Exosites 1 and 2 Are Essential for Protection of Fibrin-bound Thrombin from Heparin-catalyzed Inhibition by Antithrombin and Heparin Cofactor II*

Debra L. Becker, James C. FredenburghDagger , Alan R. Stafford, and Jeffrey I. Weitz§

From the Department of Medicine, McMaster University and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada

    ABSTRACT
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Abstract
Introduction
References

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma -thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 right-arrow Ala, Arg97 right-arrow Ala, and Arg101 right-arrow Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha 1-antitrypsin Met358 right-arrow Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.

    INTRODUCTION
Top
Abstract
Introduction
References

Heparin acts as an anticoagulant by activating antithrombin (AT),1 a serine protease inhibitor (serpin) that inhibits thrombin and factor Xa (1-5). Catalysis of AT is dependent on a unique pentasaccharide sequence found on about one-third of naturally occurring heparin chains. Pentasaccharide-containing heparin chains bind AT with high affinity and induce a conformational change in the reactive center loop of AT that facilitates its interaction with target proteinases. Whereas this conformational change is sufficient to accelerate the reactivity of AT with factor Xa, inhibition of thrombin is only effected by heparin chains that are of sufficient length to bridge the inhibitor and the proteinase (6).

In addition to catalyzing AT, heparin also activates heparin cofactor II (HCII), a naturally occurring serpin whose only target in the coagulation system is thrombin. Heparin-induced catalysis of HCII has been described as a "double bridging" effect because, in addition to the heparin bridge between thrombin and HCII, a second bridge is formed when heparin displaces the acidic amino terminus of HCII which then binds thrombin (7). In contrast to AT, HCII does not bind with high affinity to a unique sequence of heparin (8).

Interactions of thrombin with inhibitors and substrates are mediated by positively charged exosites (9-11). Exosite 1 binds fibrinogen, the fifth and sixth growth factor domains of thrombomodulin, hirudin, and the amino terminus of HCII. As a recognition site, exosite 1 is commonly employed as an initial docking site that orients the substrate or inhibitor with the active site of thrombin (12). A second positively charged region, exosite 2, is the heparin-binding site. To bridge thrombin to AT or HCII, heparin binds simultaneously to exosite 2 on thrombin and to the serpin (13). In addition to serving as docking sites, exosites 1 and 2 may also serve allosteric regulatory roles by influencing reactivity of the active site (14).

Although heparin is an effective anticoagulant, it has limitations, particularly in patients with acute coronary ischemic syndromes (15-18). These limitations have been attributed, at least in part, to the inability of the heparin-AT complex to inactivate thrombin bound to fibrin. Thrombin bound to fibrin remains enzymatically active and is protected from rapid inhibition by plasma inhibitors, in the presence of heparin (19, 20). The basis of this resistance is thought to reflect formation of a ternary thrombin-heparin-fibrin complex wherein thrombin is protected from inactivation by AT (21, 22). The ternary complex is formed because heparin augments thrombin binding to fibrin, presumably by bridging the protease to fibrin (22). Since thrombin binds fibrin via exosite 1, and heparin binds to thrombin exosite 2, we predicted that both exosites were essential not only for formation of the ternary complex but also for rendering thrombin within the complex resistant to inhibition by AT and other heparin-catalyzed inhibitors. To explore this possibility, we measured the binding of thrombin to immobilized fibrin monomer in the absence or presence of heparin, and we compared thrombin binding with the binding of thrombin variants with impaired exosites 1 or 2. In addition, protection of thrombin from inhibition by AT in the presence of fibrin was examined using the same thrombin variants. We also studied the extent to which thrombin is protected from inactivation by HCII and by alpha 1-antitrypsin Met358 right-arrow Arg, inhibitors whose mode of interaction with thrombin differs from that of AT. It was observed that only when both exosites of thrombin are ligated is fibrin-bound thrombin protected from inactivation by either the heparin-AT or heparin-HCII complex. In contrast, fibrin-bound thrombin is susceptible to inhibition by alpha 1-antitrypsin Met358 right-arrow Arg even in the presence of heparin suggesting that formation of the ternary thrombin-heparin-fibrin complex does not impair access of macromolecular inhibitors to the active site of the enzyme. The use of a fourth inhibitor, consisting of a covalent complex of heparin and AT (23), revealed that accessibility of AT-bound heparin to thrombin within the ternary complex is impaired. Thus, these studies not only elucidate the mechanism of ternary thrombin-fibrin-heparin complex assembly but also explain why thrombin within this complex is protected from inhibition by AT and HCII.

    EXPERIMENTAL PROCEDURES

Proteins and Reagents-- Human alpha -thrombin, gamma -thrombin, plasminogen-free fibrinogen, and factor Xa were obtained from Enzyme Research Laboratories (South Bend, IN). The naturally occurring thrombin variant Quick 1, with a Cys to Arg mutation at position 67 in exosite 1 (24), was kindly provided by Ruth Ann Henriksen, University of North Carolina School of Medicine. RA-thrombin (exosite 2 mutations Arg93 right-arrow Ala, Arg97 right-arrow Ala, and Arg101 right-arrow Ala) (25) was a generous gift from Charles T. Esmon, Howard Hughes Medical Institute, Oklahoma City, OK. Recombinant alpha 1-antitrypsin Met358 right-arrow Arg (mutation Met358 right-arrow Arg (26) and truncation of five amino-terminal amino acids) was a gift from Dr. R. Bischoff, Transgene S. A. (Strasbourg, France). Human plasma AT and HCII were from Affinity Biologicals (Hamilton, Ontario, Canada). The covalent antithrombin-heparin (ATH) complex (23) was generously provided by L. Berry, McMaster University. Thrombin blocked at its active site with D-Phe-Pro-Arg chloromethyl ketone (FPRCK; Calbiochem) was prepared as described previously (14). The chromogenic thrombin substrate N-rho -tosyl-Gly-Pro-Arg-rho -nitroanilide (tGPR-pNA), hexadimethrine bromide (Polybrene), gelatin-agarose, and Gly-Pro-Arg-Pro-amide (GPRP-NH2) were from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).

Heparin-- Unless otherwise specified, all experiments were performed with heparin with high affinity for AT. High affinity heparin was prepared by affinity chromatography of unfractionated heparin from porcine intestinal mucosa (grade II; Sigma) using an AT-Sepharose column (27). Human AT and cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech) were used to prepare the column according to the manufacturer's instructions. Approximately 2-5 mg of AT were immobilized per ml of Sepharose. The column (50 ml bed volume) was equilibrated with 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4 (TBS). Unfractionated heparin (10 mg) was dissolved in 1 ml of TBS and pumped onto the column at an approximate flow rate of 15 ml/h at 4 °C, followed by another 90 ml of starting buffer. Bound material was eluted with 90 ml of 2.0 M NaCl, collecting 3-ml fractions. The heparin-containing fractions, identified by Alcian blue staining (23), were pooled, precipitated with 3 volumes of ethanol, dissolved in starting buffer, and reapplied to the re-equilibrated column. After repeating the procedure three times, the final product was dissolved in 0.15 M NaCl, and its concentration was determined using a protamine sulfate turbidometric assay (28). The high affinity heparin prepared in this way had a specific activity of 280 anti-Xa units/mg when compared with the 1st International Unfractionated Heparin Standard and had an average molecular mass of 15,000 Da, as determined by high pressure liquid chromatography (29).

Preparation of Soluble Fibrin-- Plasminogen-free human fibrinogen was subjected to gelatin-agarose adsorption to remove fibronectin. Approximately 15 ml of fibrinogen (~130 µM) was mixed in a tube for 30 min with 5 ml of gelatin-agarose. After centrifugation for 10 min, the supernatant was removed and exposed a second time to a new aliquot of gelatin-agarose. The fibrinogen concentration in the supernatant was determined using a molecular weight of 340,000 and by measuring the absorbance using epsilon 2801% of 15.1 (21) after correction for light scatter at 320 nm using the relationship A280corr = A280 - 1.7 × A320 (30). Soluble fibrin (SF) was prepared by clotting fibrinogen (60-100 µM) at 37 °C for 4-6 h with 2 nM thrombin (21). The resultant fibrin was sedimented by centrifugation at 2000 × g for 5 min and transferred to dialysis tubing (12,000-14,000 molecular weight cut-off, 2.5 cm wide). After dialysis against distilled water (>200 volume) at 4 °C overnight to remove fibrinopeptides A and B, the material was dialyzed against 20 mM acetic acid for approximately 8 h until dissolved. A molecular weight of 340,000 and epsilon 2801% value of 14.0 was used to calculate the SF concentration (31), which was typically about 100 µM. Aliquots were stored at -70 °C. Prior to use, SF was neutralized with 40% v/v 1 M Tris-HCl, pH 7.5, and polymerization was blocked by the addition of 5 mM GPRP-NH2 (32).

Preparation of Fibrin Monomer-Sepharose-- Approximately 35 mg of fibrinogen was coupled to 3 ml of cyanogen bromide-activated Sepharose, as described above for AT. To block unreacted amino groups, the resin was packed in a small column (0.7 × 9 cm) and washed over 2 h with 15 ml of coupling buffer and then with 10 ml of TBS. Finally, the washed resin was removed from the column and diluted 1:2 with TBS, and the immobilized fibrinogen was converted to fibrin monomer (FM) by addition of thrombin at a final concentration of 2 nM. After 3 h incubation with gentle end-over-end mixing in a 15-ml conical tube at room temperature (33), the FM-Sepharose was transferred to a column (0.7 × 9 cm) and washed with 20 mM Tris-HCl, pH 7.4, 1 M NaCl (10 volumes) followed by TBS (10 volumes). The amount of FM conjugated to the Sepharose was determined by performing a BCA Protein Assay (Pierce) on an aliquot of the FM-Sepharose using fibrinogen as the standard and unconjugated Sepharose as a blank.

Proteinase Inhibition Assays-- The rates of inactivation of (a) thrombin, gamma -thrombin, Quick 1 dysthrombin, or RA-thrombin by AT, HCII, or alpha 1-antitrypsin Met358 right-arrow Arg and (b) factor Xa by AT were measured discontinuously under pseudo first-order rate conditions (13) in the absence or presence of heparin, or SF, or both. Either thrombin (20 nM), factor Xa (60 nM), or a thrombin variant (20-60 nM) was incubated for 5 min at room temperature in 20 mM Tris-HCl, 150 mM NaCl, 0.6% PEG 8000, pH 7.4 (TSP), containing 10 mM GPRP-NH2 and various concentrations of heparin (0-100 µM) and SF (0-12 µM). Reaction mixtures were aliquoted to 96-well round bottom microtiter plates (Fisher, Nepean, Ontario, Canada), and an equal volume of inhibitor (in a concentration at least 10-fold higher than that of the proteinase) was added to each well at time intervals ranging from 2 s to 10 min. Reactions were terminated simultaneously by the addition of 200 µl of a solution containing 222 µM tGPR-pNA and 10 mg/ml Polybrene, and residual proteinase activity was determined by the initial rate of substrate hydrolysis measured at 405 nm using a Spectra Max 340 Microplate Reader (Molecular Devices, Menlo Park, CA). The pseudo first-order rate constant of inhibition was calculated by fitting the data to the first-order rate Equation 1.
V<SUB>t</SUB>/V<SUB>o</SUB>=e<SUP><UP>−</UP>k<SUB>1</SUB>t</SUP> (Eq. 1)
where Vo represents proteinase activity at time = 0; Vt represents proteinase activity at time = t, and k1 represents the pseudo first-order rate constant. The apparent second-order rate constant (k2) of inhibition was then obtained by dividing k1 by the inhibitor concentration (13). When high concentrations of heparin were used, the rates of proteinase inactivation were too fast to be measured under these conditions. To circumvent this problem, reactions were done in the presence of tGPR-pNA, which serves as a competitive inhibitor (34). To correct for the presence of chromogenic substrate, the pseudo first-order rate constant of inhibition was given by Equation 2.
k<SUB>1</SUB>=k<SUB><UP>app</UP></SUB>×(1+[S]/K<SUB>m</SUB>) (Eq. 2)
where kapp is the apparent pseudo first-order rate constant, [S] is the concentration of competitor, and Km is the Michaelis-Menten constant of the proteinase for tGPR-pNA (13). The Km values of thrombin and factor Xa for tGPR-pNA were determined to be 14 and 34 µM, respectively. In experiments where SF reduced the rate of proteinase inactivation, fold inhibition was calculated by dividing the k2 value in the absence of SF by that measured in its presence.

Inhibition of Thrombin by Covalent ATH Complex-- Inhibition of thrombin in the presence of ATH was determined by continuous assay of thrombin activity, monitored by cleavage of a fluorescent substrate. A 2-ml solution containing 18 nM ATH and 50 µM methoxysuccinyl-Ile-Glu-Gly-Arg-7-amino-4-trifluoromethyl coumarin (AFC-67, Enzyme System Products, Dublin, CA) in TBS was incubated at room temperature with stirring in a 3-ml quartz cuvette. Fluorescence (lambda ex 400 nm and lambda em 505 nm; slit widths 10 nm) was monitored at 0.5-s intervals in a Perkin-Elmer LS 50B spectrofluorimeter. SF and heparin were added, alone or in combination, to the cuvette at final concentrations of 4 µM and 500 nM, respectively. Finally, thrombin was added to a final concentration of 2 nM. Plots of I505 versus time were analyzed by nonlinear least squares analysis of Equation 3 (25).
I<SUB>t</SUB>=(I<SUB><UP>max</UP></SUB>/k<SUB>1</SUB>)(1−e<SUP><UP>−</UP>k<SUB>1</SUB>t</SUP>) (Eq. 3)
where k1 and Imax, the maximal rate of fluorescence change, were calculated by nonlinear regression analysis using TableCurve (Jandel, San Rafael, CA).

Effects of Soluble Fibrin and GPRP-NH2 on Thrombin Chromogenic Activity-- The chromogenic activity of 5 nM thrombin in TSP was determined with 185 µM tGPR-pNA in the presence of various concentrations of GPRP-NH2 (0.5-25 mM) or SF (1-6 µM) in 96-well round bottom microtiter plates. The initial rates of substrate hydrolysis, measured as the change in absorbance at 405 nm per min, were determined over 10 min using a microplate reader and were compared with the activity of thrombin in the absence of GPRP-NH2 and SF.

Effects of Thrombin-Heparin-Fibrin Ternary Complex Formation on the Rate of tGPR-pNA Hydrolysis by Thrombin, gamma -Thrombin, or RA-Thrombin-- Chromogenic substrate reactions were performed in 96-well round bottom microtiter plates in TSP at room temperature. Solutions of 5 nM thrombin, gamma -thrombin, or RA-thrombin, SF (0 or 4 µM), 5 mM GPRP-NH2, and heparin (0 or 500 nM) were mixed and then added to a range of tGPR-pNA concentrations (0-250 µM). The initial rates of substrate hydrolysis were determined by monitoring the reaction at 405 nm using a microplate reader and were plotted versus tGPR-pNA concentration. The data were then analyzed by nonlinear regression of the Michaelis-Menten Equation 4
V=(V<SUB><UP>max</UP></SUB>)×(S)/(K<SUB>m</SUB>+S) (Eq. 4)
to solve for Km, the Michaelis-Menten constant, and Vmax, the maximum rate of substrate hydrolysis, given V, the rate of substrate hydrolysis, and S, the substrate concentration.

Effect of Heparin on Thrombin, gamma -Thrombin, or RA-Thrombin Binding to Fibrin Monomer-Sepharose-- Binding studies were carried out in 20 mM Tris-HCl, 150 mM NaCl, 0.6% PEG 8000, 0.01% Tween 20, pH 7.4, at room temperature in 1.5-ml microcentrifuge tubes (Rose Scientific, Edmonton, Alberta, Canada) in total volumes of 500 µl. FM-Sepharose (50 µl; 1.4 µM final FM concentration) was mixed with thrombin, gamma -thrombin, or RA-thrombin (100 nM final concentration) in the absence or presence of various concentrations of unfractionated heparin (25 nM to 15 µM final concentration). After mixing for 2 min, FM-agarose was sedimented by centrifugation for 1 min in a microcentrifuge at 15,000 × g. An aliquot of each supernatant was transferred to a 96-well round bottom microtiter plate, and a solution of 222 µM tGPR-pNA and 10 mg/ml Polybrene was added simultaneously to each well. Hydrolysis of the chromogenic substrate was monitored by measuring the increase in absorbance at 405 nm over time using a plate reader. The quantity of thrombin bound to FM-Sepharose was calculated by subtracting the thrombin activity in the supernatant from a control measurement of thrombin activity in the absence of FM-Sepharose. Thrombin activity was converted to thrombin concentration using the specific activity of thrombin, determined under these conditions.

Statistical Analysis-- All values reported represent the mean ± S.D. of at least two separate experiments, each done in duplicate.

    RESULTS

Thrombin Inactivation by AT-- To quantify the consequences of ternary thrombin-heparin-fibrin complex formation, the influence of SF on thrombin inactivation by AT was determined in the absence or presence of 100 nM heparin (Fig. 1A). SF produced a concentration-dependent and saturable decrease in the heparin-catalyzed rate of inactivation of thrombin. In the presence of 100 nM heparin, the addition of 6 µM SF decreased k2 by a factor of 50, from a value of 2.97 ± 1.01 × 108 to 6.04 ± 1.20 × 106 M-1 min-1. Half-maximal reduction in k2 occurred with approximately 25 nM SF. With 10 nM heparin, 6 µM SF had slightly less of an inhibitory effect, causing a 41-fold reduction in k2 at 6 µM SF (not shown). Hogg and Jackson (21) observed a 310-fold reduction at 10 nM heparin. These differences could reflect the fact that the high affinity heparin used in the present studies had lower specific activity. In the absence of heparin, less than a 2-fold reduction in the rate of thrombin inhibition occurred when SF was present at concentrations up to 6 µM.


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Fig. 1.   Reduction in the heparin-catalyzed rate of thrombin inhibition by AT in the presence of soluble fibrin. Second-order rate constants of thrombin inhibition by AT were determined under pseudo first-order conditions. A, effect of varying concentrations of soluble fibrin on the rate of thrombin inactivation by AT in the absence () or presence of 100 nM heparin (black-square). B, effect of varying heparin concentrations on the rates of thrombin inactivation by AT in the absence (open circle ) or presence 4 µM soluble fibrin (). Each point represents the mean of at least two experiments done in duplicate, and the bars represent the standard deviation.

In parallel experiments, the heparin dose-response for thrombin inhibition was determined in the presence of 4 µM SF (Fig. 1B). With all concentrations of heparin tested, SF reduced the heparin-catalyzed rate of thrombin inactivation by AT. A maximal reduction in the rate of thrombin inhibition of 58-fold occurred with 100 nM heparin. In addition to a persistent reduction in k2, 4 µM SF also altered the shape of the heparin titration curve. The heparin dose-response curve is biphasic in the absence of SF, characteristic of a surface approximation phenomenon (13, 34). In the presence of SF, there is a saturable increase in k2 with a significantly lower maximal rate of inhibition. This result suggests that SF alters the mechanism of thrombin inactivation by the AT-heparin complex.

As a control experiment, the rates of thrombin inactivation in the absence and presence of both 10 nM heparin and 4 µM SF were determined over a range of AT concentrations (not shown). As predicted, varying AT concentration from 50 to 500 nM (the range of AT concentrations used in our studies) had no effect on k2 because calculations take the inhibitor concentration into account (13). As further controls, the effects of SF or GPRP-NH2 on the chromogenic activity of thrombin for tGPR-pNA were examined. The presence of SF up to 6 µM or GPRP-NH2 up to 25 mM had no effect on the rate of tGPR-pNA hydrolysis by thrombin (data not shown). Furthermore, the effect of GPRP-NH2 on the heparin-catalyzed and uncatalyzed rates of thrombin inactivation by AT were determined to ensure that the decreases in rates were not caused by the peptide. GPRP-NH2, at concentrations up to 5 mM, caused a less than 2-fold reduction in k2 (data not shown). To account for this small effect, 5 mM GPRP-NH2 was present in all reactions, even when the sample did not contain SF.

Factor Xa Inactivation by AT-- To examine directly the effect of SF on the catalytic activity of heparin, factor Xa was substituted for thrombin. Since factor Xa binds neither heparin nor fibrin, only the heparin-fibrin interaction would remain. As with thrombin, a range of SF concentrations had no effect on the uncatalyzed rate of factor Xa inhibition by AT. In contrast to thrombin, SF produced only a modest dose-dependent reduction in the rate of factor Xa inhibition in the presence of 10 or 100 nM heparin (not shown). At 10 nM heparin, 4 µM SF reduced k2 by a factor of 6 (from a value of 4.70 ± 0.90 × 106 to 7.63 ± 1.0 × 105 M-1 min-1) with half-maximal inhibition occurring at approximately 0.4 µM SF. With 100 nM heparin, SF reduced the rate constant only 2-fold. The relatively small magnitude of these inhibitory effects suggests that the heparin-SF interaction does not compromise the ability of heparin to catalyze AT to any appreciable extent.

Thrombin Inactivation by Heparin Cofactor II-- The influence of SF on the heparin-catalyzed and uncatalyzed rates of thrombin inactivation by HCII was examined because, unlike AT which interacts solely with the active site of thrombin, the amino-terminal region of HCII requires an additional interaction with thrombin exosite 1 (35-37), the domain also responsible for the interaction of thrombin with fibrin (38). Experiments were performed by varying the concentrations of each of the following three components: SF, heparin, and HCII. Similar to AT, SF caused a dose-dependent and saturable decrease in the heparin-catalyzed rates of thrombin inactivation by HCII, although the magnitude of the effect was much greater (Fig. 2A). In contrast to the 50-fold reduction in the rate of thrombin inactivation by AT in the presence of 100 nM heparin, 6 µM SF reduced the heparin-catalyzed rate of HCII by 222-fold (from 2.14 ± 0.05 × 108 to 9.64 ± 4.49 × 105 M-1 min-1). Half-maximal inhibition occurred at approximately 5 nM SF. When the heparin concentration was reduced from 100 to 10 nM, the inhibitory effect of SF was slightly less, reaching a maximum reduction in rate of 185-fold with 6 µM SF (not shown). Similar to AT, SF did not influence the uncatalyzed rate of thrombin inhibition by HCII.


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Fig. 2.   Reduction in the heparin-catalyzed rate of thrombin inhibition by HCII in the presence of soluble fibrin. Second-order rate constants of thrombin inhibition by HCII were determined under pseudo first-order conditions. A, effect of varying concentrations of soluble fibrin on the rates of thrombin inactivation by HCII in the absence () or presence of 1 µM heparin (black-square). B, effect of varying heparin concentrations on the rates of thrombin inactivation by HCII in the absence (open circle ) or presence of 4 µM soluble fibrin (). Each point represents the mean of at least two experiments done in duplicate, and the bars represent the standard deviation.

The effect of varying concentrations of heparin on the rate of thrombin inhibition by HCII in the presence of 4 µM SF is shown in Fig. 2B. Whereas 250 nM SF caused a modest reduction in k2 values at the various heparin concentrations (not shown), 4 µM SF had a more dramatic effect. Maximal 247-fold reduction occurred at 1 µM heparin. As with AT, the addition of SF changed the shape of the heparin titration curve from a biphasic curve to a curve showing a heparin dose-dependent and saturable increase. This finding suggests that SF alters the mechanism of thrombin inactivation by the heparin-HCII complex.

As controls, the k2 values for thrombin inactivation in the absence and presence of 10 nM heparin and 4 µM SF were determined at various HCII concentrations (not shown). Over the range of HCII concentrations used in these experiments there was no systematic change in k2.

Inactivation of Thrombin Exosite Variants-- The exosites of thrombin are implicated in the protective effect of the ternary complex due to their known interactions with fibrin and heparin (38). To investigate directly the involvement of thrombin exosite 1 and exosite 2 in the mechanism of resistance of fibrin-bound thrombin to inactivation by heparin-serpin complexes, variants of thrombin with impaired exosites were compared with native thrombin in inhibition assays in the absence or presence of 4 µM SF. The exosite 1 variants utilized were gamma -thrombin, a proteolytic derivative lacking residues in exosite 1 (39), and Quick 1 thrombin, a dysthrombin characterized by mutation of Arg67 in exosite 1 to Cys (24). The exosite 2 variant, RA-thrombin, has three mutations in exosite 2 that impair heparin binding (25). Table I compares the rates of inactivation of thrombin versus the thrombin variants by AT and 100 nM heparin or HCII and 1 µM heparin in the absence or presence of 4 µM SF. In the presence of 4 µM SF, the rates of thrombin inactivation by heparin-AT and heparin-HCII were inhibited 58-fold and 247-fold, respectively, as illustrated in Figs. 1 and 2. In contrast, when the exosite 1 variants gamma -thrombin or Quick 1-thrombin were substituted for thrombin, SF only decreased the heparin-catalyzed rates of inactivation by either AT or HCII by 4- or 10-fold, respectively. Similarly, the protective effect of SF was essentially eliminated when the exosite 2 mutant, RA-thrombin, was used in place of thrombin because its rate of inactivation by heparin-AT or heparin-HCII was reduced only 2- or 7-fold, respectively. As with thrombin, SF had no effect on the uncatalyzed rates of inactivation of the thrombin variants by either AT or HCII (data not shown). These results reveal a strict requirement for the integrity of both exosites in formation of productive thrombin-heparin-fibrin ternary complexes that attenuate thrombin inhibition by either AT or HCII.

                              
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Table I
Influence of soluble fibrin on the heparin-catalyzed rate of inhibition of thrombin or thrombin variants by antithrombin and heparin cofactor II
Second-order rate constants (k2) for heparin-catalyzed inhibition of thrombin (IIa) or thrombin variants by antithrombin or heparin cofactor II were determined under pseudo first-order conditions as described under "Experimental Procedures." Experiments were performed in the absence and presence of 4 µM soluble fibrin. The values are the mean of at least four determinations ± S.D.

Binding of Thrombin and Exosite Variants to Fibrin Monomer-Sepharose-- To explore the role of the exosites of thrombin in formation of the ternary complex, the influence of heparin on the binding of thrombin exosite 1 and 2 variants to FM-Sepharose was examined. In the absence of heparin, approximately 22% thrombin or RA-thrombin bound to FM-Sepharose (Fig. 3). In contrast, less than 4% gamma -thrombin bound, confirming that thrombin binds fibrin via exosite 1. The addition of 250 nM unfractionated heparin increased thrombin binding from 22 to 69%. Although a defective exosite 1 prevents gamma -thrombin from binding in the absence of heparin, binding increased from 4 to 32% in the presence of heparin reflecting heparin-facilitated binding via its intact exosite 2. In contrast to both thrombin and gamma -thrombin, 250 nM heparin only marginally increased the binding of RA-thrombin to fibrin (from 22 to 29%), presumably because the mutations in exosite 2 decrease the enzyme's affinity for heparin, thereby preventing heparin from bridging RA-thrombin to fibrin. These results are consistent with the proposal that thrombin forms a ternary complex with fibrin and heparin and that both thrombin exosites are involved (38); exosite 1 binds fibrin directly, whereas exosite 2 binds fibrin indirectly via fibrin-bound heparin.


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Fig. 3.   Effect of 250 nM unfractionated heparin on thrombin (IIa), gamma -thrombin (gamma -IIa), or RA-thrombin (RA-IIa) binding to fibrin monomer-Sepharose. Binding of 100 nM thrombin or thrombin variant to 1.4 µM fibrin monomer-Sepharose was accomplished by determination of the concentration of free thrombin in the supernatant after pelleting the Sepharose by centrifugation. The open bars represent the percent thrombin bound in the absence of heparin; the solid bars show the percent thrombin bound in the presence of 250 nM unfractionated heparin, and the lines represent the standard deviation.

Thrombin Active Site-- To determine whether thrombin bound within the ternary thrombin-heparin-fibrin complex undergoes allosteric changes at its active site, the reactivity of thrombin and thrombin variants with a chromogenic substrate and with a heparin-independent macromolecular inhibitor was assessed. Neither SF nor heparin alone altered the Km for thrombin hydrolysis of tGPR-pNA. However, SF and heparin in combination increased Km of thrombin for tGPR-pNA 3-fold, from a value of 25 ± 1.5 to 84 ± 0.7 µM (not shown). To explore the contribution of exosite 1 and exosite 2 ligation by fibrin and heparin, respectively, to this increase in Km, gamma -thrombin or RA-thrombin was substituted for thrombin. In contrast to thrombin, the ability of gamma -thrombin or RA-thrombin to hydrolyze the chromogenic substrate was unaffected by heparin, SF, or the combination. In the presence of both heparin and SF, the Km of gamma -thrombin for tGPR-pNA increased only slightly (from 58 ± 2.7 to 63 ± 4.1 µM), whereas the Km of RA-thrombin for the substrate decreased slightly (from 29 ± 1.5 to 24 ± 2.3 µM). These results confirm those of previous studies indicating that a conformational change occurs at the catalytic site of thrombin only when the ternary complex is formed (21, 40). Furthermore, our data indicate that ligation of both exosite 1 by fibrin and exosite 2 by heparin is essential to elicit this change.

The P1 mutation Met358 right-arrow Arg in alpha 1-antitrypsin yields a potent heparin-independent inhibitor of thrombin (26) that does not require interaction with either exosite on thrombin. To confirm this, we found that alpha 1-antitrypsin Met358 right-arrow Arg inactivated both gamma -thrombin and RA-thrombin at the same rate as thrombin (4.60 ± 0.15 × 106, 5.18 ± 0.97 × 106, and 4.22 ± 0.93 × 106 M-1 min-1, respectively). The effects of 500 nM heparin and 4 µM SF alone, and in combination, on the rate of thrombin inactivation by alpha 1-antitrypsin Met358 right-arrow Arg are shown in Fig. 4. Heparin or SF alone inhibited the rate of thrombin inactivation by 1.4- and 1.7-fold, respectively. When both were added to form thrombin-heparin-fibrin ternary complexes, the rate of thrombin inactivation was reduced only 4-fold (from 4.22 ± 0.93 × 106 to 1.05 ± 0.28 × 106 M-1 min-1). Thus, the protective effect of ternary complex formation is less with alpha 1-antitrypsin Met358 right-arrow Arg than with AT or HCII, whose rates were inhibited 58- and 247-fold, respectively. These results indicate that allosteric changes at the active site induced by ternary complex formation have minimal effects on the interaction of thrombin with a macromolecular heparin-independent inhibitor.


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Fig. 4.   Influence of soluble fibrin and heparin on the rate of thrombin inhibition by alpha 1-antitrypsin Met358 right-arrow Arg or a covalent antithrombin-heparin complex (ATH). The second-order rate constants for thrombin inhibition by alpha 1-antitrypsin Met358 right-arrow Arg or ATH were determined. The experiments were repeated in the presence of 4 µM SF, 500 nM heparin, or both SF and heparin. The open bars represent alpha 1-antitrypsin Met358 right-arrow Arg, and the solid bars represent ATH. Each bar represents the mean of at least two experiments done in duplicate, and the lines represent the standard deviation.

Mechanism of Protection-- The preceding experiments demonstrate that the two thrombin exosites participate in ternary complex formation. They also confirm the proposal that heparin bridges thrombin to fibrin, effectively increasing the affinity of thrombin for fibrin (38). To begin to elucidate the mechanism by which fibrin-bound thrombin is protected from inactivation by the heparin-AT complex, we examined whether the heparin component of the ternary complex is able to activate AT. Although heparin is able to catalyze factor Xa inhibition by AT in the presence of fibrin (not shown) (21), heparin and fibrin alone do not constitute a ternary complex. Therefore, complete ternary complexes were assembled by the addition of FPRCK-thrombin to heparin and SF, and their effect on factor Xa inhibition by AT was determined. FPRCK-thrombin was added at concentrations equivalent to, or 10-fold higher than, that of heparin (10 nM), conditions under which the rate of inhibition of active thrombin by AT is reduced 42-fold. The addition of FPRCK-thrombin caused no further reduction in the rate of factor Xa inhibition than the 2-3-fold observed with SF alone (not shown). These results suggest that heparin within the ternary thrombin-heparin-fibrin complex retains its ability to bind and activate AT. Thus, formation of a ternary complex does not appear to compromise the function of either heparin or AT. Instead, the protective effects of ternary complex formation likely reside with thrombin.

Results with the heparin-independent inhibitor, alpha 1-antitrypsin Met358 right-arrow Arg, demonstrated that access of macromolecular inhibitors to the active site of thrombin is not impaired upon formation of the ternary complex, thereby making steric mechanisms of protection unlikely (Fig. 4). Comparison of the results with alpha 1-antitrypsin Met358 right-arrow Arg with those with AT-heparin or HCII-heparin also reveals that the protective effect is heparin-dependent. Therefore, the next most likely scenario involves impairment of the ability of heparin to act as a catalyst in thrombin inhibition in the presence of ternary complexes. Functional studies to address this possibility are complicated by the fact that heparin serves two roles, bridging thrombin to fibrin and bridging thrombin to AT. To circumvent this problem, we used a covalent AT-heparin complex (ATH) to ensure that these two roles were performed by separate heparin molecules. The ATH complex is prepared by incubating heparin and AT so that a Schiff base is formed that subsequently rearranges into a covalent complex (23). ATH is a more potent inhibitor of thrombin and factor Xa than heparin-catalyzed AT, presumably because of the covalent binding of heparin to AT in the ATH complex (23, 41). In support of this, the second-order rate constant for thrombin inhibition is 9.3 × 108 M-1 min-1 for ATH and 4 × 108 M-1 min-1 for AT with 100 nM heparin, the concentration of heparin that provides maximal catalysis (Fig. 1). In the presence of 500 nM heparin, the rate of inhibition by ATH decreased only 2-fold, whereas 4 µM SF caused only a 5.4-fold reduction in the rate of inhibition by ATH (Fig. 4). These findings demonstrate that fibrin has minimal effects on the heparin component of ATH. When exogenous heparin was added to bridge thrombin to fibrin thereby forming a ternary complex, there was a 66-fold reduction in the rate of thrombin inhibition by ATH, a decrease comparable to that produced by SF for AT and heparin (Fig. 1). Since the heparin moiety of ATH must still bind thrombin to catalyze inhibition (41), the reduction in the rate of thrombin inhibition reflects impaired access of the heparin moiety of ATH to thrombin within the thrombin-heparin-fibrin complex. Therefore, the major source of protection from inhibition resides in the inability of AT-associated heparin to access exosite 2 of thrombin and perform its obligatory bridging role.

    DISCUSSION

Exosites 1 and 2 of thrombin have been implicated in the formation of ternary thrombin-heparin-fibrin complexes that protect thrombin from inhibition by AT (38). To investigate the role of the thrombin exosites directly, the protective effect of the ternary complex was investigated from the perspective of both the inhibitor and the enzyme. AT, HCII, and alpha 1-antitrypsin Met358 right-arrow Arg have distinct structural requirements for inhibition of thrombin. AT and HCII, when catalyzed by heparin, require the participation of thrombin exosite 2, the heparin-binding site, whereas alpha 1-antitrypsin Met358 right-arrow Arg is heparin-independent (26, 42). HCII has an additional requirement for exosite 1 for the initial docking of its amino terminus (8). The effect of ternary complex formation on thrombin inhibition was exaggerated with HCII compared with AT and abrogated with alpha 1-antitrypsin Met358 right-arrow Arg, with 250-, 50-, and 4-fold reductions in the rate of thrombin inhibition, respectively. These data reveal that protection is observed only with heparin-catalyzed inhibitors, confirming the importance of the exosites in the protective mechanism.

In order to discern more directly the role of thrombin exosites on the protective effect of ternary complex formation, variants of thrombin with impaired exosites 1 or 2 were employed. Involvement of the exosites can be inferred from a lack of protective effect of SF on the heparin-catalyzed rate of factor Xa inhibition, because factor Xa does not bind fibrin and, in the absence of calcium (43), does not interact with heparin. Thrombin Quick 1, a dysthrombin (24), and gamma -thrombin, a proteolytic derivative of native thrombin (39), both have defects in exosite 1, whereas RA-thrombin, a recombinant variant, has mutations in exosite 2 (25). SF caused only minor reductions in the heparin-catalyzed rate of inhibition by AT for both exosite 1 and 2 thrombin variants. The lack of protection cannot be attributed to low rates of inhibition of the thrombin variants because the heparin-catalyzed rates of inhibition of gamma -thrombin and thrombin Quick 1 by AT are comparable to that of native thrombin. This cannot be said of the exosite 2 thrombin variant because the magnitude of heparin catalysis is modest. These data demonstrate that the protective effect of ternary complex formation is lost when the function of either exosite is impaired.

Both exosites on thrombin also are critical for assembly of the ternary thrombin-heparin-fibrin complex. Heparin promoted the binding of both native thrombin and gamma -thrombin to immobilized fibrin monomer, although little gamma -thrombin bound in the absence of heparin. In contrast, the binding of RA-thrombin to fibrin was only marginally enhanced in the presence of heparin, presumably because its reduced affinity for heparin prevents the heparin from bridging RA-thrombin to fibrin. Although this bridging is independent of the exosite 1-fibrin interaction, native thrombin demonstrates a greater increase in binding than gamma -thrombin, possibly reflecting a cooperative effect of binding via both exosites in the ternary complex.

Although these experiments demonstrate the interactions required to support ternary complex formation, they do not reveal the mechanism responsible for protection of thrombin within the ternary complex from inhibition by AT or HCII. A number of models can be envisioned that involve impairment of accessibility or function of thrombin, heparin, or AT (38, 44). Briefly, these models can be classified as allosteric or steric. The former model proposes that the active site of thrombin is altered in such a way as to limit its reactivity with macromolecular inhibitors. Steric models suggest physical impairment of the ability of AT or heparin to bind thrombin, heparin to bind AT, or the AT-heparin complex to bind the thrombin-heparin-fibrin complex. Models proposing functional changes at the active site of thrombin or the formation of a nonproductive AT-heparin-fibrin complex also have been suggested (38).

The possibility that formation of the ternary complex caused steric inhibition at the active site of thrombin which altered its accessibility to macromolecular inhibitors was addressed with the use of alpha 1-antitrypsin Met358 right-arrow Arg, a serpin that reacts exclusively with the active site of thrombin. The observation that ternary complex formation does not protect thrombin from inhibition by this inhibitor suggests that the active site of thrombin remains accessible to macromolecules. This concept is corroborated by previous work indicating that thrombin within the ternary complex retains its ability to convert prothrombin to prethrombin 1 (40). Although our study and those of others (40, 45) reveal structural and functional changes in the active site upon ternary complex formation, these changes do not appear to influence the reactivity of the enzyme with macromolecules. Since the protective effect is observed with heparin-dependent inhibitors, and not heparin-independent inhibitors, the results, to this point, suggest that the protective effect of the ternary complex results from limitations of heparin catalysis and not changes at the active site of thrombin.

Since heparin binds both thrombin and AT, hampered catalysis could result from impaired interaction with either protein. This was addressed with the use of factor Xa since its inhibition by AT is catalyzed by heparin solely through heparin-AT interactions. Factor Xa inhibition by heparin-AT was only slightly affected by fibrin monomer (22). These findings suggest that heparin associated with fibrin is able to catalyze AT. It could be argued, however, that heparin in a binary heparin-fibrin complex is not identical to that in a ternary thrombin-heparin-fibrin complex. To address this possibility, factor Xa inhibition also was monitored in the presence of heparin, fibrin, and FPRCK-thrombin. Since heparin bridges FPRCK-thrombin to fibrin (Fig. 3), a productive ternary complex is formed. That there was only a modest effect on factor Xa inhibition reveals that the catalytic properties of heparin are retained, even when heparin is associated with both thrombin and fibrin.

There are two mechanisms of protection of thrombin that could involve an impaired ability of heparin to catalyze thrombin inhibition by AT. The first is one in which thrombin and AT are bound with high affinity to the same heparin chain but are unable to associate with each other because of physical separation of the binding sites. In this scenario, AT is bound to the high affinity pentasaccharide sequence on heparin at a site remote from the portion of the heparin chain involved in the formation of the thrombin-heparin-fibrin complex. This mechanism is unlikely because thrombin within this ternary complex is protected from inactivation by HCII to a greater extent than by AT, even though HCII does not utilize the pentasaccharide or any other high affinity binding site on heparin (8). The second mechanism of protection involving heparin could result from impaired access of serpin-bound heparin to an already occupied heparin-binding site on thrombin. This possibility was assessed using a covalent ATH complex to ensure that the catalytic heparin species was distinct from the heparin associated with thrombin in the ternary complex. Inhibition of thrombin by the covalent ATH complex was reduced 66-fold in the presence of the ternary complex suggesting that the heparin moiety of ATH was unable to adequately bind to exosite 2 on thrombin. This is consistent with the observation that the protective effect of the ternary complex is obtained with heparin-catalyzed inhibitors, such as AT and HCII, but not with the heparin-independent alpha 1-antitrypsin Met358 right-arrow Arg. These data suggest that formation of the ternary complex increases the affinity of heparin for exosite 2 on thrombin such that heparin associated with AT or HCII is unable to bind to fibrin-bound thrombin (Fig. 5). With HCII, the effect is exacerbated because heparin increases the affinity of fibrin for exosite 1 on thrombin, a requisite binding site for HCII. Thus, although all the binary interactions between heparin, thrombin, and fibrin are strengthened in the ternary complex, the heightened heparin-thrombin interaction appears to be responsible for the impairment of AT function.


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Fig. 5.   Proposed mechanism for protection of fibrin-bound thrombin from inactivation by AT and HCII in the presence of heparin. Thrombin (IIa) is held in a ternary complex through interaction with fibrin via exosite 1 and heparin via exosite 2. A, heparin, in complex with antithrombin (AT), cannot access exosite 2 because the domain is occupied by heparin from the ternary complex. B, heparin, in complex with heparin cofactor II (HCII) has restricted access to exosite 2 on thrombin. In addition, the amino-terminal acidic domain of HCII cannot interact with exosite 1 on thrombin because this site is bound to fibrin. This additional interaction explains why fibrin-bound thrombin is more resistant to inhibition by HCII than by AT.

In forming the ternary complex, both exosites on thrombin appear to be occupied simultaneously. Likewise, both thrombin exosites are ligated when thrombin binds to thrombomodulin, where the growth factor moiety binds exosite 1 and the chondroitin sulfate moiety binds exosite 2 (46). Thrombin within the thrombomodulin-thrombin complex demonstrates impaired inhibition by AT in the presence of heparin (47), consistent with the effect of ternary complex formation. Similarly, meizothrombin des-F1, a thrombin precursor in which the covalently associated prothrombin fragment 2 occupies exosite 2, demonstrates reduced inhibition by AT in the presence of heparin (48). In each of these examples, occupation of exosite 2 on thrombin limits the ability of heparin to catalyze inhibition by AT. The consequences of ternary complex formation are analogous to the effect of reducing the affinity of thrombin for heparin. The recombinant thrombin derivative with mutations in exosite 2, RA-thrombin, exhibits a greater than 20-fold decrease in its affinity for heparin which results in a 30-fold reduction in the heparin-catalyzed rate of thrombin inhibition by AT (25).

Our studies have revealed the mechanism by which thrombin is protected from inhibition by AT in the presence of fibrin. Protection is a consequence of formation of a ternary thrombin-heparin-fibrin complex. Assembly of the ternary complex is a result of mutual interactions between thrombin, heparin, and fibrin. Revelation of the mechanism of protection may provide new avenues for development of antithrombotic agents whose activities are not compromised when thrombin binds fibrin. Although HCII is a heparin-catalyzed inhibitor of thrombin whose efficacy is impaired in the presence of fibrin, catalysis of HCII by dermatan sulfate is unaffected by fibrin (49) suggesting that, unlike heparin, dermatan sulfate does not promote ternary complex formation. Another approach would be to investigate the molecular weight dependence of heparin with respect to ternary complex formation. Since bridging of thrombin to fibrin is reduced with shorter heparin chains (22), the degree of protection may be correspondingly minimized. Our studies also suggest that heparin-independent inhibitors, such as alpha 1-antitrypsin Met358 right-arrow Arg (42) or a protease nexin-1 variant (50), may be better antithrombotic agents than heparin because they can inactivate fibrin-bound thrombin.

    ACKNOWLEDGEMENTS

We thank Michael Nesheim and Charles Esmon for many helpful discussions and Janice Rischke for high pressure liquid chromatography analyses.

    FOOTNOTES

* This work was supported in part by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario.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.

Dagger Recipient of a Research Fellowship Award from the Heart and Stroke Foundation of Canada.

§ Recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Hamilton Civic Hospitals 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.

    ABBREVIATIONS

The abbreviations used are: AT, antithrombin; ATH, covalent antithrombin-heparin complex; FM, fibrin monomer; FPRCK, D-Phe-Pro-Arg chloromethyl ketone; GPRP-NH2, Gly-Pro-Arg-Pro-amide; HCII, heparin cofactor II; IIa, thrombin; Polybrene, hexadimethrine bromide; RA-thrombin, Arg93 right-arrow Ala, Arg97 right-arrow Ala, and Arg101 right-arrow Ala thrombin; SF, soluble fibrin; tGPR-pNA, tosyl-Gly-Pro-Arg rho -nitroanilide.

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Abstract
Introduction
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