The COOH-terminal of AN EXOSITE-DIRECTED COMPETITIVE INHIBITOR OF THE ACTION OF (u-THROMBIN ON FIBRINOGEN*

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.

Hirudin, a potent 65-residue polypeptide inhibitor of a-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic Nacetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SOs)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 pM) of human a-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen.
These observations indicate that hirugen binds to a-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of athrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to a-thrombin. Studies of the effect of hirugen on the rate of inactivation of a-thrombin by antithrombin III (AT), the major plasma inhibitor of a-thrombin, indicated that binding of hirugen to a-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of a-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of a-thrombin which bind to a-thrombin and block both conversion of fibrinogen to fibrin and inactivation of a-thrombin by AT. tide (fibrinopeptide A, FPA)l from the amino terminus of the Aol chain of fibrinogen. Interestingly, interaction at exosite(s) distinct from the active site of cu-thrombin appears to determine the affinity and catalytic specificity of cr-thrombin toward fibrinogen (l-6). This exosite region of cu-thrombin appears to be lost when cu-thrombin is proteolytically modified, as evidenced by the observation that certain proteolytic nicks in cy-thrombin result in thrombin forms which have dramatically reduced competence (relative to that of ru-thrombin) to process fibrinogen, but essentially undiminished catalytic activity toward small peptide nitroanilide substrates (3,(7)(8)(9). Moreover, an antibody directed against residues 62 to 73 of the B chain of human a-thrombin can block cu-thrombincatalyzed hydrolysis of fibrinogen without altering small substrate hydrolysis (5).
Hirudin, a 65-residue polypeptide inhibitor of cy-thrombin obtained from the leech Hirudo medicinalis, appears to interact with a-thrombin at both the active site and an exosite of cu-thrombin, as evidenced by kinetic studies of the reaction of hirudin with cY-thrombin and chemically or proteolytically modified thrombins (10)(11)(12). Synthetic peptides corresponding in primary structure to the COOH-terminal domain of hirudin inhibit the ability of cu-thrombin to clot fibrinogen with little effect on the activity of a-thrombin toward small substrates (13,14). This observation suggests that the synthetic peptides corresponding to the COOH terminus of hirudin selectively bind to an exosite of cy-thrombin. Substances that reversibly block the fibrinogen-binding exosite of (Ythrombin might be expected to have more pronounced effects than reversible active site-directed inhibitors of cu-thrombin on the amount of fibrin formed in response to generation of a bolus of cu-thrombin in blood plasma. Reversible active sitedirected inhibitors would be expected to inhibit equally the rate of conversion of fibrinogen to fibrin and the rate of irreversible inactivation of ol-thrombin by antithrombin Ill (AT), since both processes require an accessible active site. It follows from this realization that the amount of fibrin clot directed inhibitor had a greater inhibitory effect on the rate of fibrinogen processing than it had on the rate of the irreversible reaction of a-thrombin with AT. Reversible exosite-directed competitive inhibitors of cythrombin such as those corresponding to the COOH-terminus of hirudin might exert their inhibitory effect so as to: (i) block the binding of fibrinogen to a-thrombin, (ii) cause fibrinogen to bind nonproductively to a-thrombin, or (iii) incrementally reduce the affinity of cu-thrombin for fibrinogen.
A study of the effect of the peptides on the initial rate of a-thrombincatalyzed release of FPA from fibrinogen could distinguish between the possible modes of action of peptides corresponding to the COOH-terminal domain of hirudin. Such a study is presented for the first time in the present paper, where we analyze the effects of hirugen, an N-acetylated synthetic peptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SOB)-Leu) corresponding in primary structure to that of the COOH-terminal domain of hirudin, on kinetic parameters for the interaction of cu-thrombin with fibrinogen, AT, and the small fluorogenic cu-thrombin substrate N-p-tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin (tos-GPR-amc).
Our observation that hirugen competitively inhibits the interaction of Lu-thrombin with fibrinogen while only modestly affecting the action of cu-thrombin on tos-GPR-amc and the reaction of a-thrombin with AT in the presence or absence of heparin, indicates that: (i) hirugen is an exosite-directed competitive inhibitor of the action of a-thrombin on fibrinogen; (ii) the exosite of cY-thrombin that binds hirugen is a critical determinant of the interaction between a-thrombin and fibrinogen; and (iii) hirugen should act in concert with AT and heparin to reduce the amount of fibrin formed during the plasma lifetime of a-thrombin. EXPERIMENTAL PROCEDURES AND RESULTS'

DISCUSSION
The analysis presented under "Results" (see Miniprint) of the effect of hirugen on the initial velocity for the cY-thrombincatalyzed release of FPA from fibrinogen indicated that binding of hirugen and fibrinogen to cu-thrombin are mutually exclusive. Analysis of the effect of hirugen as a competitive inhibitor of Lu-thrombin-catalyzed release of FPA from fibrinogen yielded a value of 0.54 (+ 0.02) pM for the dissociation constant of the a-thrombin-hirugen complex. This value is in reasonable agreement with the value (0.64 + 0.09 pM) that we obtained from an analysis of the hirugen-induced increase in the specificity constant for cu-thrombin catalysis of tos-GPRamc hydrolysis and is similar to the ICsO observed in an earlier study of the hirugen-induced increase in the activated partial thromboplastin time (14). It is unlikely that the observed competitive inhibition by hirugen of the activity of cY-thrombin toward fibrinogen is due to the binding of hirugen at the active site, since hirugen does not inhibit the activity of (Ythrombin toward tos-GPR-amc and other small peptide substrates (14). The effects of hirugen documented in the present study are consistent with the proposal that hirugen binds to cu-thrombin at a site distinct from the active site. The effect of hirugen as a pure competitive inhibitor of the interaction of Lu-thrombin with fibrinogen suggests that the same exosite that binds hirugen is also a critical determinant of the affinity of a-thrombin for fibrinogen. The observation that fibrin II binds to active site-blocked derivatives of cr-thrombin (1,(34)(35)(36)(37)(38) suggested the possibility that the fibrinogen-binding exosite of cY-thrombin is also a major determinant of the ability of a-thrombin to bind to fibrin. This hypothesis is supported by our observation that hirugen blocked the binding of (Ythrombin to fibrin II. Although hirugen appears to interact with cy-thrombin at a site distinct from the active site, the modest increase in the specificity constant (due to a decrease in the Michaelis constant) for cY-thrombin-catalyzed hydrolysis of tos-GPR-amc suggests that binding of hirugen at the exosite induces a conformational change in cY-thrombin that alters interactions at the active site so as to modestly decrease the Michaelis constant for cu-thrombin-catalyzed hydrolysis of tos-GPRamt. In accord with this conclusion is the finding that the circular dichroism of cY-thrombin is altered upon binding of a nonsulfated peptide from the COOH-terminal domain of hirudin (13).
It is interesting to note that competitive inhibition of the action of a-thrombin on fibrinogen without inhibiting small substrate hydrolysis has been reported previously for thrombomodulin (TM), a macromolecular modifier of a-thrombin which does not bind to the active site (39), and an antibody directed against an Lu-thrombin fragment thought to comprise an exosite domain (5). It is difficult to exclude the possibility, however, that the effects of these macromolecules reflect steric interactions that prevent fibrinogen from approaching an unoccupied exosite in the complex of cy-thrombin with either the antibody or TM, rather than direct competition between the macromolecule and fibrinogen for the fibrinogenbinding exosite of Lu-thrombin.
Our observation that a 12residue peptide (hirugen) competitively inhibits cY-thrombincatalyzed release of FPA from fibrinogen without inhibiting tos-GPR-amc hydrolysis provides strong evidence that the (Ythrombin:fibrinogen interaction is largely determined by interactions at a small contiguous exosite in cY-thrombin. The observation that hirugen binding to cr-thrombin effected only a 1.9-fold decrease in the rate of reaction of crthrombin with AT provides additional evidence that hirugen doesn't bind to the active site of cr-thrombin, since active sitedirected competitive inhibitors of cy-thrombin block the reaction of a-thrombin with AT (32). In accord with our conclusion that hirugen and fibrin II bind to the same exosite of a-thrombin, fibrin II monomer has been reported to effect a reduction similar to that of hirugen for the rate of reaction of cu-thrombin with AT (40).
In contrast to the modest inhibitory effects of hirugen and fibrin II monomer, TM accelerates inactivation of a-thrombin by AT in the absence of heparin (41,42). Heparin-catalyzed inactivation of cu-thrombin by AT, however, is inhibited by TM (39). Fibrin II monomer also inhibits heparin-catalyzed inactivation of cY-thrombin by AT (40). These observations in conjunction with the observations that heparin inhibits the clotting of fibrinogen by cy-thrombin (43) and decreases the affinity of a-thrombin for hirudin (44) suggested the possibility that heparin binds at the same exosite of Lu-thrombin as does fibrin(ogen), TM, and hirugen. Our observation that hirugen causes a similar reduction (1.9-to 2.4-fold) in the reaction between Lu-thrombin and AT for both the heparincatalyzed and uncatalyzed reaction suggests that: (i) hirugen alters the intrinsic reactivity of cu-thrombin with AT without preventing heparin from bridging AT to Lu-thrombin in the noncovalent termolecular complex AT:heparin:cu-thrombin; and (ii) hirugen does not bind significantly (under our experimental conditions) to the heparin-binding exosite of (Y-thrombin that is important for heparin catalysis of the inactivation of cy-thrombin by AT. In light of these conclusions, how then can we account for the inhibitory effect of heparin both on the a-thrombin-catalyzed conversion of fibrinogen to fibrin and on the interaction of a-thrombin with hirudin, as well as the ability of TM and fibrin to inhibit heparincatalyzed inactivation of cu-thrombin by AT? Perhaps, in addition to binding to the exosite involved in bridging athrombin and AT in a termolecular complex, heparin also binds to the fibrin(ogen)-binding exosite of a-thrombin. The existence of more than one heparin-binding site in o-thrombin is consistent with the observation that cu-thrombin-heparin complexes precipitate in a concentration-dependent manner resembling a cross-linking interaction (45).3 The effect of TM and fibrin II on heparin-catalyzed inactivation of cY-thrombin by AT could of course reflect steric and electronic interactions between a-thrombin-bound heparin and CYthrombin-bound TM or Lu-thrombin-bound fibrin II. Clearly, further studies will be required to validate these notions.
The effects of hirugen on the interactions of cu-thrombin with fibrinogen and AT disclosed in the present study suggest that hirugen and other exosite-directed competitive inhibitors of cu-thrombin may be useful antagonists of fibrin formation in uiuo, since they should act in concert with AT. The binding of hirugen to cy-thrombin prevents interaction between crthrombin and fibrinogen without blocking inactivation of (Ythrombin by AT. Equation 8 (see "Results" in Miniprint) can be simplified to account for the effect of hirugen on the fraction of fibrinogen (rather than tos-GPR-amc) that is converted to product (fibrin I) in the presence of AT. Hirugen and fibrinogen competitively bind to a-thrombin, thus 1/K!? = 0 and Equation 8 reduces to Equation 10. (10) In this equation, f, represents the fraction of fibrinogen con-  (11) Thus, in regions of the circulatory system where the major mode of inactivation of cr-thrombin is via reaction with AT and similarly reacting serine proteinase inhibitors, exositedirected competitive inhibitors of cY-thrombin may be more effective antagonists of fibrin clot formation than active sitedirected competitive inhibitors. 4 This conclusion has been derived previously for irreversible inactivation of an enzyme where substrate and inactivator act as competitive inhibitors (30,31).