SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992
Molecular Interactions Between Heparin Cofactor II and Thrombin
Heparin cofactor II (HC II) is a 480-amino acid glycoprotein in plasma that inhibits thrombin and chymotrypsin and is a member of the serpin family of serine protease inhibitors.1,2 HC II serves as a pseudosubstrate for its target proteases. Both thrombin and chymotrypsin attack the reactive site Leu444-Ser445 peptide bond (desig nated Pl-Pl') near the COOH-terminus of HC II, thereby forming a stable 1:1 complex in which the protease is inactive.3 Under denaturing conditions, the complex re mains covalently linked by an acyl ester bond between the active site serine of the protease and the carbonyl group at the P1 position in HC II. Heparin and dermatan sulfate increase the rate of thrombin inhibition at least 1000-fold,4 but the rate of chymotrypsin inhibition is unaffected by glycosaminoglycans.3 This review will discuss regions of HC II, including the reactive site and the NH2-terminal acidic domain, that are involved in the interaction with proteases, as well as a model for the stimulation observed with glycosaminoglycans.
REACTIVE SITE The P1 residue in the reactive site of a serpin plays a major role in determining the relative rates of inhibition of various proteases, as expected if the reactive site re sembles a substrate for the protease.5 For example, the presence of Met358 at the P1 position of α1-antitrypsin is consistent with the substrate specificity of its major tar get protease, neutrophil elastase, which preferentially
From the Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri. Reprint requests: Dr. Tollefsen, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
cleaves the peptide bond following a medium-sized hy drophobic amino acid (Fig. 1). The natural mutant α1antitrypsin Pittsburgh (Met358→Arg) is a poor inhibitor of elastase but a potent inhibitor of thrombin and certain other proteases that preferentially attack Arg-X peptide bonds.6 The presence of leucine at the P1 position in HC II explains the fact that HC II inhibits chymotrypsin more rapidly than it inhibits thrombin in the absence of a glycosaminoglycan.3 In addition, HC II fails to inhibit a variety of other proteases that preferentially cleave sub strates following a basic amino acid residue.7 We con structed a variant of recombinant HC II (rHC II) contain ing arginine in place of Leu444 at the P1 position.8 This mutation increased the basal rate of inhibition of throm bin in the absence of glycosaminoglycan ~100-fold and caused a reciprocal decrease in the ability of rHC II to inhibit chymotrypsin. rHC II (Leu444→Arg) also inhib ited Factor Xa, plasmin, and kallikrein, proteases that are not inhibited by native rHC II or plasma HC II (Fig. 2). These results emphasize the importance of the P1 residue in determining the rate of protease inhibition by HC II and suggest that HC II has evolved to be essentially inactive toward thrombin in the absence of a glycosami noglycan. The difference between the rates of inhibition of chymotrypsin and thrombin by HC II (three- to fourfold in the absence of a glycosaminoglycan) is perhaps smaller than one would expect if the P1 residue were the sole determinant of the rate. Furthermore, the primary structure of the reactive site does not explain the rate enhancement of thrombin inhibition produced by gly cosaminoglycans. These considerations suggest the pos sibility that inhibition of thrombin may be facilitated by interactions with portions of HC II apart from the reactive site. One potential site for thrombin binding is the acidic domain near the NH2-terminus of HC II (residues 54-75)
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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which contains two tyrosine sulfation sites and is similar in structure to the COOH-terminal portion of hirudin.9,10
NH2-TERMINAL ACIDIC DOMAIN The COOH-terminal portion of hirudin is rich in acidic amino acids and interacts with an area on the surface of thrombin termed "anion-binding exosite I" 11 , while the NH2-terminal domain of hirudin occupies the catalytic site. 12,13 The acidic domain of HC II contains a tandem repeat of two nearly identical sequences (E56DDDYLD and E69DDDYID) that can be aligned with E61EYLQ-COOH in hirudin, as shown in Fig. 3. Complexes of hirudin and human α-thrombin have been crystallized, and the contacts between the two proteins have been determined by x-ray diffraction analysis. Un expectedly, hydrophobic contacts appear to play a major role in stabilizing the hirudin-thrombin complex. 12,13 In
the region of greatest similarity to HC II, Tyr63 and Leu64 of hirudin form hydrophobic contacts and the α-carboxyl group of Gln65 forms an ionic bond with thrombin. The corresponding residues in one or both of the acidic re peats of HC II could interact with thrombin in a similar manner. Additional contacts occur between Phe56, Glu57, He59, and Pro60 of hirudin and anion-binding ex osite I of thrombin. However, it is difficult to predict whether analogous interactions could occur with HC II, since alignment of the two polypeptides in this region is imperfect. Hortin et al10 found that a synthetic peptide contain ing both acidic repeats of HC II (residues 54-75) com peted with the 13-residue COOH-terminal peptide of hi rudin PA (residues 54-66) for binding to thrombin, but does not affect the ability of thrombin to hydrolyze a tripeptide p-nitroanilide substrate. The affinity of HC II(54-75) for thrombin was lower than that of hirudin PA(54-66), implying that fewer intermolecular contacts
FIG. 2. Inhibition of proteases by native recombinant heparin cofactor II (rHC II) and rHC ll(Leu444→Arg). rHC II was produced in Escherichia coli and partially purified from the bacterial cell lysate. Native rHC II (142 nM), rHCll(Leu444→Arg) (76 nM), or control lysate was incubated with thrombin (15 nM), chymotrypsin (21 nM), Factor Xa (16 nM), plasmin (24 nM), or kallikrein (10 nM). After various times of incubation, the remaining protease activity was determined by adding the appropriate chromogenic substrate and measuring A405/min. (Reprinted with permission from Derechin et al.8)
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FIG. 1. Reactive site structures of selected serpins.
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FIG. 3. Alignment of the NH2-terminal acidic domain of heparin cofactor II (HC II) with hirudin. Partial amino acid sequences are shown for the COOH-terminal domains of hirudin PA and hirudin29 and for the NH2-terminal acidic domain of plasma HC II.2 Amino acid residues in hirudin that have been shown by x-ray diffraction analysis12-13 to form contacts with anion-binding exosite I of thrombin are indicated by cross-hatched symbols (hydrophobic contacts) and shaded symbols (ionic contacts). The corre sponding residues in hirudin PA and HC II are also indicated. (Reprinted with permission from Van Deerlin and Tollefsen.15)
were present. Interestingly, both HC II(54-75) and hiru din PA(54-66) slightly stimulated thrombin's amidolytic activity, apparently by inducing a conformational change in the active site cleft that lowers the Km for the sub strate. 1 0 , l 4 Thus, interaction of anion-binding exosite I of thrombin with the acidic domain of HC II could facilitate formation of the covalent thrombin-HC II complex by two mechanisms: (1) by bringing the active site of throm bin into approximation with the reactive site of HC II; and (2) by lowering the dissociation constant of the en zyme-substrate (Michaelis) complex. We have recently conducted mutagenesis experi ments with rHC II in order to examine the importance of the NH2-terminal acidic domain for the inhibition of thrombin and chymotrypsin in the presence and absence of glycosaminoglycans.15 We constructed a series of 5' deletions in the HC II cDNA and expressed the mutant proteins in Escherichia coli. Deletion of the first acidic repeat (residues 1-67) or both acidic repeats (residues 1-74) did not affect the second-order rate constant for inhibition of α-thrombin or chymotrypsin in the absence of a glycosaminoglycan (Fig. 4). In contrast, deletion of the first acidic repeat greatly diminished the ability of dermatan sulfate or heparin to stimulate the inhibition of
α-thrombin, and deletion of both acidic repeats produced a slightly greater effect. These results suggest that the NH2-terminal acidic domain of HC II, particularly the first acidic repeat, mediates the interaction between HC II and thrombin in the presence of glycosaminoglycans. Ragg et al 16,17 reached a similar conclusion based on site-directed mutagenesis of rHC II expressed in COS cells. The fact that the NH2-terminal acidic domain of HC II is not required for inhibition of thrombin in the absence of a glycosaminoglycan may be explained by the model shown in Figure 5. According to this model, the acidic domain binds intramolecularly to the glycosaminoglycan-binding site, which includes several basic amino acid residues between positions 173 and 193 of the polypep tide. 16-19 In this conformation, the acidic domain is un able to interact with anion-binding exosite I of thrombin, and the rate of thrombin inhibition depends on the fre quency of productive contacts between the catalytic site of thrombin and the reactive site peptide bond (Leu444Ser445) of HC II (Fig. 5A). In the presence of dermatan sulfate or heparin, the NH2-terminal acidic domain of HC II is displaced from the glycosaminoglycan-binding site and is free to interact with thrombin. Binding of thrombin
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HEPARIN COFACTOR II AND THROMBIN—VAN DEERLIN, TOLLEFSEN
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992
FIG. 4. Second-order rate constants for protease inhibition by native recombinant heparin cofactor II (rHC II) and Non terminal deletion mutants. Partially purified lysates of Es cherichia coli containing rHC II (45-222 nM) were incubated with α-thrombin (15 nM) or chymotrypsin (21 nM). Secondorder rate constants (k2) were calculated from the time course of protease inhibition and the initial concentration of rHC II present in each incubation. Inhibition of α-thrombin was de termined in the absence (no GAG) or presence of 25 µg/ml of heparin (+Hep) or dermatan sulfate (+DS). The height of each column represents the mean k2 value. The range of k2 values determined in two to eight separate experiments is indicated by the l-bar. No l-bar indicates a single determina tion. (Reprinted with permission from Van Deerlin and Tollefsen.15)
to the acidic domain promotes contact with the reactive site of HC II, thereby increasing the rate of inhibition (Fig. 5B). The model predicts that deletion of the acidic do main, by uncovering the glycosaminoglycan binding site, should increase the apparent affinity of rHC II for heparin. This is indeed what has been observed experi mentally. Deletion of the first acidic repeat increased the sodium chloride concentration required to elute rHC II from heparin-Sepharose. The required sodium chloride concentration was increased further by deletion of Glu69Asp72 in the second acidic repeat. Therefore, heparin and the acidic domain appear to compete for binding to the glycosaminoglycan-binding site in native rHC II. Additional support for the model in Figure 5 was obtained from experiments with the peptide hirudin(5465)-SO4, which binds tightly to anion-binding exosite I of thrombin. Hirudin(54-65)-SO4 had no effect on the rate of inhibition of α-thrombin by native rHC II in the absence of glycosaminoglycan but greatly decreased the rate of inhibition of α-thrombin in the presence of dermatan sulfate or heparin.15 These results suggest that inter-
FIG. 5. Model for inhibition of thrombin by heparin cofactor II (HC II). A: The active site serine (S) hydroxyl group of thrombin attacks the reactive site leucyl-serine (LS) peptide bond of HC II to form a covalent complex. In the absence of a glycosaminoglycan, the NH2-terminal acidic domain of HC II (-) forms ionic bonds with the glycosaminoglycan-binding site (+) and is unable to interact with thrombin. B: A gly cosaminoglycan chain displaces the NH2-terminal acidic do main of HC II from the glycosaminoglycan-binding site. The acidic domain then interacts with anion-binding exosite I of thrombin. Binding of thrombin both to the NH2-terminal acidic domain of HC II and to the glycosaminoglycan tem plate greatly increases the rate of covalent complex forma tion. C: A deletion mutant of HC II lacking the acidic domain is shown. Complex formation with thrombin can be stimulated only by the glycosaminoglycan template mechanism. D: The dermatan sulfate hexasaccharide displaces the NH2-terminal acidic domain of HC II but is of insufficient length to bind thrombin simultaneously. Complex formation can be stimu lated only by the displacement mechanism. Approximate second-order rate constants are indicated for the thrombin-HC II reaction in the presence of intact dermatan sulfate and the high-affinity hexasaccharide. (Reprinted with permis sion from Van Deerlin and Tollefsen.15)
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inhibition by native rHC II 67-fold.15 Since the hexasac charide is presumed to be of insufficient length to bind HC II and thrombin simultaneously, the stimulatory ef fect may be due primarily to displacement of the NH2terminal acidic domain. Two additional findings support this conclusion: (1) the hexasaccharide had no effect on the rate of inhibition of α-thrombin by rHC II(Δ1-74), which lacks the acidic domain; and (2) hirudin(54-65)SO4 blocked the ability of the hexasaccharide to increase the rate of inhibition of α-thrombin by native rHC II.
CONCLUSIONS The proposed mechanism for stimulation of HC II by glycosaminoglycans probably does not apply to AT III. Although HC II and AT III are homologous,2 the NH2-terminal region of AT III does not contain a hirudinlike domain. Furthermore, the heparin pentasaccharide, which comprises the minimum high-affinity binding site for AT III, does not accelerate thrombin inhibition by AT III, although it stimulates the inhibition of coagulation Factor Xa.25 Heparin chains containing ≥18 monosac charide units are required for thrombin inhibitory activ ity, perhaps because chains of this length are necessary to form a ternary complex with thrombin and AT III. 26 In contrast, low molecular weight heparin fragments (Mr < 5400) that do not stimulate thrombin inhibition by AT III have weak stimulatory activity with HC II. 26 The effect of low molecular weight heparin fragments on HC II is similar to that of the dermatan sulfate hexasaccharide and may result from displacement of the NH2-terminal acidic domain. The interaction described between the NH2-terminus of HC II and the anion-binding exosite I of thrombin is not unique. Recently, it has been shown that thrombin interacts with its platelet receptor in an analogous man ner. 27,28 The NH2-terminus of the thrombin receptor con tains a thrombin cleavage site and a region with homol ogy to the COOH-terminus of hirudin. Cleavage leads to activation of the receptor. There is evidence that interac tion of the hirudin-like domain of the receptor with the anion-binding exosite I of thrombin results in more effi cient cleavage and activation of the receptor. Clearly, the important role of the anion binding exosite I in determin ing thrombin substrate specificity is just beginning to be elucidated. We propose that stimulation of the thrombin-HC II reaction by a glycosaminoglycan can occur by the fol lowing mechanisms: (1) displacement of the NH2-terminal acidic domain from the glycosaminoglycan-binding site of HC II, allowing the acidic domain to interact with anion-binding exosite I of thrombin; and (2) simulta-
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actions between rHC II and anion-binding exosite I of thrombin take place only in the presence of a glycosaminoglycan. Experiments with 7-thrombin also suggest that an ion-binding exosite I plays an important role in the mech anism of inhibition of thrombin by HC II. γ-Thrombin is a proteolytically cleaved form of α-thrombin, which re tains the ability to hydrolyze synthetic peptide substrates, does not clot fibrinogen, and has a greatly reduced affin ity for the COOH-terminal region of hirudin.20'21 The heparin-binding site of 7-thrombin appears to be intact, since α-thrombin and 7-thrombin both elute from heparin-Sepharose at ~0.4 M sodium chloride, and heparin stimulates inhibition of both α-thrombin and γ-thrombin by antithrombin III (AT III) ~5000-fold. By contrast, dermatan sulfate and heparin stimulated inhibition of 7-thrombin by native rHC II only 14- to 33-fold.15 A similar degree of stimulation by glycosaminoglycans was observed in experiments with α-thrombin and a deleted form of rHC II lacking residues 1-74 (rHC II(Δ1-74)). Furthermore, Sheehan et al22 demonstrated that mutation of residues in the anion-binding exosite I of thrombin, especially Arg68, decreased the rate of inhibition by HC II, but not AT III, in the presence of heparin. Thus, deletion of the NH2-terminal acidic domain of rHC II or alterations of anion-binding exosite I of thrombin greatly reduce the maximum rate of reaction between the two proteins. The model shown in Figure 5 includes the possibil ity that a glycosaminoglycan chain can also function as a template to which both HC II and thrombin bind. The glycosaminoglycan-binding site on the surface of throm bin (i.e., anion-binding exosite II) has not been defined precisely, but it appears to be distinct from anion-binding exosite I 23 . Therefore, binding of thrombin to a gly cosaminoglycan chain and to the NH2-terminal acidic domain of HC II should not be mutually exclusive. The template mechanism may explain the modest increase in the rate of inhibition of α-thrombin by rHC II(Δ1-74) or 7-thrombin by native rHC II in the presence of dermatan sulfate or heparin. The difference in the abilities of the two glycosaminoglycans to stimulate thrombin inhibition suggests that heparin may form a better template than dermatan sulfate. Maimone and Tollefsen24 isolated the following hexasaccharide, which represents the smallest fragment of porcine skin dermatan sulfate that binds to HC II with high affinity: (IdoA[2-SO4]-GalNAc[4-SO4])2-IdoA[2SO4]-ATalR[4-SO4]. To test the relative importance of the template mechanism already described, we compared the ability of the high-affinity hexasaccharide and intact dermatan sulfate to stimulate thrombin inhibition by na tive rHC II and rHC II(Δ1-74). We found that the highaffinity hexasaccharide increased the rate of α-thrombin
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neous binding of HC II and thrombin to a single glycosaminoglycan chain, which functions as a catalytic template. As suggested by experiments with the highaffinity hexasaccharide, the displacement mechanism may increase the rate of formation of the covalent thrombin-HC II complex independently, although the degree of stimulation is less than that obtained with intact dermatan sulfate. In addition, rHC II molecules lacking the NH2terminal acidic domain can be stimulated weakly by fulllength glycosaminoglycan chains. Together, the dis placement and template mechanisms produce maximal stimulation (3600- to 7000-fold) of the thrombin-HC II reaction.
REFERENCES 1. Tollefsen DM, DW Majerus, MK Blank: Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 257:2162-2169, 1982. 2. Blinder MA, JC Marasa, CH Reynolds, LL Deaven, DM Tollef sen: Heparin cofactor II: cDNA sequence, chromosome localiza tion, restriction fragment length polymorphism, and expression in Escherichia coli. Biochemistry 27:752-759, 1988. 3. Church FC, CM Noyes, MJ Griffith: Inhibition of chymotrypsin by heparin cofactor II. Proc Natl Acad Sci USA 82:6431-6434, 1985. 4. Tollefsen DM, CA Pestka, WJ Monafo: Activation of heparin cofactor II by dermatan sulfate. J Biol Chem 258:6713-6716, 1983. 5. Carrell RW, J Travis: a,-Antitrypsin and the serpins: Variation and countervailation. Trends Biochem Sci 10:20-24, 1985. 6. Owen MC, SO Brennan, JH Lewis, RW Carrell: Mutation of antitrypsin to antithrombin. α1-Antitrypsin Pittsburgh (358 Met→Arg), a fatal bleeding disorder. N Engl J Med 309:694-698, 1983. 7. Parker KA, DM Tollefsen: The protease specificity of heparin cofactor II. Inhibition of thrombin generated during coagulation. J Biol Chem 260:3501-3505, 1985. 8. Derechin VM, MA Blinder, DM Tollefsen: Substitution of arginine for Leu444 in the reactive site of heparin cofactor II enhances the rate of thrombin inhibition. J Biol Chem 265:5623-5628, 1990. 9. Hortin G, DM Tollefsen, AW Strauss: Identification of two sites of sulfation of human heparin cofactor II. J Biol Chem 261:1582715830, 1986. 10. Hortin GL, DM Tollefsen, BM Benutto: Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II. J Biol Chem 264:13979-13982, 1989. 11. Fenton JW II, FA Ofosu, DG Moon, JM Maraganore: Thrombin structure and function: why thrombin is the primary target for antithrombotics. Blood Coagul Fibrinolysis 2:69-75, 1991. 12. Grutter MG, JP Priestle, J Rahuel, H Grossenbacher, W Bode, J Hofsteenge, SR Stone: Crystal structure of the thrombin-hirudin
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complex: a novel mode of serine protease inhibition. EMBO J 9:2361-2365, 1990. Rydel TJ, KG Ravichandran, A Tulindky, W Bode, R Huber, C Roitsch, JW Fenton II: The structure of a complex of recombinant hirudin and human α-thrombin. Science 249:277-280, 1990. Hortin GL, BL Trimpe: Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibi tors and substrates. J Biol Chem 266:6866-6871, 1991. Van Deerlin VMD, DM Tollefsen: The N-terminal acidic domain of heparin cofactor II mediates the inhibition of α-thrombin in the presence of glycosaminoglycans. J Biol Chem 266:20223-20231, 1991. Ragg H, T Ulshöfer, J Gerewitz: On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans. J Biol Chem 265:5211-5218, 1990. Ragg H, T Ulshöfer, J Gerewitz: Glycosaminoglycan-mediated leuserpin-2/thrombin interaction: Structure-function relationships. J Biol Chem 265:22386-22391, 1990. Blinder MA, TR Andersson, U Abildgaard, DM Tollefsen: Hep arin cofactor II Oslo. Mutation of Arg-189 to His decreases the affinity for dermatan sulfate. J Biol Chem 264:5128-5133, 1989. Blinder MA, DM Tollefsen: Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II. J Biol Chem 265:286-291, 1990. Berliner LJ: Structure-function relationships in human a- and γ-thrombins. Mol Cell Biochem 61:159-172, 1984. Stone SR, J Hofsteenge: Basis for the reduced affinity of (ßT- and γT-thrombin for hirudin. Biochemistry 30:3950-3955, 1991. Sheehan JP, QY Wu, JE Sadler: Mutagenesis of thrombin selec tively modulates inhibition of serpins heparin cofactor II (HOI) and antithrombin III (ATIII). (Abst.) Blood 78 (Suppl 1):277, 1991. Church FC, CW Pratt, CM Noyes, T Kalayanamit, GB Sherrill, RB Tobin, JB Meade: Structural and functional properties of hu man α-thrombin, phosphopyridoxylated α-thrombin, and γTthrombin. J Biol Chem 264:18419-18425, 1989. Maimone MM, DM Tollefsen: Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J Biol Chem 265:18263-18271, 1990. Choay J, M Petitou, JC Lormeau, P Sinay, B Casu, G Gatti: Structure-activity relationship in heparin: a synthetic pentasaccha ride with high affinity for antithrombin III and eliciting high antifactor Xa activity. Biochem Biophys Res Commun 116:492-499, 1983. Bray B, DA Lane, J Freyssinet, G Pejler, U Lindahl: Anti-thrombin activities of heparin: effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin. Biochem J 262:225-232, 1989. Vu T-KH, DT Hung, VI Wheaton, SR Coughlin: Molecular clon ing of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068, 1991. Vu T-KH, VI Wheaton, DT Hung, I Charo, SR Coughlin: Do mains specifying thrombin-receptor interaction. Nature 353:674677, 1991. Seemüller U, J Dodt, E Fink, H Fritz: Proteinase inhibitors of the leech Hirudo medicinalis (hirudins, bdellins, eglins). In: Barrett AJ, G Salveson (Eds): Proteinase Inhibitors. Elsevier, Amster dam, 1986, pp 337-359.
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Molecular Interactions Between Heparin Cofactor II and Thrombin
Heparin cofactor II (HC II) is a 480-amino acid glycoprotein in plasma that inhibits thrombin and chymotrypsin and is a member of the serpin family of serine protease inhibitors.1,2 HC II serves as a pseudosubstrate for its target proteases. Both thrombin and chymotrypsin attack the reactive site Leu444-Ser445 peptide bond (desig nated Pl-Pl') near the COOH-terminus of HC II, thereby forming a stable 1:1 complex in which the protease is inactive.3 Under denaturing conditions, the complex re mains covalently linked by an acyl ester bond between the active site serine of the protease and the carbonyl group at the P1 position in HC II. Heparin and dermatan sulfate increase the rate of thrombin inhibition at least 1000-fold,4 but the rate of chymotrypsin inhibition is unaffected by glycosaminoglycans.3 This review will discuss regions of HC II, including the reactive site and the NH2-terminal acidic domain, that are involved in the interaction with proteases, as well as a model for the stimulation observed with glycosaminoglycans.
REACTIVE SITE The P1 residue in the reactive site of a serpin plays a major role in determining the relative rates of inhibition of various proteases, as expected if the reactive site re sembles a substrate for the protease.5 For example, the presence of Met358 at the P1 position of α1-antitrypsin is consistent with the substrate specificity of its major tar get protease, neutrophil elastase, which preferentially
From the Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri. Reprint requests: Dr. Tollefsen, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
cleaves the peptide bond following a medium-sized hy drophobic amino acid (Fig. 1). The natural mutant α1antitrypsin Pittsburgh (Met358→Arg) is a poor inhibitor of elastase but a potent inhibitor of thrombin and certain other proteases that preferentially attack Arg-X peptide bonds.6 The presence of leucine at the P1 position in HC II explains the fact that HC II inhibits chymotrypsin more rapidly than it inhibits thrombin in the absence of a glycosaminoglycan.3 In addition, HC II fails to inhibit a variety of other proteases that preferentially cleave sub strates following a basic amino acid residue.7 We con structed a variant of recombinant HC II (rHC II) contain ing arginine in place of Leu444 at the P1 position.8 This mutation increased the basal rate of inhibition of throm bin in the absence of glycosaminoglycan ~100-fold and caused a reciprocal decrease in the ability of rHC II to inhibit chymotrypsin. rHC II (Leu444→Arg) also inhib ited Factor Xa, plasmin, and kallikrein, proteases that are not inhibited by native rHC II or plasma HC II (Fig. 2). These results emphasize the importance of the P1 residue in determining the rate of protease inhibition by HC II and suggest that HC II has evolved to be essentially inactive toward thrombin in the absence of a glycosami noglycan. The difference between the rates of inhibition of chymotrypsin and thrombin by HC II (three- to fourfold in the absence of a glycosaminoglycan) is perhaps smaller than one would expect if the P1 residue were the sole determinant of the rate. Furthermore, the primary structure of the reactive site does not explain the rate enhancement of thrombin inhibition produced by gly cosaminoglycans. These considerations suggest the pos sibility that inhibition of thrombin may be facilitated by interactions with portions of HC II apart from the reactive site. One potential site for thrombin binding is the acidic domain near the NH2-terminus of HC II (residues 54-75)
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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which contains two tyrosine sulfation sites and is similar in structure to the COOH-terminal portion of hirudin.9,10
NH2-TERMINAL ACIDIC DOMAIN The COOH-terminal portion of hirudin is rich in acidic amino acids and interacts with an area on the surface of thrombin termed "anion-binding exosite I" 11 , while the NH2-terminal domain of hirudin occupies the catalytic site. 12,13 The acidic domain of HC II contains a tandem repeat of two nearly identical sequences (E56DDDYLD and E69DDDYID) that can be aligned with E61EYLQ-COOH in hirudin, as shown in Fig. 3. Complexes of hirudin and human α-thrombin have been crystallized, and the contacts between the two proteins have been determined by x-ray diffraction analysis. Un expectedly, hydrophobic contacts appear to play a major role in stabilizing the hirudin-thrombin complex. 12,13 In
the region of greatest similarity to HC II, Tyr63 and Leu64 of hirudin form hydrophobic contacts and the α-carboxyl group of Gln65 forms an ionic bond with thrombin. The corresponding residues in one or both of the acidic re peats of HC II could interact with thrombin in a similar manner. Additional contacts occur between Phe56, Glu57, He59, and Pro60 of hirudin and anion-binding ex osite I of thrombin. However, it is difficult to predict whether analogous interactions could occur with HC II, since alignment of the two polypeptides in this region is imperfect. Hortin et al10 found that a synthetic peptide contain ing both acidic repeats of HC II (residues 54-75) com peted with the 13-residue COOH-terminal peptide of hi rudin PA (residues 54-66) for binding to thrombin, but does not affect the ability of thrombin to hydrolyze a tripeptide p-nitroanilide substrate. The affinity of HC II(54-75) for thrombin was lower than that of hirudin PA(54-66), implying that fewer intermolecular contacts
FIG. 2. Inhibition of proteases by native recombinant heparin cofactor II (rHC II) and rHC ll(Leu444→Arg). rHC II was produced in Escherichia coli and partially purified from the bacterial cell lysate. Native rHC II (142 nM), rHCll(Leu444→Arg) (76 nM), or control lysate was incubated with thrombin (15 nM), chymotrypsin (21 nM), Factor Xa (16 nM), plasmin (24 nM), or kallikrein (10 nM). After various times of incubation, the remaining protease activity was determined by adding the appropriate chromogenic substrate and measuring A405/min. (Reprinted with permission from Derechin et al.8)
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FIG. 1. Reactive site structures of selected serpins.
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FIG. 3. Alignment of the NH2-terminal acidic domain of heparin cofactor II (HC II) with hirudin. Partial amino acid sequences are shown for the COOH-terminal domains of hirudin PA and hirudin29 and for the NH2-terminal acidic domain of plasma HC II.2 Amino acid residues in hirudin that have been shown by x-ray diffraction analysis12-13 to form contacts with anion-binding exosite I of thrombin are indicated by cross-hatched symbols (hydrophobic contacts) and shaded symbols (ionic contacts). The corre sponding residues in hirudin PA and HC II are also indicated. (Reprinted with permission from Van Deerlin and Tollefsen.15)
were present. Interestingly, both HC II(54-75) and hiru din PA(54-66) slightly stimulated thrombin's amidolytic activity, apparently by inducing a conformational change in the active site cleft that lowers the Km for the sub strate. 1 0 , l 4 Thus, interaction of anion-binding exosite I of thrombin with the acidic domain of HC II could facilitate formation of the covalent thrombin-HC II complex by two mechanisms: (1) by bringing the active site of throm bin into approximation with the reactive site of HC II; and (2) by lowering the dissociation constant of the en zyme-substrate (Michaelis) complex. We have recently conducted mutagenesis experi ments with rHC II in order to examine the importance of the NH2-terminal acidic domain for the inhibition of thrombin and chymotrypsin in the presence and absence of glycosaminoglycans.15 We constructed a series of 5' deletions in the HC II cDNA and expressed the mutant proteins in Escherichia coli. Deletion of the first acidic repeat (residues 1-67) or both acidic repeats (residues 1-74) did not affect the second-order rate constant for inhibition of α-thrombin or chymotrypsin in the absence of a glycosaminoglycan (Fig. 4). In contrast, deletion of the first acidic repeat greatly diminished the ability of dermatan sulfate or heparin to stimulate the inhibition of
α-thrombin, and deletion of both acidic repeats produced a slightly greater effect. These results suggest that the NH2-terminal acidic domain of HC II, particularly the first acidic repeat, mediates the interaction between HC II and thrombin in the presence of glycosaminoglycans. Ragg et al 16,17 reached a similar conclusion based on site-directed mutagenesis of rHC II expressed in COS cells. The fact that the NH2-terminal acidic domain of HC II is not required for inhibition of thrombin in the absence of a glycosaminoglycan may be explained by the model shown in Figure 5. According to this model, the acidic domain binds intramolecularly to the glycosaminoglycan-binding site, which includes several basic amino acid residues between positions 173 and 193 of the polypep tide. 16-19 In this conformation, the acidic domain is un able to interact with anion-binding exosite I of thrombin, and the rate of thrombin inhibition depends on the fre quency of productive contacts between the catalytic site of thrombin and the reactive site peptide bond (Leu444Ser445) of HC II (Fig. 5A). In the presence of dermatan sulfate or heparin, the NH2-terminal acidic domain of HC II is displaced from the glycosaminoglycan-binding site and is free to interact with thrombin. Binding of thrombin
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992
FIG. 4. Second-order rate constants for protease inhibition by native recombinant heparin cofactor II (rHC II) and Non terminal deletion mutants. Partially purified lysates of Es cherichia coli containing rHC II (45-222 nM) were incubated with α-thrombin (15 nM) or chymotrypsin (21 nM). Secondorder rate constants (k2) were calculated from the time course of protease inhibition and the initial concentration of rHC II present in each incubation. Inhibition of α-thrombin was de termined in the absence (no GAG) or presence of 25 µg/ml of heparin (+Hep) or dermatan sulfate (+DS). The height of each column represents the mean k2 value. The range of k2 values determined in two to eight separate experiments is indicated by the l-bar. No l-bar indicates a single determina tion. (Reprinted with permission from Van Deerlin and Tollefsen.15)
to the acidic domain promotes contact with the reactive site of HC II, thereby increasing the rate of inhibition (Fig. 5B). The model predicts that deletion of the acidic do main, by uncovering the glycosaminoglycan binding site, should increase the apparent affinity of rHC II for heparin. This is indeed what has been observed experi mentally. Deletion of the first acidic repeat increased the sodium chloride concentration required to elute rHC II from heparin-Sepharose. The required sodium chloride concentration was increased further by deletion of Glu69Asp72 in the second acidic repeat. Therefore, heparin and the acidic domain appear to compete for binding to the glycosaminoglycan-binding site in native rHC II. Additional support for the model in Figure 5 was obtained from experiments with the peptide hirudin(5465)-SO4, which binds tightly to anion-binding exosite I of thrombin. Hirudin(54-65)-SO4 had no effect on the rate of inhibition of α-thrombin by native rHC II in the absence of glycosaminoglycan but greatly decreased the rate of inhibition of α-thrombin in the presence of dermatan sulfate or heparin.15 These results suggest that inter-
FIG. 5. Model for inhibition of thrombin by heparin cofactor II (HC II). A: The active site serine (S) hydroxyl group of thrombin attacks the reactive site leucyl-serine (LS) peptide bond of HC II to form a covalent complex. In the absence of a glycosaminoglycan, the NH2-terminal acidic domain of HC II (-) forms ionic bonds with the glycosaminoglycan-binding site (+) and is unable to interact with thrombin. B: A gly cosaminoglycan chain displaces the NH2-terminal acidic do main of HC II from the glycosaminoglycan-binding site. The acidic domain then interacts with anion-binding exosite I of thrombin. Binding of thrombin both to the NH2-terminal acidic domain of HC II and to the glycosaminoglycan tem plate greatly increases the rate of covalent complex forma tion. C: A deletion mutant of HC II lacking the acidic domain is shown. Complex formation with thrombin can be stimulated only by the glycosaminoglycan template mechanism. D: The dermatan sulfate hexasaccharide displaces the NH2-terminal acidic domain of HC II but is of insufficient length to bind thrombin simultaneously. Complex formation can be stimu lated only by the displacement mechanism. Approximate second-order rate constants are indicated for the thrombin-HC II reaction in the presence of intact dermatan sulfate and the high-affinity hexasaccharide. (Reprinted with permis sion from Van Deerlin and Tollefsen.15)
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inhibition by native rHC II 67-fold.15 Since the hexasac charide is presumed to be of insufficient length to bind HC II and thrombin simultaneously, the stimulatory ef fect may be due primarily to displacement of the NH2terminal acidic domain. Two additional findings support this conclusion: (1) the hexasaccharide had no effect on the rate of inhibition of α-thrombin by rHC II(Δ1-74), which lacks the acidic domain; and (2) hirudin(54-65)SO4 blocked the ability of the hexasaccharide to increase the rate of inhibition of α-thrombin by native rHC II.
CONCLUSIONS The proposed mechanism for stimulation of HC II by glycosaminoglycans probably does not apply to AT III. Although HC II and AT III are homologous,2 the NH2-terminal region of AT III does not contain a hirudinlike domain. Furthermore, the heparin pentasaccharide, which comprises the minimum high-affinity binding site for AT III, does not accelerate thrombin inhibition by AT III, although it stimulates the inhibition of coagulation Factor Xa.25 Heparin chains containing ≥18 monosac charide units are required for thrombin inhibitory activ ity, perhaps because chains of this length are necessary to form a ternary complex with thrombin and AT III. 26 In contrast, low molecular weight heparin fragments (Mr < 5400) that do not stimulate thrombin inhibition by AT III have weak stimulatory activity with HC II. 26 The effect of low molecular weight heparin fragments on HC II is similar to that of the dermatan sulfate hexasaccharide and may result from displacement of the NH2-terminal acidic domain. The interaction described between the NH2-terminus of HC II and the anion-binding exosite I of thrombin is not unique. Recently, it has been shown that thrombin interacts with its platelet receptor in an analogous man ner. 27,28 The NH2-terminus of the thrombin receptor con tains a thrombin cleavage site and a region with homol ogy to the COOH-terminus of hirudin. Cleavage leads to activation of the receptor. There is evidence that interac tion of the hirudin-like domain of the receptor with the anion-binding exosite I of thrombin results in more effi cient cleavage and activation of the receptor. Clearly, the important role of the anion binding exosite I in determin ing thrombin substrate specificity is just beginning to be elucidated. We propose that stimulation of the thrombin-HC II reaction by a glycosaminoglycan can occur by the fol lowing mechanisms: (1) displacement of the NH2-terminal acidic domain from the glycosaminoglycan-binding site of HC II, allowing the acidic domain to interact with anion-binding exosite I of thrombin; and (2) simulta-
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actions between rHC II and anion-binding exosite I of thrombin take place only in the presence of a glycosaminoglycan. Experiments with 7-thrombin also suggest that an ion-binding exosite I plays an important role in the mech anism of inhibition of thrombin by HC II. γ-Thrombin is a proteolytically cleaved form of α-thrombin, which re tains the ability to hydrolyze synthetic peptide substrates, does not clot fibrinogen, and has a greatly reduced affin ity for the COOH-terminal region of hirudin.20'21 The heparin-binding site of 7-thrombin appears to be intact, since α-thrombin and 7-thrombin both elute from heparin-Sepharose at ~0.4 M sodium chloride, and heparin stimulates inhibition of both α-thrombin and γ-thrombin by antithrombin III (AT III) ~5000-fold. By contrast, dermatan sulfate and heparin stimulated inhibition of 7-thrombin by native rHC II only 14- to 33-fold.15 A similar degree of stimulation by glycosaminoglycans was observed in experiments with α-thrombin and a deleted form of rHC II lacking residues 1-74 (rHC II(Δ1-74)). Furthermore, Sheehan et al22 demonstrated that mutation of residues in the anion-binding exosite I of thrombin, especially Arg68, decreased the rate of inhibition by HC II, but not AT III, in the presence of heparin. Thus, deletion of the NH2-terminal acidic domain of rHC II or alterations of anion-binding exosite I of thrombin greatly reduce the maximum rate of reaction between the two proteins. The model shown in Figure 5 includes the possibil ity that a glycosaminoglycan chain can also function as a template to which both HC II and thrombin bind. The glycosaminoglycan-binding site on the surface of throm bin (i.e., anion-binding exosite II) has not been defined precisely, but it appears to be distinct from anion-binding exosite I 23 . Therefore, binding of thrombin to a gly cosaminoglycan chain and to the NH2-terminal acidic domain of HC II should not be mutually exclusive. The template mechanism may explain the modest increase in the rate of inhibition of α-thrombin by rHC II(Δ1-74) or 7-thrombin by native rHC II in the presence of dermatan sulfate or heparin. The difference in the abilities of the two glycosaminoglycans to stimulate thrombin inhibition suggests that heparin may form a better template than dermatan sulfate. Maimone and Tollefsen24 isolated the following hexasaccharide, which represents the smallest fragment of porcine skin dermatan sulfate that binds to HC II with high affinity: (IdoA[2-SO4]-GalNAc[4-SO4])2-IdoA[2SO4]-ATalR[4-SO4]. To test the relative importance of the template mechanism already described, we compared the ability of the high-affinity hexasaccharide and intact dermatan sulfate to stimulate thrombin inhibition by na tive rHC II and rHC II(Δ1-74). We found that the highaffinity hexasaccharide increased the rate of α-thrombin
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neous binding of HC II and thrombin to a single glycosaminoglycan chain, which functions as a catalytic template. As suggested by experiments with the highaffinity hexasaccharide, the displacement mechanism may increase the rate of formation of the covalent thrombin-HC II complex independently, although the degree of stimulation is less than that obtained with intact dermatan sulfate. In addition, rHC II molecules lacking the NH2terminal acidic domain can be stimulated weakly by fulllength glycosaminoglycan chains. Together, the dis placement and template mechanisms produce maximal stimulation (3600- to 7000-fold) of the thrombin-HC II reaction.
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