From the Department of Medicine, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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The proteolytic formation of thrombin is
catalyzed by the prothrombinase complex of blood coagulation. The
kinetics of prethrombin 2 cleavage was studied to delineate
macromolecular substrate structures necessary for recognition at the
exosite(s) of prothrombinase. The product, -thrombin, was a linear
competitive inhibitor of prethrombin 2 activation without significantly
inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and
-thrombin compete for binding to the exosite without restricting
access to the active site of factor Xa within prothrombinase.
Inhibition by
-thrombin was not altered by saturating concentrations
of low molecular weight heparin. Furthermore, proteolytic removal of
the fibrinogen recognition site in
-thrombin only had a modest effect on its inhibitory properties. Both
-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp148 and
separated into component domains. The C-terminal-derived
2
fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the
1 fragment
was without effect. As the
2 fragment lacks the fibrinogen
recognition site, the P1-P3 residues or the intact cleavage site,
specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the
fibrinogen recognition or heparin-binding sites, and spatially removed
from structures surrounding the scissile bond.
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INTRODUCTION |
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The conversion of prothrombin to the serine proteinase,
-thrombin, is pivotal for the maintenance of hemostasis. The
specific proteolytic activation of prothrombin is catalyzed by the
prothrombinase complex, which assembles through reversible interactions
between the trypsin-like serine proteinase, factor Xa, and the cofactor protein, factor Va, on appropriate phospholipid surfaces in the presence of Ca2+ ions (6, 7). Although factor Xa itself can
catalyze prothrombin cleavage, the macromolecular interactions which
stabilize prothrombinase lead to a profound increase, by a factor of
~100,000, in the catalytic efficiency of prothrombin activation
(6-8).
A major fraction of the increased catalytic efficiency observed upon assembly of factor Xa into the prothrombinase complex likely arises as a result of the influence of factor Va on the catalyst (6). However, the molecular basis for the ability of factor Va to enhance the catalytic efficiency of factor Xa within prothrombinase is poorly understood as is the basis for the narrow and distinctive macromolecular substrate specificity of factor Xa, despite its high degree of homology with trypsin (6, 9). These two aspects of enzymic function appear closely related since the increased rate of prothrombin activation that results from the incorporation of factor Xa into the prothrombinase complex is not accompanied by changes in the rate of cleavage of synthetic peptidyl substrates, in the reaction with active site-directed reagents or even in the rate constant for inhibition by macromolecular inhibitors such as antithrombin III (10-12).
Suggestions for the importance of specific macromolecular recognition, by prothrombinase, through interactions at extended recognition sites removed from the active site of factor Xa (exosites) were initially derived from studies with tick anticoagulant peptide (TAP)1 (13). Work with a mutant derivative of TAP suggested that the selective modulation of such exosite interactions following the assembly of factor Xa into prothrombinase could lead to large changes in affinity and kinetic mechanism in the interaction of the enzyme with macromolecules (14). However, the significance of these findings toward prothrombin activation by prothrombinase has required documentation by appropriate functional studies.
The interpretation of kinetic studies of prothrombin activation is complicated by the fact that the conversion of prothrombin to thrombin involves the cleavage of two peptide bonds and is the sum of two consecutive enzyme-catalyzed reactions (6, 15). However, kinetic interpretations are simplified by the use of prethrombin 2 as a substrate analog, which requires cleavage at a single site to yield thrombin (16). The kinetics of recognition and cleavage of this bond in prethrombin 2 are established to be indistinguishable from the cleavage of the same site in intact prothrombin (15).
Studies with reversible inhibitors targeting the active site of factor Xa have provided evidence for a significant contribution from exosite interactions within prothrombinase in the recognition of prethrombin 2 (17). Active site-directed reversible inhibitors as well as oligopeptidyl alternate substrates are classical noncompetitive inhibitors of macromolecular substrate cleavage by prothrombinase despite their established ability to compete for substrate binding to the active site (17). In contrast, thrombin is a competitive product inhibitor of prethrombin 2 activation but does not interfere with oligopeptidyl substrate cleavage by prothrombinase (17). These findings indicate that the affinity and binding specificity for prethrombin 2 is determined by interactions at exosites rather than by interactions between elements surrounding the scissile bond and the active site of the protease (17). Thus, competitive inhibition of macromolecular substrate cleavage by thrombin is achieved by competition for the initial exosite interaction between the substrate and prothrombinase.
It therefore follows that the structural features of prethrombin 2 that determine substrate affinity, through interactions with the exosite, are spatially distinct from residues surrounding the scissile bond. Following cleavage, the polypeptide sequence N-terminal to the scissile bond is not released, but retained in the two chain product, thrombin, through a disulfide bond (18). Since thrombin competes for prethrombin 2 binding without obscuring access to the active site of prothrombinase, it also follows that the interaction between thrombin and prothrombinase is achieved by product domains spatially distinct from the P1-P32 residues found in the A-chain of thrombin. We have used proteolytic derivatives of thrombin and prethrombin 2 as well as ligands established to bind to specific sites in the substrate and product, to test these predictions and delineate the regions of the substrate that contribute to binding specificity through interactions with the prothrombinase exosite.
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EXPERIMENTAL PROCEDURES |
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Materials
Hepes, Trizma (Tris base), L--phosphatidylserine
(PS), L-
-phosphatidylcholine (PC),
p-amidinophenylmethanesulfonyl fluoride (APMSF), bovine
fibrinogen (Fraction I, Type IV), and soybean trypsin inhibitor
immobilized on Sepharose were from Sigma. Glu-Gly-Arg chloromethylketone (EGR-CH2Cl) was from Calbiochem (La
Jolla, CA). Succinimidyl acetylthioacetate and Oregon
Green488 iodoacetamide was purchased from Molecular Probes
(Eugene, OR). Bovine chymotrypsin (N
-p-tosyl-L-lysine
chloromethyl ketone-treated) and trypsin (TPCK-treated) were from
Worthington (Freehold, NJ). Peptidyl substrates
H-D-Phe-pipecolyl-Arg p-nitroanilide (S2238) and
methoxycarbonylcyclohexyl-Gly-Gly-Arg p-nitroanilide
(Spectrozyme Xa, SpXa) were from Chromogenix (Molndal, Sweden) and
American Diagnostica (Greenwich, CT), respectively. Stock solutions
(~4 mM) of either substrate were prepared in water and
concentrations were determined using E342 = 8270 M
1 cm
1 (19). Polyethylene
glycol Mr = 8000 (PEG) was obtained from J. T. Baker (Danvers, MA). All kinetic studies were performed in 20 mM Hepes, 0.15 M NaCl, 2 mM
CaCl2, 0.1% (w/v) PEG, pH 7.4 (assay buffer) at 25° C.
Phospholipid vesicles (PCPS) composed of 75% (w/w) PC and 25% (w/w)
PS were prepared as described previously (20). The concentration of
phospholipid was determined after oxidation by a colorimetric phosphate
assay and is stated as the concentration of inorganic phosphate (21).
The concentration of unfractionated heparin (ESI, Cherry Hill, NJ) was
calculated assuming an average molecular weight of 20,000. Low
molecular weight heparin (LMW heparin) was obtained from Celsus
Laboratories (Cincinnati, OH). The average molecular weight (3,000) of
this preparation is consistent with the predominant species being
composed of 5 disaccharides. HirugenTM, a synthetic peptide comprising
the C-terminal region of hirudin
(N-acetyl-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-SO3-Leu) (22), originally manufactured by Biogen (Cambridge, MA) was a gift from
Drs. S. R. Hanson and L. A. Harker (Emory University).
Proteins
Prothrombin and factor X were purified from bovine plasma as
described previously (23). Bovine factor X was converted to factor Xa
by using the purified activator from Russell's viper venom and further
purified by chromatography using benzamidine Sepharose (24, 25).
Kinetic titration of factor Xa with
p-nitrophenyl-p'-guanidinobenzoate (26),
typically yielded 1.12-1.15 mol of active sites/mol of factor Xa.
Bovine factor Va was purified using an established procedure (27) and
recombinant TAP was produced and purified as described (28).
Prethrombin 2 and -thrombin were prepared as described previously
(16, 18). Gel-filtration chromatography of prethrombin 2 on Sephadex
G-50 in 20 mM Hepes, 2.5 M sodium chloride, pH
7.4 (16), resolved prethrombin 2 from a polymeric form eluting at the
void volume. The aggregated species was discarded as it was found to be
a potent inhibitor of prethrombin 2 cleavage by prothrombinase.
Fibrin-Sepharose was prepared as described and extensively precycled
prior to use (29). Buffer components of the hirugen preparation were
removed by preparative reversed phase HPLC (Aquapore C-18, ABI, San
Jose, CA) and elution with a gradient of increasing CH3CN
in 0.1% (v/v) trifluoroacetic acid. Multiple peaks were identified by
monitoring absorbance at 215 nm. Heterogeneity in the sample was
confirmed by microbore reversed phase HPLC. However, amino acid
analysis was consistent with the primary structure of hirugen and the
basis for sample heterogeneity could not be determined by further
analysis using either laser desorption (MALDI) or electron spray mass
spectrometry. As the individual species identified by HPLC could not be
adequately resolved on a preparative scale, a single pool was prepared,
lyophilized, and dissolved in assay buffer. Peptide content was
established by amino acid analysis.
Fragments of thrombin and prethrombin 2 were analyzed by MALDI mass spectrometry and by automated Edman degradation at the Emory University Microchemical facility. For N-terminal sequence analysis, fragments were separated by SDS-PAGE following disulfide bond reduction with dithiothreitol and transferred to Immobilon PSQ membranes (Millipore, Bedford, MA) by electroblotting in a semidry apparatus (Hoeffer, San Francisco, CA) as described previously (30). Protein sequence was determined from excised membrane fragments.
Preparation of Thrombin Derivatives
Inactivation of -thrombin with APMSF to yield
-IIai was performed as described previously (17).
For the preparation of bovine T-thrombin,
-thrombin
in assay buffer (22 µM, 45 ml) was incubated with 0.7 µM trypsin for 3 h at room temperature. The reaction
was quenched with 10 µM soybean trypsin inhibitor,
dialyzed against 20 mM Tris-PO4, pH 5.8, 40 mM NaCl, 0.1% (w/v) PEG for 4 h at 4 °C and
applied to a column (1.5 × 12.5 cm) of S-Sepharose equilibrated
in the same buffer. Bound protein was eluted (4 ml/min, 120 min) with a
linear gradient of increasing NaCl (40-700 mM) in 20 mM Tris-PO4, 0.1% (w/v) PEG, pH 5.8. All
fractions containing protein exhibited equivalent specific activities
to
-thrombin in the cleavage of S2238. Fractions from the leading
peak, containing
T-thrombin with an estimated
contamination of 5% undigested material determined by SDS-PAGE were
pooled, dialyzed against 20 mM Hepes, 40 mM
NaCl, 0.1% (w/v) PEG, pH 7.4, and subject to affinity chromatography using a 4.5 × 17-cm column of fibrin-Sepharose equilibrated in the same buffer. Bound protein was eluted with a linear gradient of
increasing NaCl (40-600 mM) in 20 mM Hepes,
0.1% (w/v) PEG, pH 7.4. The
T-thrombin containing
fractions were pooled and reapplied to a second fibrin-Sepharose column
with isocratic elution to remove traces of remaining
-thrombin. The
flow-through fractions were characterized by the same specific activity
as
-thrombin toward S2238 with ~1% of the specific activity of
-thrombin in a fibrinogen clotting assay (18). Pooled material was
concentrated by ultrafiltration in a stirred cell (YM10, Amicon,
Danvers, MA) to a concentration of ~100 µM, inactivated
by the addition of 1 mM APMSF followed by brief incubation
at room temperature and dialyzed against assay buffer. The resulting
preparation of inactivated
T-thrombin
(
T-IIai) possessed <0.01% catalytic activity when compared with
-thrombin. Protein sequencing of the fragments resolved by SDS-PAGE yielded the expected sequence for bovine
T-thrombin (3-5, 31). In addition, the mass of
T-thrombin was determined by MALDI mass spectrometry and
found to be consistent with removal of the undecapeptide
(Ile68-Arg77A).3
Bovine -thrombin was prepared by treatment of
-thrombin (27 µM, 15 ml) in 0.25 M sodium phosphate buffer,
pH 6.5, with 4.2 nM chymotrypsin for 4 h at room
temperature. The digest, terminated by addition of 10 µM
TPCK, was dialyzed against 20 mM MES, 80 mM
NaCl, pH 6.5, and applied to a column (1.5 × 8 cm) of S-Sepharose equilibrated in the same buffer. Elution with a linear gradient (4 ml/min, 130 min) of increasing NaCl (80-800 mM) in 20 mM MES, pH 6.5, resulted in the separation of chymotrypsin,
undigested thrombin, and two peaks of cleaved material. Protein in the
two resolved peaks of cleaved material appeared identical by SDS-PAGE, protein sequencing, and specific activity measured with S2238. The
cleaved material was pooled, concentrated, inactivated with AMPSF, and
dialyzed into assay buffer as described above. Protein sequence
analysis indicated a single cleavage at Trp148 consistent
with the expected result for the action of chymotrypsin on human
thrombin to yield
-thrombin (32). On this basis, the resulting
inactivated preparation was designated
-IIai.
The two chymotryptic fragments of thrombin were isolated by HPLC
separation of the products generated following chymotryptic cleavage of
-thrombin. Thrombin (30 mg) treated with chymotrypsin (as above) was
dialyzed against 20 mM Tris, 30% (v/v) CH3CN,
pH 9.0, for 4 h at room temperature. Following clarification by
centrifugation (50,000 × g, 20 min), aliquots (3 ml)
were fractionated by cation exchange HPLC (Aquapore Cation 7 µm,
0.46 × 22.2 cm, ABI). Elution (1 ml/min, 30 min) with a linear
gradient of increasing NaCl (0-350 mM) in 20 mM Tris, 30% (v/v) CH3CN, pH 9.0, resolved two
peaks corresponding to the two thrombin fragments. Material from each of the peaks accumulated from successive runs was pooled, dialyzed against 0.1% (v/v) trifluoroacetic acid, concentrated by
lyophilization, and further purified by reversed phase HPLC. Each of
the pools was dialyzed against Buffer A (20 mM
NEt3-PO4, pH 2.5) and fractionated in ~1-mg
aliquots using an Aquapore Phenyl column (0.46 × 22.2 cm, ABI).
Bound protein was eluted (1 ml/min) with a biphasic gradient of
increasing Buffer B (20 mM
NEt3-PO4, 80% (v/v) CH3CN, pH 2.5)
of 0-24% Buffer B in 25 min followed by 30-37% Buffer B in 80 min.
Analysis by SDS-PAGE confirmed quantitative separation of the two
fragments which were identified as
1-thrombin and
2-thrombin based on N-terminal sequence analysis and mass
spectrometry. Pools were prepared for each of fragments, lyophilized,
dialyzed against assay buffer, and clarified by centrifugation
(50,000 × g, 20 min). Dialysis resulted in variable
amounts of protein loss due to precipitation. The final concentrations
were in the range of 15 µM for
1-thrombin and 40 µM for
2-thrombin.
Preparation of Prethrombin 2 Derivatives
The two chymotryptic fragments of prethrombin 2 were prepared by
treating prethrombin 2 (27 µM, 5 ml) in 0.25 M sodium phosphate, pH 6.5, with 1 nM
chymotrypsin for 3 h at room temperature. The reaction mixture was
dialyzed against Buffer A (above) and fragments were separated by
reversed phase HPLC as described above for the thrombin fragments. The
resulting peptides were lyophilized, dialyzed against assay buffer, and
clarified by centrifugation (50,000 × g, 20 min). The
fragments were designated 1-prethrombin 2 and
2-prethrombin 2 on
the basis of SDS-PAGE, N-terminal sequence analysis, and mass
spectrometry.
The purity of all protein preparations was judged by SDS-PAGE (33).
Protein concentrations were determined using the following molecular
weights and extinction coefficients
(E2800.1%): bovine factor Xa,
45,300, 1.24 (34, 35); bovine factor Va, 168,000, 1.74 (27, 36); bovine
prethrombin 2, 37,400, 1.95 (31); bovine -thrombin, 37,400, 1.95 (18); trypsin, 23,800, 1.58 (37); chymotrypsin 24,200, 1.88 (37). The
concentrations of the thrombin and prethrombin 2 derivatives were
determined using molecular weights determined from the primary
structure and extinction coefficients calculated by the method of Gill
and von Hippel (38):
T-thrombin, 34,100, 1.89;
-thrombin 37,400, 1.85;
1-thrombin or
1-prethrombin 2, 23,000, 1.83;
2-thrombin or
2-prethrombin 2, 12,450, 1.90.
Preparation of a Fluorescent Derivative of Factor Xa
Factor Xa with a fluorescent probe tethered to the active site via a peptidyl chloromethyl ketone was prepared using the procedure developed by Bock (39). In the first step, the acetothioester derivative of EGR-CH2Cl was prepared by minor modifications to described procedures (39). Purification of the acetothioacetyl adduct of EGR-CH2Cl (ATA-EGR-CH2Cl) was performed by application to a column (1.5 × 120 cm) of S-Sepharose equilibrated in 50 mM sodium phosphate, pH 2.5, with isocratic elution. The resulting ATA-EGR-CH2Cl was identified, concentrated, and characterized as described (39). Factor Xa in 0.5 M Tris, 0.1% (w/v) PEG, pH 7.5, was inactivated by sequential additions of ATA-EGR-CH2Cl and the resulting modified factor Xa (ATA-EGR-Xa) was separated from excess unreacted inhibitor by chromatography on a G-25 column (40). Traces of uninhibited factor Xa were depleted by affinity chromatography using soybean trypsin inhibitor Sepharose (41). In the final step, a fluorophore was incorporated into the active site by reacting ATA-EGR-Xa (60 µM, 3 mg) with 680 µM Oregon Green488 iodoacetamide in 0.1 M Hepes, 0.15 M NaCl, 87 mM NH2OH, pH 7.0, for 75 min at room temperature. Unreacted dye and buffer components were separated from the protein by gel filtration as described previously (40), to yield the fluorescent adduct of factor Xa ([OG488]-EGR-Xa). The concentration of the fluorescent adduct was determined with a colorimetric protein assay (BCA assay, Pierce, Rockford, IL) using a standard curve prepared with known concentrations of ATA-EGR-Xa (40).
Discontinuous Measurements of the Initial Rate of Thrombin Formation
The initial rate of thrombin formation by the action of prothrombinase on prethrombin 2 was determined as described previously (16). Reaction mixtures (290 µl) were prepared in assay buffer containing 25 nM factor Va, 60 µM PCPS, the indicated concentrations of prethrombin 2 and inhibitor at 25° C. Thrombin formation was initiated by the addition of 5 nM factor Xa (10 µl, 150 nM). Aliquots (10 µl) were withdrawn either before initiation (t = 0 datum) or serially at t = 0.5, 1, 1.5, 2, and 3 min following the addition of factor Xa, and mixed with 90 µl of 20 mM Hepes, 0.15 M NaCl, 0.1% (w/v) PEG, 50 mM EDTA, 2 µM TAP, pH 7.4, to quench thrombin formation. Aliquots (10 µl) of each quenched sample were diluted further in the same buffer but lacking TAP in wells of a 96-well plate and the initial velocity of S2238 hydrolysis was determined by continuously monitoring the change in absorbance at 405 nm in a kinetic plate reader (Molecular Devices, Sunnyvale, CA) following the addition of 200 µM S2238 in 20 mM Hepes, 0.15 M NaCl, 0.1% (w/v) PEG, 50 mM EDTA, pH 7.4. The concentration of thrombin formed as a function of time was determined by interpolation from the linear dependence of initial velocity of S2238 hydrolysis on known concentrations of thrombin performed as a control with each experiment. The initial, steady state rate of thrombin formation in the initial reaction mixture was determined from the slope of the linear appearance of thrombin as a function of time. Control experiments established that inhibitors added to the initial stage of the assay were sufficiently dilute to have no noticeable effect on the activity of thrombin toward S2238. When inhibition studies were performed using fragments purified by HPLC and dialysis, control experiments using appropriate volumes of the dialysate ruled out trace solvent contamination as a trivial explanation for the results. For kinetic measurements in the presence of hirugen, the final concentration of factor Xa was increased to 10 nM and aliquots were quenched at 0, 1, 2, 3, 4, and 6 min following initiation.
The concentrations of factor Va and PCPS were chosen to be saturating relative to the concentration of factor Xa and to the measured equilibrium dissociation constants for the binary interactions that lead to the assembly of the prothrombinase complex (42). It is therefore valid to normalize reaction rates by dividing by the concentration of factor Xa which determines the concentration of prothrombinase. The validity of this assumption was established in separate control experiments documenting the linear dependence of reaction rate on the concentration of factor Xa under these conditions.
Steady State Measurements of Fluorescence Anisotropy
Fluorescence anisotropy was measured in T-format using a
SLM8000C fluorescence spectrophotometer (SLM Instruments, Urbana, IL).
Anisotropy was measured in 1 × 1-cm2 quartz cuvettes
using ex = 490 nm and
em = 520 nm with
long pass filters (Schott KV-520) in the emission beam. Reaction
mixtures (2 ml) in assay buffer containing the indicated concentration of [OG488]-EGR-Xa, 50 µM PCPS and the
indicated concentration of
-IIai were titrated with
microliter additions of a stock solution of factor Va prepared in assay
buffer. Following each addition, the reaction mixture was gently mixed
and anisotropy was measured at 25° C by manually rotating the
excitation polarizer, integrating the signal at each of the two
positions for 10 s and averaging 5-8 successive readings.
Anisotropy was calculated (43), following the subtraction of a
scattering blank from a reaction mixture containing identical reactant
concentrations except that factor Xa previously inactivated with APMSF
(Xai) was used instead of [OG488]-EGR-Xa. When
necessary,
r at each concentration of titrant was
calculated by subtracting the anisotropy observed in the absence of
factor Va. Displacement experiments were performed by preforming
prothrombinase in assay buffer using 28.8 nM
[OG488]-EGR-Xa, 50 µM PCPS and either 20 or
60 nM factor Va. Anisotropy was measured following
titration with increasing concentrations of Xai. Following
corrections for scattering,
r at each addition of titrant was calculated by subtraction of the anisotropy from that of parallel reaction mixtures lacking factor Va.
Data Analysis
Data were analyzed according to the indicated equations by nonlinear least squares regression analysis using the Marquardt algorithm (44). Alternative models were eliminated on the basis of poorer fits on the basis of criteria described previously (45). The fitted constants are presented ± 95% confidence limits and one of at least two similar experiments performed using different protein preparations is presented in each case.
Inhibition of Prethrombin 2 Activation by Thrombin and Derivatives-- Initial velocity measurements of prethrombin 2 activation by prothrombinase using increasing concentrations of substrate at different fixed concentrations of thrombin derivatives or determined at one fixed concentration of prethrombin 2 and increasing concentrations of inhibitor were analyzed according to the rate expression for linear competitive inhibition (46), to yield fitted values for Km, Vmax, and Ki.
Inhibition of Prethrombin 2 Activation by Hirugen-- Initial rates of thrombin formation by prothrombinase at increasing concentrations of prethrombin 2 in the presence of different fixed concentrations of hirugen were adequately described using Equation 1 (46) which assumes that 1 mol of hirugen (I) reversibly binds per mole of prethrombin 2 (S) and the resulting SI complex cannot bind to prothrombinase,
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(Eq. 1) |
Fluorescence Measurements of the Factor Xa and Va Interaction-- Fluorescence titrations of prothrombinase assembly obtained at one or more fixed concentrations of [OG488]-EGR-Xa, a fixed and saturating concentration of PCPS with varying concentrations of factor Va were analyzed using the model and experimental considerations previously developed for the assembly of prothrombinase (42). In this case, titration curves describe the equilibrium dissociation constant for the interaction between [OG488]-EGR-Xa and factor Va on the membrane surface (42). The dependence of fluorescence anisotropy on the concentration of factor Va was analyzed according to Equation 2,
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(Eq. 2) |
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RESULTS |
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Kinetics of Inhibition of Prothrombinase by
-Thrombin--
Previous studies have shown that
-thrombin acts
as an exosite-directed product inhibitor of macromolecular substrate
cleavage by prothrombinase (17). Thrombin, inactivated with APMSF
(
-IIai) was tested for its ability to inhibit the cleavage
of either a tripeptidyl substrate (SpXa) or the macromolecular
substrate analog, prethrombin 2, by prothrombinase (Fig.
1). Even though either substrate was
present at approximately the same multiple of Km (~0.7 × Km), increasing concentrations of
-IIai yielded significant inhibition of prethrombin 2 activation (Ki = 2.02 ± 0.11 µM)
with a minor effect (Ki
80 µM) on the initial rate of SpXa hydrolysis (Fig. 1). Initial velocity studies
using increasing concentrations of prethrombin 2 at different fixed
concentrations of
-IIai (not shown) yielded linear competitive inhibition with Ki = 2.15 ± 0.3 µM (Table I). Thus,
-IIai and prethrombin 2 bind in a mutually exclusive fashion
to prothrombinase. Because
-IIai has a minor effect on
peptidyl substrate hydrolysis by prothrombinase, competitive
inhibition of prethrombin 2 cleavage is achieved without restricting access to the active site of factor Xa within the prothrombinase complex. Such observations, in part, form the basis for
the previous suggestion that the affinity of the enzyme complex for
macromolecular substrates such as prethrombin 2 is determined by
binding interactions at exosites and not the active site of factor Xa
within prothrombinase (17).
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Influence of Thrombin on the Assembly of the Prothrombinase
Complex--
Factor Xa assembled into the ternary prothrombinase
complex with saturating concentrations of factor Va and phospholipid
membranes catalyzes prethrombin 2 cleavage with greatly increased
catalytic efficiency compared with factor Xa in solution or saturated
with membranes (16). Since the three enzyme species only exhibit minor
differences in the kinetics of hydrolysis of SpXa and other peptidyl
substrates (11), inhibition of the assembly of prothrombinase by
-IIai could provide a trivial explanation for the selective
inhibition of macromolecular substrate cleavage.
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Dissection of Interaction Sites of Thrombin and Prethrombin 2 with
Prothrombinase--
A schematic representation of prethrombin 2, -thrombin, and known proteolytic derivatives is provided in Fig.
3A. The corresponding products
isolated on a preparative scale were analyzed by SDS-PAGE (Fig.
3B). Activation of the single chain zymogen, prethrombin 2, converts it to the two chain
-thrombin, which retains the P1-P3
residues (Fig. 3A). Functional and structural studies have established the presence of sites in
-thrombin, removed from the
P1-P3 residues, that play a role in the diverse macromolecular interactions of the protease (50). The fibrinogen recognition site and
the heparin-binding site in
-thrombin (Fig. 3A), are at
least partially expressed in prethrombin 2 as well (51).
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Kinetics of Exosite-dependent Product Inhibition in the
Presence of Hirugen--
Hirugen binds to the fibrinogen recognition
site and thereby competitively inhibits interactions between
macromolecules and -thrombin at this site (22). Kinetic studies with
hirugen were used to further test the conclusions derived from
inhibition studies with
T-IIai and
-IIai.
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Localization of a Domain That Imparts Binding Specificity for
Prethrombin 2--
Cleavage of thrombin by chymotrypsin at
Trp148 (-thrombin, Fig. 3A) yields two
peptides that remain noncovalently associated (32, 52). The cleavage
site separates thrombin into approximate hemispheres, each bearing
elements of the catalytic triad, that can be reassociated following
dissociation and separation to reconstitute enzymatic activity (52).
The N-terminal domain (
1-thrombin) bears the P1-P3 sites of the
product, the intact fibrinogen recognition site as well as some
residues involved in heparin binding. The C-terminal domain
(
2-thrombin) bears elements of the heparin-binding site (Fig.
3A). By analogy, cleavage at the same site in prethrombin 2 yields similar species denoted as
1- and
2-prethrombin 2 (Fig. 3A). While the
2 fragments from prethrombin
2 and thrombin are chemically identical,
1-prethrombin 2 retains the
intact scissile bond acted upon by prothrombinase.
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DISCUSSION |
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Productive recognition of prethrombin 2 by prothrombinase proceeds through interactions at exosites on the enzyme complex followed by binding of substrate structures surrounding the scissile bond to the active site of factor Xa prior to cleavage and product release as illustrated in Scheme I (17). Substrate affinity is determined by the bimolecular interaction between the substrate and enzymic exosite(s) rather than by binding interactions at the active site which are unfavorable and instead contribute to maximum catalytic rate (17). One hallmark for this type of mechanism is that thrombin acts as a linear competitive inhibitor of macromolecular substrate cleavage without obscuring access of small molecule inhibitors and oligopeptidyl substrates to the active site of factor Xa within prothrombinase (17). The present results bear out predictions that necessarily follow from these conclusions and indicate that binding to the exosite(s) in prothrombinase is mediated by a domain of prethrombin 2 and thrombin distinct from the residues immediately surrounding the cleavage site. As illustrated in Scheme I, binding specificity is conferred by structures present in the C-terminal region of prethrombin 2 or thrombin, physically separable from the domain containing the P1-P3 sites or the intact scissile bond and distinct from the fibrinogen recognition or heparin-binding sites.
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The results obtained with T-IIai,
-IIai, and the purified
2 fragment support the
conclusion that the fibrinogen recognition site is not directly
involved in the interaction of prethrombin 2 or thrombin with the
prothrombinase exosite. The strongest evidence to suggest this derives
from the ability of the
2 fragment, which lacks the fibrinogen
recognition site, to reproduce the inhibitory properties of
-IIai. While
T-IIai does display
reduced affinity for prothrombinase, the changes in
Ki are modest when compared with the large reduction
in fibrinogen cleavage or hirudin binding which accompanies proteolysis
in the fibrinogen recognition site (3-5). However, a role for the
fibrinogen recognition site in exosite binding is suggested by the
ability of hirugen to directly inhibit prethrombin 2 cleavage by
increasing the Km and by saturating concentrations of hirugen to reduce the ability of
-IIai to act as a
product inhibitor. Given the observations with the thrombin derivatives, it seems unlikely that the fibrinogen recognition site is
directly involved in the binding of the macromolecular substrate/product to the exosite(s) in prothrombinase. Instead, the
data are more consistent with the possibility that hirugen binding to
thrombin or prethrombin 2 elicits changes at sites distinct from the
fibrinogen recognition site which in turn modulate binding to
prothrombinase. Extensive allosteric linkage has been documented
between the fibrinogen recognition site and the binding of ligands such
as heparin, antithrombin III, fragment 2, and Na+ to other
sites in thrombin (55-57). Recent binding measurements imply that
hirugen and a fragment 2 peptide behave as competitive ligands even
though they are known to bind to distinct sites on thrombin (56). It is
also possible that the effects observed at high concentrations of
hirugen in the present study are related to secondary interactions of
the highly charged peptide. We are unable to distinguish between these
possibilities.
Structure-function studies of substrate specificity, of the type described, are susceptible to interpretation problems arising from unanticipated effects of protein fragments on the stability of the prothrombinase complex or from inhibition derived from the ability of protein fragments containing Arg-X residues to act as alternate substrates. In the present study, direct equilibrium binding measurements of prothrombinase assembly, at reactant concentrations approaching those used in the kinetic measurements, were used to exclude inhibitory effects arising from the destabilization of the enzyme complex. In addition, all inhibitory proteolytic derivatives acted as selective inhibitors of prethrombin 2 activation with a minor effect on synthetic peptidyl substrate hydrolysis by prothrombinase. This fact rules out significant alternate substrate effects as a trivial explanation for the present findings.
The ability of thrombin to act as a linear competitive inhibitor of
prethrombin 2 cleavage catalyzed by prothrombinase implies that
prethrombin 2 and thrombin bind to the enzyme complex in a mutually
exclusive fashion. The similarities in the known structures of
prethrombin 2 and thrombin (51) and the substrate-product relationship
between the two suggests that they are likely to compete for
interactions at the same site in the enzyme complex as illustrated in
Scheme I. While, alternate, more complicated explanations cannot be
adequately excluded at present, this interpretation is supported by the
fact that the 2 fragment from either species exhibits equivalent
inhibitory properties toward prethrombin 2 activation.
The fragment 1 domain mediates the binding of prothrombin to membranes (58) and the fragment 2 region has been shown to be responsible for the interaction between the substrate and factor Va (59). Since prothrombin binding to factor Va and membranes does not involve the active site of factor Xa within prothrombinase, they represent potential exosite interactions. However, these domains are absent in purified prethrombin 2 and thrombin. Thus, it is possible that the exosite interactions inferred in this and previous work (17) involves specific recognition of the macromolecular substrate by sites in factor Xa itself that are removed from the active site. This possibility is supported by prior work documenting the inhibition of macromolecular but not oligopeptidyl substrate cleavage by prothrombinase with a monoclonal antibody specific for factor Xa (60). However, it remains possible that previously unidentified interactions between prethrombin 2 or thrombin and other sites in the prothrombinase complex also contribute to substrate/product binding.
Explanations for the narrow and distinctive substrate specificity of factor Xa or prothrombinase have, thus far, been sought from the active site geometry based on the x-ray structure of factor Xa, by mutagenesis studies of residues surrounding the active site and by studies with synthetic peptidyl substrates (9, 61, 62). The results of this and previous studies (17) suggest that while such approaches may describe the properties of factor Xa in solution, they are inadequate for assessing the basis for the substrate specificity of the prothrombinase complex. The affinity of prothrombinase for the macromolecular substrate is not determined by binding of the substrate to the active site of factor Xa but rather by interactions between extended macromolecular recognition sites, distinct from the active site, and substrate regions spatially distinct from structures immediately surrounding the scissile bond.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jan Pohl of the Emory University Microchemical Facility for N-terminal sequencing, amino acid composition, and the mass spectrometry analyses. We thank Dr. Steven Olson for helpful discussions regarding the heparin experiments and Drs. Pete Lollar, Paul Bock, and William Church for critical review of the manuscript.
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FOOTNOTES |
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* 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.
This work is dedicated to the memory of our late colleague, Dr. Stuart Stone.
Present address: COR Therapeutics Inc., 256 E. Grand Ave., South
San Francisco, CA 94080.
§ Supported by Grant HL-52883 from the National Institutes of Health. To whom all correspondence should be addressed: Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia, 310 Abramson, 324 South 34th St., Philadelphia, PA 19104. Tel.: 215-590-3346; Fax: 215-590-3660.
1
The abbreviations used are: TAP, recombinant
wild type tick anticoagulant peptide; APMSF,
p-amidinophenylmethanesulfonyl fluoride; ATA-EGR-CH2Cl, acetothioacetyl derivative of
EGR-CH2Cl; ATA-EGR-Xa, factor Xa inactivated with
ATA-EGR-CH2Cl; EGR-CH2Cl,
glutamyl-glycyl-arginyl chloromethylketone; IIai, thrombin
inactivated with APMSF; LMW heparin, low molecular weight heparin
(average Mr = 3,000); [OG488]-EGR-Xa, adduct of ATA-EGR-Xa and Oregon
Green488 iodoacetamide; PEG, polyethylene glycol; PC,
L--phosphatidylcholine; PS,
L-
-phosphatidylserine; PCPS, small unilamellar vesicles
composed of 75% (w/w) PC and 25% (w/w) PS; S2238,
H-D-phenylalanyl-L-pipecolyl-L-arginyl
p-nitroanilide; SpXa,
methoxycarbonyl-cyclohexyl-glycyl-glycyl-arginyl
p-nitroanilide; Xai, factor Xa inactivated with
APMSF; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; TPCK,
tosyl-L-1-phenylalanine chloromethyl ketone; MES,
4-morpholineethanesulfonic acid; HPLC, high performance liquid
chromatography; PAGE, polyacrylamide gel electrophoresis.
2 Nomenclature of Schechter and Berger (1).
3
The residues of -thrombin have been numbered
after the corresponding amino acids in chymotrypsin using the
convention of Bode et al. (2). Cleavage of human
-thrombin by trypsin at Arg67, Arg77A, and
Lys149E leads to the loss of the
Ile68-Arg77A peptide and yields
T-thrombin (3-5). Tryptic digestion of bovine thrombin
results in cleavage at Arg67 and Arg77A but not
at Glu149E. We have designated this derivative of bovine
thrombin, which lacks a significant part of the fibrinogen recognition
site, as
T-thrombin.
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REFERENCES |
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