(Received for publication, May 28, 1997, and in revised form, June 13, 1997)
From the Department of Haematology, University of
Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom,
the § Department of Microbiology and Immunology, Institute
of Molecular Biology, Jagiellonian University, Kraków PL-31120,
Poland, the ¶ Boston University School of Medicine, Boston,
Massachusetts 02118-2394, the
Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, Georgia 30205, and
the ** Department of Clinical Biochemistry, Christchurch Hospital,
Christchurch, New Zealand
Antithrombin, the principal plasma inhibitor of
coagulation proteinases, circulates in a form with low inhibitory
activity due to partial insertion of its reactive site loop into the
A--sheet of the molecule. Recent crystallographic structures reveal
the structural changes that occur when antithrombin is activated by the
heparin pentasaccharide, with the exception of the final changes, which
take place at the reactive center itself. Here we show that the side
chain of the P1 Arg of
-antithrombin is only
accessible to modification by the enzyme peptidylarginine
deiminase on addition of the heparin pentasaccharide, thereby
inactivating the inhibitor, whereas the natural P1 His
variant, antithrombin Glasgow, is unaffected, indicating that only the
P1 Arg becomes accessible. Furthermore, the deimination of
P1 Arg converts antithrombin to a form with 4-fold higher
affinity for the heparin pentasaccharide, similar to the affinity found
for the P1 His variant, due to a lowered dissociation rate
constant for the antithrombin-pentasaccharide complex. The results
support the proposal that antithrombin circulates in a constrained
conformation, which when released, in this study by perturbation of the
bonding of P1 Arg to the body of the molecule, allows
the reactive site loop to take up the active inhibitory conformation
with exposure of the P1 Arg.
The plasma serpin (1), antithrombin, is the major inhibitor of the serine proteinases of the coagulation network, especially thrombin and factor Xa (2). This inhibitory activity is stimulated by the complexing of antithrombin with the sulfated polysaccharide, heparin. In particular, the activity against factor Xa is mediated by a core pentasaccharide component of heparin. Antithrombin has an initial low affinity for heparin, but this immediately changes to a high affinity on initial binding, the change occurring concomitantly with the activation of inhibitory function (3).
The interaction of the heparin pentasaccharide with antithrombin and the associated mechanism of conformational activation of inhibition has recently been revealed in the crystal structure of a dimer of antithrombin complexed with the pentasaccharide (4). Linkage between the two antithrombin molecules in the dimer directly involves the reactive site loop of the inhibitory component, with a consequent constraint in the movement at its reactive site. Hence, while showing the commencement of the movement of the reactive site loop that results in activation, the crystal structure does not show the change that occurs at the reactive center itself.
There is, however, a good model of the likely unconstrained active
conformation of the reactive site loop of antithrombin. This is
provided by the structure of the closely related serpin, 1-antitrypsin (5), in which the loop is fixed in the
optimal canonical inhibitory conformation present in all other families of serine proteinase inhibitors. The transition of antithrombin to this
conformation would require a shift of the side chain of the
P1 arginine from an internally oriented position where it is hydrogen-bonded to the body of the molecule (6), to an external and
exposed orientation, as found for the P1 residue of
1-antitrypsin. Furthermore, the perturbation of the
bonding of the side chain of P1 Arg in antithrombin, as
occurs in the natural variant antithrombin Glasgow (P1 Arg
to His), is accompanied by a shift from low to high affinity binding of
heparin (7). This has led to the proposal (6-8) that antithrombin is,
in part, held in a conformation with initial low heparin affinity by
the constraints on reactive site loop movement imposed by the internal
bonding at the side chain of P1 Arg.
To check these proposals, normal antithrombin and the Glasgow P1 His variant were each incubated, with and without the heparin pentasaccharide, with the enzyme peptidylarginine deiminase (hereafter referred to as deiminase) (EC. 3.5.3.15), which acts on the side chain of arginine residues to produce the amino acid citrulline. We show that this enzyme interacts with the P1 Arg side chain, which is only vulnerable to modification in the presence of the heparin pentasaccharide. The deimination of the P1 Arg converts antithrombin to a form with higher affinity for the heparin pentasaccharide.
Materials
Peptidylarginine deiminase was purchased from PanVera Corp., Madison, WI. Heparin pentasaccharide was a gift from Maurice Petitou, Sanofi Recherche, Toulouse, France. Thrombin was a gift from Professor Stuart Stone, Monash University, Melbourne, Australia. Human neutrophil elastase was prepared as described previously (9). Factor Xa was purchased from Boehringer Mannheim (Lewes, UK). The chromogenic substrate S22381 was purchased from Quadratech (Epsom, UK). Plasma from a patient containing the variant antithrombin Glasgow was a gift from Dr. Isobel Walker, Glasgow, UK. Ampholine PAGplates for isoelectric focusing and Superdex 75 were purchased from Pharmacia Biotech Inc. (St. Albans, UK).
Methods
Purification of AntithrombinsNormal -antithrombin was
purified from time-expired, slow bleed units of human plasma as
described previously (10), using a combination of heparin-Sepharose and
anion exchange chromatography. Antithrombin Glasgow was purified as
described (7), with the high affinity variant eluting later on the salt
gradient from heparin-Sepharose, distinct from the peak for normal
-antithrombin. The forms of antithrombin were pure as judged by PAGE
(data not shown).
Antithrombin (10 µM) was incubated with the deiminase at 37 °C at a molar ratio of 50:1 (inhibitor:enzyme) in 100 mM Tris-HCl, 5 mM CaCl2, pH 7.4, for up to 16 h in the presence or absence of heparin pentasaccharide (50 µM). The reaction was stopped using 50 mM EDTA. The deiminated antithrombin was separated from both the heparin pentasaccharide and the deiminase using gel filtration chromatography on a Superdex-75 column (1.6 × 35 cm = 70 ml) in 50 mM Tris-HCl, 2 M NaCl, 10 mM citrate, 5 mM EDTA, pH 7.4. The eluted antithrombin was concentrated using centrifugal ultrafiltration on a 10-kDa molecular mass cutoff device and buffer-exchanged into 50 mM Tris-HCl, 10 mM citrate, 5 mM EDTA, pH 7.4.
Cleavage of AntithrombinAntithrombin (control and deiminated) at 4 mg/ml in 20 mM Tris-HCl, pH 8.0, was incubated with heparin pentasaccharide (50 µM) for 5 min at 37 °C, following which 120 ng of human neutrophil elastase was added. The mixture was allowed to incubate for 1 h at 37 °C, following which an equal volume of reducing SDS-PAGE sample buffer was added and the whole boiled for 5 min. SDS-PAGE was run on 10-20% Tris-Tricine gels (11), following which proteins were electroblotted onto polyvinylidene difluoride membranes (12). The band corresponding to the low molecular mass subunit of cleaved antithrombin was sequenced by Alta Biosciences, Birmingham, UK.
Activity MeasurementsAntithrombin (200 nM) was incubated with thrombin (5 nM) for 30 min at 37 °C, following which residual activity was measured against 40 µM S2238 substrate, all in 20 mM NaH2PO4, 250 mM NaCl, 0.1 mM EDTA, 0.1% (m/v) polyethylene glycol 8000, pH 7.4 (buffer with ionic strength of 0.3 (I0.3 buffer)). The time of incubation and concentration of antithrombin were chosen to ensure that normally active antithrombin should inactivate thrombin to completion at the end of the incubation period. This criterion was seen to be fulfilled for control samples.
Determination of Equilibrium Binding Constants and Rapid Kinetics for the Interaction between Antithrombin and Heparin PentasaccharideThe equilibrium binding constants for the interaction between the antithrombins and the heparin pentasaccharide were determined by fluorometric titrations in I0.3 buffer as described (13). The rapid kinetics of heparin pentasaccharide binding to antithrombin in I0.3 buffer were determined using stopped flow fluorometry essentially as described (3).
Electrophoresis and Isoelectric FocusingPolyacrylamide gel electrophoresis was carried out on 7.5% Tris/glycine gels (14). Isoelectric focusing was carried out on precast Ampholine PAGplates (Pharmacia) using ampholytes in the range from pH 4 to pH 6.5.
Incubation of antithrombin with deiminase at molar ratios of enzyme:inhibitor greater than 1:50, in the presence of heparin pentasaccharide, resulted in complete inactivation of antithrombin, as judged by its ability to inhibit thrombin under defined assay conditions, within 2 h. Lower enzyme:inhibitor ratios such as 1:100 and 1:200 resulted in less inactivation and longer times of incubation were required. No inactivation was found in the absence of heparin pentasaccharide.
The site and number of residues of arginine modified by deiminase in
-antithrombin was deduced by comparison with the effects of
deiminase on antithrombin Glasgow, a natural variant of antithrombin with a mutation of P1 Arg to His. Antithrombin, in the
presence of heparin pentasaccharide, was modified such that its
mobility in isoelectric focusing gels became more anodal, while
antithrombin Glasgow was not changed in its mobility (Fig.
1). Since no Arg residues were therefore
modified in antithrombin Glasgow and as it only differs from normal
antithrombin at the P1 position, these data strongly
indicate that
-antithrombin is modified at a single site only, which
is likely to be the P1 Arg. The site of modification was
further examined using cleaved deiminase-treated antithrombin (deiminated antithrombin) and control
-antithrombin. Each of these
was cleaved within their reactive site loops, using human neutrophil
elastase (15), thus generating the C-terminal fragment of antithrombin,
which was electroblotted to polyvinylidene difluoride after separation
on SDS-PAGE. This permitted N-terminal sequencing at the reactive site
of antithrombin, since the sequence from the cleavage point of human
neutrophil elastase should begin: AVIAGRSLNPNR, with the
R representing the reactive site P1 Arg.
N-terminal sequencing of control
-antithrombin showed the expected
sequence, but sequencing of deiminated antithrombin revealed that the
reactive site arginine had indeed been changed to a citrulline residue.
Arg-399 was not altered, however. This confirmed the earlier
hypothesis, generated by comparison with antithrombin Glasgow, that the
reactive site residue had been modified and all the evidence indicates
this is the only residue altered.
Heparin Affinity of Deiminase-modified Antithrombin
Since it
was previously shown that antithrombin Glasgow, which has a
substitution of its P1 Arg residue, had increased affinity for heparin, as judged by heparin-Sepharose affinity
chromatography (7), the affinity of both antithrombin Glasgow and
deiminated antithrombin were measured in comparison with normal
-antithrombin. This was carried out using fluorometric
titrations against the heparin pentasaccharide and revealed that
both antithrombin Glasgow and deiminated antithrombin had higher
affinity for the pentasaccharide than
-antithrombin, the gain being
about 4.4-fold in both cases (Table
I).
|
The reason for the higher affinity binding of the pentasaccharide was
investigated by determining the rapid kinetics of the interaction
between the antithrombins and the heparin. It has been shown previously
(3, 16) that antithrombin binds heparin in a two-step procedure, which
may be evaluated using stopped flow fluorometry to assess the speed at
which the antithrombin increases its intrinsic fluorescence upon
binding heparin. When the kinetics of heparin pentasaccharide binding
to antithrombin Glasgow were evaluated in this way, it was found that
the increase in its affinity for pentasaccharide was due to a lower
dissociation rate constant for the interaction (Fig.
2 and Table I). This is most accurately
and easily measured by stopped flow experiments at low heparin
concentrations at ionic strengths of 0.3 (3). Thus, this technique was
applied directly to the deiminated antithrombin which was isolated in
limited quantities, and it was shown to have the same profile as
antithrombin Glasgow, with its dissociation rate constant lowered to a
similar degree, while its association rate constant was unaffected, as
for antithrombin Glasgow (Fig. 2 and Table I).
The results of this study answer two central questions with
respect to the heparin activation of antithrombin. First, it was shown
that the side chain of P1 Arg only becomes available for modification by the deiminase after the addition of the
pentasaccharide. This is in keeping with the prediction (5) that the
release of the reactive site loop from its insertion into the
A--sheet will allow the loop to adopt the canonical inhibitory
conformation found in
1-antitrypsin (Fig.
3). There is clear evidence that binding
of the pentasaccharide results in an exposure of P1 Arg in
a uniquely accessible position, the accessibility of this arginine side
chain paralleling that of the P1 Arg of soybean trypsin
inhibitor, which is fixed in the exposed canonical inhibitory
conformation and is similarly uniquely available for deiminase
modification (17). Confirmation that it is the P1 Arg that
is modified by the deiminase in the pentasaccharide-activated
antithrombin, is provided by amino acid sequencing of the reactive site
loop after deliberate cleavage with human neutrophil elastase, which
shows that there is a citrulline at position P1. The
evidence strongly indicates that only the P1 Arg is
affected, as shown in parallel experiments with antithrombin Glasgow,
which varies only in having P1 His and does not change in
electrophoretic mobility on incubation with the deiminase, with or
without added pentasaccharide.
The concept that the conformation of antithrombin was constrained with
respect to inhibitory activity and that heparin released this
constraint to give optimal activity as inherently present in
1-antitrypsin arose from the finding that the mutant
1-antitrypsin Pittsburgh, with an arginine at
P1, was a potent thrombin inhibitor (18). The reactive site
loop of antithrombin becomes more exposed in the presence of heparin,
as shown by its increased susceptibility to proteinase cleavage (15,
19), by NMR studies (20), and fluorescence studies (21). All of these
studies fit with a reactive loop rearrangement and activation as
modeled in Fig. 3.
The second question we have addressed relates to these concomitant
changes in activation and heparin affinity; specifically whether the
linkage of the side chain of P1 Arg to the body of the
molecule is an inherent contributor to the structural constraints that
limit the inhibitory activity and heparin affinity of circulating antithrombin. This was proposed based on the findings that
antithrombins in which the P1 Arg has been replaced by a
His (7, 22) or Pro (8) had an increased heparin affinity. This view was
strengthened by the crystallographic finding of the internal
orientation and hydrogen bonding of the P1 Arg to the body
of the molecule (6). If this bonding is a significant contributor to
the structural constraints of inhibitory antithrombin, then
modification of the side chain should release the constraint with a
predictable increase in heparin affinity, as is indeed demonstrated
here. The P1-deiminated antithrombin had a 4-fold increase
in pentasaccharide affinity, due to a lowered dissociation constant,
paralleling that present in antithrombin Glasgow, for which we have
also shown (but not published) a similar 3-fold increased affinity for
full-length heparin. The current structures of
pentasaccharide-complexed active and latent antithrombins (4) show the
conformational basis for this increased affinity. The pentasaccharide
induces a series of changes at its binding site on antithrombin, with a
tilting of the D-helix, a shift at the commencement of the A-helix and the induction of a new 2-turn helix. These changes bring a series of
side chains into a stable alignment for linkage to the pentasaccharide. This conformational change at the heparin binding site is accompanied by a transition at 30 Å distance, which releases the reactive site
loop from the A--sheet, the two changes being linked by a series of
intervening conformational shifts. Reactive loop expulsion is therefore
linked to the transition to the high affinity state for heparin. A
recent mutation of antithrombin, which stabilized the molecule in a
reactive loop expelled form, had high affinity for heparin (21), most
likely because it tended to be stabilized in the high affinity state.
We propose that a similar mechanism occurs for antithrombin Glasgow and
deiminated antithrombin, stabilizing the molecule into a high affinity
state and thus lowering the dissociation constant for heparin
binding.
Taken together, the recent series of structures of antithrombin (4, 6,
23, 24), and the data presented here, allow an overall view of the
mechanism of heparin activation to be formed. Tempting though it is to
describe this in terms of a sequential series of changes radiating from
the heparin binding site, it is better to consider the transition as
the consequence of the release of the broader constraints that hold the
molecule in its low activity conformation. These constraints include:
the partial insertion of the reactive site loop into the A--sheet,
the stability of the partially opened A-
-sheet, and, as shown here,
the bonding of P1 Arg to the body of the molecule.
Perturbation of any of these constraints, as has already been
demonstrated with loop insertion (21) and A-
-sheet stability (25),
will result in the reversion toward the unconstrained, high heparin
affinity conformation. Thus the addition of heparin is just a special
example of an induced loss of constraint, which results in reversion to a relaxed-loop inhibitory conformation. The initial low affinity of
antithrombin for the pentasaccharide reflects the equilibrium between
the unconstrained loop-expelled conformation, in which the
pentasaccharide is tightly bound, and the constrained loop-inserted conformation, with more ready dissociation of the pentasaccharide. It
follows from this that the loss of a structural constraint, as with
perturbations of P1 Arg, will favor the loop-expelled, unconstrained conformation with the associated decrease in the pentasaccharide dissociation rate constant, as observed here.
All of these findings fit with the deductions, from the initial finding
of a P1 Arg mutant of 1-antitrypsin (18),
that
1-antitrypsin acts as a model for heparin activated
antithrombin. This earlier finding, that P1
Arg-
1-antitrypsin had a thrombin inhibitory activity
greater than antithrombin in the absence of the pentasaccharide, has
been reinforced by a recent study of Hopkins and
colleagues.2 They have shown
that a more extensive mutation of the reactive site loop of
1-antitrypsin to give the reactive center
P4-P3
sequence present in antithrombin,
IAGRSLN, results in an increase in inhibitory activity against factor
Xa, an enzyme which is more sensitive to the reactive site loop
conformation of antithrombin than thrombin, approximating to that of
pentasaccharide activated antithrombin. Both functionally and
structurally the model shown in Fig. 3 therefore provides a satisfying
explanation of the changes that occur at the reactive
center of antithrombin on heparin activation. In particular, these
changes involve a shift at the heparin binding site to give the high
affinity conformation with a concomitant breaking of the conformational
constraint contributed by the linkage of P1 Arg to the body
of the molecule, together with a movement of the side chain of
P1 Arg to an exposed external orientation, likely to
resemble the P1 canonical orientation seen in
1-antitrypsin.
We thank Dr. Isobel Walker for her gift of plasma containing the antithrombin Glasgow variant.