Eur. J. Biochem. 188,61-66 (1990) 0 FEBS 1990

Use of fragments of hirudin to investigate thrombin-hirudin interaction Stanley DENNIS, Andrew WALLACE, Jan HOFSTEENGE and Stuart R. STONE

Friedrich Miescher-lnstitut, Basel, Switzerland

(Received August 24,1989) - EJB 89 1050

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methonine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg- NH-Ph). The N-terminal fragment (residues 1 - 52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected a-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of a-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.

Hirudin is a polypeptide of 65 or 66 amino acids [I - 31 that acts as a slow, tight-binding inhibitor of thrombin [4, 51. Hirudin was originally isolated from the medicinal leech Hirudo medicinalis and recombinant forms of this inhibitor are now available [3, 6-91, Hirudin isolated from leeches contains a sulphated tyrosine at position 63; in the re- combinant forms, Tyr-63 is not sulphated and this results in a somewhat lower affinity of thrombin for the recombinant form [5 , 101.

The interaction between thrombin and hirudin involves a number of regions on both molecules [I0 - 131. Indeed, hirudin is unique among inhibitors of serine proteases in that interac- tion with the primary specificity pocket of its target protease appears not to be important for complex formation [5, lo]. The C-terminal region, residues 57 - 65, and the two N-terminal residues of hirudin have been shown to be important for its interaction with thrombin [lo, 11, 13, 141. Regions of throm- bin which interact with hirudin have been identified in studies using modified thrombins [I 21, peptide-specific antibodies [15] and chemical modification [16]. Areas of thrombin identified in these studies were a positively charged surface loop and an apolar binding site.

Studies on the kinetic mechanism of hirudin inhibition [4] indicated that the rate-limiting first step in the formation of the complex is dependent on ionic strength and is not influ- enced by the binding of the substrate at the active site. In the second step, hirudin is bound at the active site. It has been

Correspondence to S. R. Stone, Friedrich Miescher-Institut, P. 0. Box 2543, CH-4002 Basel, Switzerland

Abbreviations. r-hir, recombinant hirudin; [Asn52+Met]hirudin, recombinant hirudin with Am52 replaced by methionine; r-hir(1- 52), the fragment of recombinant hirudin comprising residues 1 - 51 plus a C-terminal homoserine; r-hir(53 - 65), the fragment of re- combinant hirudin comprising residues 53 - 65; Pip, pipecolyl; Tos,

Enzymes. Trypsin (EC 3.4.21.4); thrombin (EC 3.4.21.5); elastase tosyl.

(EC 3.4.21.36).

proposed that the first, ionic-strength-dependent step involves the interaction of the negatively charged C-terminal region of hirudin with a positively charged surface loop on thrombin [12, 141. The extent to which the interaction taking place in the first step facilitates the second step is not clear. Studies by Mao et al. [17] indicated that the binding of a C-terminal fragment of hirudin induces a conformational change in thrombin. However, the contribution of this conformational change to the formation of the thrombin-hirudin complex has not been evaluated.

In the present study, fragments of hirudin have been used to investigate the regions of thrombin and hirudin that interact with each other. In addition, the effect of the binding of the C-terminal fragment of hirudin on the binding of the rest of the hirudin molecule to thrombin has been evaluated.

EXPERIMENTAL PROCEDURES

Mu teriuls

The substrates DPhe-Pip-Arg-NH-Ph and Tos-Gly-Pro- Arg-NH-Ph were from Kabi, Molndal, Sweden, and Boehringer-Mannheim, Mannheim, FRG, respectively. Ala- Phe-Arg-CH2C1 was a gift from Dr. Elliot Shaw (Friedrich Miescher-Institut, Basel, Switzerland). Bovine trypsin and porcine pancreatic elastase were from Worthington, Freehold, NJ. SDSjPAGE molecular mass markers were from Bio-Rad, Richmond, CA. All other chemicals were of the highest grade available commercially. Human a-thrombin and human anti- thrombin 111 were purified and characterised as described previously [IS].

Amidolytic ussay of thrombin

Assays were performed as described previously [4, 181 at 37°C in 50 mM Tris/HCl buffer, pH 7.8, containing 0.1 M NaCl and 0.1 YO poly(ethy1ene glycol) M, 6000. For slow, bind-

62

ing-inhibition experiments involving recombinant hirudin (r-hir) or the mutant [Asn52+Met]hirudin and for the inacti- vation by antithrombin 111, the substrate DPhe-Pip-Arg-NH- Ph was present at a concentration of 100 pM and the a- thrombin concentration was 20 or 25 pM. For the determi- nation of kinetic parameters by the progress-curve method of Duggleby and Morrison [19, 201, the initial concentration of DPhe-Pip-Arg-NH-Ph was varied over 12 - 70 pM (six differ- ent concentrations) with an a-thrombin concentration of 0.2 nM. For initial velocity-inhibition experiments, the sub- strates were present at the indicated concentrations and 50 pM a-thrombin was used.

Expression and characterisation of the mutant hirudin (Asn52 + Metlhirudin

Site-directed mutagenesis of the hirudin gene to mutate Asn52 to Met52 and subsequent expression of the mutant [Asn52+Met]hirudin in Escherichia coli were performed as described previously [lo]. [Asn52+Met]Hirudin was purified by ion-exchange and reversed-phase chromatography; its structure and purity were verified by amino acid analysis, peptide mapping and peptide sequencing [lo].

Production of the hirudin fragments r-hir(1- 52) and r-hir (53 - 65)

[Asn52+Met]Hirudin was cleaved with CNBr as de- scribed by Gross (211 and two fragments were obtained as expected; the N-terminal and C-terminal fragments are re- ferred to as r-hir(1-52) and r-hir(53 -65). These fragments could be readily separated from each other by reversed-phase HPLC using a 10-40% gradient of acetonitrile in 0.1% (by vol.) trifluoroacetic acid with a CI8 column (Vydac, Hesperia, CA). It was not possible, however, to separate r-hir(53 - 65) from traces of the parent molecule ([Asn52 +Met]hirudin). The r-hir(53 - 65) used in this study was synthesised using 9-fluorenylmethoxycarbonyl-protected amino acids and a Milligen 9050 peptide synthesizer. The amino acid analyses of both r-hir(1-52) and r-hir(53 -65) yielded the expected compositions; r-hir(1- 52) contained a single homoserine. Further characterisation of r-hir( 1 - 52) by peptide mapping and N-terminal sequencing confirmed the proposed structure. A complete sequence analysis of r-hir(53 - 65) established its identity. The concentrations of r-hir(1- 52) and r-hir(53 - 65) were determined by amino acid analysis [22].

Partial proteolysis qf a-thrombin by trypsin and pancreatic elastase

Partial proteolysis experiments were performed at 37 "C in the buffer used for the amidolytic assays. Human a-thrombin was present at a concentration of 2.95 pM (2 pg in a reaction vol. of 25 pl). r-Hir, r-hir(1- 52) and r-hir(52 - 65) were added at concentrations that would result in greater than 99% of the a-thrombin being present in the form of a complex with these molecules. The concentrations used were 3.5 pM, 10 pM and 250 pM for r-hir, r-hir(1- 52) and r-hir(53 - 65), respectively. The reaction was started by the addition of 2% (by mass) trypsin or pancreatic elastase and stopped by the addition of Ala-Phe-Arg-CH2C1 at a concentration of 4.0 pM in the case of trypsin and by heating to 95°C for elastase. The extent of proteolysis of a-thrombin was monitored by SDSjPAGE under reducing conditions on 15% polyacrylamide gels [23]. Coomassie blue was used to stain the polypeptide chains.

DATA ANALYSIS

The formation of the thrombin-hirudin complex can be represented by the following scheme:

ki E + I G E I

where E, I, and El represent thrombin, hirudin and the throm- bin-hirudin complex, respectively. The dissociation constant of the complex (K,) can be related to the association (k,) and dissociation (k,) rate constants by the expression KT = k2/ kl . [Asn52+Met]Hirudin and r-hir were slow, tight-binding inhibitors of thrombin and progress-curve data obtained in the presence of DPhe-Pip-Arg-NH-Ph were fitted to Eqn (4) of [4] using non-linear regression; estimates of k,, k2 and K, were calculated as previously described [4, 101. These values are reported together with their standard errors. It should be noted, however, that determination of kinetic parameters by non-linear regression analysis of progress-curve data leads to an underestimation of the standard errors [20].

Initial velocity experiments in which both the substrate and r-hir(1- 52) were varied established that r-hir(1- 52) was a competitive inhibitor of thrombin (Fig. 1). These data were fitted by weighted linear regression [24] to the equation de- scribing competitive inhibition [25] to yield an estimate of the inhibition constant (KJ . For a competitive inhibitor, the inhibition constant will equal the dissociation constant of the compound. In subsequent experiments, the substrate was fixed and the data were fitted to the Dixon equation [25] to yield an estimate of the apparent inhibition constant (K,,). The true value was obtained by applying the following relationship: K,' = K,(1 + SjK,), where S represents the concentration of substrate and K, its Michaelis constant.

The kinetic parameters of thrombin with the substrate DPhe-Pip-Arg-NH-Ph in the absence and presence of r-hir(53 - 65) were determined by progress-curve kinetics es- sentially as described by Duggleby and Morrison [19, 201.

The second-order rate constant for the inactivation of thrombin by antithrombin I11 was determined in the presence of the substrate DPhe-Pip-Arg-NH-Ph as previously described [18]. Five concentrations of antithrombin I11 ranging over 1.4 - 7.0 pM were used.

k2

RESULTS

Production of hirudin fragments

The amino acid sequence of hirudin does not contain a methionyl residue. Therefore, it was possible, by introducing such a residue into r-hir at a particular position, to produce a molecule that could be cleaved by CNBr into two exactly defined fragments. A methionine was subsituted for Asn52 to produce the mutant [Asn52+Met]hirudin. This form of hirudin exhibited inhibitory properties that were very similar to r-hir; the inhibition constant ( K I ) for the thrombin- [Asn52+Met]hirudin complex was slightly lower than that observed for r-hir (122 fM compared with 231 fM; Table 1). Cleavage of [Asn52+Met]hirudin resulted in the production of two fragments as expected; the N-terminal and C-terminal fragments are referred to as r-hir(1- 52) and r-hir(53 - 65). These fragments were isolated and characterized chemically as decribed in Experimental Procedures.

63

Table 1. Binding constants for the complexes between thrombin and different forms ofhirudin Assays were performed and the data were analyzed as described in Experimental Procedures and Results. The estimates of the dissociation constant (K,) for each form of hirudin are given together with their standard errors. The value for AG; was calculated from the relationship AG; = RT In(&) where R is the gas constant and T is the absolute temperature. The values for r-hir were determined previously [lo]. [Asn52+Met]Hirudin was a slow, tight-binding inhibitor of thrombin and the determined association and dissociation rate constants were (1.21 4 0.02) x lo8 M- ' . S - ' and (1.47 0.07) x lo5 s-' , respectively

Form of hirudin Ki Unit - AG;

kJ . mol-

r-Hir [Asn52 + Metlhirudin Hir(1- 52) Hir(1-52) [in the presence of 20 pM hir(53-65)] Hir(53 - 65)

231 f 6 fM 75.0 122 f 6 fM 76.6 24.4 f 1.0 nM 45.2 9.7 * 0.8 nM 47.6 3.18 +_ 0.19 PM 32.6

O ' O 6 Z

0'051 0. 04

I 0. 00 0. 05 0. 10 0. 15 0. 20

0.00-

1/15] (pM-l)

Fig. 1. Effect ofr-hir(1-52) on the amidolytic activity ofcc-thrombin. Assays were performed as described in Experimental Procedures with the Tos-Gly-Pro-Arg-NH-Ph in the presence of 0 nM (A), 48.1 nM (0), 96.3 nM (0) and 144.4 nM ( V ) r-hir(1- 52). The lines represent the best fit of the data to the equation for competitive inhibition; the estimates obtained from the analysis of the data were: k,,, = 265 6 s- ' , K , = 10.0 0.5 pM and K, = 24.4 f 1.0 nM

Effect of r-hir(1-52) and r-hir(53-65) on the amidolytic activity of thrombin

r-Hir(1- 52) was a competitive inhibitor of thrombin as shown in Fig. 1. The KI value for r-hir(1- 52) determined from these data was 24.4 1 .O nM (Table 1). r-hir(53 - 65) caused a slight stimulation of the amidolytic activity with low concen- trations (< 10 pM) of the substrates Tos-Gly-Pro-Arg-NH- Ph and DPhe-Pip-Arg-NH-Ph. Therefore, the kinetics of the hydrolysis of DPhe-Pip-Arg-NH-Ph were studied in the pres- ence of 25 pM r-hir(53 - 65) which represents a concentration eightfold higher than the dissociation constant for this peptide (Table 1). Progress-curve data were analyzed by the method of Duggleby and Morrison [19, 201 and the results are given in Table 2. The kinetic constants for DPhe-Pip-Arg-NH-Ph in the absence of r-hir(53 - 65) corresponded well to values previously determined [18, 261. The presence of r-hir(53 -65) did not significantly change the value for k,,, but caused a 60% reduction in the value of K, (Table 2).

The N-terminal fragment of hirudin was a slightly better inhibitor of thrombin in the presence of r-hir(53 - 65) (data not shown). The Kl value for r-hir(1-52) in the presence of 20 pM r-hir(53-65) was 9.7 f 0.8 nM (Table 1). This 60% decrease in the value of KI in the presence of r-hir(53 - 65) is

Table 2. Kinetic parameters fo r the hydrolysis of DPhe-Pip-Arg-NH- Ph by thrombin in the absence andpresence ofr-hir(53--65) Assays were performed as described in Experimental Procedures and the data were analysed by the method of Duggleby and Morrison [19, 201. The estimates of the parameters are given together with their standard errors

Concentration K , k,,, (kcatiKm) x lo-* of r-hir(53 -65)

PM w S - I M - 1 . s - l

0 3.4 f 0.1 205 f 1 0.60 _+ 0.10 25 1.4 f 0.1 2 1 6 1 1 1.58 +0.13

of the same magnitude as the decrease in the value of K, caused by the peptide.

Effect of r-hir(53 - 65) on the inhibition of thrombin by r-hir and antithrombin III

The effect of increasing concentrations of r-hir(53 - 65) on the observed value of the inhibition constant for r-hir [K,(obs)] is shown in Fig. 2. Increasing concentrations of r-hir(53 - 65) would be expected to increase K,(obs) for two reasons: the peptide would directly compete with r-hir for a binding site, and it would also cause a decrease in the K, value for the substrate resulting in the substrate being a more effective competitor with r-hir. The effect is described by Eqn. (1):

KI(0bs) = Ki(1 + SjK,,) (1 + J/Kj) (1)

where J is the concentration of r-hir(53 - 65), K,, is the ob- served K, value at the particular concentration of r-hir(53 - 65), and Kj is the dissociation constant for r-hir(53 - 65). The value of K,, will be given by Eqn (2):

(2) KrnIKJIJ + Km2

1 + Kj/J K,, ==

where K,, and K,, are the Michaelis constants for the sub- strate at zero and infinite concentrations of r-hir(53 - 65), respectively. Substitution of Eqn (2) into Eqn (1) and re- arrangement yields Eqn (3):

Values for KI(obs) obtained at different concentrations of

64

0 0. 0 10.0 20. 0 30. 0 40. 0

chit- (53-65) OJM)

Fig. 2. EjJect of r-hir(52--65) on the observed dissociation constant K,(obs) fo r r-hir. Assays were performed as described in Experimental Procedures and the estimates of K,(obs) were determined by non- linear regression. These estimates were weighted according to the squared inverse of their standard errors and fitted by non-linear regression to Eqn (3) (see Results). The line represents the best fit of the data to this equation; the analysis yielded estimates of 0.22 f 0.01 pM and 3.2 f 0.2 pM for the dissociation constants of r-hir and r-hir(53 -65), respectively

Fig. 3. Effect of hirudin and hirudin fragments on the cleavage of a-thrombin by trypsin. Cleavage of a-thrombin with trypsin was performed as described in Experimental Procedures. Lanes 1, 3, 5 and 7 contain a-thrombin prior to trypsin treatment. The effects of treatment with trypsin for 120 rnin in the absence of hirudin (lane 2) and in the presence of r-hir (lane 4), r-hir(1- 52) (lane 6) and r-hir(53 - 65) (lane 8) are also shown. Molecular mass markers of the indicated size were loaded in lane 0

r-hir(53 - 65) were weighted according to the inverse square of their standard error and fitted to Eqn (3) by non-linear regression. For this analysis, the values of K,, and Km2 were fixed at previously determined values of the K, for the sub- strate in the absence and presence of r-hir(53 - 65), respective- ly (Table 2). The data of Fig. 2 fitted well to this equation and the analysis yielded values of 216 f 11 fM and 3.2 0.2 pM for the dissociation constants of r-hir and r-hir(53 -65), re- spectively. These values correspond well to those previously determined for r-hir [lo] and C-terminal fragments of hirudin

In contrast to its effect on the interaction of thrombin with r-hir, r-hir(53 - 65) did not significantly affect the inactivation of thrombin by antithrombin 111. The second-order rate con- stant for the inactivation by antithrombin 111 in the absence of peptide was determined to be 10.2 f 0.1 mM-' . s-' which is in agreement with previously determined values [18,26,28]. In the presence of 31.2 FM r-hir(53-65), the value of this constant was 11.4 & 0.1 mM-' . s-'.

~ 7 1 .

Fig. 4. Effect of hirudin and hirudin fragments on the cleavage of a-thrombin by pancreatic elastase. Cleavage of a-thrombin with pan- creatic elastase was performed as described in Experimental Pro- cedures. Lanes 1 - 3 show a-thrombin prior to treatment with elastase, and after treatment for 30 min and 60 min. The effects of r-hir (lane 4), r-hir(1- 52) (lane 5) and r-hir(53 - 65) (lane 6) on the cleavage by elastase for 60 min are also shown. Molecular mass markers of the indicated size were loaded in lane 0

Effect of r-hir(1-52) and r-hir(53-65) on theproteolytic cleavage of thrombin by trypsin and elastase

Trypsin cleaves human a-thrombin at Arg73 of the B- chain to form /&-thrombin [29]. This conversion is observed as a reduction in the apparent molecular mass of the major band in SDSjPAGE from 36 kDa to 24 kDa. Further cleav- ages may occur at Arg62 and Lysl54 to produce yT-thrombin and results in the appearance of bands with lower apparent molecular masses. As shown in Fig. 3 (lanes 1 and 2), incu- bation of a-thrombin with 2% (by mass) trypsin for 2 h at 37°C results in complete conversion of a-thrombin to PT- thrombin and 7,-thrombin. r-Hir was able to block completely the conversion of a-thrombin to proteolytically degraded forms (Fig. 3, lanes 3 and 4). A similar effect was observed with r-hir(53 - 65) (Fig. 3, lanes 7 and 8). In contrast, r-hir(1- 52) did not protect a-thrombin from degradation by trypsin (Fig. 3, lanes 5 and 6). A small amount of protection by r-hir(1- 52) was observed when shorter time periods were used (data not shown). At the concentrations used in the above experiment, r-hir, r-hir(1- 52) and r-hir(53 - 65) did not inhibit the amidolytic activity of trypsin.

Pancreatic elastase cleaves the B-chain of a-thrombin after Ala150 to form &-thrombin [30]. Fig. 4 (lanes 1 - 3) shows the effect of incubation of a-thrombin with 2% (by mass) porcine pancreatic elastase for 30 min and 60 min at 37 "C; a-thrombin was completely converted to 8-thrombin by 60 min. r-Hir and r-hir(1- 52) were able to protect a-thrombin from cleavage by elastase (Fig. 4, lanes 4 and 5) . r-Hir(53 - 65) afforded a lesser degree of protection (Fig. 4, lane 6). The amidolytic activity of pancreatic elastase was not inhibited by r-hir, r-hir(1- 52) and r-hir(53-65) at concentrations used in the above experiment.

DISCUSSION

The first step in the formation of the thrombin-hirudin complex is an ionic-strength-dependent step that does not involve the active site of thrombin [4]. In a subsequent, faster step, hirudin combines with the active site. The ionic-strength- dependent interactions between hirudin and thrombin have been shown to occur predominantly with the C-terminal re- gion of hirudin [lo, 141. Data from the present study indicate that the binding of the C-terminal region causes a confor- mational change that affects the active site of thrombin and slightly facilitates the binding of the rest of the hirudin mol-

65

ecule. Chromatographic and spectroscopic studies by Mao et al. [17] also indicate that the binding of this region of the hirudin molecule causes a conformational change in thrombin. In the presence of r-hir(53 - 65), the Michaelis constant for the substrate DPhe-Pip-Arg-NH-Ph was 60% lower (Table 2). This change in the active site region also favoured the combi- nation of r-hir(1-52) with thrombin; its K, value was also 60% lower in the presence of the peptide which corresponds to an increase of 2.4 kJ . mol-' in the binding energy of r-hir(1- 52) (Table 1). This effect was not observed with all ligands that interact with the active site of thrombin. The rate of inactivation of thrombin by antithrombin I11 was not altered by r-hir(53 - 65). Given that the increase in affinity for r-hir(1- 52) caused by r-hir(53 - 65) is small, the question arises as to whether the cooperativity in the binding of the C-terminal and N-terminal regions would be greater if the two regions were covalently linked. The data presented in Table 1 exclude such a possibility. The sum of the binding energies for r-hir(1- 52) and r-hir(53-65) is slightly greater than the binding energy observed for [Asn52-+Met]hirudin. Thus, a greater cooperativity in the binding of the two regions in the intact molecule cannot be expected.

Cleavage of a-thrombin by trypsin at Arg73 produces /&-thrombin; further cleavage at Arg62 and Lys154 yields ?,-thrombin [29]. The region containing Arg62 and Arg73 corresponds to a surface loop in a-thrombin [31] and has been termed the p-loop of thrombin [32]. Results presented in Fig. 3 suggest that hirudin binds to this loop via its C-terminal re- gion. r-hir and r-hir(53-65) were able to protect a-thrombin against cleavage by trypsin. In contrast, r-hir(1- 52) provided no such protection (Fig. 3). The proposal that hirudin binds to the j-loop of thrombin is supported by the results of a number of other experiments. Alteration of the structure of this loop in &thrombin results in a decrease in the affinity for hirudin [12]. Peptide-specific antibodies, that bind to the sequence between the trypsin cleavage sites at Arg62 and Arg73, compete with hirudin for a binding site [15]. Hirudin was also able to protect lysyl residues in this area of a-throm- bin from chemical modification [16]. The p-loop of thrombin is rich in basic residues and would make an ideal counterpart to the negatively charged C-terminal region of hirudin in an ionic interaction. Thus, data obtained using a wide variety of different techniques suggest that hirudin binds to the p-loop region of a-thrombin. However, it is difficult to exclude an allosteric mechanism for the effects of r-hir and r-hir(53 - 65). It is possible that these molecules could bind at another site on u-thrombin and cause a conformational change around Arg73 that affects its susceptibility to trypsinolysis. Confor- mational changes in a-thrombin caused by the binding of hirudin [33] and a C-terminal peptide [17] have been observed. The unequivocal identification of the areas of interaction be- tween hirudin and thrombin must await the solution of the crystal structure of the enzyme-inhibitor complex. The recent determination of the structure of human a-thrombin should facilitate the achievement of this goal [31].

Pancreatic elastase cleaves u-thrombin at Ala150 to pro- duce &-thrombin [30]. r-hir and r-hir(1- 52) were able to com- pletely protect a-thrombin from elastase cleavage while r-hir(53 - 65) afforded a lesser degree of protection (Fig. 4). The results imply that r-hir binds to an area of thrombin in the vicinity of Alal5O via a region on the N-terminal side of residue 53. As discussed above, however, it is not possible to exclude an allosteric mechanism for the effects of r-hir and r-hir(1- 52). The partial protection obtained with r-hir(53 - 65) suggests that either a portion of this molecule is bound in

the vicinity of Ala150 or that the conformational change caused by r-hir(53 - 65) [17] has reduced the susceptibility of this region to cleavage. The proposal that hirudin binds to thrombin in the region of Alal50 is consistent with the results obtained by Chang [16], which indicate that r-hir protects Lysl54 from chemical modification. Kinetic studies indicate, however, that &-thrombin has only a slightly lower affinity for hirudin compared with a-thrombin; a decrease in binding energy of 2.4 kJ . mol-' was observed with &-thrombin [12]. Apparently, the change in structure caused by the elastase cleavage has only a minor effect on the binding of hirudin.

Thrombomodulin is a receptor for thrombin found on the surface of vascular endothelial cells [34]. It is also thought to be bound to the p-loop of thrombin [15,26] and a number of similarities exist with respect to the effects of thrombomodulin and r-hir(53 - 65) on the catalytic properties of thrombin. Both thrombomodulin and the C-terminal fragment of hirudin block the cleavage of fibrinogen [17, 18, 27, 35-37] and slightly decrease the K, value for tripeptidylnitroanilide substrates [18] (Table 2). However, the effect of the two mol- ecules on the inactivation of thrombin by antithrombin 111 is different. Thrombomodulin stimulates the rate of inactivation [18, 38, 391 whereas r-hir(53-65) is without effect.

The authors thank C. Servis, D. A. Rennex and G. Rovelli for reading the manuscript.

REFERENCES 1. Bagdy, D., Barabas, E., Graf, L., Peterson, T. E. & Magnusson,

S. (1976) Methods Enzymol. 45, 669-678. 2. Dodt, J., Machleidt, W., Seemiiller, U., Maschler, R. & Fritz, H.

(1986) Biol. Chem. Hoppe-Seyler 367, 803 - 81 1. 3. Harvey, R. P., Degryse, E., Stefani, L., Schamber, F., Cazenave,

J.-P., Courtney, M., Tolstoshev, P. & Lecocq, J.-P. (1986) Proc. Natl Acad. Sci. USA 83, 1084- 1088.

4. Stone, S. R. & Hofsteenge, J. (1986) Biochemistry 25,4622 -4628. 5. Dodt, J., Kohler, S. & Baici, A. (1988) FEBS Lett. 229, 87-90. 6. Bergmann, C., Dodt, J., Kohler, S., Fink, E. & Gassen, H. G.

(1986) Biol. Chem. Hoppe-Seyler 367,731 -740. 7. Fortkamp, E., Rieger, M., Heisterberg-Moutses, G., Schweitzer,

S. & Sommer, R. (1986) DNA ( N Y ) 5, 511 -517. 8. Loison, G., Findeli, A., Bernard, S., Nguyen-Juilleret, M.,

Marquet, M., Riehl-Bellon, N., Carvallo, D., Guerry-Santos, L., Brown, S. W., Courtney, M., Roitsch, C. & Lemoine, Y. (1988) Bio-technology 6, 72-77.

9. Meyhack, B., Heim, J., Rink, H., Zimmermann, W. & Marki, W. (1987) Thromb. Res. 7, 33.

10. Braun, P. J., Dennis, S., Hofsteenge, J. & Stone, S. R. (1988) Biochemistry 27, 6517 -6522.

11. Chang, J.-Y. (1983) FEBSLett. 164, 307-3313, 12. Stone, S. R., Braun, P. J. & Hofsteenge, J. (1987) Biochemistry

13. Wallace, A., Dennis, S., Hofsteenge, J. & Stone, S. R., Biochemis-

14. Stone, S. R., Dennis, S. & Hofsteenge, J. (1989) Biochemistry 28,

15. NoC, G., Hofsteenge, J., Rovelli, G. & Stone, S. R. (1988) J. Biol.

16. Chang, J.-Y. (1989) J . Biol. Chem. 264,7141 -7146. 17. Mao, S. J. T., Yates, M. T., Owen, T. J. & Krstenansky, J. L.

18. Hofsteenge, J., Taguchi, H. &Stone, S. R. (1986) Biochem. J . 237,

19. Duggleby, R. G. & Morrison, J. F. (1977) Biochim. Biophys. Acta

20. Duggleby, R. G. & Morrison, J. F. (1978) Biochim. Biophys. Acta

21. Gross, E. (1967) Methods Enzymol. I I , 238-255.

26,4611-4624.

try, in the press.

6857-6863.

Chem. 263, 11729-11735.

(1988) Biochemistry 27,8170-8173.

243 -251.

481,297-312.

526,398 -409.

66

22. Knecht, R. & Chang, J.-Y. (1987) Anal. Chem. 58, 2375-

23. Laemmli, U. K . (1970) Nature 227,680-685. 24. Cornish-Bowden, A. & Endrenyi, L. (1981) Biochem. J . 193,

25. Fromm, H. J. (1975) Enzyme kinetics, Springer-Verlag, Berlin. 26. Hofsteenge, J., Braun, P. J. & Stone, S. R. (1988) Biochemistry

27. Krstenansky, J. L., Owen, T. J., Yates, M. T. & Mao, S. J. T.

28. Olson, S. T. & Shore, J. D. (1982) J . Biol. Chem. 257, 14891-

29. Braun, P. J., Hofsteenge, J., Chang, J.-Y. & Stone, S. R. (1988)

30. Kawabata, S., Morita, T., Iwanaga, S. & Igarashi, H. (1985) J .

31. Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R. &

2379.

1005 - 1008.

27,2144-2151.

(1987) J . Med. Chem. 30,1688-1691.

14895.

Thromb. Res. 50,273 - 283.

Biochem. (Tokyo) 97, 325-331.

Hofsteenge, J., EMBO J . 8, 3467-3475.

32. Fenton, J . W., 11, & Bing, D. H. (1986) Semin. Thromb. Hemo-

33. Konno, S., Fenton, J. W., 11, & Villanueva, G. B. (1988) Arch.

34. Esmon, C. T. (1989) J . Biol. Chem. 264,4743 -4746. 35. Esmon, C. T., Esmon, N . L. &Harris, K. W. (1982) J . Biol. Chem.

36. Jakubowski, H. V., Kline, M. D. & Owen, W. G. (1986) J . Biol.

37. Krstenansky, J. L. & Mao, S. J. T. (1987) FEBS Lett. 211, 10-

38. Bourin, M.-C., Boffa, M.-C., Bjork, I. & Lindahl, U. (1986) Proc.

39. Preissner, K. T., Delvos, U. & Muller-Berghaus, G. (1987) Bio-

stusis 12, 200 - 208.

Biochem. Biophys. 267,158 - 166.

257,7944-7947.

Chern. 261, 3876-3882.

16.

Natl Acad. Sci. USA 83, 5924-5928.

chemistry 26, 2521 -2528.