From the Hamilton Civic Hospitals Research Centre and
Department of Medicine, McMaster University, Hamilton, Ontario L8V 1C3,
Canada and the ¶ Vascular Therapeutics Inc.,
Mountain View, California 94040
Received for publication, November 3, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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In buffer systems, heparin and low molecular
weight heparin (LMWH) directly inhibit the intrinsic factor
X-activating complex (intrinsic tenase) but have no effect on the
prothrombin-activating complex (prothrombinase). Although chemical
modification of LMWH, to lower its affinity for antithrombin (LA-LMWH)
has no effect on its ability to inhibit intrinsic tenase,
N-desulfation of LMWH reduces its activity 12-fold. To
further explore the role of sulfation, hypersulfated LA-LMWH was
synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent
inhibitor of intrinsic tenase than LA-LMWH; it also acquires
prothrombinase inhibitory activity. A direct correlation between the
extent of sulfation of LA-LMWH and its inhibitory activity against
intrinsic tenase and prothrombinase is observed. In plasma-based assays
of tenase and prothrombinase, sLA-LMWH produces similar prolongation of
clotting times in plasma depleted of antithrombin and/or heparin
cofactor II as it does in control plasma. In contrast, heparin has no
effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on
various components of tenase and prothrombinase was examined, its
inhibitory activity was found to be cofactor-dependent
(factors Va and VIIIa) and phospholipid-independent. These studies
reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase.
Coagulation is initiated when tissue factor, a cell surface
protein found on nonvascular cells, is exposed by vascular injury. Tissue factor binds factor VII/VIIa
(f.VII/VIIa),1 forming
the extrinsic tenase complex that activates f.X (1). f.Xa generates
sufficient thrombin through prothrombinase, the phospholipid
membrane-bound complex of f.Xa and f.Va, to induce local aggregation of
platelets and activate f.V and f.VIII (2). f.Xa generated via extrinsic
tenase is insufficient to sustain hemostasis because tissue factor
pathway inhibitor rapidly inactivates tissue factor-bound f.VIIa in a
f.Xa-dependent fashion (1, 3). To overcome this limitation,
additional f.Xa is generated by intrinsic tenase, the phospholipid
membrane-bound complex of f.IXa and f.VIIIa. f.IX can be activated by
extrinsic tenase or by f.XIa, generated by thrombin cleavage of f.XI
(1).
The critical role of intrinsic tenase and prothrombinase in coagulation
makes these enzyme complexes attractive targets for inhibition.
Prothrombinase and intrinsic tenase share similar properties, with each
complex consisting of a vitamin K-dependent serine protease
and a nonproteolytic cofactor protein. The reactions are
calcium-dependent and require a negatively charged
phospholipid surface for optimal expression of activity (4-7).
Heparin and low molecular weight heparin (LMWH) act as anticoagulants
by activating antithrombin, which inactivates f.Xa and thrombin (8). In
buffer systems, heparin and LMWH also inhibit intrinsic tenase activity
in an antithrombin-independent fashion (9, 10). In plasma systems,
however, the antithrombin-dependent anticoagulant effects of
heparin and LMWH predominate.
The purpose of this study was to investigate methods for modifying
heparin so as to maximize its antithrombin-independent effects.
Starting with a size-restricted LMWH to capitalize on its
decreased propensity to bind to plasma proteins, a property that endows
it with pharmacokinetic advantage over unfractionated heparin (11, 12),
LMWH was chemically modified to reduce its affinity for antithrombin
1700-fold (from a Kd value of 25 nM to
43 µM) by periodate oxidation (13). Like LMWH, this low
affinity LMWH (LA-LMWH), which we termed Vasoflux, inhibited intrinsic
tenase but had no effect on prothrombinase, thereby confirming that its
ability to inhibit intrinsic tenase is not dependent on its affinity
for antithrombin. When LA-LMWH was N-desulfated, however,
most of its activity was lost, suggesting that its ability to inhibit
intrinsic tenase is charge-dependent. To explore this possibility, LA-LMWH was progressively hypersulfated, and the inhibitory activities of these hypersulfated LA-LMWH (sLA-LMWH) compounds against intrinsic tenase and prothrombinase were examined. Herein, we demonstrate that upon sulfation, LA-LMWH becomes a more
potent inhibitor of intrinsic tenase and acquires the ability to
inhibit prothrombinase.
Materials
Human f.V, f.Va, and f.IXa were obtained from Hematologic
Technologies Inc. (Essex Junction, VT), whereas f.X, f.Xa, prothrombin, and L- For glycosaminoglycans, unfractionated grade 1 sodium heparin (184 units/mg) from porcine intestinal mucosa was purchased from
Sigma, whereas the LMWH enoxaparin was from Rhône-Poulenc-Rorer Canada (Montreal, Canada).
A series of low affinity low molecular weight heparins (LA-LMWH) and
hypersulfated LA-LMWH derivatives (sLA-LMWH) were used in this study
(see Table I). A LMWH fraction (mean molecular weight 5000) was
prepared from unfractionated heparin by nitrous acid depolymerization,
and its affinity for antithrombin was reduced by sodium periodate
oxidation, as previously described (13). The resultant LA-LMWH was
subjected to ultrafiltration using a 3000-Da cut-off membrane and
lyophilized. The LA-LMWH was O-sulfated using a modification
of the method described by Nagasawa et al. (18). Briefly,
200 mg of LA-LMWH dissolved in 5 ml of water was converted into acid
form by ion exchange at 0 °C. After neutralization with
tributylamine and lyophilization, the sample was dissolved in 20 ml of
dry N,N-dimethylformamide. With constant
stirring, 2 g of trimethylamine sulfur trioxide was added, and the
mixture was incubated for varying times and temperatures to obtain the hypersulfated LA-LMWH derivatives (sLA-LMWH) listed in Table I. At the
end of each reaction, 50 ml of double distilled water was added,
and the solution was dialyzed three times (12 h each) against 1000 ml
of 5% NaCl and then three times against 1000 ml of double distilled
water. After lyophilization, each sample was subjected to elemental
analysis, and the number of sulfate residues/disaccharide was
calculated based on the percentage of carbon relative to sulfur. Five
sLA-LMWH derivatives were prepared and are designated sLA-LMWH-S1 to
-S5. LA-LMWH was N-desulfated (N-DS-LA-LMWH) using the
solvolytic desulfation method described by Inoue and Nagasawa (19).
Methods
Effect of Glycosaminoglycans on the Activity of
Prothrombinase--
To examine the effects of glycosaminoglycans on
the activation of prothrombin by the prothrombinase complex, the rate
of thrombin generation was assayed using a modification of the method
of Barrow et al. (9). Reactions were performed in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl containing
0.1% polyethylene glycol (TSP buffer). Two times concentrated stock
solution A was made in TSP buffer so as to give final reaction
concentrations of 24 µM PCPS vesicles, 0.24 nM f.Va, 1.2 µM prothrombin, and 4 mM CaCl2. 50 µl of stock A was mixed with 30 µl of TSP and 10 µl of glycosaminoglycan at final concentrations
ranging from 1-1000 µg/ml. Reactions were initiated by addition of
10 µl of 1 nM f.Xa. Control samples lacking glycosaminoglycan were run in parallel. 10-µl aliquots were removed at 30-s intervals into the wells of a 96-well microtitre plate containing 10 µl of 10 mM EDTA, pH 7.4, to quench the
activation reaction. At the end of the time course, the activity of
thrombin in each well was determined by adding 190 µl of 200 µM tGPR-pNA containing 0.1 mg/ml polybrene. Chromogenic
substrate hydrolysis was monitored at 405 nm at 10-s intervals for 5 min at 23 °C in a Spectramax 340 plate reader (Molecular Devices,
Sunnyvale, CA).
Effect of Glycosaminoglycans on the Activity of Intrinsic
Tenase--
The effects of glycosaminoglycans on the activity of
intrinsic tenase were determined in a similar fashion. Two times
concentrated stock B was prepared so as to contain final reaction
concentrations of 300 nM f.X, 4 mM
CaCl2, 24 µM PCPS, 4 nM f.IXa,
and 0.4 nM f.VIII. f.VIII was activated to f.VIIIa in stock
B by incubation with 10 nM thrombin for 1 min. 50 µl of
stock B was mixed with 30 µl of TSP containing glycosaminoglycan at
final concentrations ranging from 1 to 1000 µg/ml. Reactions in each
well were initiated by the addition of 10 µl of 8 nM
f.IXa, and 10-µl aliquots were removed at 30-s intervals as described
above. f.Xa activity was assayed using S-2222 or Chromozym.X.
Effect of sLA-LMWH-S5 on Components of the Prothrombinase
Complex--
Individual components of the prothrombinase complex were
systematically removed or substituted to examine their susceptibility to inhibition by sLA-LMWH. In all cases, the rate of prothrombin activation was examined in the absence or presence of sLA-LMWH-S5 (S5),
the most potent of the sLA-LMWH derivatives. The final reactant conditions were as follows: (a) absence of f.Va (1.2 µM prothrombin, 100 nM f.Xa, 24 µM PCPS, and 4 mM CaCl2),
(b) absence of phospholipid (1.2 µM
prothrombin, 4 mM CaCl2, 12 nM
f.Va, and 10 nM f.Xa), (c) absence of cofactor,
phospholipid, and calcium (1.2 µM prothrombin and 100 nM f.Xa), (d) substitution of f.Va with f.V (1.2 µM prothrombin, 4 mM CaCl2, 24 µM PCPS, 8 nM f.V, and 0.2 nM
f.Xa), and (e) chromogenic activity of f.Xa (1 nM f.Xa and
200 µM S-2222). Assays were performed as described above
except for reactions with conditions b and c.
Because the rate of generation of thrombin in these systems was slow,
even with increased reactant concentrations, subsampling was performed
at 15- and 30-min intervals, respectively.
Effect of sLA-LMWH-S5 on Components of the Intrinsic Tenase
Complex--
In an analogous series of assays, the effect of S5 on
various components of intrinsic tenase also was examined. The final reactant conditions were as follows: (a) absence of cofactor
(4 mM CaCl2, 400 nM f.X, 24 µM PCPS, and 1 µM f.IXa), (b)
absence of phospholipid (4 mM CaCl2, 300 nM f.X, 16 nM f.VIIIa, and 1 µM
f.IXa), (c) absence of phospholipid and cofactor (4 mM CaCl2, 400 nM f.X, and 1 µM f.IXa), (d) substitution of f.VIIIa with f.VIII (4 mM CaCl2, 300 nM f.X, 24 µM PCPS, 16 nM f.IXa, and 20 nM
f.VIII), and (e) chromogenic activity of f.IXa (0.1 µM f.IXa with 1 mM Pefa-IXa in the presence
of 30% ethylene glycol).
Data Analysis--
Rates of chromogenic substrate cleavage were
obtained from the slope of the linear portion of the
A405 versus time plot. Standard curves of thrombin or f.Xa activity, with the respective chromogenic substrate, were used to convert slopes to enzyme concentration. The
rates of activation were calculated by linear regression analysis of
thrombin or factor Xa concentration versus time (Quattro
Pro, version 5.0, Borland International Inc. Scott's Valley, CA).
Comparison with control reactions performed in the absence of
glycosaminoglycan, run in parallel, gave a rate relative to the
control. The concentration of glycosaminoglycan that produced 50%
inhibition of the f.X or prothrombin activation rate
(IC50) was calculated to compare the different
glycosaminoglycans. All reactions were performed in duplicate and means
from three to six experiments were calculated for each analysis.
Activated Partial Thromboplastin Time--
Glycosaminoglycan was
added to 50-µl aliquots of control plasma, plasma immunodepleted of
antithrombin, or plasma immunodepleted of both antithrombin and heparin
cofactor II (Affinity Biologicals Inc., Hamilton, Canada). 50 µl of
Thrombosil (Ortho Clinical Diagnostics, Raritan, NJ) was added, and the
sample was incubated for 5 min. Clotting was initiated with 50 µl of
20 mM CaCl2, and the time to clot formation was
determined on a ST4 coagulometer (Stago Diagnostica, Parsippany, NJ).
f.Xa Clotting Time--
100 µl of 1 nM f.Xa in 10 mM CaCl2, 0.1 mg/ml bovine serum albumin,
0.05% cephalin, 10 mM Hepes-NaOH, pH 6.8 was added to 50 µl of plasma containing varying concentrations of glycosaminoglycan, and the time for clot formation was monitored using an ACL 30000 centrifugal coagulation analyzer (Beckman-Coulter Inc., Fullerton, CA).
Assays were performed in normal plasma, plasma immunodepleted of
antithrombin, or plasma immunodepleted of both antithrombin and heparin
cofactor II.
Binding Studies--
Fluorescein labeling was performed by
incubating 10 mg of S5 with 15 mg of 5'-fluorescein isothiocyante
(Sigma) in 2.5 ml of 1.0 M Na2CO3,
pH 9.0, for 5 h at 23 °C. After centrifugation at
13,000 × g for 5 min, 1 ml of the supernatant was
applied to duplicate PD-10 columns (Millipore Corp., Bedford, MA),
equilibrated with H2O, and eluted with H2O
under gravity. 0.5-ml fractions were collected, frozen, and
lyophilized. Recovered material was pooled, weighed, and dissolved in
20 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TS), to a
concentration of 10 mg/ml.
The affinity of fluorescein-sLA-LMWH-S5 (Fl-S5) for f.IXa and f.Xa was
determined by monitoring fluorescence of Fl-S5 upon titration with
f.IXa or f.Xa. 50 nM of Fl-S5 in TS containing 2 mM CaCl2 was stirred with a microstirbar in a
1-ml cuvette and maintained at 23 °C with a circulating waterbath.
Fluorescence was monitored in an LS50B luminescence spectrophotometer
(Perkin Elmer, Norwalk, CT) in time drive with
The affinities of other heparin derivatives for f.IXa and f.Xa were
determined in competition experiments by monitoring displacement of
Fl-S5 from each enzyme. A solution of 50 nM Fl-S5 and 80 nM f.IXa or 100 nM f.Xa was titrated with
unlabeled S5 or LA-LMWH. Using Imax, the change
in fluorescence intensity between fully bound and unbound Fl-S5, the
corrected intensity values after each addition were used to calculate
the concentration of bound fluorescein-S5 (PL1)
using I/Imax × [Fl-S5]i, where [Fl-S5]i is the initial
concentration of the fluorophore. Likewise, the free enzyme
concentration (P) was determined using the equation,
Kd × I/(Imax Statistical Analysis--
Values for IC50 were
calculated based on the means of three to six experiments, each
performed in duplicate. Means and standard deviations were determined
using Quattro Pro. The correlation between the degree of sulfation of
LMWH and mean IC50 values for prothrombinase and intrinsic
tenase was determined using a test of rank correlation (Pearson) and by
one-way analysis of variance using Minitab (Release 11.11, Minitab
Inc., State College, PA). A value of p < 0.05 was
considered statistically significant.
Effect of Glycosaminoglycans on Intrinsic Tenase--
To examine
the antithrombin-independent effect of LMWH on intrinsic tenase, a
LA-LMWH was prepared as previously described (13). LA-LMWH produced
50% inhibition of the initial velocity of f.X activation
(IC50) of 16.3 ± 6.1 µg/ml (Fig.
1A). A commercial LMWH
(enoxaparin) with normal antithrombin affinity, inhibited intrinsic
tenase with an IC50 value of 13.2 ± 7.7 µg/ml
(Table I), a value comparable with the
IC50 value of 6 µg/ml reported for LMWH by other
investigators (9). To investigate the influence of sulfation of LA-LMWH
on inhibition of tenase activity, LA-LMWH was N-desulfated
by solvolysis. N-DS-LA-LMWH had 12-fold lower inhibitory activity, with
an IC50 value of 166 ± 25 µg/ml. These findings
suggest that the ability of LMWH to inhibit intrinsic tenase is
independent of its affinity for antithrombin but dependent on its
charge.
To further investigate the importance of charge, progressively
hypersulfated LA-LMWH derivatives were synthesized, and their inhibitory activity was compared with that of the starting material (Table I). The most highly sulfated LA-LMWH, designated S5, was 32-fold
more potent than LA-LMWH, inhibiting intrinsic tenase with an
IC50 value of 0.47 ± 0.2 µg/ml. As illustrated in
Fig. 1A, increasing the sulfation of LA-LMWH produces a
progressive reduction in IC50 values. When a plot of the
number of sulfate residues/disaccharide versus
IC50 is subjected to regression analysis (not shown), the
correlation coefficient is Effect of Glycosaminoglycans on Prothrombinase--
The inhibitory
effect of the series of hypersulfated LA-LMWH derivatives on
prothrombinase activity was examined to determine whether increased
inhibitory activity against intrinsic tenase conferred inhibitory
properties against prothrombinase (Fig. 1B). At
concentrations up to 1000 µg/ml, unfractionated heparin had no effect
on prothrombinase function, consistent with the results of Barrow
et al. (9). Likewise, neither
enoxaparin nor LA-LMWH had inhibitory activity against prothrombinase
at these concentrations. In contrast, sLA-LMWH inhibited prothrombinase
in a concentration-dependent fashion, and its inhibitory
activity increased with progressive sulfation, as reflected by a
reduction in IC50 values (Table I). Maximum inhibition was
effected by S5, which inhibited prothrombinase with an
IC50 value of 30 ± 16 µg/ml. Linear regression
analysis of a plot of the number of sulfate residues/disaccharide
versus IC50 values yielded a correlation
coefficient of Mode of Disruption of Prothrombinase and Intrinsic Tenase by
S5--
As the intrinsic tenase and prothrombinase complexes are
composed of multiple components, various reactants could serve as targets for modulation by sLA-LMWH. To reveal the susceptible component(s), the influence of S5 on partially reconstituted activation complexes was examined. S5 was used in these experiments because it
exhibited the most potent inhibitory effects (Table I).
When all components were present, S5 produced
dose-dependent inhibition of both intrinsic tenase (Fig. 2)
and prothrombinase (Fig. 3). Similar
inhibitory effects were evident in assays devoid of phospholipid. Thus,
the IC50 values for S5 on intrinsic tenase in the presence
or absence of phospholipid were 0.47 ± 0.2 and 1.0 ± 0.7 µg/ml, respectively. Comparable IC50 values for
inhibitory activity of S5 against prothrombinase also were found in the
presence or absence of phospholipid (30 ± 16.3 and 68 ± 22 µg/ml, respectively). In contrast, in systems devoid of cofactor
(f.Va/f.VIIIa), S5 had no inhibitory effect, suggesting that S5
interferes with the cofactor activity of f.Va and f.VIIIa in their
respective enzyme complexes. S5 also had no inhibitory activity in
prothrombinase systems devoid of cofactor and phospholipid or of
cofactor, phospholipid, and calcium. For intrinsic tenase, despite
increases in reactant concentrations, rates of activation remained slow
in a system lacking f.VIIIa and PCPS and were unmeasurable in a system
devoid of f.VIIIa, PCPS, and calcium (data not shown). The data
obtained in partially reconstituted systems support the concept that
the cofactor-enzyme interaction is the predominant target of S5 in both
prothrombinase and intrinsic tenase.
When f.V or f.VIII was substituted for f.Va or f.VIIIa, an initial lag
phase was seen on plots of thrombin or f.Xa generation versus time, reflecting the positive feedback effect of
thrombin or f.Xa on f.V or f.VIII activation. When the linear part of
the curve was analyzed to give an apparent rate of activation, S5 had a
similar inhibitory effect in systems using f.V and f.VIII as it did in
those using their activated counterparts, suggesting that S5 has no
effect on cofactor activation.
The effect of S5 on the chromogenic activity of f.Xa and f.IXa also was
examined. The hydrolysis of S-2222 by f.Xa is unaffected by S5.
Although S5 produced some inhibition of f.IXa-mediated hydrolysis of
Pefa IXa, this assay is of limited value because f.IXa has almost no
activity against chromogenic amide substrates (21-23).
Affinity of LA-LMWH and sLA-LMWH for f.IXa and f.Xa--
To begin
to explore why S5 has greater activity against intrinsic tenase than
prothrombinase, its affinity for f.IXa and f.Xa was determined and
compared with that of LA-LMWH, which only has inhibitory activity
against intrinsic tenase. Binding affinities were determined from
competition experiments where fluorescein-labeled sLA-LMWH-S5 was
displaced from f.IXa or f.Xa by unlabeled S5 or LA-LMWH (Fig.
4). LA-LMWH displaced Fl-S5 from f.IXa
and f.Xa with Ki values of 1000 and 9300 nM, respectively. In contrast, S5 displaced Fl-S5 from
f.IXa and f.Xa with Ki values of 115 and 555 nM, respectively, consistent with its more potent
inhibitory effect. The greater than 5-fold higher affinity of S5 for
f.IXa relative to f.Xa could explain why S5 inhibits intrinsic tenase
more effectively than prothrombinase.
Effect of S5 on Coagulation Assays--
To determine whether the
inhibitory activity of S5 observed in buffer systems also occurs in
plasma systems, the effects of S5 on the APTT and f.Xa clotting time
were examined (Fig. 5). The APTT was used
as a measure of both intrinsic tenase and prothrombinase, whereas the
f.Xa clotting time was used as an index of prothrombinase activity. To
verify that S5 was acting independent of plasma protease inhibitors,
its anticoagulant activity in plasma fractions immunodepleted of
antithrombin and/or of both antithrombin and heparin cofactor II was
compared with that in control plasma. S5 produces
concentration-dependent prolongation of the APTT and f.Xa
clotting time in control plasma, plasma immunodepleted of antithrombin,
and plasma depleted of both antithrombin and heparin cofactor II. These
data confirm that S5 inhibits coagulation in an antithrombin- and
heparin cofactor II-independent manner and are consistent with its
inhibitory activity against intrinsic tenase and prothrombinase. In
contrast, concentrations of heparin that produce similar prolongations
of the APTT and f.Xa clotting time in control plasma have no effect in
either immunodepleted plasma, indicating that the anticoagulant effects of heparin are antithrombin-dependent.
Intrinsic tenase and prothrombinase complexes are critical for
thrombin generation in the process of blood coagulation and, as such,
are ideal targets for inhibitors of blood coagulation. Because these
multicomponent complexes are assembled from intrinsic and activated
components, several approaches can be used for their inhibition. These
include direct inactivation of the enzyme or cofactor or disruption of
the capacity of the complex to assemble productively. Our results
demonstrate that unfractionated heparin and LMWH directly inhibit
intrinsic tenase, consistent with the results of Barrow et
al. (9). However, we have extended their findings in two important
ways. First, we demonstrate that reducing the affinity of LMWH for
antithrombin does not affect its ability to inhibit intrinsic tenase.
Second, we demonstrate that progressive hypersulfation of LA-LMWH
increases its potency of inhibition of intrinsic tenase and enables
inhibition of prothrombinase. This endows sLA-LMWH with greater
activity because it acts at two critical sites in the coagulation system.
Mechanism--
The kinetics of f.X activation in the presence of
unfractionated heparin have established that heparin inhibits intrinsic tenase in a noncompetitive fashion (9). The data reported here demonstrate that inhibition of the activation complexes involves the
cofactor and enzyme but not the phospholipid surface. Because intrinsic
tenase and prothrombinase are homologous complexes, it is conceivable
that the site of action of sLA-LMWH is the same for both systems. Our
data suggest that sLA-LMWH disrupts the interaction of the enzyme with
its cofactor, inferring that one or both of these components bind
sLA-LMWH. That heparin binds to f.IXa is well known (24-26). Likewise,
calcium-dependent heparin binding to f.Xa has recently been
described (27). The competitive binding studies reported here reveal
that S5 binds over 5-fold more tightly to f.IXa than to f.Xa,
consistent with the greater inhibitory activity of all
glycosaminoglycans tested against tenase rather than prothrombinase.
These results suggest that the enzymes within the activation complexes
represent at least part of the target for glycosaminoglycan binding and
subsequent disruption of complex assembly. The binding site for heparin
on f.IXa has not been formally identified but is presumed to be at a
site homologous to the heparin-binding domain of f.Xa and thrombin
because arrangements of basic residues in the protease domains are
largely preserved (21, 28-31). It is notable that the putative
heparin-binding sites on f.Xa and f.IXa also comprise respective f.Va
and f.VIIIa binding sites (32-35). In addition to the enzyme component
of the activation complexes, f.V and f.VIII also bind heparin,
providing additional targets for interference by glycosaminoglycans
(10, 36).
Although previous studies also suggested that the interaction of f.IXa
and f.VIIIa were the likely target for glycosaminoglycan inhibition,
direct binding studies did not reveal disruption of binding of the two
factors on phospholipid surfaces (9). One possible explanation for this
paradox is that the glycosaminoglycan may interfere with interaction of
the substrate (either f.X or prothrombin) with an exosite that is only
expressed on the enzyme in the presence of its cofactor. The existence
of such an exosite, or alternate substrate binding mode, has been
suggested for prothrombinase (37-39) and tenase (33, 40-42). Further
studies will be necessary to reveal the precise mechanism by which
glycosaminoglycans inhibit these complexes.
Heparin also directly inhibits activation of the f.VIII-von Willebrand
factor complex by thrombin (10). This may reflect competition between
heparin and f.VIII for thrombin binding because, in addition to exosite
1, exosite 2, the heparin-binding domain, has also been implicated in
f.VIII recognition by thrombin (43). Although inhibition of f.VIII
activation by heparin may cause additional attenuation of intrinsic
tenase activity, it is not the predominant mechanism because sLA-LMWH
had the same effect regardless of whether f.VIII or f.VIIIa was used.
Role of Sulfation--
A series of variably sulfated LA-LMWH
derivatives was used to investigate the structural requirements for
inhibition of intrinsic tenase so that more potent inhibitors could be
identified. In our assay of intrinsic tenase activity, LA-LMWH produced
50% inhibition of the initial velocity of fX activation at 16.3 µg/ml. A LMWH with normal antithrombin affinity inhibited intrinsic
tenase to a similar extent, with an IC50 value of 13.2 µg/ml (Table I). In contrast, N-DS-LMWH had less inhibitory effect,
with a 12-fold increase in the IC50 value to 166 µg/ml.
These findings indicate that the inhibition of intrinsic tenase by LMWH
is independent of the antithrombin-binding pentasaccharide sequence.
That the inhibitory activity is dependent on the charge of the
glycosaminoglycan is supported by the observation that the potency for
inhibition of intrinsic tenase and prothrombinase by LA-LMWH and
sulfated derivatives thereof correlates with the number of sulfate
residues/disaccharide. Furthermore, a dextran sulfate whose sulfate
content is equivalent to that of S5 has similar inhibitory activity
against intrinsic tenase and prothrombinase, indicating that this
activity does not require the heparin backbone. In contrast, Barrow
et al. (9) demonstrated that less sulfated carbohydrates
such as dermatan sulfate, chondroitin sulfate, or keratin sulfate
inhibit intrinsic tenase with IC50 values more than 2 orders of magnitude higher than those of heparin or LMWH.
Whereas the reaction of heparin with antithrombin is dependent on
charge and a specific arrangement of saccharides (44, 45), interaction
of heparin with thrombin is a charge-dependent phenomenon
that does not involve specific saccharide sequences (46). Because
sulfation of heparin increases its affinity for thrombin (47), a
similar effect would be anticipated for glycosaminoglycan interactions
with the homologous enzymes of the tenase and prothrombinase complex.
This is what is observed because the affinities of S5 for f.IXa and
f.Xa are 8- and 16-fold higher, respectively, than those of the LA-LMWH
starting material. Therefore, the increased potency of sulfated
glycosaminoglycans in disrupting the activation complexes is consistent
with increased affinity for the enzyme and/or the cofactor.
The results obtained in the present study are similar to those reported
for DHG, a depolymerized glycosaminoglycan from the sea cucumber
(48). DHG was reported to inhibit both tenase and prothrombinase
activities in a dose-dependent manner that involved the
respective cofactors. It was observed that both f.IXa and f.VIIIa bound
to immobilized DHG. However, DHG also accelerated thrombin inhibition
by heparin cofactor II (49). DHG displays a higher molecular weight
than S5 (12, 500, and 5000, respectively) and a lower degree of
sulfation (2.6 and 3.9 sulfate residues/disaccharide, respectively)
(50). A phosphorothioate oligonucleotide that inhibits intrinsic tenase
and activates heparin cofactor II has recently been described (36). Our
observation that S5 retains its anticoagulant activity in plasma
immunodepleted of antithrombin and heparin cofactor II indicates that
serpin activation is not the means by which S5 exerts its inhibitory
effects on coagulation.
As a potent inhibitor of intrinsic tenase and prothrombinase, sLA-LMWH
inhibits coagulation in a novel fashion. sLA-LMWH can be added to a
growing list of enzyme complex inhibitors, which include active site
blocked f.Xa and f.IXa (51, 52), f.IXa antibodies (53), and active site
directed f.Xa inhibitors (54-57). sLA-LMWH may have advantages over
other agents because it simultaneously attenuates f.Xa and thrombin
generation, with selectively greater inhibition of f.Xa generation by
intrinsic tenase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin were obtained from Enzyme Research Laboratories (South
Bend, IN). Recombinant f.VIII (Kogenate) was from Bayer Inc.
(Etobicoke, Canada). Albumin-free plasma-derived f.VIII, a gift from
Dr. E. Saenko (Holland Laboratory, American Red Cross, Rockville, MD),
was used in some experiments. The purity of f.V and f.VIII preparations
were confirmed by SDS-polyacrylamide gel electrophoresis analysis
(14).
-Phosphatidylcholine (Type III-E from egg yolk),
L-
-phosphatidyl-L-serine (from bovine
brain), tosyl-D-Gly-Pro-Arg-p-nitroanilide (tGPR-pNA),
bovine cephalin, and dextran sulfate (molecular weight average 5000;
catalogue number D7037) were obtained from Sigma. Chromozym.tPA
(N-methylsulfonyl-D-phenyl-Ala-Gly-Arg-4-nitranilide-acetate) and Chromozym.X
(N-methoxycarbonyl-D-norleucyl-glycyl-L-arginine-4-nitroaniline-acetate) was obtained from Roche Molecular Biochemicals. S-2444
(L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide-hydrochloride) and S-2222
(N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride) were obtained from Chromogenix A.B. (Molndal, Sweden). Pefachrom-IXa
(CH3SO2-D-CHG-Gly-Arg-p-nitoranilide
hydrochloride) was obtained from Pentapharm Ltd. (Basel, Switzerland).
Polybrene (hexadimethrine bromide) was purchased from Aldrich.
Unilamellar phosphatidylcholine-phosphatidylserine (PCPS; 75%/25%,
w/w) vesicles were prepared using a modification of the methods of
Barenholz et al. (15) and others (16) and assayed using an
inorganic phosphate assay (17).
ex of 492 nm,
em of 535 nm (slit widths of 5 and 20 nm,
respectively), and a 515-nm cut-off filter in the emission beam, as
1-10-µl aliquots of f.IXa or f.Xa were added to the cuvette.
Additions were made at 1-2-min intervals, allowing the fluorescence
signal to stabilize between additions. At the end of the titration,
intensity values were determined from the time drive profiles. The
ratio I/Io was calculated by dividing
the fluorescence intensity after each addition (I) by the
initial intensity (Io). Plots of
I/Io versus titrant
concentration were analyzed by nonlinear regression fit to a binding
isotherm equation, which yielded values for Kd and
maximal fluorescence change (20).
I), where Kd is the dissociation constant determined above. By the mass action relationship,
PL2, the concentration of the unlabeled
heparin/enzyme complex, is given by Po
PL1
P, where
Po is the total concentration of the enzyme. A
plot of L2/PL2
versus 1/P has a slope of Ki, the
dissociation constant for the unlabeled heparin
(L2) with the enzyme.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Influence of heparin derivatives on
prothrombinase and intrinsic tenase. The influence of sLA-LMWH-S5
( ), LA-LMWH (
), unfractionated heparin (
), or N-DS-LMWH (
)
on activation of f.X and prothrombin by their respective complexes was
analyzed. A, the rates of activation of 300 nM
f.X by intrinsic tenase (4 nM f.IXa, 0.4 nM
f.VIIIa, 24 µM PCPS, and 4 mM
CaCl2) were determined in the presence of varying
concentrations of glycosaminoglycan. The ordinate is expressed as the
rate of f.X activation relative to that of a control carried out in the
absence of glycosaminoglycan. B, the rates of activation of
1.2 µM prothrombin by prothrombinase (1 nM
f.Xa, 0.24 nM f.Va, 24 µM PCPS, and 4 mM CaCl2) were determined in the presence of
varying glycosaminoglycan concentrations. The ordinate is expressed as
the rate of prothrombin activation relative to that in the absence of
glycosaminoglycan.
Influence of variably sulfated LA-LMWH derivatives on intrinsic tenase
and prothrombinase
0.86, a value that on one-way analysis of
variance is highly significant (p < 0.001), supporting
the concept that the potency of LMWH derivatives is dependent on their
degree of sulfation. A dextran sulfate, which contained 3.9 sulfate
residues/disaccharide, inhibited intrinsic tenase with an
IC50 value of 0.4 µg/ml, a value similar to that of S5.
This observation provides further evidence that the extent of sulfation
is an important determinant of potency in this system.
0.92 (not shown), which on one-way analysis of
variance was highly significant (p < 0.001). Dextran
sulfate inhibited prothrombinase with an IC50 value of 35 µg/ml, further highlighting the importance of sulfation for
expression of this activity. The IC50 values against
prothrombinase were 2 orders of magnitude higher than those for
intrinsic tenase, indicating that all of the hypersulfated
carbohydrates have greater inhibitory activity against intrinsic tenase
than prothrombinase.
View larger version (16K):
[in a new window]
Fig. 2.
Influence of sLA-LMWH-S5 on fully and
partially reconstituted intrinsic tenase. The influence of varying
concentrations of S5 on the rate of f.X activation was determined in
series of reactions where the constituents of intrinsic tenase were
selectively omitted or replaced. The reactions contained f.IXa,
f.VIIIa, and PCPS ( ), f.IXa, f.VIII, and PCPS (
), f.IXa and
f.VIIIa (
), f.IXa and PCPS (
), and f.IXa chromogenic activity
(
). Final reactant conditions are given under "Methods."
View larger version (18K):
[in a new window]
Fig. 3.
Influence of sLA-LMWH-S5 on fully and
partially reconstituted prothrombinase. The influence of varying
concentrations of S5 on prothrombin activation was determined in series
of reactions where the constituents of prothrombinase were selectively
omitted or replaced. The reactions were f.Xa, f.Va, and PCPS ( ),
f.Xa, f.V, and PCPS (
) f.Xa and PCPS (
), f.Xa and f.Va (
),
f.Xa (
), and f.Xa chromogenic activity (
). Final reactant
conditions are given under "Methods."
View larger version (12K):
[in a new window]
Fig. 4.
Displacement of Fl-S5 from f.IXa or f.Xa by
LA-LMWH or S5. The initial fluorescence intensity
(Io) of 50 nM Fl-S5 in the presence
of 80 nM f.IXa ( ) or 100 nM f.Xa (
) was
monitored at
ex of 492 nm and
em of 535 in a fluorimeter. Subsequent intensity values (I) were
determined as the sample was titrated with LA-LMWH (A) or S5
(B). Values for I/Io are
plotted versus the concentration of the titrant.
Displacement data were used to calculate Ki values
for LA-LMWH or S5 binding to f.IXa or f.Xa, as described under
"Methods."
View larger version (22K):
[in a new window]
Fig. 5.
Effect of heparin and S5 on clotting
assays. Clotting assays were performed in control plasma ( ),
plasma immunodepleted of antithrombin (
), or plasma immunodepleted
of both antithrombin and heparin cofactor II (
). S5 (A)
or heparin (B) were tested at varying concentrations in the
APTT assay. S5 (C) or heparin (D) were tested at
varying concentrations in the f.Xa clotting time assay. 0.1 unit/ml
heparin is equivalent to 0.5 µg/ml.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael Nesheim and Laszlo Bajzar for many helpful discussions, Marilyn Johnston for providing coagulation assays, and Dr. Evgueni Saenko for generously supplying purified f.VIII.
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FOOTNOTES |
---|
* This work was supported by Grant MT-3992 from the Medical Research Council of Canada and the Ontario Research and Development Challenge Fund (to J. I. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a British Society of Haematology Fellowship Award and of funding from the Chest, Heart and Stroke Association, the Kerr-Fry Award from the University of Edinburgh, Scotland, and the Myre-Sim Fellowship from the Royal College of Physicians of Edinburgh, Scotland. Present address: Dept. of Haematology, Royal Infirmary of Edinburgh, Edinburgh, Scotland.
Present address: Neoparin, Inc., 14274 Wicks Blvd., San
Leandro, CA 94577.
** Present address: Imetrx, Inc., 801 Hermosa Way, Menlo Park, CA 94025.
Recipient of a Career Investigator Award from the Heart and
Stroke Foundation of Ontario and holds the Heart and Stroke Foundation of Ontario/J. F. Mustard Chair in Cardiovascular Research at McMaster University. To whom correspondence should be addressed: Hamilton Civic
Hospitals Research Centre, 711 Concession St., Hamilton, ON L8V 1C3,
Canada. Tel.: 905-574-8550; Fax: 905-575-2646; E-mail: jweitz@thrombosis.hhscr.org.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010048200
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ABBREVIATIONS |
---|
The abbreviations used are: f., factor; APTT, activated partial thromboplastin time; Fl-S5, fluorescein-labeled S5; LMWH, low molecular weight heparin; LA-LMWH, LMWH with low affinity for antithrombin; sLA-LMWH, sulfated LA-LMWH; S5, sLA-LMWH-S5; PCPS, phosphatidylcholine-phosphatidylserine; DHG, depolymerized holothurian glycosaminoglycan.
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