From the Department of Medicine, McMaster University and the Henderson Research Centre, Hamilton, Ontario L8V 1C3, Canada
Received for publication, January 17, 2003
, and in revised form, March 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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The activity of thrombin is primarily regulated through inhibition by antithrombin in a reaction that is enhanced by heparin (3). Although the heparin-antithrombin complex readily inhibits fluid-phase thrombin, thrombin that remains bound to fibrin after clotting is protected from inactivation by the heparin-antithrombin complex. This protection reflects, at least in part, heparin-mediated bridging of thrombin onto fibrin to form a ternary thrombin-heparin-fibrin complex (4, 5).
Fibrin-bound thrombin retains its ability to cleave fibrinogen (4, 6), a phenomenon that provides a plausible explanation for the in vitro observation that thrombi induce activation of platelets and trigger coagulation (7). The concept that fibrin-bound thrombin contributes to the procoagulant activity of thrombi is supported by the recent demonstration that human thrombi obtained from pathological specimens harbor active thrombin (8). Sequestered thrombin may be generated by activated factor X (factor Xa) bound to platelets within the thrombus. Like fibrin-bound thrombin, factor Xa within the platelet-bound prothrombinase complex also is protected from inactivation by the heparin-antithrombin complex (9, 10). Consequently, thrombus-associated factor Xa may further contribute to the procoagulant activity of thrombi by triggering local thrombin generation (11).
Thrombin binds to fibrin via a domain distinct from the active site of the enzyme. Thrombin possesses two electropositive domains (termed exosites) that bracket the catalytic site. Exosite 1 serves as the initial docking site that orients substrates and inhibitors within the active site cleft (12). The second electropositive domain (exosite 2) binds heparin, other glycosaminoglycans, and prothrombin fragment 2 (13). In addition to acting as binding sites, the two exosites on thrombin may also modulate thrombin enzymatic activity. Thus, ligand binding to either exosite alters the active site environment (14), effecting conformational changes that alter thrombin substrate specificity (2, 6). Moreover, there is evidence for allosteric linkage between the two exosites because ligand binding to one exosite can influence the binding properties of the other (15).
Thrombin utilizes exosite 1 to dock fibrinogen as it is converted to fibrin. Fibrinogen is composed of duplicated A-, B
-, and
-chains held together by disulfide bonds. Two chromatographically distinct forms of fibrinogen, distinguished by the structure of their
-chains, can be isolated from human plasma (16). Most fibrinogen molecules contain two
A-chains, each composed of 411 amino acids, and are thus designated
A/
A. About 10% of circulating fibrinogen molecules contain a variant of the native
-chain (termed
') composed of 427 amino acids. This longer
'-chain results from alternative mRNA polyadenylation and has an acidic hirudin-like 20-amino acid sequence at its COOH terminus (17, 18). Fibrinogen heterodimers containing one
A- and one
'-chain are designated
A/
'.
Conversion of A/
A- or
A/
'-fibrinogen to fibrin requires sequential thrombin-mediated release of fibrinopeptides A and B from the NH2 termini of the A
- and B
-chains of fibrinogen, respectively. Current thinking is that once thrombin-mediated fibrinopeptide release is effected, some of the thrombin remains bound to the resultant fibrin via exosite 1 (16). Functionally,
A/
A- and
A/
'-fibrin are distinguished by their thrombin-binding properties (16). Binding of thrombin to
A/
A-fibrin occurs through a single class of low affinity binding sites that have been localized to the NH2 termini of the
- and
-chains, proximal to the fibrinopeptide cleavage sites (19). Thrombin binds to these low affinity sites with Kd values that range from 1 to 3.4 µM (20). In addition to low affinity thrombin binding,
A/
'-fibrin also displays high affinity thrombin binding that has been attributed to the COOH terminus of the
'-chain. The Kd value for this interaction is 0.18 µM (16).
To further explore the high affinity interaction of thrombin with A/
'-fibrin, we measured the affinity of thrombin for a synthetic 20-amino acid peptide analog of the COOH terminus of the
'-chain. Using thrombin variants or DNA aptamers directed against the exosites, we demonstrated that thrombin binding to this
'-chain peptide is mediated by exosite 2. Building on this observation, we then explored the role of exosites 1 and 2 in the interaction of thrombin with
A/
A- and
A/
'-fibrin. These studies suggest that both exosites on thrombin contribute to the high affinity interaction of thrombin with
A/
'-fibrin. In contrast, the low affinity interaction of thrombin with
A/
'- or
A/
A-fibrin is mediated solely by exosite 1.
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EXPERIMENTAL PROCEDURES |
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Isolation of A/
A- and
A/
'-FibrinogenHuman fibrinogen was fractionated on a DEAE-Sepharose column (1.5 x 11.3 cm) (31). After diluting fibrinogen with water and 39 mM Tris and 5 mM phosphoric acid (pH 8.8) (buffer A) at a 1:1:1 ratio, 180 mg of fibrinogen was applied to the column at a flow rate of 5 ml/min. A concave pH gradient from buffer A to 500 mM Tris and 500 mM phosphoric acid (pH 4) was used to separate
A/
A- from
A/
'-fibrinogen (32). 10-ml fractions were collected, and their absorbance at 280 nm was determined using a Beckman Gold 168 detector.
A/
A- and
A/
'-fibrinogen fractions were precipitated by addition of ammonium sulfate to 50%, incubated for 3 h at 4 °C, and subjected to centrifugation at 2000 x g for 15 min at 23 °C. Pellets were resuspended in Tris-buffered saline (TBS; 20 mM Tris-HCl and 150 mM NaCl (pH 7.4)) containing 0.01% Tween 20 (TBS/Tween) and incubated for 60 min at 37 °C prior to dialysis against TBS/Tween. Fibrinogen concentrations were determined using a Beckman DU 7400 spectrophotometer by measuring absorbance at 280 and using Mr = 340,000 and
(33). Aliquots were stored at 70 °C. The integrity of isolated
A/
A- and
A/
'-fibrinogen fractions was assessed by SDS-PAGE (34) on 415% polyacrylamide gels (Ready-Gel, Bio-Rad, Mississauga, Ontario, Canada) under reducing conditions (Fig. 1A). The gels were then transferred to nitrocellulose and subjected to immunoblot analysis using an antibody directed against the
'-chain (Fig. 1B). Two bands are seen in the
-chain region of
A/
'-fibrinogen (Fig. 1A, lane 2), but only the higher molecular weight band of the doublet was recognized by the antibody against the
'-chain (Fig. 1B, lane 2). In contrast, the single
-chain band of
A/
A-fibrinogen (Fig. 1A, lane 1) was not recognized by the antibody (Fig. 1B, lane 1).
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Preparation of Des-B142-
A/
A- and Des-B
142-
A/
'-Fibrinogen
A/
A- or
A/
'-fibrinogen (8.4 µM; in TBS containing 12 mM EDTA) was incubated with 2.3 µM protease III fraction from C. atrox venom for 90 min at 23 °C. Reactions were terminated by addition of FPRck to 10 µM. After 10 min, 20% (v/v) ethanol was added, and the solution was maintained on ice for 30 min prior to centrifugation at 14,000 x g for 4 min. Pellets were resuspended in 1 ml of TBS and dialyzed against TBS. Protein concentrations were determined as described above, and the material was stored in aliquots at 70 °C. The integrity of des-B
142-
A/
A- or des-B
142-
A/
'-fibrinogen was assessed by SDS-PAGE analysis. Immunoblot analysis using an antibody against the
'-chain was used to ensure that treatment with C. atrox venom had no effect on the COOH-terminal region of the
'-chain of
A/
'-fibrinogen. Upon SDS-PAGE, the
-chain lacking B
142 migrated with the
-chain (Fig. 1A, lanes 3 and 4). Immunoblot analysis demonstrated that the
'-chain of des-B
142-
A/
'-fibrinogen was still recognized by the antibody against the
'-chain (Fig. 1B, lane 4). NH2-terminal sequence analysis of the
-chain of des-B
142-
A/
A- or des-B
142-
A/
'-fibrinogen confirmed that the
-chains of both species were cleaved at the
4243 bond. In contrast, the sequences of the NH2 termini of the
- and
-chains corresponded to those of native fibrinogen (data not shown).
Labeling of ProteinsFluorescent derivatives of -IIa,
-IIa, and RA-IIa (f-FPR-
-IIa, f-FPR-
-IIa, and f-FPR-RA-IIa, respectively) were prepared by incubation with a 5-fold molar excess of fluorescein-FPRck until there was no detectable chromogenic activity against tGPR-pNA. Samples were then dialyzed against TBS, and thrombin concentrations were determined using Mr = 37,000 and
280 1% = 18. A fluorescent derivative of the
'-peptide (f-
'-peptide) was generated by incubating the peptide with a 10-fold molar excess of fluorescein isothiocyanate in 1 M NaHCO3 (pH 9) for 90 min at 23 °C. Unreacted fluorescein isothiocyanate was removed by passing the material over a 10-ml Sephadex G-10 column equilibrated with TBS, and the concentration was determined as described (15).
-IIa was radiolabeled with Na125I (PerkinElmer Life Sciences) using IODO-BEAD (Pierce) as described (35). Specific radioactivity ranged from 0.7 to 1.5 x 109 cpm/mg. Radiolabeled
-IIa migrated with its unlabeled counterpart when subjected to SDS-PAGE on 415% polyacrylamide gels under reducing conditions. Radiolabeled
-IIa bound to fibrin monomer-Sepharose (36) and to heparin-Sepharose with affinities similar to those of unlabeled
-IIa, thereby confirming the integrity of exosites 1 and 2, respectively. Only the fraction of 125I-
-IIa that bound to the columns was used. Active site-blocked 125I-
-IIa was prepared by incubating 125I-
-IIa with a 10-fold molar excess of FPRck, followed by dialysis against TBS. When prepared in this fashion, 125I-FPR-
-IIa had no activity against tGPR-pNA (37). For binding studies, trace amounts of 125I-FPR-
-IIa were combined with FPR-
-IIa.
Methods
'-Peptide Binding to f-FPR-IIaf-FPR-
-IIa, f-FPR-
-IIa, or f-FPR-RA-IIa (at a concentration of 100 nM in 1 ml of TBS) was added to a semi-micro quartz cuvette, and samples were stirred with a mini-stir bar and maintained at 23 °C using a circulating water bath. Initial fluorescence (Io) was monitored at 1-s intervals using a PerkinElmer LS 50B luminescence spectrometer at excitation and emission wavelengths of 492 and 535 nm, respectively, with a 515-nm cutoff filter in the emission beam and excitation and emission slit widths of 10 nm. Samples were then titrated with 110-µl aliquots of 50 µM
'-peptide containing 100 nM f-FPR-IIa to prevent fluorescent probe dilution. The signal was allowed to stabilize before each addition, and fluorescence intensity values (I) were obtained from the time drive profile. I/Io values were plotted against the concentration of
'-peptide, and binding was analyzed by nonlinear regression (Table Curve, Jandel Scientific, San Rafael, CA) in Equation 1,
![]() | (Eq. 1) |
Experiments were then performed to examine the ability of DNA aptamers directed against exosite 1 or 2 (HD1 and HD22, respectively) to displace the '-peptide from
-IIa. The fluorescence of a 1-ml sample containing 1 µM thrombin was monitored before and after addition of 50 nM f-
'-peptide. The sample was subsequently titrated with 12-µl aliquots of HD1 or HD22 to a final concentration of 5.6 or 4.4 µM, respectively. Data were analyzed as described above.
Thrombin Binding to FibrinBinding studies were performed with either radiolabeled or unlabeled thrombin. When labeled thrombin was used, 125I-FPR--IIa (08 µM) was added to microcentrifuge tubes containing 2 mM CaCl2 in the absence or presence of 2 µM
A/
A- or
A/
'-fibrinogen. Clotting was initiated by addition of 10 nM
-IIa; and after a 60-min incubation at 23 °C, fibrin was pelleted by centrifugation at 14,000 x g for 4 min. Two 10-µl aliquots of supernatant were removed and counted for radioactivity. The concentration of unbound thrombin in the supernatant was determined by comparison with a standard curve prepared in the absence of fibrinogen, and the concentration of the bound thrombin was then calculated by subtraction.
Binding of unlabeled thrombin to fibrin was assessed in 1.5-ml microcentrifuge tubes to which were added 2 mM CaCl2, 30 nM -,
-, or RA-IIa, and 010 µM
A/
A- or
A/
'-fibrinogen to a total volume of 50 µl. Because
-IIa has minimal procoagulant activity, atroxin was added to 5% (v/v) to initiate clotting. After a 60-min incubation at 23 °C, fibrin was pelleted by centrifugation at 14,000 x g for 4 min, and two 10-µl aliquots of supernatant were removed. The chromogenic activity of thrombin in each of these samples was assessed by their addition to the wells of a 96-well plate prefilled with 200 µl of 200 µM tGPR-pNA. Substrate hydrolysis was monitored at 405 nm in a plate reader (Molecular Devices, Sunnyvale, CA). The thrombin concentration was determined by comparison with a standard curve, and the concentration of bound thrombin was then calculated by subtraction. In all cases, values were divided by the fibrinogen concentration to determine the number of moles of thrombin bound per mol of fibrin (
). For each point in the titration, these values were then plotted against the concentration of unbound protein. Scatchard plots were also constructed, and depending on whether these appeared linear, reflecting a single class of binding sites, or curved downward, reflecting two classes of binding sites, data were analyzed by nonlinear regression analysis (Table Curve, Jandel Scientific) of a one-site (Equation 2) or two-site (Equation 3) binding equation,
![]() | (Eq. 2) |
![]() | (Eq. 3) |
Effect of Exosite 1- or 2-directed Ligands on Thrombin Binding to FibrinConcentrations of hirudin-(5465) (50 µM), heparin cofactor II-(5475) (100 µM), HD1 (1 µM), h-F2 (64 µM), '-peptide (236 µM), and HD22 (0.25 µM), determined to be saturating in separate experiments were added to a series of microcentrifuge tubes containing 1 µM
A/
A-fibrinogen or 0.35 µM
A/
'-fibrinogen, 2 mM CaCl2, and 25 nM 125I-FPR-
-IIa. Clotting was initiated by addition of atroxin to 5% (v/v), and after a 60-min incubation at 23 °C, fibrin was pelleted by centrifugation at 14,000 x g for 2 min. Duplicate 10-µl aliquots of supernatant were then removed and counted for radioactivity. The amount of 125I-FPR-
-IIa bound to fibrin in the absence or presence of competitor was calculated by subtraction. Data were displayed by plotting the concentration of 125I-FPR-
-IIa bound to fibrin in the presence of each concentration of competitor as a percentage of that bound in the absence of competitor.
Statistical MethodsUnless otherwise indicated, experiments were performed at least three times in duplicate. Results are presented as the mean ± S.E. Student's t test was used to evaluate significance.
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RESULTS |
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To further explore this concept, we performed reverse titrations to examine the ability of exosite 1- or 2-directed DNA aptamers to displace the f-'-peptide from
-IIa. The fluorescence intensity of 50 nM f-
'-peptide decreased by 6% after addition of
-IIa (Fig. 3A). Titration with HD22, an aptamer directed against exosite 2, restored the fluorescence intensity to initial background levels, indicating displacement of the f-
'-peptide from
-IIa. In contrast, HD1, an exosite 1-directed aptamer, had no effect (Fig. 3B). These data support the concept that the interaction of
-IIa with the
'-peptide is mediated by exosite 2.
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Binding of 125I--IIa to
A/
A- and
A/
'-FibrinThe interactions of 125I-FPR-
-IIa with clots formed from
A/
A- and
A/
'-fibrinogen were compared to examine the influence of the
'-chain on binding. Gel electrophoretic analysis verified that the
A/
A- and
A/
'-fibrinogen preparations contained one and two
-chains, respectively (Fig. 1). 125I-FPR-
-IIa bound to both forms of fibrin in a concentration-dependent and saturable fashion. Scatchard analysis of the data for the interaction of 125I-FPR-
-IIa with
A/
A-fibrin (Fig. 4A) reveals a straight line, consistent with a single class of binding sites. The Kd of 2.3 ± 0.32 µM, determined by nonlinear regression analysis of the direct plot, is in good agreement with previously reported results (20). Binding to
A/
'-fibrin was also saturable, but the direct plot revealed a steeper increase in binding at lower 125I-FPR-
-IIa concentrations, indicating a higher affinity interaction. The Scatchard plot for the interaction of 125I-FPR-
-IIa with
A/
'-fibrin is nonlinear (Fig. 4B), indicating heterogeneous binding sites or negative cooperativity. A double-reciprocal plot of 1/bound versus 1/free yields a straight line, whereas a plot of bound2/free versus bound yields a sigmoidal curve (data not shown). These findings are indicative of binding site heterogeneity (39). Accordingly, the data were fit to a two-site model (Equation 3) by nonlinear regression analysis, yielding Kd values of 107 ± 36 nM and 1.5 ± 0.24 µM for the high and low affinity binding sites, respectively. These results are consistent with the Kd values of 180 nM and 3.4 µM reported previously (16).
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To confirm the role of the '-chain in the high affinity interaction of
-IIa with fibrin, we examined the influence of the
'-peptide on 125I-FPR-
-IIa binding to
A/
'-fibrin. 2 µM
A/
'-fibrinogen was clotted in the presence of 125I-FPR-
-IIa (08 µM) in the absence and presence of 50 µM
'-peptide (Fig. 5). The direct plot for the data in the presence of the
'-peptide closely resembles that for 125I-
-IIa binding to
A/
A-fibrin. Scatchard analysis of the binding data in the presence of the
'-peptide yields a straight line, consistent with a single class of binding sites with a Kd of 2.8 ± 0.12 µM, a value comparable to that for the low affinity interaction of
-IIa with
A/
A-fibrin. These findings suggest that the
'-peptide eliminates the high affinity interaction of
-IIa with
A/
'-fibrin by competing with its
'-chain for access to exosite 2 on
-IIa. The data also support the concept that the
'-peptide serves as a good surrogate for the
'-chain of intact fibrin. The
'-peptide had no effect on the Kd of 125I-FPR-
-IIa binding to
A/
A-fibrin (data not shown).
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Binding of -IIa,
-IIa, or RA-IIa to
A/
A- and
A/
'-Fibrin Thrombin and thrombin variants were used to further explore the contribution of exosites 1 and 2 to the interaction of thrombin with
A/
A- or
A/
'-fibrin. These experiments were performed with unlabeled active thrombin species where unbound thrombin was detected by measuring chromogenic activity in clot supernatants. Active
-IIa bound to
A/
A-fibrin with a Kd value (2.25 ± 0.05 µM) comparable to that of 125I-FPR-
-IIa (Table I). RA-IIa, whose exosite 1 is intact, bound to
A/
A-fibrin with a Kd value of 3.11 ± 0.02 µM, an affinity similar to that of
-IIa. In contrast,
-IIa, a variant with a cleaved exosite 1, did not bind to
A/
A-fibrin. These findings are consistent with the concept that the low affinity interaction of
-IIa with
A/
A-fibrin is mediated solely by exosite 1.
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Experiments were then repeated using A/
'-fibrin in place of
A/
A-fibrin.
-IIa bound to
A/
'-fibrin with a Kd of 80 ± 30 nM, reflecting the high affinity interaction with the
'-chain. Because the affinity was measured by titrating
-IIa with fibrinogen, two-site binding of
-IIa to
A/
'-fibrin could not be discriminated. RA-IIa bound to
A/
'-fibrin with a Kd of 2.02 ± 0.03 µM, an affinity similar to that of
-IIa for
A/
A-fibrin. These findings suggest that without a functional exosite 2, RA-IIa cannot bind to the
'-chain and can interact only with low affinity binding sites on
A/
'-fibrin.
To further investigate the importance of exosite 2 in the interaction of thrombin with A/
'-fibrin, the binding of
-IIa to
A/
'-fibrin was examined.
-IIa bound to
A/
'-fibrin with a Kd of 5.54 ± 0.02 µM. Because
-IIa lacks a functional exosite 1, its interaction with
A/
'-fibrin must be mediated by exosite 2. Furthermore,
-IIa must bind to the
'-chain of
A/
'-fibrin because
-IIa does not bind to
A/
A-fibrin. These concepts are supported by the observation that the
'-peptide inhibited
-IIa binding to
A/
'-fibrin in a concentration-dependent fashion (Fig. 6).
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Binding of -IIa to Des-
1542-
A/
A- or Des-
1542-
A/
'-FibrinAlthough both
-IIa and
-IIa bound to the
'-chain on
A/
'-fibrin, the affinity of
-IIa for
A/
'-fibrin was 69-fold higher than that of
-IIa (Kd values of 0.08 ± 0.03 and 5.54 ± 0.02 µM, respectively). These findings raise the possibility that the high affinity interaction of
-IIa with
A/
'-fibrin requires both exosites on thrombin, wherein exosite 2 is ligated by the
'-chain of fibrin and exosite 1 by the NH2 termini of
- and
-chains. To explore this possibility, the binding of
-IIa to fibrin generated from des-B
142-
A/
A- or des-B
142-
A/
'-fibrinogen was measured.
-IIa bound des-
1542-
A/
A-fibrin with a Kd value of 7.67 ± 0.09 µM (Table I). Thus, the affinity of
-IIa for
A/
A-fibrin was reduced 3.4-fold when the
1542 sequence was removed, consistent with the concept that this sequence represents at least part of the low affinity thrombin-binding site on
A/
A-fibrin (16, 40). Although the affinity of
-IIa for des-
1542-
A/
'-fibrin was 2.2-fold higher than that for des-
1542-
A/
A-fibrin (Kd values of 3.51 ± 0.07 and 7.67 ± 0.09 µM, respectively), the affinity of
-IIa for des-
1542-
A/
'-fibrin was 44-fold lower than that for
A/
'-fibrin. The reduction in affinity of
-IIa for des-
1542-
A/
'-fibrin was verified using 125I-FPR-
-IIa, where the Scatchard plot reveals a single class of binding sites (Fig. 7). These findings suggest that removal of a low affinity binding site on
A/
'-fibrin has profound effects on the high affinity interaction of thrombin. This concept is supported by binding studies with RA-IIa and
-IIa. RA-IIa bound to des-
1542-
A/
A- and des-
1542-
A/
'-fibrin with similar affinities (Kd values 11.72 ± 0.05 and 10.93 ± 0.03 µM, respectively). These findings were expected because without a functional exosite 2, RA-IIa can interact only with low affinity binding sites on fibrin. However, when the affinity of RA-IIa for
A/
A- or
A/
'-fibrin is compared with its affinity for des-
1542-
A/
A- or des-
1542-
A/
'-fibrin, it is apparent that removal of the
1542 sequence from
A/
A- and
A/
'-fibrin reduces their affinity for RA-IIa by 3.8- and 5.4-fold, respectively.
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As expected, -IIa did not bind to des-
1542-
A/
A-fibrin. However,
-IIa bound to des-
1542-
A/
'-fibrin with an affinity similar to that for
A/
'-fibrin (Kd values of 5.86 ± 0.02 and 5.54 ± 0.02 µM, respectively). Thus, removal of a low affinity thrombin-binding site had no effect on the interaction of
-IIa with the binding site on the
'-chain of
A/
'-fibrin or des-
1542-
A/
'-fibrin. These findings support the concept that binding of
-IIa to the
'-chain is independent of exosite 1.
Effect of Exosite 1- or 2-directed Ligands on -IIa Binding to
A/
A- or
A/
'-FibrinTo verify the contribution of the two exosites on
-IIa to its interaction with fibrin, binding assays were performed in the absence or presence of ligands directed at either exosite 1 or 2 of
-IIa, and the ability of these ligands to block
-IIa binding was determined. When ligands directed to exosite 1, hirudin-(5465), HD1, and heparin cofactor II-(5475), were present at saturating concentrations (50, 1, and 100 µM, respectively),
-IIa binding to
A/
A-or
A/
'-fibrin was reduced by 7080% (Fig. 8). These findings highlight the importance of
-IIa exosite 1 binding to either form of fibrin. To address the role of exosite 2, the experiment was then repeated with saturating concentrations of
'-peptide, h-F2, and HD22 (236, 64, and 0.25 µM, respectively). The
'-peptide produced almost 100% reduction in
-IIa binding to
A/
'-fibrin. In contrast, the
'-peptide reduced
-IIa binding to
A/
A-fibrin by 50%. Because the interaction of
-IIa with
A/
A-fibrin is not expected to involve exosite 2 directly, these results suggest an allosteric linkage between the two exosites. Similar results were obtained with h-F2 and HD22, although the extent of reduction was less with both forms of fibrin.
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DISCUSSION |
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Consistent with the previous report (16), -IIa binds to
A/
'-fibrin via two classes of binding sites: high affinity binding with a Kd of 0.107 µM and low affinity binding with a Kd of 1.5 µM. Exosite 2 contributes to the interaction of thrombin with
A/
'-fibrin because RA-IIa binds to
A/
A- and
A/
'-fibrin with Kd values similar to those for the low affinity interaction of
-IIa with
A/
'- and
A/
A-fibrin. These findings suggest that because its exosite 2 function is impaired, RA-IIa does not bind
A/
'-fibrin with high affinity. In contrast, the exosite 1 variant
-IIa binds only
A/
'-fibrin. The interaction of
-IIa with
A/
'-fibrin likely reflects exosite 2-mediated binding to the
'-chain because the
'-peptide attenuates binding.
Further evidence that exosite 2 mediates the interaction of thrombin with the '-chain comes from the demonstration that
-IIa and
-IIa bind the
'-peptide with similar affinities, a finding consistent with recently reported data (24). In contrast, RA-IIa, an exosite 2 variant, does not bind. Furthermore,
-IIa bound to the
'-peptide is displaced by an exosite 2-directed aptamer, but not by an aptamer that binds to exosite 1.
Despite its intact exosite 2, -IIa binds to
A/
'-fibrin with an affinity lower than that of
-IIa. These findings raise the possibility that exosite 1 also contributes to the high affinity interaction of
-IIa with
A/
'-fibrin. Several observations support this concept. First, the affinity of
-IIa for des-
1542-
A/
'-fibrin is 44-fold lower than that for intact
A/
'-fibrin. Thus, abolishing exosite 1-mediated interaction with the low affinity binding site on the NH2 terminus of the
-chain markedly reduces the affinity of
-IIa for
A/
'-fibrin. In contrast, removal of these low affinity binding sites from
A/
A-fibrin produces only a 3.4-fold reduction in its affinity for
-IIa. Second,
-IIa binds to des-
1542-
A/
'-fibrin with an affinity similar to that for intact
A/
'-fibrin. Likewise, removal of the NH2 termini of the
-chains from
A/
'-fibrin reduces the affinity of RA-IIa to the same extent as removal of these low affinity binding sites from
A/
A-fibrin.
The concept that both exosites on thrombin are necessary for its high affinity interaction with A/
'-fibrin provides an explanation for the results of thrombin binding studies with dysfibrinogen Naples I. When clotted, fibrinogen Naples I (B
A68T) exhibits reduced affinity of both high and low affinity binding interactions (40). These findings suggest that attenuation of exosite 1-mediated interaction with the mutant NH2 terminus of the
-chain impairs high affinity binding mediated by the
'-chain.
The proposal that both exosites are necessary for the high affinity interaction of -IIa with fibrin is supported by studies using synthetic exosite 1- or 2-directed ligands. Exosite 1-directed ligands reduce
-IIa binding to either
A/
A- or
A/
'-fibrin by 80100%, thereby confirming that this exosite is important for both the high and low affinity interaction of
-IIa with fibrin. Exosite 2-directed ligands reduce
-IIa binding to
A/
'-fibrin by 60100%, supporting the concept that exosite 2 is important for the interaction of
-IIa with
A/
'-fibrin. However, exosite 2 ligands also produce modest reductions in
-IIa binding to
A/
A-fibrin, despite the fact that this interaction is mediated by exosite 1. These findings can be rationalized by our previous observation that there is allosteric linkage between the two exosites such that binding of a ligand to one exosite alters the affinity of the opposite exosite for its ligands (15). A recent study has challenged this concept based on the observation that prothrombin fragment 2, which binds to exosite 2 on thrombin, does not influence the binding of hirudin-(5465) to exosite 1 (41). This discrepancy likely reflects differences in assay technique because our current findings indicate that exosite 2-directed ligands, including prothrombin fragment 2, attenuate the binding of
-IIa to
A/
A-fibrin via exosite 1.
Both prothrombin fragment 2 and the '-chain of fibrinogen possess clustered anionic residues that likely mediate its interaction with the electropositive exosite 2 domain on thrombin (22, 42, 43). Although no homology between the sequences that bind to exosite 1 or 2 can be discerned (22), the two exosites appear to have functionally distinct roles. In general, exosite 1 on thrombin is utilized by substrates, cofactors, and inhibitors to gain direct access to the active site of the enzyme. In contrast, exosite 2 binds heparin or prothrombin fragment 2, both of which are ligands that recruit tertiary reactants, antithrombin or factor V, respectively. This suggests that exosite 2 functions primarily as an anchoring site, whereas exosite 1 serves to align substrates or inhibitors. This is consistent with the observation that exosite 2 functions are retained when thrombin is complexed by antithrombin (35). The exosite 2-mediated anchoring of thrombin to
'-chain of fibrinogen enhances the capacity of fibrin to harbor catalytically active thrombin. Thrombin bound in this fashion would remain active and protected from inhibition by heparin-antithrombin because the heparin-binding site of the bound thrombin is ligated by the
'-chain. The observation of a correlation between plasma levels of
A/
'-fibrinogen and the risk of coronary artery disease supports this contention (30).
From a structural perspective, our data suggest that the COOH-terminal portion of the '-chain must be in close proximity to the NH2 termini of the
- and
-chains of cross-linked
A/
'-fibrin to bind both thrombin exosites simultaneously. Supporting this concept are recent data revealing the proximity of the COOH terminus of the
-chain to polymerization site "a" (4446). The putative exosite 1-binding site that resides on the
-chain is located near the NH2-terminal "A" knob that binds to site "a" (19). The COOH terminus of the
-chain is untethered and flexible (44), and the
'-chain is 16 residues longer than the
-chain. Thus, the
' terminus could bind to exosite 2 of thrombin already bound to the
-chain via exosite 1. In this model, thrombin is likely bound first to the NH2 terminus of the
-chain via exosite 1 after cleavage of fibrinopeptide A. This initiates polymerization and brings the
'-chain into proximity of thrombin residing on the other strand of the protofibril.
In summary, the data suggest that the high affinity interaction of thrombin with A/
'-fibrin requires simultaneous ligation of both exosites on thrombin. Whereas exosite 2 binds to the
'-chain, exosite 1 interacts with the NH2 terminus of the
-chain. Occupation of both exosites may induce conformational changes in the active site of thrombin. Such changes occur upon formation of the ternary thrombin-heparin-fibrin complex or when thrombin binds to thrombomodulin via exosite 1 and the chondroitin sulfate moiety of thrombomodulin binds to exosite 2. Conformational changes in the active site of thrombin or occupation of the heparin-binding site may limit its reactivity with serpins, thereby protecting thrombin from inhibition (5, 36).
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FOOTNOTES |
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Recipient of a career investigator award from the Heart and Stroke Foundation of Ontario and holds the Heart and Stroke Foundation of Ontario/J. Fraser Mustard Chair in Cardiovascular Research and the Canada Research Chair in Thrombosis at McMaster University. To whom correspondence should be addressed: Henderson Research Centre, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.: 905-574-8550; Fax: 905-575-2646; E-mail: jweitz{at}thrombosis.hhscr.org.
1 The abbreviations used are: IIa, thrombin; RA-IIa, thrombin R93A/R97A/R101A; HPLC, high pressure liquid chromatography; tGPR-pNA, tosyl-Gly-Pro-Arg p-nitroanilide; FPRck, D-Phe-Pro-Arg chloromethyl ketone; h-F2, human prothrombin fragment 2; TBS, Tris-buffered saline; f-, fluorescein isothiocyanate-modified; FPR-, FPRck-inactivated.
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