readily discernible amounts
present in plasma on incubation at 37°C for 72 hours. More rapid
conversion occurs on incubation of isolated antithrombin at 41°C or
50°C, but the appearance on electrophoresis of free latent
antithrombin is preceded by the formation, in reciprocal proportions,
of a new slow band. This slow component is shown to be a heterodimer of
active and latent antithrombin. It can be isolated as a single stable
band either by incubation of antithrombin or by mixing equimolar
proportions of active and latent antithrombin under the same conditions
that give overnight crystallization of the active/latent antithrombin
heterodimer. Similarly, equimolar addition of latent antithrombin to
plasma results electrophoretically in a quantitative shift to the
slower heterodimer mobility. Clinically, the presence of latent
antithrombin is potentially deleterious, because its linkage to form
the heterodimer results in inactivation of the otherwise normal
molecule linked to the latent antithrombin. In the case of
 -antithrombin, because the dimer readily dissociates, there is only
a 11% additive loss of activity, but with  -antithrombin the dimer
appears more stable, with the additive loss of activity from the normal
component being 21%, increasing to 33% on stabilization of the
dimer with heparin. This linked and selective loss of activity of
-antithrombin provides an explanation for the unexpected severity of
thrombotic episodes in heterozygotes with conformationally unstable antithrombins.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANTITHROMBIN IS THE principal
inhibitor in the plasma of the coagulation proteases thrombin and
factor Xa.1 As is the case with other members of the serpin
family of protease inhibitors, antithrombin is a monomer that functions
by exposing its reactive site loop as a pseudosubstrate for the target
protease. Cleavage at the reactive center results in entrapment of the
protease, with movement of the cleaved reactive center peptide loop
together with the bound protease, such that the loop forms an extra
sixth strand in the middle of the A -pleated sheet of the molecule. This movement of the cleaved loop, with its incorporation into the body
of the molecule, can also be induced without cleavage of the reactive
loop to give the inactive 6-stranded form of antithrombin.2 This 6-stranded intact form of antithrombin has an identical
crystallographic structure to that of the physiological latent
conformation3 of plasminogen activator inhibitor-1 (PAI-1).
For this reason, it is referred to as the latent form, even though with
antithrombin this latent transition is irreversible.
The 3-dimensional structure of antithrombin was independently
determined in 1994 by 2 different groups,4,5 but in each instance there was the unexpected finding that the protein had crystallized not as a monomer but as a heterodimer of antithrombin. There is now a consensus agreement between the 2 groups that this dimer
is formed by the linkage of a molecule of active antithrombin to a
second molecule of antithrombin in the inactive latent conformation. The puzzling implication from this finding in both laboratories was
that, in each case, some of the antithrombin, which was freshly prepared for crystallization, must have spontaneously converted to the
latent form. This apparent evidence that the latent conversion of
antithrombin could spontaneously take place at room temperature was
dismissed rather unconvincingly at the time as being a crystallographic artifact. One reason for this dismissal was the inability to
demonstrate the appearance in the crystallization solutions of a
component with the characteristically increased electrophoretic
mobility of the latent form.
We show here that normal antithrombin does indeed undergo a slow
conversion to the latent form even under in vivo conditions. However,
this conversion is masked by the immediate linkage of the newly formed
latent molecule to a molecule of active antithrombin to give a dimer
that unexpectedly has an electrophoretic mobility close to that of the
monomeric normal component. Hence, in past investigations, the presence
of latent antithrombin has not been detected electrophoretically until
it appears as the free species, and this does not occur until the
concentration of latent antithrombin exceeds 50% of the total
antithrombin. It is now apparent that, in the past, stored samples of
what had been believed to be active antithrombin could actually have
contained a substantial proportion of the latent form, as is
demonstrated here with a commercially fractionated preparation. The
presence of undetected latent antithrombin provides an explanation for
puzzling inconsistencies in previous experimental results. However, we
believe that the most significant implications of our findings relate
to the mechanism of onset of thrombosis. The latent form not only is in
itself inactive, but also its dimerization with an otherwise active
molecule is shown to give an amplification of loss of inhibitory
activity. The effects of this loss of activity is exacerbated by the
dimerization occurring preferentially to the most active inhibitor of
coagulation proteases in the plasma, the glycosaminoglycan-bound
-glycoform of antithrombin. This augmentation of the loss of
inhibitory activity consequent to the latent conversion is of
particular relevance to the unexpectedly severe thrombotic episodes
associated with conformationally unstable variants of antithrombin.
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MATERIALS AND METHODS |
Antithrombin and derivatives.
Normal  -antithrombin was isolated from fresh frozen plasma using
precipitation with dextran sulfate and calcium chloride, 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 EDTA,
0.15 mol/L NaCl, pH 7.4). The mixture was then fractionated on
heparin-Sepharose as previously described6,7 with residual
heparin being removed by Q-Sepharose (Amersham Pharmacia Biotech,
Uppsala, Sweden) anion exchange chromatography. The
concentration of antithrombin was determined with an extinction
coefficient of 6.5.8 Latent antithrombin was prepared as
previously described9 by incubating freshly prepared native
antithrombin (final concentration, 1 mg/mL) with 0.25 mol/L trisodium
citrate and 10 mmol/L Tris-HCl at pH 7.4 and 60°C for 16 hours. The
sample was exchanged into 20 mmol/L Tris, 10 mmol/L sodium citrate, pH
7.4, by ultrafiltration on an Amicon (Beverly, MA) concentrator
using a YM30 membrane, after which it was purified on
heparin-Sepharose and ion-exchange chromatography as described above.
The latent-antithrombin was eluted at approximately 0.4 mol/L NaCl on
heparin-Sepharose chromatography. Confirmation of the latent-state was
assessed by transverse urea gradient (TUG) gel electrophoresis and
thermal stability measurement as previously described.10
Cleaved antithrombin was prepared by incubating 5 µmol/L antithrombin
with 50 nmol/L porcine pancreatic elastase for 4 hours at 37°C in
50 mmol/L Tris-HCl, pH 8.5, containing 0.15 mol/L NaCl and 0.1%
polyethylene glycol (molecular weight [Mr] 8,000, wt/vol). The product was analyzed by N-terminal sequencing, and the
reactive center loop cleavage sites were identified corresponding to
residues 388-389 and 389-390, respectively.
Commercial antithrombin concentrate.
Bio-Products Laboratory (Elstree, UK) dried Antithrombin III in a 30 g
container, prepared and heat-treated in 1992, was kindly supplied by Dr
Elaine Gray (UK National Institute for Biological Standards and
Control, South Mimms, UK). The concentrate had been stored in the dark
at refrigerated temperature. A second sample, currently (1999)
available from the manufacturers for therapeutic use, was also tested.
Incubation and thermal stress.
Normal -antithrombin was incubated, with the addition of 20%
vol/vol glycerol, at 0.5 mg/mL in buffer A (50 mmol/L Tris, 50 mmol/L
NaCl, pH 7.5) at 50°C, and aliquots taken at different time
intervals were analyzed by native polyacrylamide gel electrophoresis (PAGE; see Fig 1A) and activity assay. Incubation of
isolated antithrombin Wibble (T85M; see Fig 1B) was performed at
50°C in buffer B (50 mmol/L Tris buffer, pH 7.4, with 50 mmol/L
KCl) as in Beauchamp et al10 and at a concentration of 0.2 mg/mL. Incubations of normal antithrombin at 37°C and 41°C were
performed in buffer B at 0.2 mg/mL (see Fig 1E). Aliquots taken at
different incubation times were snap-frozen in liquid nitrogen. To
prepare the dimer, the latent form was mixed with - or
-antithrombin in 1:1 stoichiometric proportion to a concentration of
1 mg/mL in buffer A and incubated at 37°C for 5 minutes (see Figs
1D and 3D). Competitive displacement from the dimer of -antithrombin
by -antithrombin was performed by sequentially adding
-antithrombin to the -dimer previously prepared as described
above by the addition of 3 µg each of -antithrombin and latent
antithrombin to a volume of 30 µL (see Fig 3D). Two bags of standard
donor plasma (300 mL each, collected in adenosine-citrate-dextrose) were incubated at 37°C for 0 or 72 hours and fractionated on
heparin-Sepharose with a 0.1 to 2 mol/L NaCl gradient. Fractions from
the peaks were pooled (as shown in Fig 2B) and analyzed on native PAGE
(see Fig 3AI). The protein from the native gel was then transferred to
a nitro-cellulose membrane, and the bands with the same mobility as
latent antithrombin were excised and applied onto a nonreducing sodium
dodecyl sulfate (SDS) gel and probed by Western blotting (see Fig
3AII). A parallel check on loss of activity of plasma during 72 hours
of incubation at 37°C was separately performed by the incubation of
1 mL of plasma in a stoppered microfuge tube. Activity before and after
incubation was determined as described.11 To check if dimer
can form in human plasma, 5 µL whole plasma was incubated with 0.9 µg latent antithrombin at 37°C for 5 minutes before loading onto
native PAGE with subsequent analysis by Western blotting (see Fig 3B).
PAGE.
SDS-PAGE was performed in 10% (wt/vol) polyacrylamide as previously
described.10,11 All samples for SDS-PAGE were boiled for 3 minutes under nonreducing conditions before electrophoresis. Nondenaturing PAGE was performed as previously described7
but modified to obtain high resolution by use of an 8% gel and with the running time extended to 90 minutes. Isoelectric focusing was
performed on precast Ampholine PAGplates (Pharmacia) using ampholytes
in the range of pH 4 to pH 6.5. The gels were stained with 0.25%
Commassie Blue R250.
Western blot analysis.
Proteins were separated by gel electrophoresis, blotted to
nitro-cellulose, and immunostained with rabbit anti-antithrombin antibodies (Dako, Ely, UK), followed by alkaline phosphatase-coupled goat antirabbit IgG (Dako), with detection by incubating the membrane in a phosphate substrate solution consisting of 0.15 mg/mL
5-bromo-4-chloro-3-indolyl phosphate (Sigma, St Louis, MO)
and 0.3 mg/mL nitroblue tetrazolium (Sigma) in 0.1 mol/L Tris, pH 9.5, containing 100 mmol/L NaCl and 5 mmol/L MgCl2.
Determination of loss of inhibitory activity.
Second order rate constants of inhibition of human thrombin (Sigma) and
bovine factor Xa (Boehringer Mannheim, Ltd, East Sussex, UK) were
determined using discontinuous chromogenic assays described by Olson et
al1 using S-2238 and S-2222, respectively. Antithrombin isoforms or were mixed with latent antithrombin derived from the -isoform to 1 µmol/L each, so that the final total
antithrombin concentration was similar to that in plasma and the
proteinase concentration was 100 nmol/L. Stoichiometries of inhibition
were determined to be equivalent for and and were unaffected by the addition of latent antithrombin. Reactions were performed at room
temperature in the presence of either 0.1 mg/mL polybrene or
unfractionated heparin (Sigma). Values given in the presence of heparin
were derived from the slope of kobs versus molar heparin concentration based on the average molecular weight of 14,000 Daltons
and an occurrence of the high-afinity pentasaccharide sequence in one
third of the chains. Table 1
shows results obtained with factor Xa; similar results were obtained
with thrombin (not shown). The reported percentage loss in inhibitory
activity is the percentage decrease in rate of inhibition for the
active monomer consequent to the addition of inactive latent monomer.
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RESULTS |
Dimer in vitro.
The recognition of the ready formation of latent-active heterodimers
came during studies of the changes in normal (-) antithrombin on
prolonged incubation at 50°C. This incubation results in almost full conversion to the latent form by 96 hours
(Fig 1A), but it was also observed that the
appearance of the latent band was preceded by the initial formation of
a previously unidentified slower electrophoretic band that diminished
as the latent band increased. This reciprocity in appearance from an
initial slow electrophoretic component to the faster band of latent
antithrombin was also evident (Fig 1B) on incubation of the
conformationally unstable variant,10 antithrombin Wibble
(Thr85Met). Incubation of this variant at pH 8.0 for 24 hours gave
almost complete conversion to the electrophoretically faster latent
form, but in retrospect, careful alignment shows that this is preceded
by the appearance of the new slow component.
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| Fig 1.
Nondenaturing PAGE electrophoreses. (A) Normal ()
antithrombin incubated at 50°C at pH7.4 (buffer A) and in 20%
glycerol showing initial appearance at 12 hours of slow dimer band with
complete conversion to latent form by 96 hours. A similar result but
with more rapid change occurred under the same conditions in the
absence of glycerol. L, latent control; + L, 1:1 mixture of
and latent antithrombin. (B) A time sequence of incubation of the
unstable antithrombin Wibble variant (50°C, pH 7.4, buffer
B).10 Careful alignment shows how the transition to the
latent form is preceded, in reciprocal proportions, by the formation of
the slow dimeric component. Trace polymer bands are also present with
the position of the loop-sheet dimer and trimer arrowed. (C)
Isoelectric focusing of the stable slow electrophoretic component (lane
3), prepared as in (A), confirms its heterodimeric composition, with
separation into equal bands of -antithrombin and latent
antithrombin. Lanes 1 and 4, -antithrombin; lane 2, latent standard.
(D) Mixing of antithrombins (left) and (right) 1:1 with latent
antithrombin immediately gives the single slow dimeric component that
retains its electrophoretic integrity even after 7 days at room
temperature. (E) Incubation of normal -antithrombin in buffer B (pH
7.4, 50 mmol/L KCl) showing conversion over 54 hours at 37°C to the
slow dimeric form, with acceleration at 41°C to give the appearance
of the free latent band at 48 hours.
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The unidentified slow band was prepared as a single component (Fig 1A)
by incubation of -antithrombin at 50°C for 24 hours in the
presence of 20% glycerol to minimize polymerization. Even after
storage at room temperature, the incubated antithrombin maintained its
characteristic single slow electrophoretic mobility. However, on
isoelectric focusing electrophoresis (Fig 1C) and also on
heparin-Sepharose affinity chromatography
(Fig 2A), the single band split into 2 equal components with the characteristic mobilities of normal
-antithrombin and of latent antithrombin. The identity of each was
confirmed by native PAGE electrophoresis and activity measurements. The
deduction from these results that the slow band is a heterodimer of
latent and -antithrombin was confirmed by the mixing of equimolar
proportions of latent and active -antithrombin, as shown in Fig 1A.
The rapidity of formation and stability of the heterodimeric band is
shown in Fig 1D, with immediate formation of the dimer on mixing of
latent with either - or -antithrombin and its subsequent
stability as a single electrophoretic band, even after 7 days of
standing at room temperature.
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| Fig 2.
Heparin-Sepharose affinity chromatography. Three results
are aligned but that in (C; adapted and reprinted with permission from
Chang and Harper12) was performed using a
different affinity heparin and consequently has noncomparable elution
concentrations. (A) Chromatography of the slow component (lane 4, Fig
1A) gives a result identical to that obtained with a crystal of the
heterodimer,16 with separation into equivalent peaks of
-antithrombin and latent antithrombin. (B) Elution profile of
antithrombin from plasma incubated for 72 hours at 37°C. The first
peak, including fraction 1, had no thrombin-inhibitory activity.
(Reprinted with permission.12) (C) Previous larger scale
heparin-Sepharose chromatography of a pasteurized commercial
concentrate of antithrombin.12 The inactive L (latent)
forms make up 40% of the total antithrombin, but the presence of
additional peaks reinforces other unpublished evidence that the latent
transition may also involve minor stable intermediate forms. All of
these forms apparently dimerize, as shown by the single band with dimer
mobility on electrophoresis of the concentrate (Fig 3CII).
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To demonstrate that significant conversion to the latent form could
occur at physiological pH and temperatures, active -antithrombin samples in pH 7.4 (50 mmol/L KCl) buffer were incubated at 37°C and
41°C for 54 hours (Fig 1E). The result at 37°C shows no
presence of free latent antithrombin even after 54 hours, but clearly
there has been a substantial transition to the slower dimeric form. At
41°C, the transition is more rapid, with complete conversion to the
dimer by 24 hours and appearance of the free latent band at 48 hours.
Formation of dimer in plasma.
In a previous study,10 we had shown the presence of trace
amounts of latent antithrombin in normal donor plasma. To demonstrate that the latent transition can occur in vivo, a donor plasma bag was
incubated at 37°C for 72 hours. Measurement before and after incubation of a separate tube of plasma showed a 15% loss of thrombin inhibitory activity over the 72 hours. Antithrombin from the incubated bag, together with that from a matching unincubated control bag, was
then isolated by standard heparin-Sepharose chromatography (Fig 2B).
The first elution peak (fraction 1 in Fig 2B), which showed no
inhibitory activity to thrombin and was known to cover the elution of
both latent and reactive-loop cleaved antithrombin, was collected in
each of the 72-hour and 0-hour plasma incubations. Native PAGE
electrophoresis (Fig 3AI) showed a faint
band with the mobility of latent or cleaved antithrombin in the
unincubated sample, but a much denser band in the same position in the
72-hour incubated sample. Furthermore, in fraction 2, representing the overlap with the main -antithrombin peak, the unincubated sample shows the presence of a band of -antithrombin, whereas the 72-hour incubated sample shows a clear band in the position of the dimer. To
determine the proportions of latent versus cleaved antithrombin in
fraction 1, the fast-running band was eluted from each gel (Fig 3AI)
and subjected to SDS-PAGE electrophoresis (Fig 3AII). This showed in
each the presence of similar trace amounts of cleaved antithrombin, but
a much denser band is present in the 72-hour sample in the position of
intact antithrombin. The combination of these findings of an inactive
component with increased mobility on native electrophoresis but normal
mobility on SDS-PAGE electrophoresis confirms its identity as latent
antithrombin.
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| Fig 3.
(A) (I) Native-PAGE of aliquots of fractions 1, 2, and 3 (Fig 2B) from 0- and 72-hour incubations of plasma at 37°C. Bands
with the characteristic fast mobility of both the latent and cleaved
forms of antithrombin are seen faintly in the 0-hour sample and
strongly in the 72-hour sample. (II) Western blot of SDS-PAGE of the
eluted band 1 and an intact normal control () shows the predominant
presence of intact antithrombin in the 72-hour sample with the presence
of some cleaved (Cld) antithrombin at 0 and 72 hours. The combined
results (I) and (II), together with the absence of inhibitory activity,
confirm the identity of the latent component. (B) Western
immunostaining of a native PAGE of plasma shows that normal plasma (P)
has a major band that aligns with -antithrombin standards (). The
addition of latent antithrombin (L) in near equimolar proportions to
the control plasma (P + L) fails to show the presence of the free
latent band, but results in a quantitative shift of the major band from
the position to the characteristic slower mobility of the
/latent heterodimer. (C) Commercial antithrombin concentrate. (I)
SDS-PAGE confirms that the concentrate (conc) described in Chang and
Harper12 has a single major component with the molecular
weight of intact antithrombin. (II) Native PAGE illustrates how the
electrophoretic mobility of the concentrate could be readily mistaken
for that of active -antithrombin; alignment with an + L
standard (with excess ) confirms that the concentrate is
predominantly /latent dimer. (D) Native PAGE showing addition of
increasing amounts of -antithrombin (to 4.3 µg) to preformed
-dimer (3 µg latent:3 µg , with incubation for 5 minutes) to
give complete displacement of the -antithrombin with formation of
the -dimer (d). The position of the -dimer (d) is indicated
by the dotted arrow.
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To assess whether latent antithrombin would combine with active
antithrombin in vivo, previously prepared latent antithrombin was added
to a donor plasma sample at a concentration calculated to slightly
exceed the concentration of the normal component. Native PAGE
electrophoresis with Western blotting showed (Fig 3B) that this
addition of latent antithrombin shifted the position of the major band
from that of -antithrombin to the slower mobility characteristic of
the dimer.
Dimer in antithrombin concentrates.
Earlier studies had shown12 that an antithrombin
concentrate commercially prepared in 1992 contained as much as 40% in
the latent form (Fig 2C). To demonstrate how this concentration of latent antithrombin could have been masked by formation of the dimer, a
sample of the concentrate was electrophoresed in SDS-PAGE (Fig 3CI)
that showed a single band compatible with the presence of a sole
component of intact antithrombin. Electrophoresis in native PAGE of the
plasma concentrate (Fig 3CII) also showed just a single major band;
however, the inclusion on the gel of a control of mixed
-antithrombin plus latent antithrombin shows that the concentrate
moves on electrophoresis not as -antithrombin but as its dimer with
latent antithrombin. A check of the current concentrate from the same
manufacturer showed on native PAGE a main band of -antithrombin,
but, in addition, a band, scanning at 36% of the total, in the
position of the dimer. This implies a presence of 18% latent antithrombin.
Dissociability, -antithrombin, heparin, and loss of
activity.
Although the mixing of latent and normal antithrombin shows that they
form an electrophoretically stable dimer, the separability of the
component molecules on heparin-Sepharose chromatography and by
isoelectric focusing show that the dimer is readily dissociable. To
assess the degree to which the transition to the latent conformation of
an abnormal antithrombin in a heterozygote may affect the inhibitory activity of normal antithrombin, the second order rate constants were
determined for inhibition of thrombin and factor Xa before and after
the addition of previously prepared latent antithrombin. The results
for factor Xa are shown in Table 1, which also tabulates the percentage loss of activity of the active inhibitory component. On
addition of equimolar proportions of latent antithrombin to active
-antithrombin, there is an 11% loss of activity, compatible with
the ready dissociability of the dimer. An assessment was also made of
the loss of activity of active -antithrombin both on addition of
equimolar proportions of latent antithrombin and at the 10:1 molar
proportion representative of that in the plasma. As Table
1 shows, there is a 21% loss of activity on the addition of latent antithrombin to an equimolar solution of -antithrombin and
a 47% loss when the proportion of latent to -antithrombin is
increased to 10:1. In each case, the loss of activity is considerably further increased in the presence of long-chain (unfractionated) heparin. In keeping with this increased affinity of association with
-antithrombin and hence decreased dissociation, Fig 3D confirms that
latent antithrombin preferentially dimerizes with -antithrombin, with the sequential addition of -antithrombin giving a displacement of -antithrombin from the preformed heterodimer.
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DISCUSSION |
The results shown here are an end-point in a 15-year study of the
conformational changes that result in the dysfunction of antithrombin.
When the study began,13 it was known that antithrombin, as
with the serpins in general, had 2 conformational forms: 1 being the
active inhibitor with an intact reactive center and the other being the
inactive cleaved form.14 At that time, many of the studies
were performed using antithrombin samples that had been stored for long
periods or purchased from biochemical suppliers or derived from
commercially fractionated plasma concentrates. Careful measurement of
such samples almost invariably showed deficits in the range of 10% to
40% between the antigenic concentration of antithrombin and its
activity. Yet, only a small proportion of the loss of activity could be
shown to be due to cleavage of the antithrombin, so clearly some other
inactive form was present. We believed that a contribution to this
deficit in activity could come from a conformational transition of
antithrombin to give an incorporation of its reactive center into the
body of the molecule, analogous to the changes that occur in the
cleaved inhibitor.15 The structural confirmation for this
shift of an intact serpin from a 5- to a 6-stranded form was
subsequently provided3 by latent PAI-1. That the same
change could occur in antithrombin was further confirmed by later
structural studies.2,5 These showed that antithrombin
spontaneously crystallized as a dimer, with 1 molecule being in the
6-stranded latent form.
Subsequently, it was demonstrated12 that the large deficit
in activity in some commercial concentrates of antithrombin was due to
the presence of up to 40% of the antithrombin in the latent conformation (Fig 2C). This transition to the inactive latent form took
place during the pasteurization of the plasma concentrates, and it was
shown that similar conditions, with incubation at 60°C in the
presence of citrate, could be used for the quantitative preparation of
latent antithrombin.9 This latent form was found to have a
clearly identifiable increase in electrophoretic mobility, as in lane 1 of Fig 1A. However, the perplexing feature was that this
characteristically fast band could not be seen in solutions in which
there was good reason to believe latent antithrombin would be present
(eg, the mother liquor from which the dimer
crystallized).16 Moreover, the plasma concentrates of
antithrombin, as the manufacturers pointed out, showed on
electrophoresis only a single band in a position compatible with that
of normal -antithrombin and no evidence of a band in the position of
latent antithrombin (Fig 3C). The possibility that these and other
similar anomalies were due to the formation of the active/latent
heterodimer was considered. However, it was excluded because of the
assumption that such a dimer would have the same slow-moving mobility
as observed with the loop-sheet linked polymers17 that are
known to form under the same conditions that produce the latent
transition (see arrows in Fig 1B).
The failure to identify the dimer and hence the difficulty in
demonstrating the presence of latent antithrombin in plasma except as a
late in vitro occurrence led to the conclusion that the latent
transition of antithrombin was of biochemical rather than biological
significance. This conclusion, and the results on which it is based,
must be revised in view of the findings here that latent antithrombin
as soon as it is formed can dimerize with active antithrombin and will
only become apparent as a free component when its concentration exceeds
50% of the total antithrombin. In particular, the demonstration that
the dimerization can occur in vivo has direct implications for the
mechanism of onset of thrombosis, particularly for heterozygous
carriers of conformationally unstable variants.7,10,18 For
our own part, the presence of undetected latent antithrombin in the
samples used as starting material in the experiments of 10 years ago
explains the otherwise puzzling discrepancies between results obtained
then15 and those in subsequent experiments years
later9,19 using freshly prepared material. Even with such
freshly prepared or short-storage samples, evidence can now be seen of
the appearance of latent antithrombin in its dimeric form (see starting
sample in Fig 1E).
Recognition and characterization of dimer.
The formation of the dimer as a precursor to the appearance of free
latent antithrombin is apparent on the prolonged incubation of
antithrombin not only at raised temperatures (Fig 1A and B), but also
at 37°C (Fig 1E). The isolated dimeric band can readily be
dissociated into equivalent proportions of and latent forms on
isoelectric focussing (Fig 1C), and heparin-Sepharose chromatography (Fig 2A) gives a separation identical to that previously obtained on
heparin-Sepharose chromatography of the dimeric crystals of antithrombin.16 The final and convincing evidence of the
constitution of the dimer comes from the independent demonstration by
our colleague Lei Jin2 that the mixing of equimolar
proportions of previously prepared -antithrombin and latent
antithrombin leads to overnight crystallization of the dimer, with
confirmation of the structure (as in
Fig 4) by x-ray diffraction.
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| Fig 4.
Ribbon depictions of current crystallographic
structures of antithrombin showing the sequential conformational
changes in antithrombin leading to the exacerbation of loss of
inhibitory activity by dimerization. (A) Initiation by irreversible
transition from 5-stranded (-sheet A) active antithrombin (left),
with insertion of the reactive site loop (red), to give the inactive
6-stranded latent form (right). The transition is accelerated at
increased body temperature particularly in the presence of
conformationally destabilizing mutations. Apposition of the 2 forms, as
shown, results in the induction of -strand conformation in the
reactive site loop of the active inhibitory molecule with immediate
linkage to the vacated strand in the C-sheet of the latent molecule.
(B) The absence of a carbohydrate sidechain at Asn135 in
-antithrombin ( - - -) at the interface of the 2 molecules
explains the preferential linkage by the latent molecule to the
-isoform. The radius of the negatively charged carbohydrate is
indicated in red on the latent molecule. (C) The crystallographic
(charge-contour) structure of the dimer complexed with the core
pentasaccharide of heparin (green) demonstrates how full-length heparin
(modelled yellow) can link the 2 molecules through a continuous
cationic (blue) site to stabilize the dimer. Crystal structures shown
are of dimers of latent and -antithrombin (PDB , ) at 2.6Å
and complexed with heparin pentasaccharide at 2.9Å (PDB ) plus the
completed (but awaiting publication) structure of dimeric
-antithrombin at 2.6Å. (Figures were prepared by T.R. Dafforn, A.M.
Lesk, and L. Jin, using MOLSCRIPT34 and
GRASP.35 Figures are adapted and reprinted from Current
Opinion in Structural Biology, Volume 8, R. W. Carrell and B. Gooptu, Conformational changes and disease Including Serpins, Prions,
and Alzheimer's, page 799, Copyright 1998, with permission from
Elsevier Science.27)
|
|
Latent transition and the dimer in vivo.
An indication that the latent transition could occur in vivo came from
the studies of a conformationally unstable antithrombin variant (T85M).
Bruce et al7 had previously performed incubations at
37°C and 41°C of both normal -antithrombin and of another unstable variant (N187D) and demonstrated the appearance of free latent
antithrombin as a late in vitro finding with the unstable variant but
found no apparent latent antithrombin in the normal control. In
retrospect, it can be seen that incubation at physiologic pH and at
37°C as well as 41°C results in a steady conversion of normal
-antithrombin to the dimeric band. This is shown here in a repeat
incubation of normal -antithrombin (Fig 1E) that closely parallels
the results obtained by Bruce et al in 1994.7
To show that the transition to latent antithrombin could occur in vivo,
antithrombin from donor plasma was isolated by heparin-Sepharose chromatography before and after incubation at 37°C for 72 hours. Incubation over this period resulted in a 15% loss of antithrombin activity with the appearance on heparin-Sepharose chromatography of a
substantially increased inactive antithrombin component (fraction 1, Fig 2B) with the electrophoretic mobilities on SDS-PAGE and native PAGE
of latent antithrombin (Fig 3A). The identity was further confirmed by
electrophoresis of the overlapping fraction 2, which gave a predominant
dimeric band (Fig 3AII). This electrophoresis also incidentally
confirms the presence10 in the control unincubated plasma
of a trace but significant band of latent antithrombin. All of these
results taken together show that the latent conversion does occur in
vivo, although with the likelihood of rapid elimination from the
circulation of this intact 6-stranded molecular form, as also occurs
with protease-complexed antithrombin.20
Evidence that latent antithrombin will form a heterodimer with active
antithrombin in the plasma is shown by the mixing of latent
antithrombin at near equimolar proportions with the active antithrombin
in donor plasma. The consequence (Fig 3B) is a quantitative shift in
the position of the main band of antithrombin from that of
-antithrombin to the slower mobility characteristic of the dimer.
The shift in mobility is slight, and the failure to identify it has
been at the cost of both experimentalists and processors of transfusion
concentrates of antithrombin. The concentrate shown in Fig 3C is
substantially a solution not of active antithrombin but of the
heterodimer. As discussed below, even small amounts of latent
antithrombin may have deleterious effects, and this unrecognized
contamination, which is still present in some current concentrates, may
have contributed to the inconsistent outcome of previous trials of
antithrombin therapy.21-23
-antithrombin, heparin, and the onset of thrombosis.
The realization that the spontaneous transition of antithrombin to the
latent conformation could be greatly accelerated by small increases in
body temperature explains the sudden onset of thrombosis, at times of
infection, observed in families with conformationally unstable
antithrombins. Nevertheless, there is still a discrepancy between the
severity and nature of the thrombotic events associated with the
conformational variants, as compared with those resulting from a simple
genetic deficiency of antithrombin. Such genetic deficiency, with a
50% deficit in plasma antithrombin, results in an increased risk of
thrombosis, but this is usually not life-threatening, particularly
before full adult life. By contrast, the presence of a conformationally
unstable antithrombin, with a much lesser deficiency of inhibitory
activity, predisposes to atypically severe thromboses, as seen, during
a bout of pneumonia, in a 10-year-old with ileo-femoral thrombosis from
a family heterozygous for an unstable variant.10 This
unusually severe pattern of thrombosis can only be partly explained by
the loss of activity of the variant antithrombin. An additional
contributory cause could be the formation of longer-chain polymers,
but, although these are known to accompany the conformational
transition of antithrombin (Fig 1B), there is no reason to believe that
such polymers are in themselves thrombogenic. The finding with an
unstable variant of nearly 10% of the plasma antithrombin in the
latent form suggests the alternative possibility of additive
inactivation, because the latent monomer links to a normal molecule to
give the heterodimer. However, these dimers are readily dissociable (Figs 1C and 2A), and, as shown here, there is only an additional 11%
loss of the inhibitory activity of the normal (-antithrombin) component in the dimer (Table 1). Even with a complete
transition of the variant antithrombin, the overall deficit in activity
is not significantly greater than that of the common genetic
deficiency. Clearly, something else is happening.
Comparisons of known structures of antithrombin suggested the
likelihood that latent antithrombin will preferentially link to the
minor -isoform of antithrombin24 that lacks a
carbohydrate sidechain situated close to the interface of the 2 molecules in the dimer (Fig 4B). Such preferential binding will have
considerable functional significance, because, although this isoform
constitutes only 5% to 10% of the total plasma antithrombin, the
greater affinity of -antithrombin for heparin25 binds it
to the endothelial wall and makes it the primary inhibitor of
thrombosis in the circulation.26 Confirmation that latent
antithrombin does preferentially link to -antithrombin is shown by
the electrophoretic competition studies in Fig 3D. The tighter linkage
of the latent form to -antithrombin is further supported by
inhibitory assays, with a doubled additive loss of activity by the
dimer with -antithrombin as compared with that with -antithrombin
(Table 1). Another recent crystallographic structure2 of the latent-active dimer shows each molecule
complexed to a 5-saccharide heparin fragment. It is apparent from this
structure (Fig 4C) that linkage of the 2 molecules in the dimer will be stabilized by the longer glycosaminoglycan templates present on the
heparans lining the microcirculation, and this is supported by the
substantially increased loss of inhibitory activity by the antithrombin
dimer in the presence of long-chain heparin (Table 1).
The potential physiological consequences of the combination of these
effects is particularly significant with -antithrombin, in which the
likely in vivo ratio could result in a 65% loss of activity of the
heparin-bound inhibitor (Table 1). This preferential loss
of -antithrombin function will significantly contribute to the
severe clinical consequences of the latent transition, because it is
the inhibitory activity of endothelial bound -antithrombin, rather
than that of the total circulating antithrombin, that is the critical
factor in the prevention of thrombosis.26
Conclusion: Thrombosis and conformational disease.
The conversion of active antithrombin to the latent form is a slow
process that in health is likely to only contribute to the natural
senescence and turnover of the protein in the plasma. However, the
acceleration of the latent transition at times of stress may be a
contributory factor in the increased risk of the thrombosis that
accompanies ill-health. The real threat is the fulminant transition of
antithrombin that can occur in carriers of unstable variants,
particularly during fever and ill health.7,10 Our study
completes, in structural detail, the sequential changes that follow
from this initial transition and that typify the conformational diseases in general.27 A feature of such conformational
diseases is not just that they result from an aberrant change in
folding of a protein, but even more so from the self-association that is a consequence of this misfolding. The striking finding here is the
extraordinary selectivity of that association. When we added latent
antithrombin to plasma, we fully expected it to form multiple
associations with a variety of plasma proteins that could fill the
vacant -strand space in latent antithrombin (Fig 4A). But, as Fig 3B
shows, linkage was almost wholly confined to that with active
antithrombin. It is this remarkable favoring of self-association and
the subsequent consequences of polymer and fibril formation that
characterizes a diverse group of degenerative diseases, including Alzheimer's dementia28 and the prion
encephalopathies.29 There is good evidence that these
conformational diseases are similarly initiated by the sequence of
changes that parallel those shown in Fig 4: with a conformational
transition and subsequent associative propagation,30 with
selective involvement of glycoforms, and with stabilization of the
formed complexes by heparin-like glycosaminoglycans.31-33
| |
ACKNOWLEDGMENT |
The authors thank Linda Butler for expert technical assistance and
Arthur Lesk, Tim Dafforn, and Lei Jin for help in the preparation of
the figures.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted June 25, 1999.
Supported by the Wellcome Trust, the British Heart Foundation, and the
EC. J.A.H. has a National Institutes of Health training fellowship (HL 0992702).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Robin W. Carrell, PhD, FRCP, Structural
Medicine, Cambridge Institute for Medical Research, Wellcome Trust/MRC
Building, Hills Road, Cambridge CB2 2XY, UK.
| |
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TOP
ABSTRACT
INTRODUCTION