(Received for publication, July 19, 1996, and in revised form, September 17, 1996)
From the Departments of Pharmacology and
Pathology and Laboratory Medicine, ¶ Center for Thrombosis
and Hemostasis, The University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599
A heparin cofactor II (HCII) mutant with an Arg
substituted for Leu444 at the P1 position (L444R-rHCII) was
previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol.
Chem. 265, 5623-5628). The present study characterizes the
effect of glycosaminoglycans on the substrate versus
inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry
of inhibition of L444R-rHCII with -thrombin (compared with minus
glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a
mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate
decreased the stoichiometry of inhibition of L444R-rHCII with both
proteinases. SDS-polyacrylamide gel electrophoresis showed no
proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin
in the absence or the presence of glycosaminoglycan or with
-thrombin and dermatan sulfate. In contrast, greater than 75% of
the L444R-rHCII was converted to a lower molecular weight form when
incubated with
-thrombin/heparin. A time course of
-thrombin
inhibition by L444R-rHCII/heparin showed a rapid but transient
inhibition with approximately 80% of the
-thrombin activity being
regained after 6 h of incubation. In contrast, all other
combinations of inhibitor, proteinase, and glycosaminoglycan resulted
in complete and sustained inhibition of the proteinase. Heparin
fragments of 8-20 polysaccharides in length rapidly accelerated
L444R-rHCII inhibition of both
-thrombin and
R93A,R97A,R101A-thrombin. After extended incubations,
R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with
all the heparin fragments, but approximately 30-50% of
-thrombin
activity remained with fragments long enough to bridge HCII-thrombin.
These results collectively indicate that ternary complex formation,
mediated by heparin, increases L444R-rHCII inactivation by
-thrombin.
Serine proteinase inhibitors
(serpins)1 are a highly conserved family of
proteins whose primary role is to regulate the activity of a wide
variety of serine proteinases involved in processes such as blood
coagulation, fibrinolysis, inflammation, and cancer metastasis (1, 2, 3).
Serpins inhibit their target proteinase by functioning as suicide
substrates. The serpin contains a peptide bond, termed P1-P1 by the
nomenclature of Schechter and Berger (4), within an exposed region,
called the reactive site loop, that is recognized as a substrate by the
proteinase (2). The initial binding of the serpin and proteinase
results in the formation of a Michaelis complex, which can then
partition between a kinetically stable intermediate, resulting in
proteinase inhibition, and a cleaved inactive inhibitor (3, 5, 6).
Although the sequence of the amino acids in the reactive site loop and
on the P1-P1
residues in particular determines in part the proteinase
specificity of the serpin, they also determine the partitioning of the
serpin into an inhibitor versus substrate pathway with a
given proteinase.
Heparin cofactor II (HCII), protein C inhibitor, protease nexin-1,
plasminogen activator inhibitor-1, and antithrombin III (AT) belong to
a subgroup of serpins whose inhibitory activity is accelerated upon
binding to glycosaminoglycans, such as heparin, heparan sulfate, and
dermatan sulfate (Refs. 1, 2, 3 and 7, 8, 9) and references therein). AT has
a P1-P1 Arg-Ser reactive site, and this serpin is important for
regulating hemostasis because it inhibits thrombin and most other
coagulation proteinases in the presence of heparin or heparan sulfate.
Glycosaminoglycan-accelerated thrombin inhibition by AT requires the
formation of a ternary complex in which heparin binds AT and thrombin
simultaneously (10). Thrombin inhibition by AT in the presence of
heparin is accelerated more than 1,000-fold. In contrast to AT, the
target proteinase of HCII is believed to be only thrombin (11, 12), a
pluripotent serine proteinase with additional biological roles in
inflammation and wound healing. The P1-P1
bond of HCII
(Leu444-Ser445) is an unusual thrombin
substrate sequence because all coagulation proteinases prefer Arg at
the P1 position (13, 14). Thus, in the absence of glycosaminoglycan,
HCII is a poor thrombin inhibitor. However, HCII is a exceptional
thrombin inhibitor in the presence of heparin or dermatan sulfate,
either of which can accelerate the thrombin inhibition rate more than
9,000-fold (7, 15, 16, 17). Although heparin can simultaneously bind
thrombin and HCII, ternary complex formation does not appear to be
absolutely required for thrombin inhibition by HCII (18).
Mutations in the reactive site loop of AT (19),
1-antichymotrypsin (20, 21), and protein C inhibitor
(22) have been shown to affect the inhibitor versus
substrate properties of the serpin. In addition, exogenous factors such
as heparin and ionic strength (23) and the addition of peptides that
insert into the
-sheet of a serpin (24) can also change the serpin
from an inhibitor to a substrate. An HCII mutant with an Arg
substituted for Leu444 at the P1 position (L444R-rHCII) was
previously engineered and characterized by Derechin et al.
(25). Consistent with thrombin preferring a P1 Arg residue, the
thrombin inhibition rate of the mutant in the absence of
glycosaminoglycan was about 100-fold higher than wild type recombinant
HCII (wt-rHCII). Because a P1 Leu to Arg mutation in HCII caused
increased thrombin inhibitory activity in the absence of
glycosaminoglycans (25), we became interested in determining whether
this mutation altered HCII's inhibitor versus substrate
properties in the absence or the presence of glycosaminoglycans. In
this report we demonstrate that (a) although L444R-rHCII is
a better thrombin inhibitor, it is also a better thrombin substrate,
(b) heparin promotes L444R-rHCII inactivation by
-thrombin in a mechanism dependent upon ternary complex formation,
(c) dermatan sulfate does not promote L444R-rHCII inactivation, and (d) the effect of heparin on inactivation
is manifested only with L444R-rHCII and not with wt-rHCII.
Human wild type recombinant HCII (cDNA kindly provided by Dr. Douglas M. Tollefsen, Washington University School of Medicine, St. Louis, MO) was previously expressed in the baculovirus expression system and characterized (17). To obtain L444R-rHCII, site-directed mutagenesis was performed by the method of Kunkel (26) on a XhoI-EcoRI HCII cDNA cassette subcloned in the pBluescript SK+ mutagenesis and cloning vector (Stratagene). Positive clones were identified by DNA sequencing (Sequenase®, version 2.0, U. S. Biochemical). A XhoI-EcoRI cassette containing the mutation was subcloned into full-length HCII cDNA in the pGEM®-3 blue vector (Promega). Full-length HCII cDNA was then subcloned into the baculoviral transfer vector pVL1392 (PharMingen) via flanking EcoRI sites and co-transfected with linearized Autographica californica nuclear polyhedrosis virus (AcNPV) into Spodoptera frugiperda (Sf9, InVitrogen) insect cells (27). The infectious medium was collected 4 days post-transfection and used in plaque assays as described in PharMingen protocols. Individual plaques were isolated and amplified by infection of Sf9 cells in 24-well plates. Production of rHCII was determined by immunoblot analysis of whole cell lysates from plaque-infected cells; positive clones were amplified and stored at 4 °C. Sf9 cells were maintained in spinner flasks in Grace's medium (JRH Scientific) supplemented with 10% fetal bovine serum (HiClone), 0.3 g/liter L-glutamine (Life Technologies, Inc.), and 50 µg/ml gentamicin (Life Technologies, Inc.).
Protein Expression and PurificationHigh FiveTM insect
cells (InVitrogen) grown at 27 °C in serum free Ex-Cell 405 medium
with L-glutamine (JRH Scientific) were used to express
rHCII. Typically, two T150 flasks of High FiveTM cells were infected
with viral stock (200 µl/flask). The cell supernatant was collected
after 2 days and centrifuged at 350 × g for 5 min to
remove cell debris. The medium was diluted with an equal volume of HPN
buffer, pH 6.5 (20 mM Hepes, 0.1% polyethylene glycol
8000, 0.05% NaN3), and batch adsorbed with 0.5 ml of
heparin-Sepharose beads (Pharmacia Biotech Inc.) for 1 h at
4 °C. rHCII was eluted from the heparin-Sepharose with 0.5 M NaCl in HPN buffer, pH 7.4, after two washes in 75 mM NaCl in HPN, pH 6.5. The heparin-Sepharose eluate was
diluted in HPN buffer to a final concentration of 50 mM
NaCl, pH 7.8, and batch adsorbed with 0.5 ml of Q-Sepharose (Sigma) for 1 h at 4 °C. After two washes in
50 mM NaCl, the protein was eluted with 0.5 M
NaCl in HPN buffer, pH 7.8. The eluate was stored at 80 °C.
A direct enzyme-linked immunosorbent assay using a mouse anti-HCII monoclonal antibody and a goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma) was used as described previously to measure rHCII concentrations (17). Human plasma HCII, purified as described previously (28) was used as a standard. Assays were performed in 96-well microtiter plates and color development was monitored at 405 nm on a Vmax microplate reader (Molecular Devices). For immunoblot analysis, rHCII was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (29) and electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was probed with the anti-HCII monoclonal antibody, followed by goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma) as described previously (17). Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Corp.).
Inhibition Rate ConstantsHuman -thrombin was prepared
from blood plasma prothrombin as detailed previously (30). Wild type
recombinant thrombin and R93A,R97A,R101A-thrombin were kindly provided
by Dr. Charles T. Esmon (31). R93A,R97A,R101A-thrombin is a recombinant
thrombin that contains three point mutations in anion-binding exosite
2, the putative glycosaminoglycan-binding site, that results in
significantly reduced heparin affinity (31). Compared with wild type
recombinant thrombin, R93A,R97A,R101A-thrombin inhibition in the
presence of heparin or dermatan sulfate with HCII was not dramatically changed (Table I).2 Chromogenic substrate
was tosyl-Gly-Pro-Arg-p-nitroanilide (Boehringer Mannheim).
Heparin (Diosynth, The Netherlands) and dermatan sulfate (Calbiochem;
nitrous acid-treated to remove contaminating heparin and heparan
sulfate) were used for the glycosaminoglycan-accelerated thrombin
inhibition assays. All assays were performed at room temperature in
96-well microtiter plates (previously coated with 2 mg/ml bovine serum
albumin), and color development was monitored at 405 nm on a
Vmax microplate reader. Second order inhibition rates (k2, M
1 min
1)
were obtained as follows. In the absence of glycosaminoglycan, 200 nM wt-rHCII or 25 nM L444R-rHCII were incubated
with 1 nM thrombin in the presence of 50 µg/ml polybrene
(Sigma) and 2 mg/ml bovine serum albumin in HNPN, pH
7.4 (20 mM Hepes, 150 mM NaCl, 0.1%
polyethylene glycol 8000, 0.05% NaN3) for a range of time periods. For glycosaminoglycan-accelerated thrombin inhibition, 5 nM HCII, and 0.5 nM thrombin were incubated
with increasing amounts of heparin or dermatan sulfate for 20-60 s.
Inhibition was quenched with 150 µM
tosyl-Gly-Pro-Arg-p-nitroanilide and 3 mg/ml polybrene.
Second order inhibition rate constants were measured in triplicate on
two to three different preparations of rHCII. The rates were obtained
under pseudo-first order reaction conditions ([I] >>> [E]) and
were calculated using the equation k2 = (
ln
a)/t[I], where a is the residual
proteinase activity, t is the association time, and [I] is
the HCII concentration (32).
|
Experiments were performed in triplicate in 96-well bovine serum albumin-coated microtiter plates at room temperature in a reaction buffer containing 2 mg/ml bovine serum albumin in HNPN, pH 7.4. Stoichiometry of inhibition (SI) was determined by incubating increasing amounts of rHCII with 1 nM thrombin for 100 min in the absence or the presence of 5 µg/ml heparin or 20 µg/ml dermatan sulfate. This incubation time was greater than three half-lives of inhibition of the lowest inhibition rate (20, 22). The reaction was quenched with tosyl-Gly-Pro-Arg-p-nitroanilide and polybrene, and the residual thrombin activity was measured as described above. SI values were calculated by extrapolating the initial slope of a plot of thrombin activity on the y axis versus the ratio of rHCII/thrombin concentration on the x axis.
Visualization of rHCII-Thrombin Complexes and rHCII Proteolysis ProductsHCII (100 nM) was incubated with thrombin (10 nM) in HNPN, pH 7.4, in the absence of glycosaminoglycan or in the presence of either 5 µg/ml heparin or 20 µg/ml dermatan sulfate for 1, 5, and 30 min at room temperature. The reaction was terminated by the addition of 5 µM D-Phe-Pro-Arg-chloromethyl ketone (Calbiochem), which inactivates uncomplexed thrombin, and Laemmli sample buffer. SDS-PAGE and immunoblotting with an anti-HCII monoclonal antibody were performed as described above.
Extended Time Course StudiesHCII (10 nM) and thrombin (1 nM) were incubated in HNPN, pH 7.4 in the absence of glycosaminoglycan or in the presence of 5 µg/ml heparin or 20 µg/ml dermatan sulfate for various time points up to 18 h, and the residual thrombin activity was measured as described above. To determine the affect of ternary complex formation on substrate versus inhibitor properties of HCII, 1 nM thrombin and 10 nM HCII were incubated for 18 h in the presence of heparin fragments (1.4 µM, 3.4-8.4 µg/ml; Stago Diagnostica, kindly provided by Dr. Michel M. Canton) ranging from 8 to 20 oligosaccharides in length (HF8, HF12, HF16, and HF20). A low evaporation lid coated with anti-fogging agent (Molecular Devices) was used to minimize evaporation.
L444R-rHCII was generated, expressed, and purified as described under "Experimental Procedures." In a typical preparation, 50-130 µg of protein were obtained from two T150 flasks of High Five cells after sequential heparin- and Q-Sepharose batch adsorption of the conditioned medium. Immunoblot analysis and SDS-PAGE showed that L444R-rHCII co-migrated with wt-rHCII as a single band of approximately 70 kDa.
The data comparing the inhibition rate constants of L444R-rHCII and
wt-rHCII with -thrombin in the absence and the presence of
glycosaminoglycan are summarized in Table I. As shown
previously by Derechin et al. (25), replacement of the P1
leucine of HCII with an arginine greatly increases the thrombin
inhibition rate in the absence of glycosaminoglycan. Compared with
wt-rHCII, L444R-rHCII had a 54-fold higher rate of inhibition of
-thrombin. However, in the presence of optimal concentrations of
either heparin or dermatan sulfate, the maximal inhibition rate of
L444R-rHCII was approximately 5-fold lower than for wt-rHCII.
Wild type rHCII and L444R-rHCII were then tested for their ability to
inhibit wild type recombinant thrombin compared with R93A,R97A,R101A-thrombin, a recombinant thrombin mutant with defective heparin binding and heparin-mediated ternary complex formation with AT
(31). Overall, the rate constants of inhibition by wt-rHCII were not
substantially different when comparing -thrombin,
R93A,R97A,R101A-thrombin, and wild type recombinant thrombin (Table I).
For L444R-rHCII, all the inhibition rates were slightly higher with
R93A,R97A,R101A-thrombin as compared with
-thrombin and wild type
recombinant thrombin. However, dermatan sulfate-accelerated inhibition
of wild type recombinant thrombin was slightly higher but not
significantly different than that of R93A,R97A,R101A-thrombin.
The SI, the number of serpin molecules
consumed before an inactivated serpin-proteinase complex forms,
reflects the tendency of a serpin to behave as a substrate. The
titration curves for L444R-rHCII and wt-rHCII with -thrombin and
R93A,R97A,R101A-thrombin are shown in Fig. 1. In the
absence of glycosaminoglycan, approximately seven molecules of
L444R-rHCII are required to inhibit
-thrombin before a stable
complex is formed. Dermatan sulfate decreased the SI value to 2.5, whereas heparin increased it to 14.5, suggesting that heparin promoted
a more substrate-like behavior for L444R-rHCII. With
R93A,R97A,R101A-thrombin, the SI values in the absence of glycosaminoglycan (SI = 7.5) and in the presence of dermatan
sulfate (SI = 2.5) were comparable with those obtained with
-thrombin. In contrast, the addition of heparin (SI = 3) to
the R93A, R97A,R101A-thrombin inhibition reaction had the opposite
effect it did with
-thrombin and mimicked the results obtained with
dermatan sulfate. Wild type rHCII had an SI value ranging from 1.8 to 3 in either the presence of heparin or dermatan sulfate with either
thrombin variant. SI assays were also performed with wild type
recombinant thrombin, and the data agree with those obtained for
-thrombin (data not shown).
Immunoblot Analysis of Thrombin-HCII Reactions
The substrate
activity of wt-rHCII and L444R-rHCII in the absence and the presence of
glycosaminoglycan was visualized by SDS-PAGE and immunoblot analysis
(Fig. 2). Incubation of wt-rHCII with -thrombin or
R93A,R97A,R101A-thrombin in the presence of heparin or dermatan sulfate
resulted in a stable bimolecular complex with no apparent proteolysis
of the inhibitor (Fig. 2). Although heparin and dermatan sulfate
accelerated complex formation between wt-rHCII and either thrombin
variant, they had no effect on substrate activity. In the absence of
glycosaminoglycan, neither complex formation nor proteolysis of
wt-rHCII was visualized. In contrast, L444R-rHCII had moderate
substrate activity with
-thrombin in the absence of
glycosaminoglycan. After 30 min of incubation, approximately 10-25%
of L444R-rHCII is converted to a lower molecular weight form,
consistent with proteolysis at the reactive site loop. Heparin
accelerated the conversion of L444R-rHCII to a lower molecular weight
form, with approximately 90% of the L444R-rHCII being cleaved by
-thrombin after 30 min of incubation. Interestingly, dermatan
sulfate prevented L444R-rHCII cleavage by
-thrombin. Incubation of
R93A,R97A,R101A-thrombin with L444R-rHCII in the absence or the
presence of dermatan sulfate yielded results identical to those
observed with
-thrombin. However, heparin failed to facilitate the
proteolytic inactivation of L444R-rHCII by R93A,R97A,R101A-thrombin as
it did with
-thrombin. Longer exposures of the autoradiograph revealed SDS-PAGE-stable complexes between L444R-rHCII and thrombin for
all combinations except L444R-rHCII/
-thrombin/heparin (data not
shown).3
Extended Time Course of HCII-Thrombin Reactions
The HCII and
thrombin variants were incubated for various time periods up to 18 h in order to determine the time course and stability of thrombin
inhibition. Shown in Fig. 3 is a representative plot of
thrombin inhibition by L444R-rHCII in the absence and the presence of
glycosaminoglycan. Inhibition of R93A,R97A,R101A-thrombin by
L444R-rHCII occurs rapidly and is sustained over a 18-h period. Likewise, the inhibition of -thrombin by L444R-rHCII in the absence of glycosaminoglycan and in the presence of dermatan sulfate is also
sustained during the course of the experiment. In the presence of
heparin,
-thrombin is rapidly inhibited during the first 30-60 min,
but activity is slowly regained thereafter. Within 6 h of incubation, approximately 75-80% of
-thrombin activity is regained and is stable for at least 18 h. The instability of
-thrombin inhibition is due to the dissociation of L444R-rHCII/
-thrombin complexes promoted by heparin. There was no remaining thrombin activity
at the 18-h time point for wt-rHCII with either thrombin derivative in
the presence of heparin (data not included).
Role of Heparin and Heparin Fragments on the Substrate Activity of L444R-rHCII
To determine whether heparin bridging of -thrombin
to L444R-rHCII was a factor in the substrate-like behavior of
L444R-rHCII, thrombin inhibition assays with heparin fragments of 8-20
polysaccharides in length (HF8, HF12, HF16, and HF20) were performed.
The minimal heparin length required for ternary complex formation is
thought to be between 18 and 26 polysaccharides (16,
33, 34, 35).4 Thus, we postulated that the
heparin-mediated inactivation of L444R-rHCII by
-thrombin would
decrease with decreasing heparin fragment length. We chose an 18-h
incubation time to insure that the reaction process reached completion
with regards to either proteinase inhibition or serpin inactivation
with release of thrombin. Both
-thrombin and
R93A,R97A,R101A-thrombin were essentially completely inhibited after
18 h of incubation in the presence of wt-rHCII and HF12, HF16,
HF20, or heparin; approximately 5-10% of activity remained with
the HF8, consistent with the lower inhibition rate of wt-rHCII with a
short heparin fragment (Fig. 4). The same results
were obtained for L444R-rHCII with R93A,R97A,R101A-thrombin. In
contrast, approximately 30 and 46% of
-thrombin activity remained when incubated with L444R-rHCII and HF16 or HF20, respectively, as
compared with approximately 5% remaining activity with HF8 or HF12.
Control experiments verified that all of the heparin fragments
accelerated thrombin inhibition by L444R-rHCII as found previously with
other heparin-binding serpins (16). However, unlike HF8 and HF12, which
supported complete
-thrombin inhibition by L444R-rHCII, HF16- and
HF20-accelerated
-thrombin inhibition leveled off after 30 min of
incubation, which would be consistent with inactivation of L444R-rHCII
(data not included).
Mutations in the reactive site loop of numerous serpins have
altered their proteinase specificity and inhibitory activity (Refs. 19, 20, 21, 22, 36, 37; for a review see Ref. 38). The regions studied
most intensively have been the P1-P1 residues and the residues of the
hinge region (P9-P15), where the reactive site loop turns and joins the
A
-sheet as strand 5A. A common prediction from these studies would
be that increased inhibitory activity (measured by k2
values) would give lower SI values. Furthermore, increased substrate
activity would be correlated with reduced inhibitory activity. In 1990, Derechin et al. (25) prepared an HCII mutant with a P1
residue substitution of Leu to Arg with the expectation that this form
of rHCII would be a superb thrombin inhibitor (measured by
k2 values). In the absence of glycosaminoglycans, their
mutant was about 100-fold more active than wild type recombinant HCII.
However, the maximal rate enhancement of thrombin inhibition by
glycosaminoglycans was lower with L444R-rHCII than with
wt-rHCII.5 We became interested in further
exploring the mechanism of action of this "ideal" thrombin
inhibitor in the absence and the presence of glycosaminoglycans. The
present study shows that substitution of an Arg at the P1 Leu position
of rHCII increases its thrombin inhibitory activity in the absence of
glycosaminoglycan but also increases its substrate activity. Although
heparin and dermatan sulfate both accelerate
-thrombin inhibition by
L444R-rHCII to levels slightly lower than with wt-rHCII, heparin, but
not dermatan sulfate, promotes the proteolytic inactivation of
L444R-rHCII by
-thrombin.
Heparin-induced proteolytic inactivation of a related serpin, AT, was
described previously by Olson (23). AT was shown to be an inhibitor in
the presence of heparin at physiological ionic strength, but at lower
ionic strength, heparin promoted the substrate pathway of AT with
thrombin (23). Low ionic strength presumably increased heparin binding
to an antithrombin-thrombin intermediate complex, which normally
exhibits low heparin affinity (39, 40, 41, 42). Furthermore, only heparin
molecules long enough to bridge thrombin and AT promoted the substrate
pathway. This study showed that the formation of cleaved AT occurred
through a substrate pathway different than that of the slow spontaneous
dissociation of stable antithrombin-thrombin complexes (43). Our
results suggest that unlike AT, heparin affects both the proteolytic
inactivation of excess L444R-rHCII by thrombin and accelerates the slow
dissociation of L444R-rHCII-thrombin complexes at physiological ionic
strength. The result of extended time course incubations of L444R-rHCII with -thrombin in the presence of heparin reflects the slow release of active thrombin from ternary complexes composed of L444R-rHCII, thrombin, and heparin. One plausible explanation is that the
heparin-promoted ternary complex places L444R-rHCII in a favorable
position for cleavage by
-thrombin; thus, initially, inactivation of
L444R-rHCII and inhibition of thrombin are occurring simultaneously and
over time, the remaining L444R-rHCII that is complexed with thrombin is
slowly cleaved, releasing active thrombin.
AT requires ternary complex formation not only for heparin-accelerated
thrombin inhibition but also for heparin-accelerated inactivation by
thrombin (10, 23, 33). With L444R-rHCII, ternary complex formation
mediated by heparin appears necessary only for the proteolytic
inactivation by -thrombin. With R93A,R97A,R101A-thrombin, which is
defective in glycosaminoglycan binding and ternary complex formation,
heparin accelerates its inhibition by L444R-rHCII but not L444R-rHCII
inactivation. Dermatan sulfate, which cannot mediate ternary complex
formation (18), does not promote inactivation of L444R-rHCII,
regardless of which proteinase is used. The requirement of ternary
complex formation for L444R-rHCII inactivation is further supported by
the data with small heparin oligosaccharides of 8 or 12 units. These
heparin fragments are unable to bridge both HCII and thrombin, and they
accelerate
-thrombin inhibition by L444R-rHCII (and wt-rHCII)
without promoting L444R-rHCII inactivation. L444R-rHCII inactivation by
-thrombin is promoted by heparin oligosaccharides
16 units,
close to the putative minimal length capable of bridging HCII to
thrombin (34, 35).4 The detrimental inactivating action of
the longer heparin oligosaccharides on L444R-rHCII was absent with
R93A,R97A,R101A-thrombin. Thus, ternary complex formation between
heparin, thrombin, and L444R-rHCII, rather than heparin binding to
L444R-rHCII per se, appears to cause the substrate-like activity of
L444R-rHCII.
The differences observed between this study and Olson's study (23) are not surprising because HCII and AT appear to follow different mechanisms of heparin-accelerated thrombin inhibition. Ternary complex formation is absolutely required for the heparin-accelerated inhibition of thrombin by AT (10). Binding of heparin to AT also results in a conformational change in the reactive site loop that slightly increases the rate of thrombin inhibition (44, 45, 46). Glycosaminoglycan-accelerated thrombin inhibition by HCII occurs through an allosteric mechanism, whereby binding of the glycosaminoglycan to HCII facilitates the interaction of a hirudin-like amino-terminal domain of HCII with anion-binding exosite 1 of thrombin (18). Ternary complex formation occurs in the presence of heparin but makes only a minor contribution in HCII inhibition of thrombin. Dermatan sulfate, which binds thrombin only weakly and thus does not participate in ternary complex formation, is as effective as heparin in accelerating thrombin inhibition (15, 47). Thus, the ability of a glycosaminoglycan to form a ternary complex contributes only minimally to HCII inhibition of thrombin, implying that heparin and dermatan sulfate employ similar mechanisms of catalysis of the inhibition reaction. However, our results suggest different effects of the two glycosaminoglycans in terms of binding to and inactivation of L444R-rHCII by thrombin. It is possible that ternary complex formation, mediated by heparin, induces a conformational change in the reactive site loop that is more readily detected in L444R-rHCII as an increase in susceptibility to proteolytic inactivation.
The presence of Leu in the P1 site of HCII has always presented a
dilemma, because thrombin strongly favors cleavage after Arg residues.
In a previous molecular modeling study, we found no major binding
constraints with the binding pocket of thrombin for macromolecular
substrates containing Pro-Arg versus Pro-Leu at the P2-P1
positions (32), indicating that either P1 residue could be
accommodated. The recognition sites of different macromolecular substrates and inhibitors of thrombin were compared, and it was found
that the P1 residue was almost exclusively Arg, whereas Pro was
preferred at the P2 residue (48, 49). Thus, the L444R-rHCII reactive
site sequence would be an ideal thrombin recognition sequence because
it has a P2-P1-P1 sequence of Pro-Arg-Ser. HCII may have evolved with
a P1 Leu residue in order to remain essentially inactive in the absence
of glycosaminoglycans (i.e. while circulating in blood). In
its glycosaminoglycan-bound active form, HCII compensates for its lack
of an ideal P1 residue by the presence of its hirudin-like acidic
domain, which interacts with anion-binding exosite 1 of thrombin.
HCII-thrombin inhibitory activity is believed to be localized to
proteoglycan-rich surfaces. Because HCII and thrombin both bind
glycosaminoglycans, their co-localization to these sites might result
in ternary complex formation concomitant with the inhibition reaction.
Therefore, the P1 Leu would render HCII relatively resistant to
inactivation by thrombin in the presence of proteoglycans that might in
fact mediate ternary complex formation.
We thank Douglas M. Tollefsen and his laboratory group for sharing data on L444R-rHCII and for helpful advice during our investigation. We are most appreciative of this generosity. We thank Charles T. Esmon for providing recombinant wild type and mutant thrombins and Michel M. Canton for providing the heparin fragments. We also thank Dougald M. Monroe and Susannah J. Bauman for critical review of the manuscript.