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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4572-4580
Rapid Activation of Protein C by Factor Xa and Thrombin in the
Presence of Polyanionic Compounds
By
Alireza R. Rezaie
From the Cardiovascular Biology Research Program, Oklahoma Medical
Research Foundation, Oklahoma City, OK.
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ABSTRACT |
A recent study indicated that negatively charged substances such as
heparin and dextran sulfate accelerate thrombin activation of
coagulation factor XI by a template mechanism. Because the serine
proteinase of the natural anticoagulant pathway, activated protein C,
can bind heparin, it was reasonable to think that these compounds may
also bind protein C (PC) and accelerate its activation by thrombin or
other heparin binding plasma serine proteinases by a similar mechanism.
To test this, PC activation by thrombin and factor Xa (fXa) was studied
in the presence of these polysaccharides. With thrombin in the absence
of thrombomodulin (TM), these polysaccharides markedly reduced the
Km for PC and Gla-domainless PC (GDPC) activation in the
presence of Ca2+. With TM containing chondroitin sulfate,
heparin did not influence PC activation by thrombin, but with TM
lacking chondroitin sulfate, the characteristic high-affinity PC
interaction at low Ca2+ (~50 to 100 µmol/L) was
largely eliminated by heparin. In EDTA, heparin enhanced thrombin
activation of GDPC by reducing the Km, but it inhibited PC
activation by increasing the Km. PC activation in EDTA was
insensitive to the presence of heparin if the exosite 2 mutant,
R93,97,101A thrombin, was used for activation. These results suggest
that, when the Gla-domain of PC is not fully stabilized by
Ca2+, it interacts with the anion binding exosite 2 of
thrombin and that heparin binding to this site prevents this
interaction. Additional studies indicated that, in the presence of
phospholipid vesicles, heparin and dextran sulfate dramatically
accelerate PC activation by fXa by also reducing the Km.
Interestingly, on phospholipids containing 40%
phosphatidylethanolamine, the activation rate of near physiological PC
concentrations (~80 nmol/L) by fXa in the presence of dextran sulfate
was nearly comparable to that observed by the thrombin-TM complex. The
biochemical and potential therapeutical ramifications of these findings
are discussed.
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INTRODUCTION |
FACTOR Xa (fXa) AND THROMBIN are
trypsin-like serine proteinase that play pivotal roles in the clotting
cascad.1 fXa is the enzyme of the prothrombinase complex
that activates prothrombin to generate thrombin.2 Thrombin
plays a dual function in the coagulation cascade. As a coagulant
enzyme, thrombin activates fibrinogen, platelets, and fV, fVIII, fIX,
fXI, and fXIII.3-5 As an anticoagulant enzyme, thrombin
binds to thrombomodulin (TM) and activates the vitamin
K-dependent serine proteinase zymogen, protein C (PC) to activated
protein C (APC). APC downregulates the coagulation cascade by
inactivating the procoagulant cofactors Va and VIIIa by limited
proteolysis.6,7 PC activation by the thrombin-TM complex
occurs optimally on membrane surfaces, but binding of the
epidermal-like growth factor domains 4-6 of TM (TM4-6) to the anion
binding exosite 1 of thrombin is sufficient to change the
macromolecular specificity of thrombin, and accounts for most of the
cofactor function of TM for PC activation.8,9 fXa is not
known to play a significant role in PC activation. Although there is a
report demonstrating that bovine fXa can bind rabbit TM and rapidly
activate bovine PC, the result has not been confirmed for human fXa in
a homologous system.10
Heparin is a naturally occurring glycosaminoglycan commonly used as an
anticoagulant drug. It is believed that the primary antithrombotic
effect of heparin is through acceleration of the inhibition of the
coagulation serine proteinases, including thrombin, fIXa, fXa, fXIa,
fXIIa, and kallikrein by antithrombin.11,12 Results from
several laboratories suggest that heparin accelerates antithrombin
inhibition of thrombin, fIXa, and fXIa by a bridging mechanism, whereas
in inhibition of fXa, kallikrein, and possibly fXIIa, a heparin induced
conformational change in the reactive site loop of antithrombin
accounts for the acceleration.12-14
In a recent study, it was demonstrated that heparin and a similar
negatively charged substance, dextran sulfate, dramatically enhanced
the thrombin activation of fXI by a template mechanism, analogous to
acceleration of thrombin inhibition by antithrombin.15,16 This result suggested that heparin-like compounds may facilitate the
assembly of other enzyme-substrate complexes in plasma for rapid
activation if both proteins contained a binding site for heparin. The
anion binding exosite 2 of thrombin, located above the active site
pocket, contains 11 basic residues that are thought to constitute the
heparin binding site of thrombin.17 Structural data
indicate that 7 of the basic residues of this region are conserved in
fXa,18 so that fXa can also bind heparin. The recent crystal structure of activated Gla-domainless PC (GDPC) also showed that PC contains several basic residues that are clustered in the same
three-dimensional location analogous to the anion binding exosite 1 of
thrombin.19 It is also known that heparin accelerates APC
inhibition by the serpin, PC inhibitor, by a template
mechanism.20 Based on these observations, it was
rationalized that heparin-like compounds could also accelerate PC
activation by thrombin and fXa, if the heparin binding site on PC is
not masked by the noncatalytic domain.
In the present study, this possibility was tested by measuring the
initial rates of PC activation by thrombin and fXa in the absence or
presence of heparin and dextran sulfate. Kinetic analysis indicated
that these polysaccharides markedly accelerate PC and GDPC activation
by both thrombin and fXa in the presence of Ca2+ by
reducing the Km. Kinetic analysis in the presence of
oligosaccharides containing 6 to 64 saccharide units indicated that a
chain length with a minimum of 14 to 18 saccharide units was required
for the acceleration of PC activation. In EDTA, the polysaccharides
accelerated GDPC activation by thrombin, but they inhibited full-length
PC activation by thrombin and eliminated the characteristic higher rate
of PC activation by the thrombin-TM complex lacking chondroitin sulfate
in low Ca2+ buffer.21,22 These findings are
discussed in the context of a model for PC activation under conditions
in which the Gla-domain of PC is not stabilized by Ca2+
ions.
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MATERIALS AND METHODS |
Materials.
Plasma-derived human PC was purchased (Haematologic Technologies
Inc, Essex Junction, VT) or purified from plasma as
described.23 Human plasma fXa,24 bovine plasma
antithrombin,25 human recombinant GDPC,26
rabbit TM,27 human recombinant TM4-6,26 and
human recombinant soluble TM containing or lacking the chondroitin
sulfate moiety28 were prepared as described previously.
Phospholipid vesicles containing 80% phosphatidylcholine and 20%
phosphatidylserine (PC/PS) or 40% phosphatidylcholine, 20%
phosphatidylserine, and 40% phosphatidylethanolamine (PC/PS/PE) were
prepared as described previously.29 Unfractionated heparin
from porcine intestinal mucosa, sodium salt (169.2 USP U/mg), dextran
sulfate with an average molecular weight of 8,000, and polybrene were
purchased from Sigma (St Louis, MO). Spectrozyme PCa
(SpPCa) was purchased from American Diagnostica (Greenwich, CT). The
oligosaccharides, ranging in size from 6 to 18 saccharide units, were
generous gifts from Dr Ingemar Björk (Swedish University of
Agricultural Sciences, Uppsala, Sweden) and high-affinity heparin
fragments with 22 to 64 saccharide units were generous gifts from Dr
Steven Olson (University of Illinois-Chicago, Chicago, IL).
PC activation.
The initial rate of PC or GDPC (0.75 µmol/L) activation by thrombin
(5 nmol/L) was studied as a function of different dextran sulfate or
heparin concentrations (0 to 500 µg/mL). The activation reactions
were performed in 96-well plates in 0.1 mol/L NaCl, 0.02 mol/L
Tris-HCl, pH 7.5 (TBS), buffer containing 1 mg/mL bovine serum albumin
(BSA) and 2.5 mmol/L Ca2+ or 100 µmol/L EDTA
for 10 minutes at room temperature, after which 50 µg/mL antithrombin
and 1 U/mL (~5.9 µg/mL) heparin was added to each well to inhibit
thrombin activity. At this concentration of antithrombin, the activity
of thrombin was rapidly inhibited, whereas the amidolytic activity of
APC remained stable for more than 15 minutes. The amidolytic activity
of APC in the activation reactions was monitored by hydrolysis of 200 µmol/L SpPCa in TBS buffer containing 1 mg/mL BSA. The rate of
hydrolysis was measured at 405 nm at room temperature in a Vmax kinetic
plate reader (Molecular Devices, Menlo Park, CA). The concentrations of
active PC derivatives in reaction mixtures were determined by reference
to a standard curve that was prepared by total activation of PC or GDPC
at the time of each experiment. This was accomplished by total
activation of 1 µmol/L of each PC derivative with 10 nmol/L thrombin
in complex with 100 nmol/L TM and 2.5 mmol/L Ca2+ for 90 minutes at 37°C. Under these experimental conditions, all PC
zymogen was completely activated in less than 30 minutes. When a
kinetic analysis was performed to determine values for Km
and kcat of PC activation, the initial rates of activation were measured as a function of PC or GDPC concentration with 20 nmol/L
thrombin in the presence of 2.5 mmol/L Ca2+ or 10 nmol/L
thrombin in the presence of 100 µmol/L EDTA for 10 minutes at room
temperature. For the Km and kcat analysis in the presence of 25 µg/mL heparin, the activation conditions were the
same, except that 2 to 5 nmol/L thrombin was used in the reactions.
PC activation in the presence of TM was performed essentially as
described above. In this case, the reactions were performed with 5 mmol/L Ca2+ and 1 nmol/L thrombin in complex with 200 nmol/L TM or TM4-6. Similarly, the Ca2+ dependence of PC
activation in the presence of TM was studied with 1 µmol/L PC that
had been previously dialyzed against Chelex-treated (Bio-Rad, Hercules,
CA) TBS buffer, 1 nmol/L thrombin, 200 nmol/L TM
containing or lacking chondroitin sulfate, and Ca2+
concentrations of 0 to 5 mmol/L. The reactions continued for 15 minutes
at room temperature and the initial rates of activation were determined
as described above.
PC activation by fXa.
The initial rate of 0.75 µmol/L human PC activation on 100 µg/mL
PC/PS or PC/PS/PE phospholipid vesicles by 5 nmol/L human fXa in the
presence of 25 µg/mL heparin or dextran sulfate or by 50 nmol/L fXa
without polysaccharides was determined in TBS buffer containing 1 mg/mL
BSA, 0.1% PEG 8000, and 5 mmol/L Ca2+. After inhibition of
fXa activity with 100 µg/mL antithrombin and 5 U/mL heparin, the
activation rates were measured by the rate of hydrolysis of SpPCa as
described above. For a detailed kinetic analysis in the presence of 100 µg/mL phospholipid vesicles, PC (0.1 to 12.5 µmol/L) was incubated
with fXa (5 nmol/L) in the presence of 25 µg/mL heparin or dextran
sulfate in TBS buffer containing 1 mg/mL BSA, 0.1% PEG 8000, and 5 mmol/L Ca2+ for 10 minutes at room temperature. The
Km and kcat values were determined from the
initial rates of PC activation as described above.
Data analysis.
The Km and kcat values for PC and GDPC
activation by thrombin were calculated from the Michaelis-Menten
equation using Enzfitter computer program (R.J. Leatherbarrow,
Elsevier, Biosoft, London, UK). All data presented are the
average of at least two to three independent measurements ± SD.
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RESULTS |
The effect of dextran sulfate and heparin on PC or (GDPC) activation by
thrombin was studied both in the presence of Ca2+ and EDTA.
In the presence of Ca2+ and an optimum concentration of
heparin or dextran sulfate (~20 to 25 µg/mL), the activation rate
of PC by thrombin was enhanced approximately fivefold by heparin and
approximately 38-fold by dextran sulfate
(Fig 1A). In contrast to acceleration of
activation in Ca2+, the polysaccharides inhibited PC
activation by thrombin in EDTA (Fig 1B). However, with GDPC, the
polysaccharides accelerated the activation rates both in
Ca2+ and EDTA. The acceleration of GDPC activation in
Ca2+ was more efficient than in EDTA. Under the same
experimental conditions as in PC activation, at the optimum
concentration of the polysaccharides (~20 to 25 µg/mL), the GDPC
activation rate was enhanced approximately 20-fold by heparin and
approximately 45-fold by dextran sulfate in the presence of
Ca2+ (Fig 2A). However, in the
presence of EDTA, heparin and dextran sulfate accelerated GDPC
activation approximately 10-fold and approximately twofold,
respectively (Fig 2B). The optimum concentration of the polysaccharides
in the presence of EDTA was approximately 5 µg/mL. These results
suggest that Ca2+ binding on GDPC is required for optimal
acceleration of activation and that the Gla-domain of PC is responsible
for the polysaccharides inhibition of PC activation by thrombin in
EDTA.
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| Fig 1.
Heparin and dextran sulfate concentration dependence of
PC activation by thrombin. (A) Human plasma-derived PC (0.75 µmol/L) was incubated with thrombin (5 nmol/L) in TBS buffer containing 2.5 mmol/L Ca2+, 1 mg/mL BSA, 0.1% PEG 8000, and indicated
concentrations of heparin ( ) or dextran sulfate ( ) at room
temperature for 10 minutes. After inactivation of thrombin activity by
antithrombin, the initial rate of PC activation was measured by an
amidolytic activity assay using SpPCa as described under the Materials
and Methods. Less than 15% substrate was activated in all reactions. (B) The same as (A), except that TBS buffer contained 100 µmol/L EDTA, instead of Ca2+.
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| Fig 2.
Heparin and dextran sulfate concentration dependence of
GDPC activation by thrombin. (A) Recombinant GDPC (0.75 µmol/L) was incubated with thrombin (5 nmol/L) in TBS buffer containing 2.5 mmol/L
Ca2+, 1 mg/mL BSA, 0.1% PEG 8000, and indicated
concentrations of heparin () or dextran sulfate () at room
temperature for 10 minutes. After inactivation of thrombin activity by
antithrombin, the initial rate of PC activation was measured by an
amidolytic activity assay using SpPCa as described under the Materials
and Methods. Less than 15% substrate was activated in all reactions. (B) The same as (A), except that TBS buffer contained 100 µmol/L EDTA, instead of Ca2+.
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It was previously demonstrated that, at saturating Ca2+
(>500 µmol/L), normal PC and GDPC, whether prepared by
enzymatic30 or by the recombinant DNA
methods,26 are activated by thrombin with similar kinetic
parameters in solution. It is also known that, in the absence of TM, PC
or GDPC activation by thrombin is accelerated by EDTA, but GDPC is
activated much slower than wild-type PC in EDTA.30 The
kinetic basis for the lower rate of GDPC activation by thrombin in the
absence of Ca2+ has been demonstrated to be primarily due
to elevated Km of GDPC for thrombin in the
reactions.30 The observation that the polysaccharides in
EDTA inhibited PC activation but not GDPC activation suggests that the
lower Km of thrombin for PC in the absence of
Ca2+ might be due to binding of the acidic Gla-domain of PC
to the heparin binding exosite of thrombin and that the binding of
heparin to this site prevents this interaction.
To test the hypothesis that binding of the polysaccharides to the
heparin binding exosite 2 of thrombin is responsible for the
acceleration of PC activation in the presence of Ca2+ and
inhibition of the reaction in the presence of EDTA, the initial rates
of PC activation were measured with the anion binding exosite 2 mutant
R93,97,101A thrombin, which was previously shown to be unable to bind
heparin.31 In the presence of EDTA, heparin neither inhibited full-length PC activation (Fig
3A) nor accelerated GDPC activation by this mutant thrombin (Fig 3B).
This result supports the hypothesis that the Gla-domain of PC and
heparin are interacting with exosite 2 of thrombin in EDTA. In the
presence of Ca2+, heparin accelerated activation of both PC
derivatives by the mutant thrombin approximately twofold (data not
shown). The optimum heparin concentration for acceleration of reaction
in Ca2+ was elevated approximately 10-fold with the exosite
2 mutant of thrombin. This result is consistent with the previous
observation that heparin accelerated the inactivation rate of this
mutant by antithrombin approximately twofold and that the optimum
heparin concentration was also elevated approximately
10-fold.31 This result also suggests that Ca2+
binding to GDPC is required for acceleration of PC activation by
heparin. Dextran sulfate in the presence of Ca2+
accelerated PC or GDPC activation rate by the mutant approximately 15-fold, ie, approximately 2.5-fold to threefold lower than that by
thrombin (data not shown). The optimum dextran sulfate concentration for acceleration of PC activation by the mutant was similar to that of
wild-type thrombin (~25 µg/mL).
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| Fig 3.
Initial rate of plasma PC and recombinant GDPC activation
by the exosite 2 mutant thrombin R93,97,101A in the presence of EDTA.
The initial rate of PC (A) or GDPC (B) activation was measured with 10 nmol/L thrombin mutant in the presence ( ) or absence ( ) of
heparin in TBS buffer containing 100 µmol/L EDTA, 1 mg/mL BSA, and
0.1% PEG 8000. After 15 minutes of incubation at room temperature, the
thrombin activity was inhibited by antithrombin and the rate of PC
activation was determined by an amidolytic activity assay as described
under the Materials and Methods. Less than 5% PC was activated at all
substrate concentrations.
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To determine whether dextran sulfate uses anion binding exosite 1 or
whether relatively efficient acceleration of PC or GDPC activation by
R93,97,101A thrombin in the presence of Ca2+ is due to
ability of dextran sulfate to recognize the basic Arg and Lys residues
of exosite 2 with a specificity that might be different from that of
heparin, PC activation by the thrombin-TM4-6 complex was studied in the
absence and presence of dextran sulfate. Dextran sulfate (10 µg/mL = 1.25 µmol/L) did not compete with the TM4-6 (0.78 to 50 nmol/L) for
binding on the anion binding exosite 1, because the Kd(app)
for the thrombin-TM4-6 complex was determined to be 7.6 nmol/L in the
absence and 6.4 nmol/L in the presence of dextran sulfate. These
results, together with the observation that there was an approximately
2.5-fold to threefold decrease in dextran sulfate acceleration of PC or
GDPC activation by the mutant thrombin (see above), strongly suggest
that dextran sulfate binds on the same heparin binding exosite 2, but
that the specificity determinants for binding of these polysaccharides on this site are different. This is consistent with a previous study
that the chondroitin sulfate moiety of TM binds on exosite 2 with a
specificity that is also different from that of heparin.32 Furthermore, it has been demonstrated that dextran sulfate accelerates thrombin inhibition by antithrombin by a template mechanism similar to
heparin,33 but the acceleration rate by dextran sulfate is approximately 10-fold lower than that of low-affinity heparin. Taken
together, therefore, these results suggest that these polysaccharides bind to exosite 2 of thrombin with some degree of specificity and that
this site is not simply a cationic region that interacts nonspecifically with all anionic polymers.
Heparin effect on PC activation by the thrombin-TM complex.
The effect of heparin on PC activation by thrombin in complex with the
TM forms containing or lacking the chondroitin sulfate moiety was
determined. It has been shown previously that the
Ca2+-dependence of PC activation by thrombin in complex
with TM containing or lacking the chondroitin sulfate moiety
differs.21,22 In the presence of TM lacking chondroitin
sulfate, PC activation exhibits a distinct optimum reaching to a
maximum at approximately 100 µmol/L Ca2+, with a
considerable decrease occurring at physiological Ca2+
concentrations. In contrast, the Ca2+-dependence of PC
activation by thrombin in complex with TM containing chondroitin
sulfate is a simple hyperbolic relationship, reaching saturation at
approximately 500 µmol/L Ca2+.21,22 With TM
containing chondroitin sulfate, heparin did not influence PC activation
by thrombin (data not shown), but with TM lacking chondroitin sulfate,
the characteristic peak rate of PC activation at low Ca2+
was largely abolished by heparin (Fig 4).
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| Fig 4.
The Ca2+-dependence of PC activation by the
thrombin-soluble TM complex containing or lacking chondroitin sulfate.
Human PC (1 µmol/L) was activated by thrombin (1 nmol/L) in the
presence of recombinant soluble TM (200 nmol/L) lacking chondroitin
sulfate in the absence () or presence () of approximately 0.8 µmol/L heparin in TBS buffer containing 1 mg/mL BSA, 0.1% PEG 8000, and indicated concentrations of Ca2+. After 15 minutes of
activation at room temperature, the thrombin activity was inhibited by
antithrombin and the amount of PC generated was determined in an
amidolytic activity assay described under the Materials and Methods. PC
activation by the thrombin-TM complex containing chondroitin sulfate
() under the same experimental conditions is also shown.
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PC activation on the phospholipid vesicles.
To examine whether the polysaccharides can also accelerate PC
activation on membrane surfaces, the initial rate of PC activation by
thrombin was determined in the presence of 100 µg/mL phospholipid vesicles and 5 mmol/L Ca2+. Heparin accelerated the PC
activation rate by thrombin approximately fivefold in the presence of
PC/PS and approximately 10-fold in the presence of PC/PS/PE vesicles
(Fig 5A). The acceleration of PC activation
by dextran sulfate in the presence of PE containing phospholipids
approached approximately 45-fold (Fig 5B). In general, PC activation
rate by thrombin on PE containing phospholipids was slightly higher
(<2-fold) than on PC/PS vesicles. In the presence of heparin or
dextran sulfate however, thrombin activated PC twofold to threefold
more efficiently on PE containing phospholipids (Fig 5). It is
previously shown that the physiological function of activated PC (fVa
inactivation) is improved on PE containing phospholipids.29 The results here now suggest that PC is also activated more efficiently on this type of phospholipids.
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| Fig 5.
Initial rate of plasma PC activation by thrombin on
phospholipids in the presence and absence of the polysaccharides. (A) PC (0.75 µmol/L) was incubated with 5 nmol/L thrombin on 100 µg/mL PC/PS (, ) or PC/PS/PE (, ) phospholipid vesicles in the
absence (, ) or the presence (, ) of 25 µg/mL heparin in
TBS buffer containing 5 mmol/L Ca2+, 1 mg/mL BSA, and
0.1% PEG 8000. At indicated time intervals, the rate of PC activation
was measured by an amidolytic activity assay as described under the
Materials and Methods. (B) The same as (A), except that 25 µg/mL
dextran sulfate was used in the reactions.
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To determine the kinetic step that may be influenced by the
polysaccharides in the presence of Ca2+, the Km
and kcat constants for PC activation were determined from
the initial rates of activation as a function of increasing PC
concentrations in the presence of 100 µg/mL PC/PS/PE vesicles and 5 mmol/L Ca2+ (Table 1). Under
these experimental conditions, no kinetic analysis for PC activation in
the absence of the polysaccharide was possible, because the reaction
rate remained linear for up to 12.5 µmol/L PC concentration (the
highest concentration available). These results suggest that the effect
of the polysaccharides in the acceleration of PC activation is at
least partly, if not entirely due to lowering the Km of PC
for thrombin in the reactions. Similarly, kinetic analysis indicated
that heparin and dextran sulfate inhibited PC activation by thrombin in
EDTA by increasing the Km of PC in the reactions. In
contrast to a Km of 4.5 ± 0.7 µmol/L for PC in 100 µmol/L EDTA, as determined in this study, no Km
determination in the presence of heparin or dextran sulfate was
possible (the highest PC concentration was 12.5 µmol/L).
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Table 1.
Kinetic Parameters for PC Activation by Thrombin and fXa
on PC/PS/PE Vesicles in the Presence of Heparin or Dextran Sulfate
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The observation that the polysaccharides lowered the Km for
PC activation suggested that these compounds may act as templates to
which both thrombin and PC bind. To further investigate this possibility, PC activation by thrombin was studied in the presence of
oligosaccharides containing 6, 10, 14, or 18 saccharide units or the
high-affinity heparin fragments containing 22, 35, 50, and 64 saccharide units. All of these fragments bind thrombin with similar
affinity, because Olson et al34 previously used these
oligosaccharide fragments to characterize the heparin binding site of
thrombin and demonstrated that only 5 to 6 charged residues contained
within a 3-disaccharide binding site of heparin account for
most of the binding energy of heparin in interaction with thrombin. No
effect on PC activation by thrombin was observed with either 6- or
10-unit oligosaccharides (up to 300 µmol/L) either in solution using
GDPC or on the phospholipid vesicles using full-length PC as the
substrate (data not shown). An approximately 2.5-fold enhancement of PC
activation rate was observed with the 14-unit oligosaccharide, and the
accelerating effect was increased with increasing heparin chain length
up to 50 saccharide units. These results are consistent with a template
mechanism for heparin in acceleration of PC activation by thrombin. A
similar optimal concentration for unfractionated heparin and the 50 or
64 saccharide-long high-affinity heparin (~1 µmol/L) was observed
for acceleration of GDPC activation by thrombin, and both types of
heparin accelerated the reaction approximately 20-fold. The
unfractionated heparin preparations are mixtures of high- and
low-affinity polysaccharide chains and, therefore, the accelerating
effect of heparin is not dependent on the presence of the highly
sulfated, high-affinity pentasaccharide fragment of heparin.
PC activation by fXa.
fXa has the capacity to bind heparin35; however, fXa is not
known to play a significant role in PC activation. To examine if polysaccharides play a role in PC activation by fXa, the initial rate
of PC activation by fXa was determined in the presence of these
compounds. Interestingly, as shown in Fig
6, human fXa on phospholipid vesicles rapidly activated human PC in the
presence of 25 µg/mL heparin or dextran sulfate. Similar to thrombin,
PC activation by fXa in the presence of either heparin (Fig 6A) or dextran sulfate (Fig 6B) on the PC/PS/PE vesicles was twofold to
threefold better than that on the PC/PS vesicles. Comparison of the
initial rates of PC activation by thrombin in Fig 5, and by fXa in Fig
6, indicates that fXa in the presence of the polysaccharides activated
PC twofold to fourfold better than thrombin on both types of the
phospholipid vesicles. Kinetic analysis indicated that relative to
thrombin, a lower Km of PC for fXa on both types of
membranes, accounts for the higher catalytic efficiency of fXa in PC
activation in the presence of the polysaccharides (Table 1). Because it
was not possible to determine the kinetic constants for PC activation
by fXa in the absence of the polysaccharides or phospholipids, it is
not known whether the accelerating effect of dextran sulfate or heparin
was only to lower the Km for PC, or they also improved the
kcat of the reactions. Similar to the reactions with
thrombin, no acceleration of PC activation by fXa was observed with the
oligosaccharides containing either 6 or 10 saccharide units. A minimum
chain length of 14 saccharide units was also required to observe any
significant effect on PC activation by fXa.
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| Fig 6.
Initial rate of PC activation by fXa on phospholipids in
the presence and absence of the polysaccharides. (A) PC (0.75 µmol/L) was incubated with 5 nmol/L fXa on 100 µg/mL PC/PS (, ) or
PC/PS/PE (, ) phospholipid vesicles in the absence (, ) or
the presence (, ) of 25 µg/mL heparin in TBS buffer containing
5 mmol/L Ca2+, 1 mg/mL BSA, and 0.1% PEG 8000. At
indicated time intervals, the rate of PC activation was measured by an
amidolytic activity assay as described under the Materials and Methods.
(B) The same as (A), except that 25 µg/mL dextran sulfate instead of
heparin was used in the reactions.
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To demonstrate how rapidly PC is activated by fXa in the presence of
the polysaccharides, the initial rate of PC activation on phospholipid
vesicles by thrombin in complex with full-length rabbit TM and by fXa
in the presence of heparin or dextran sulfate was compared at near
physiological concentration of PC (~80 nmol/L). On the PC/PS/PE
vesicles, under conditions of less than 10% substrate utilization,
activation of PC in the presence of dextran sulfate was essentially
identical by either fXa or the thrombin-rabbit TM complex
(Fig 7). However, on PC/PS vesicles,
activation by the thrombin-rabbit TM complex was approximately
threefold to fourfold better than that by fXa in the presence of
dextran sulfate (Fig 7).
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| Fig 7.
Comparison of the initial rate of PC activation by fXa in
the presence of dextran sulfate and the thrombin-rabbit TM complex on
phospholipids. PC (80 nmol/L) activation was carried out on 100 µg/mL
PC/PS (, ) or PC/PS/PE (, ) phospholipids with 1 nmol/L fXa
in the presence of 25 µg/mL dextran sulfate (, ) or 1 nmol/L
thrombin in complex with 100 nmol/L rabbit TM (, ) in TBS buffer
containing 5 mmol/L Ca2+, 1 mg/mL BSA, and 0.1% PEG
8000. At indicated time intervals, the rate of PC activation was
measured by an amidolytic activity assay as described under the
Materials and Methods.
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A similar (within 75% to 80%) acceleration of PC activation by both
thrombin and fXa was observed on the phospholipid vesicles, if the
concentration of heparin was reduced to 1.25 µg/mL (~0.2 U/mL),
which is the lowest range of heparin concentrations in plasma during
antithrombotic therapy.36 A threefold to fourfold rate
enhancement of GDPC activation by thrombin was also observed with
dermatan sulfate and heparan sulfate in Ca2+ buffer over a
broad concentration range (10 to 300 µg/mL, data not shown). In the
presence of phospholipids and any of these glycosaminoglycans, fXa
activated PC approximately twofold better than thrombin under identical
conditions (data not shown). PC activation by thrombin in the presence
of Ca2+ requires the cofactor function of TM, and therefore
a trace contamination of fXa with thrombin could not result in a
significant APC generation. Nevertheless, PC activation by fXa was also
performed in the presence of hirudin, TM, and other appropriate
controls, which all ruled out this possibility. Acceleration of PC
activation by the polysaccharides were abolished if 50 µg/mL
polybrene was included in the reactions.
| |
DISCUSSION |
It is shown in this study that heparin and a similar compound, dextran
sulfate, markedly accelerate PC activation by thrombin and fXa. The
role of heparin-like compounds as anticoagulant agents have been
studied extensively.36 It is widely accepted that the
primary anticoagulant function of heparin is the catalysis of
antithrombin inhibition of the plasma serine proteinases, mainly thrombin and fXa.14,37 However, heparin possesses other
functions that can influence coagulation. It was recently reported that heparin-like compounds can regulate the coagulation cascade by accelerating the activation of fXI by thrombin.15,16 It has also been reported that heparin may function as an anticoagulant by
direct inhibition of the activation of the fVIII-von Willebrand factor
complex by thrombin.38 It was also recently demonstrated that heparin accelerates fV inactivation by activated PC.39 Based on this observation, a novel anticoagulant mechanism for heparin
was proposed.39 The results of this study now suggest that
the anticoagulant function of the heparin-like compounds may also
involve the acceleration of PC activation by fXa and thrombin.
In contrast to acceleration of thrombin inhibition by antithrombin that
requires high-affinity heparin containing a specific pentasaccharide
for optimal effect,33 the magnitude of the acceleration of
PC activation by thrombin with the unfractionated heparin was identical
to the effect observed by the homogeneous high-affinity heparin at the
equimolar concentrations. The commercial unfractionated heparin is a
mixture of the high- and low-affinity polysaccharide chains, and the
high-affinity fraction constitutes only about one third of the
molecules.40,41 It is known that low-affinity heparin
potentiates the anticoagulant action of high-affinity heparin in
plasma.42 The results of this study suggest that the
low-affinity heparin that binds antithrombin poorly may contribute to
the anticoagulant effect of therapeutic heparin by catalyzing PC
activation, particularly on the surfaces of the activated platelets.
The observation that the polysaccharides have an optimal concentration
for acceleration of the reactions, with the activities decreasing at
concentrations above and below this value, suggests that these
negatively charged substances might serve as templates to which both
thrombin and PC bind. Further support for a template mechanism for the
polysaccharides in acceleration of PC activation by thrombin is
provided by the observation that these compounds lower the
Km for PC in the reactions. This resembles the mechanism of
heparin acceleration of thrombin inhibition by antithrombin, in which
heparin lowers the Kd for the analogous enzyme-inhibitor Michaelis complex formation by a template mechanism.43
Finally, this proposal is consistent with the observation that a
minimum chain length for the oligosaccharides was required for
acceleration of the reactions. However, in contrast to thrombin, the
mechanism by which heparin or dextran sulfate accelerates PC activation by fXa was not determined with certainty. The observations that the
Km for PC activation by fXa in the presence of the
polysaccharides was very low (Table 1) and that the oligosaccharides
containing 6 or 10 saccharide units did not accelerate the reaction
suggest that the primary effect of these compounds may also be through an approximation effect by a template mechanism. However, a bell-shaped concentration dependence curve for heparin acceleration was not observed for PC activation by fXa. Although this could be due to the
weak nature of the fXa-heparin interaction, other mechanisms for the
polysaccharides acceleration of PC activation by fXa cannot be ruled
out in this study. Particularly, in a different study, it was
previously reported that another polysaccharide, the pentosan polysulfate, accelerated PC activation by fXa on phospholipids by
increasing the Vmax value fourfold with no influence on the Km value.44 Because, in this study, it was not
possible to accurately determine the kinteic constants for PC
activation by fXa in the absence of the polysaccharide, such a modest
effect on the Vmax of activation by the polysaccharides used in the
current study cannot be ruled out.
At the biochemical level, the results of this study provide insight
into several aspects of PC activation by thrombin in the absence or the
presence of TM. First, it is known that, in the absence of TM, thrombin
exhibits a low Km for PC in the presence of EDTA (1.2 µmol/L).30 The observation that heparin and dextran sulfated both accelerated GDPC activation, but inhibited full-length PC
activation in EDTA suggests that the Gla-domain of PC interacts with
the anion binding exosite 2 of thrombin lowering the Km for PC in EDTA and that heparin binding to this site prevents this interaction. Further support for this hypothesis was provided by the
observation that heparin in the presence of EDTA neither accelerated
nor inhibited PC activation with the exosite 2 mutant R93,97,101A
thrombin, which is known to not bind heparin.31 Second, it
is known that the Ca2+-dependence of PC activation by
thrombin in complex with TM containing or lacking the chondroitin
sulfate differs.21,22 The Ca2+-dependence of PC
activation with TM containing chondroitin sulfate is a simple
saturation curve with a Kd(app) of approximately 300 µmol/L Ca2+, whereas with TM lacking chondroitin sulfate
it has a characteristic high peak of activation at approximately 50 to
100 µmol/L Ca2+, which decreases thereafter to a more
normal rate at physiological Ca2+ levels. A low
Km for PC at approximately 50 to 100 µmol/L
Ca2+ accounts for the high rate of activation by
thrombin-TM lacking chondroitin sulfate.22 The result of
this study showed that heparin eliminated this peculiar
Ca2+-dependence of PC activation, suggesting that the
Gla-domain of PC, when present as in not fully Ca2+
stabilized conformer, binds to the anion binding exosite 2 of thrombin
similar to that in EDTA and that heparin prevents this interaction. It
should be noted that the Gla-domain has a Kd(app) of
approximately 250 to 300 µmol/L for Ca2+
binding30; therefore, the Gla-domain of PC is not expected
to be in the fully stabilized conformer at 50 to 100 µmol/L
Ca2+. Furthermore, because the occupancy of the
high-affinity Ca2+ binding site in the protease domain
[Kd(app) ~50 to 100 µmol/L] is a requirement for the
cofactor function of TM,45 it follows that the consequence
of the Gla-domain interaction with the anion binding exosite 2 of
thrombin (lower Km) is only manifested at approximately 50 to 100 µmol/L Ca2+. This hypothesis is also consistent
with the previous results that the Ca2+-dependence of PC
activation by the R93,97,101A thrombin-TM complex follows a simple
saturation curve whether or not TM contains the chondroitin sulfate
moiety.31
It has been reported that platelet factor 4 stimulates the cofactor
activity of TM containing chondroitin sulfate at low Ca2+
and changes the Ca2+-dependence of PC activation from
simple saturation to a distinct high peak at the lower
Ca2+.46 This stimulatory effect of platelet
factor 4 was not observed with GDPC and was minimal when TM lacking
chondroitin sulfate was used in the activation assay. The authors of
this report hypothesized several possibilities for their observation
but did not consider the PC activation model in low Ca2+
described above. The current results indicate that cationic platelet factor 4 binding to the chondroitin sulfate moiety of TM results in a
thrombin-TM complex in which the exosite 2 remains unoccupied by TM and
that the Gla-domain of PC binds to this site, lowers the
Km, and enhances the PC activation rate by thrombin.
Finally, the observation that the acceleration of both PC and GDPC
activation by dextran sulfate and heparin required Ca2+
provides further insight into the role that Ca2+ may play
in PC activation and APC function. It was previously demonstrated that
Ca2+ binding to a high-affinity site on the protease domain
of PC induces a conformational change in the activation peptide of PC that is required for recognition and rapid activation of PC by the
thrombin-TM complex.45,47 The results of this study suggest that the Ca2+-induced conformational changes in the
protease domain also involves the basic residues of PC that interact
with dextran sulfate and heparin. Such a conformational change could
provide binding sites critical for interaction of PC with the
thrombin-TM complex for rapid zymogen activation. In support of this
hypothesis, it was recently shown that a PC mutant in which
Lys37, Lys38, and Lys39
(chymotrypsin numbering) are substituted was activated normally by
thrombin, but the activation rate was no longer accelerated by
TM.48 In a previous study with an antithrombin sensitive mutant of APC (T99Y), it was also demonstrated that heparin
acceleration of the APC T99Y inhibition by antithrombin was
Ca2+-dependent.49 Taken together, these results
suggest that, in contrast to thrombin, in which basic residues of the
heparin binding site are masked by the noncatalytic prothrombin
fragment 2 domain in its zymogen form, these residues are available in
PC and may be critical for interaction with cofactors and other
macromolecules in both the active and zymogen forms of the molecule.
| |
FOOTNOTES |
Submitted October 14, 1997;
accepted February 2, 1998.
Supported by a grant awarded by the National Heart, Lung, and Blood
Institute of the National Institutes of Health (Grant No. P01 HL 54804 to A.R.R.).
Address reprint requests to Alireza R. Rezaie, PhD, Oklahoma Medical
Research Foundation, Cardiovascular Biology Research, 825 NE 13th St,
Oklahoma City, OK 73104.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
I thank Drs Charles and Naomi Esmon for the plasma proteins, Dr Deborah
Stearn-Kurosawa for reading of the manuscript and useful discussions,
Dr Steven Olson for the high-affinity heparin fractions, Dr Ingemar
Björk for the oligosaccharides, Dr Omid Safa for the phospholipid
vesicles, and Mei Cheng for preparation of the figures.
| |
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Abstract
Introduction
Abstract