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1992. 43:9-16 1992 by Annual Reviews Inc.
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ANTITHROMBINS: Their Potential
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as Antithrombotic Agents Jeffrey I. Weitz, M.D., and Jack Hirsh, M.D.
Department of Medicine, McMaster University, and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada L8V 1C3 KEY WORDS:
thrombin inhibitors, anticoagulants, heparin
ABSTRACT The inhibition of thrombin is the key to the prevention and treatment of thrombotic disorders. Although heparin is an extremely effective anti coagul�nt, it has certain limitations that are not shared by newer thrombin inhibitors. As a result, these novel inhibitors may have advantages over heparin in certain clinical settings.
INTRODUCTION Thrombin plays a central role in hemostasis and thrombogenesis. It acti vates platelets, converts fibrinogen to fibrin, and activates factor XIII, which then stabilizes the fibrin. In addition, thrombin amplifies coagu lation by activating factors V and VIII, factors that accelerate both the generation of prothrombinase and the subsequent activation of pro thrombin. Thus, the inhibition of thrombin is the key to preventing and treating thromboembolic disorders. Although heparin is an effective thrombin inhibitor, it has limitations that are not shared by newer throm bin inhibitors. As a result, these novel inhibitors may have certain advan tages over heparin. This chapter focuses on the effects of heparin and these new thrombin inhibitors on the hemostatic process.
MECflANISM OF ACTION OF HEPARIN Heparin acts as an anticoagulant by catalyzing the inactivation of throm bin and activated factor X (factor Xa) by antithrombin III ( 1). Upon 9 0066-4219/92/0401-0009$02.00
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10
WEITZ & HIRSH
binding to lysine sites on antithrombin III, heparin produces a con formational change at the active site that markedly accelerates the rate at which antithrombin III inhibits the coagulation enzymes (1). Heparin then dissociates from the enzyme-inhibitdr complex and can catalyze other antithrombin III molecules (1). Although the heparin-antithrombin III complex can inactivate a number of coagulation enzymes 0), recent studies in plasma suggest that heparin blocks coagulation primarily by catalyzing the inhibition of thrombin (2, 3). By preventing thrombin-mediated activation of factors V and VIII, heparin blocks the feedback amplification of coagulation that is initiated by thrombin (4).
HEPARIN PHARMACOKINETICS Heparin is poorly absorbed from the gastrointestinal tract, and must be given by intravenous or subcutaneous injection. The mechanisms of hep arin clearance are complex and poorly understood. Heparin binds to a number of plasma proteins other than antithrombin III. These include vitronectin, histidine-rich glycoprotein, and fibronectin (5). As a result, the plasma concentration of these heparin-binding proteins may influence the amount of heparin that is active in anticoagulation. This may explain the marked variability in the amount of heparin required to achieve a therapeutic heparin level in patients with thrombotic disorders (6). After intravenous injection, heparin is eliminated rapidly as a result of equilibration. This phase is followed by a more gradual disappearance, which can best be explained by a combination of a saturable and a non saturable, first-order mechanism of clearance (7). The saturable phase of heparin clearance is thought to be the result of heparin binding to receptors on endothelial cells and macrophages. Once bound, the heparin is inter nalized, depolymerized, and metabolized into smaller, less sulfated deriva tives (8, 9). When heparin binds to endothelial cells, it displaces platelet factor 4, which can then inactivate circulating heparin ( 10). The slower, nonsaturable mechanism of heparin clearance probably reflects renal excretion. Once the heparin dose exceeds the saturating concentration, a greater proportion is cleared by this mechanism. As a result of this complex mechanism of heparin clearance, the anti coagulant response to therapeutic doses of heparin is not linear. Instead, increasing amounts of heparin produce a disproportionate increase in both the intensity and duration of the anticoagulant effect as the saturable mechanism of clearance is overcome. This explains why the apparent biologic half-life of heparin is dose dependent. Thus, with intravenous
ANTITHROMBINS
II
Annu. Rev. Med. 1992.43:9-16. Downloaded from www.annualreviews.org Access provided by Michigan State University Library on 02/03/15. For personal use only.
boluses of 25, 100, and 400 U /kg, the apparent half-lives are 30, 60, and 150 min, respectively (11). The decreased bioavailability of heparin when it is administered sub cutaneously can also be explained on the basis of the saturable clearance mechanism. Heparin that gradually enters the circulation from the sub cutaneous depot site is rapidly cleared by the saturable mechanism. Thus, achievement of a steady state is delayed, and 10 to 15% more heparin is required to achieve a therapeutic effect when heparin is given by the subcutaneous rather than the intravenous route. POTENTIAL LIMITATIONS OF HEPARIN
Heparin is an extremely effective anticoagulant for the treatment of arterial and venous thromboembolism (12). However, heparin is limited in terms of its ability to inhibit the propagation of venous thrombi and to prevent rethrombosis after successful coronary thrombolysis. Thus, venographic evidence of propagation of thrombi in the deep veins of the legs is seen in about 10% of patients during heparin therapy (13). Further, unless maintenance anticoagulant therapy is given to patients with deep vein thrombosis, clot extension occurs in 29 to 47% of patients treated with a 5- to l 4-day course of heparin (14, 15). In addition, heparin does not totally prevent rethrombosis of the infarct-related artery after successful thrombolysis with tissue plasminogen activator. The anticoagulant effect of heparin is modified by platelets, fibrin, vas cular surfaces, and plasma proteins. Platelets limit the anticoagulant effect of heparin in two ways. First, factor Xa that is generated on the platelet surface is protected from inhibition by heparin-antithrombin III (16, 17). Second, platelets release the heparin-neutralizing protein, platelet factor 4 (10). Fibrin binds thrombin and protects it from inactivation by heparin antithrombin III (18,19). Thus, much higher concentrations of heparin are needed to inhibit thrombin bound to fibrin than are required to inactivate the free enzyme (19). Thrombin also binds to subendothelial matrix proteins, where it is again protected from inhibition by heparin (20). These observations not only explain why heparin is less effective than hirudin at preventing arterial and venous thrombosis in experimental animals (21, 22), but they also suggest that antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings. Novel Thrombin Inhibitors
The new thrombin inhibitors include low-molecular-weight heparins (LMWHs), heparinoids, and a number of antithrombin III-independent
12
WEITZ & HIRSH
compounds. These agents have a number of real or potential advantages over standard heparin and each is discussed in turn.
Annu. Rev. Med. 1992.43:9-16. Downloaded from www.annualreviews.org Access provided by Michigan State University Library on 02/03/15. For personal use only.
Low-Molecular- Weight Heparins Two observations prompted the development of LMWHs. The first was the finding that LMWH fractions prepared from standard heparin produce less prolongation of the activated partial thromboplastin time (APTT) but retain the ability to catalyze the inhibition of factor Xa. The second was the observation in experimental animals that LMWH produces less bleeding than standard heparin for an equivalent antithrombotic effect (23, 24). Recent studies provide an explanation for these differences. Commercial heparin is heterogenous and consists of molecules ranging in molecular weight from 3,000 to 30,000 (mean molecular weight, 15,000). The anticoagulant activity of heparin is attributed to a pentasaccharide sequence with a high affinity for antithrombin III (25). Only one third of the heparin molecules contain this sequence. The remaining two thirds have little anticoagulant activity and may contribute to the hemorrhagic complications of heparin because they interfere with platelet function (26) or enhance vessel wall permeability (27). The anticoagulant activity of heparin is the result of its interaction with antithrombin III. To inhibit thrombin, heparin serves as a template and must bind to both antithrombin III and thrombin (28). In contrast, heparin binding to antithrombin III alone is sufficient to accelerate the inhibition of factor Xa. Since heparin molecules with fewer than 18 saccharides are unable to bind thrombin and antithrombin III simultaneously, they cannot accelerate the inactivation of thrombin. However, these molecules retain the ability to catalyze factor Xa inhibition. The LMWHs currently in use are fragments of commercial heparin that have been produced by chemical or enzymatic depolymerization and have molecular weights ranging from 3,000 to 6,000. Unlike the antithrombin activity, the anti-factor Xa activity of heparin does not depend on molec ular weight, and the LMWHs are less able to catalyze the inhibition of thrombin relative to their activity against factor Xa. The LMWHs also produce less prolongation of the APTT than does standard heparin because prolongation of this clotting test depends more on the anti thrombin than on the anti-factor Xa activity of heparin. Like standard heparin, the LMWHs also are heterogeneous in their molecular weight distribution. Moreover, LMWHs with similar mean molecular weights may differ in the proportion of high- and low-molecular weight components. As a result, each LMWH has a unique anticoagulant profile. The LMWHs have a number of potential advantages over standard
ANTITHROMBINS
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heparin. First, at low doses, the LMWHs have greater bioavailability than standard heparin because they exhibit less binding to plasma proteins and to endothelial cells. In addition, LMWHs are not neutralized by platelet factor 4. LMWHs have a longer half-life that is independent of dose, and thus produce more predictable results than those obtained with standard heparin. This explains why once-daily subcutaneous injection is effective for the prophylaxis or treatment of venous thrombosis. Second, for an equivalent antithrombotic effect, the LMWHs may produce less bleeding than standard heparin even in the perioperative period (29). Heparin0 ids
Two heparinoids are currently being investigated. These are dermatan sulfate and ORG 10172, which is a mixture of dermatan sulfate, chon droitin sulfate, and heparan sulfate. Dermatan sulfate catalyzes heparin cofactor II (30), which is a secondary inhibitor of thrombin. Since heparin cofactor II only inhibits thrombin, unlike antithrombin III, dermatan sulfate has minimal anti-factor Xa activity. In contrast, because ORG 10172 contains small amounts of heparan sulfate, this preparation has both antithrombin and anti-factor Xa activity. Limited information is available concerning the clinical effectiveness of these heparinoids. In the setting of cardiopulmonary bypass surgery, dermatan sulfate may be a useful alternative to heparin for patients with a history of heparin-associ ated thrombocytopenia or sensitivity to protamine.
ANTITHROMBIN III-INDEPENDENT INHIBITORS Several antithrombin III-independent inhibitors are now available. These include hirudin, hirudin fragments, argatroban, and the peptide chloro methyl ketone inhibitor, D-phe-pro-argCH2Cl (PPACK) and its deriva tives. Although all of these inhibitors bind directly to thrombin, they have different mechanisms of action, as is described below. The potential advantage of the antithrombin III-independent inhibitors is that, unlike heparin, these agents can reach and inactivate thrombin that is bound to fibrin (19). As a result, these inhibitors have proven to be more effective than heparin in experimental animal models of venous and arterial throm bosis (21, 22), and as adjuncts to tissue plasminogen activator-induced thrombolysis using a variety of model systems (31, 32). These observations illustrate the importance of thrombin inhibition in achieving an optimal antithrombotic effect. Hirudin and Its Derivatives
Hirudin is a potent and specific thrombin inhibitor initially isolated from the medicinal leech and now available through recombinant DNA tech-
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14
WEITZ & HIRSH
nology. It forms an essentially irreversible, stoichiometric complex with thrombin. Analysis of the crystal structure of the thrombin-hirudin com plex illustrates the extensive contact that hirudin makes with thrombin as it binds to both the active center and the substrate recognition site of the enzyme (33). Some of these contact points may be inaccessible when thrombin is bound to fibrin, a fact that would explain why hirudin is a slightly less effective inhibitor of clot-bound thrombin than some of the other antithrombin III-independent inhibitors ( 19). Hirugen is a synthetic dodecapeptide comprising residues 53 to 64 of the carboxy-terminal region of hirudin (34). Unlike hirudin, this peptide does not interact with the catalytic center of thrombin. Instead, hirugen binds to the nearby substrate recognition site and thereby blocks thrombin from interacting with its substrates. By adding D-phe-pro-arg-pro-(gIY)4 to the amino-terminal region, researchers have converted hirugen from a weak competitive inhibitor to a potent bivalent inhibitor known as hirulog (35). Like hirudin, this inhibitor blocks both the active center and the substrate recognition site. However, active site inhibition is transient because once complexed, thrombin can slowly cleave the pro-arg bond on the amino terminal extension and thereby convert hirulog to a hirugen like species. Argatroban
Argatroban, a synthetic argmme derivative, is a relatively weak com petitive inhibitor of the enzyme (36). Argatroban interacts with the active site of thrombin and has a half-life of only a few minutes. PPACK and Its Derivatives
The tripeptide chloromethyl ketone, PPACK, irreversibly inhibits throm bin by alkylating the active center histidine (37). Since thrombin binds to fibrin through a site distinct from its catalytic center, PPACK readily inhibits clot-bound thrombin (19). Recently, a PPACK derivative, D-phe pro-arg-borate, has been developed that is a more specific inhibitor of thrombin than the parent molecule (38).
Literature
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Rosenberg, R. D. 1987. The heparin antithrombin system: a natural anti coagulant mechanism. In Hemostasis and Thrombosis: Basic Principles and Clinical Practice, ed. R. W. Colman, J. Hirsh, V.l. Marder, E. W. Salzman, pp. 1373-92. Philadelphia: Lippincott. 2nd ed.
2. Ofosu, F. A., Hirsh, 1., Esmon, C. T., Modi, G. J., Smith, L. M., et at. 1989. Un fractionated heparin inhibits throm bin-catalyzed amplification reactions of coagulation more efficiently than those catalyzed by factor Xa. Biochem. J. 257: 143-50 3. Beguin, S., Lindhout, T., Hemker, H. C.
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1988. The mode of action of heparin in plasma. Thromb. Haemostasis 60: 45762 Ofosu, F. A., Sie, P., Modi, G. J., Fer nandez, F., Buchanan, M. R., et al. 1987. The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin. Biochem. J. 243: 579-88 Lindahl, U., Hook, M. 1978. Gly cosaminoglycans and their binding to biological macromolecules. Annu. Rev. Biochem. 47: 385-417 Hirsh, J., van Aken, W. G., Gallus, A. S., Dollery, C. T.,.Cade, J. F., et al. 1976. Heparin kinetics in venous thrombosis and pulmonary embolism. Circulation 53: 691-95 deSwart, C. A. M., Nijmeyer, B., Roel ofs, J. M. M., Sixma, J. J. 1982. Kinetics of intravenously administered heparin in normal humans. Blood 60: 1251-58 Glimelius, B., Busch, C., Hook, M. 1978. Binding of heparin on the surface of cultured human endothelial cells. Thromh. Res. 12: 773-82 Mahadoo, J., Hiebert, L., Jaques, L. B. 1977. Vascular sequestration of heparin. Thromb. Res. 12: 79-90 Dawes, J., Smith, R. c., Pepper, D. S. 1978. The release, distribution and clear ance of human fJ-thromboglobulin and platelet factor 4. Thromb. Res. 12: 85161 Olsson, P., Lagergren, H., Ek, S. 1963. The elimination from plasma of intra venous heparin: an experimental study on dogs and humans. Acta Med. Scand. 173: 619-30 Hirsh, J. 1991. Heparin. N. Engl. J. Med. 324: 1565-74 Marder, V. J., Soulen, R. L., Atich artakarn, V., Budzynski, A. Z., Paru lekar, S., et al. 1977. Quantitative veno graphic assessment of deep vein thrombosis in the evaluation of strep tokinase and heparin therapy. J. Lab. c/in. Med. 89: 1018-29 Hull, R., Delmore, T., Genton, E., Hirsh, J., Gent, M., et al. 1979. Warfarin sodium versus low-dose heparin in the long-term treatment of venous throm bosis. N. Engl. J. Med. 301: 855-58 Lagerstedt, C. I., Olsson, C. G., Fagher, B. 0., Oqvist, B. W., Albrechtsson, U. 1985. Need for long-term anticoagulant treatment in symptomatic calf-vein thrombosis. Lancet 2: 515-18 Marciniak, E. 1973. Factor X. inac tivation by antithrombin III. Evidence for biological stabilization of factor X. by factor V-phospholipid complex. Br. J. Haematol. 24: 391-400
15
17. Walker, F. J., Esmon, C . T. 1979. The effects of phospholipid and factor V. on the inhibition of factor X. by anti thrombin III. Biochem. Biophys. Res. Commun. 90: 641--47 18. Hogg, P. J., Jackson, C. M. 1989. Fibrin monomer protects thrombin from inac tivation by heparin-antithrombin III: implications for heparin efficacy. Proc. Natl. Acad. Sci. USA 86: 3619-23 19. Weitz, J. I., Hudoba, M., Massel, D., Maraganore, J., Hirsh, J. 1990. Clot bound thrombin is protected from inhi bition by heparin-antithrombin III but is susceptible to inactivation by anti thrombin III-independent inhibitors. J. c/in. Invest. 86: 385-91 20. Bar-Shavit, R., Eldor, A., Vlodavsky, 1. 1989. Binding of thrombin to suben dothelial extracellular matrix: pro tection and expression of functional properties. J. Clin. Invest. 84: 1096-1104 21. Heras, M., Chesebro, 1. H., Penny, W. J., Bailey, K. R., Badimon, L., et al. 1989. Effects of thrombin inhibition on the development of acute platelet thrombus deposition during angioplasty in pigs: heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation 79: 657-65 22. Agnelli, G., Pascucci, c., Cosmi, B., Nenci, G. G. 1990. The comparative effects of recombinant hirudin (CGP 39393) and standard heparin on throm bus growth in rabbits. Thromb. Hae mostasis 63: 204--7 23. Andersson, L. 0., Barrowdiffe, T. W., Holmer, E., Johnson, E. A., Sims, G. E. 1976. Anticoagulant properties of hep arin fractionated by affinity chro matography on matrix-bound anti thrombin III and by gel filtration. Thromb. Res. 9: 575 24. Carter, C. J., Kelton, J. G., Hirsh, J., Cerskus, A. L., Santos, A. V., et al. 1982. between relationship the The hemorrhagic and antithrombotic prop erties of low molecular weight heparins and heparin. Blood 59: 1239 25. Casu, B., Oreste, P., Torri, G., Zoppetti, G., Choay, 1., et al. 1981. The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity con taining the minimal antithrombin 111binding sequence. Biochem. J. 197: 599609 26. Salzman, E. W., Rosenberg, R. D., Smith, M. H., Lindon, J. N., Favreau, L. 1980. Effect of heparin and heparin fractions on platelet aggregation. J. C/in. Invest. 65: 64--73 27. Blajchman, M. A., Young, E., Ofosu, F. A. 1989. Effects of unfractionated
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29.
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WEITZ & HIRSH heparin, dermatan sulfate and low molecular weight heparin on vessel wall permeability in rabbits. Ann. NY Acad. Sci.556: 245-54 Olson, S. T., Shore, J. D. 1982. Dem onstration of a two-step reaction mech anism for inhibition of (i-thrombin by antithrombin III and identification of the step affected by heparin. J. Bioi. Chern. 257: 14891 Levine, M. N., Hirsh, J., Gent, M., Tur pie, A. G. G., LeClerc, J. 1991. A ran domized trial comparing enoxaparine low molecular weight heparin with stan dard unfractionated heparin in patients undergoing elective hip surgery. Ann. Intern. Med. 114: 545-51 Ofosu, F. A., Modi, G. J., Smith, L. M., Cerskus, A. L., Hirsh, J., et al. 1984. Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity by complementary pathways. Blood 64: 741-47 Yasuda, T., Gold, H. K., Yaoita, H., Leinbach, R. c., Guerrero, J. L., et al. 1990. Comparative effects of aspirin, a synthetic thrombin inhibitor and a mon oclonal antiplatelet glycoprotein lIb/IlIa antibody on coronary artery reperfusion, reocclusion and bleeding with recombinant tissue-type plas minogen activator in a canine prep aration. J. Am. Call. Cardio/. 16: 71422 Klement, P., Hirsh, J., Maraganore, J.,
Fenton, J., Weitz, J. 1990. Effects of heparin and hirulog on t-PA induced thrombolysis in a rat model. Fibrinolysis 4: 9 33. Rydel, T. J., Ravichandran, K. G., Tulinsky, A., Bode, W., Huber, R., et al 1990. The structure of a complex of recombinant hirudin and human (i thrombin. Science 249: 277-80 34. Maraganore, J. M., Chao, B., Joseph, M. L., Jablonski, J., Ramachandron, K. L. 1989. Anticoagulant activity of syn thetic hirudin fragments. J. Bioi. Chem. 264: 8692-98 35. Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandron, K. L., Fenton, J. W. II. 1990. Design and char acterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 29: 7095-7101 36. Kikumoto, R., Tamao, Y., Tezuka, T., Tonomura, S., Hara, H., et al. 1984. Selective inhibition of thrombin by (2R,4R) - 4 - methyl- I[NZ- [(3- methyl1,2,3,4 - tetrahydro - 8 - quinolinyl) sul fonyl] - L - arginyl)] - 2 - piper idinecarboxylic acid. Biochemistry 23: 85-90 37. Kettner, C., Shaw, E. 1979. D-Phe-Pro ArgChzCI. A selective affinity label for thrombin. Thromb. Res. 14: 969-73 38. Kettner, C., Mersinger, L., Knabb, R. 1990.The selective inhibition of throm bin by peptides of boroarginine. J. Bioi. Chem.265: 18289-97 .
1992. 43:9-16 1992 by Annual Reviews Inc.
Annu. Rev. Med. Copyright ©
Further
Quick links to online content All rights reserved
ANTITHROMBINS: Their Potential
Annu. Rev. Med. 1992.43:9-16. Downloaded from www.annualreviews.org Access provided by Michigan State University Library on 02/03/15. For personal use only.
as Antithrombotic Agents Jeffrey I. Weitz, M.D., and Jack Hirsh, M.D.
Department of Medicine, McMaster University, and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada L8V 1C3 KEY WORDS:
thrombin inhibitors, anticoagulants, heparin
ABSTRACT The inhibition of thrombin is the key to the prevention and treatment of thrombotic disorders. Although heparin is an extremely effective anti coagul�nt, it has certain limitations that are not shared by newer thrombin inhibitors. As a result, these novel inhibitors may have advantages over heparin in certain clinical settings.
INTRODUCTION Thrombin plays a central role in hemostasis and thrombogenesis. It acti vates platelets, converts fibrinogen to fibrin, and activates factor XIII, which then stabilizes the fibrin. In addition, thrombin amplifies coagu lation by activating factors V and VIII, factors that accelerate both the generation of prothrombinase and the subsequent activation of pro thrombin. Thus, the inhibition of thrombin is the key to preventing and treating thromboembolic disorders. Although heparin is an effective thrombin inhibitor, it has limitations that are not shared by newer throm bin inhibitors. As a result, these novel inhibitors may have certain advan tages over heparin. This chapter focuses on the effects of heparin and these new thrombin inhibitors on the hemostatic process.
MECflANISM OF ACTION OF HEPARIN Heparin acts as an anticoagulant by catalyzing the inactivation of throm bin and activated factor X (factor Xa) by antithrombin III ( 1). Upon 9 0066-4219/92/0401-0009$02.00
Annu. Rev. Med. 1992.43:9-16. Downloaded from www.annualreviews.org Access provided by Michigan State University Library on 02/03/15. For personal use only.
10
WEITZ & HIRSH
binding to lysine sites on antithrombin III, heparin produces a con formational change at the active site that markedly accelerates the rate at which antithrombin III inhibits the coagulation enzymes (1). Heparin then dissociates from the enzyme-inhibitdr complex and can catalyze other antithrombin III molecules (1). Although the heparin-antithrombin III complex can inactivate a number of coagulation enzymes 0), recent studies in plasma suggest that heparin blocks coagulation primarily by catalyzing the inhibition of thrombin (2, 3). By preventing thrombin-mediated activation of factors V and VIII, heparin blocks the feedback amplification of coagulation that is initiated by thrombin (4).
HEPARIN PHARMACOKINETICS Heparin is poorly absorbed from the gastrointestinal tract, and must be given by intravenous or subcutaneous injection. The mechanisms of hep arin clearance are complex and poorly understood. Heparin binds to a number of plasma proteins other than antithrombin III. These include vitronectin, histidine-rich glycoprotein, and fibronectin (5). As a result, the plasma concentration of these heparin-binding proteins may influence the amount of heparin that is active in anticoagulation. This may explain the marked variability in the amount of heparin required to achieve a therapeutic heparin level in patients with thrombotic disorders (6). After intravenous injection, heparin is eliminated rapidly as a result of equilibration. This phase is followed by a more gradual disappearance, which can best be explained by a combination of a saturable and a non saturable, first-order mechanism of clearance (7). The saturable phase of heparin clearance is thought to be the result of heparin binding to receptors on endothelial cells and macrophages. Once bound, the heparin is inter nalized, depolymerized, and metabolized into smaller, less sulfated deriva tives (8, 9). When heparin binds to endothelial cells, it displaces platelet factor 4, which can then inactivate circulating heparin ( 10). The slower, nonsaturable mechanism of heparin clearance probably reflects renal excretion. Once the heparin dose exceeds the saturating concentration, a greater proportion is cleared by this mechanism. As a result of this complex mechanism of heparin clearance, the anti coagulant response to therapeutic doses of heparin is not linear. Instead, increasing amounts of heparin produce a disproportionate increase in both the intensity and duration of the anticoagulant effect as the saturable mechanism of clearance is overcome. This explains why the apparent biologic half-life of heparin is dose dependent. Thus, with intravenous
ANTITHROMBINS
II
Annu. Rev. Med. 1992.43:9-16. Downloaded from www.annualreviews.org Access provided by Michigan State University Library on 02/03/15. For personal use only.
boluses of 25, 100, and 400 U /kg, the apparent half-lives are 30, 60, and 150 min, respectively (11). The decreased bioavailability of heparin when it is administered sub cutaneously can also be explained on the basis of the saturable clearance mechanism. Heparin that gradually enters the circulation from the sub cutaneous depot site is rapidly cleared by the saturable mechanism. Thus, achievement of a steady state is delayed, and 10 to 15% more heparin is required to achieve a therapeutic effect when heparin is given by the subcutaneous rather than the intravenous route. POTENTIAL LIMITATIONS OF HEPARIN
Heparin is an extremely effective anticoagulant for the treatment of arterial and venous thromboembolism (12). However, heparin is limited in terms of its ability to inhibit the propagation of venous thrombi and to prevent rethrombosis after successful coronary thrombolysis. Thus, venographic evidence of propagation of thrombi in the deep veins of the legs is seen in about 10% of patients during heparin therapy (13). Further, unless maintenance anticoagulant therapy is given to patients with deep vein thrombosis, clot extension occurs in 29 to 47% of patients treated with a 5- to l 4-day course of heparin (14, 15). In addition, heparin does not totally prevent rethrombosis of the infarct-related artery after successful thrombolysis with tissue plasminogen activator. The anticoagulant effect of heparin is modified by platelets, fibrin, vas cular surfaces, and plasma proteins. Platelets limit the anticoagulant effect of heparin in two ways. First, factor Xa that is generated on the platelet surface is protected from inhibition by heparin-antithrombin III (16, 17). Second, platelets release the heparin-neutralizing protein, platelet factor 4 (10). Fibrin binds thrombin and protects it from inactivation by heparin antithrombin III (18,19). Thus, much higher concentrations of heparin are needed to inhibit thrombin bound to fibrin than are required to inactivate the free enzyme (19). Thrombin also binds to subendothelial matrix proteins, where it is again protected from inhibition by heparin (20). These observations not only explain why heparin is less effective than hirudin at preventing arterial and venous thrombosis in experimental animals (21, 22), but they also suggest that antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings. Novel Thrombin Inhibitors
The new thrombin inhibitors include low-molecular-weight heparins (LMWHs), heparinoids, and a number of antithrombin III-independent
12
WEITZ & HIRSH
compounds. These agents have a number of real or potential advantages over standard heparin and each is discussed in turn.
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Low-Molecular- Weight Heparins Two observations prompted the development of LMWHs. The first was the finding that LMWH fractions prepared from standard heparin produce less prolongation of the activated partial thromboplastin time (APTT) but retain the ability to catalyze the inhibition of factor Xa. The second was the observation in experimental animals that LMWH produces less bleeding than standard heparin for an equivalent antithrombotic effect (23, 24). Recent studies provide an explanation for these differences. Commercial heparin is heterogenous and consists of molecules ranging in molecular weight from 3,000 to 30,000 (mean molecular weight, 15,000). The anticoagulant activity of heparin is attributed to a pentasaccharide sequence with a high affinity for antithrombin III (25). Only one third of the heparin molecules contain this sequence. The remaining two thirds have little anticoagulant activity and may contribute to the hemorrhagic complications of heparin because they interfere with platelet function (26) or enhance vessel wall permeability (27). The anticoagulant activity of heparin is the result of its interaction with antithrombin III. To inhibit thrombin, heparin serves as a template and must bind to both antithrombin III and thrombin (28). In contrast, heparin binding to antithrombin III alone is sufficient to accelerate the inhibition of factor Xa. Since heparin molecules with fewer than 18 saccharides are unable to bind thrombin and antithrombin III simultaneously, they cannot accelerate the inactivation of thrombin. However, these molecules retain the ability to catalyze factor Xa inhibition. The LMWHs currently in use are fragments of commercial heparin that have been produced by chemical or enzymatic depolymerization and have molecular weights ranging from 3,000 to 6,000. Unlike the antithrombin activity, the anti-factor Xa activity of heparin does not depend on molec ular weight, and the LMWHs are less able to catalyze the inhibition of thrombin relative to their activity against factor Xa. The LMWHs also produce less prolongation of the APTT than does standard heparin because prolongation of this clotting test depends more on the anti thrombin than on the anti-factor Xa activity of heparin. Like standard heparin, the LMWHs also are heterogeneous in their molecular weight distribution. Moreover, LMWHs with similar mean molecular weights may differ in the proportion of high- and low-molecular weight components. As a result, each LMWH has a unique anticoagulant profile. The LMWHs have a number of potential advantages over standard
ANTITHROMBINS
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heparin. First, at low doses, the LMWHs have greater bioavailability than standard heparin because they exhibit less binding to plasma proteins and to endothelial cells. In addition, LMWHs are not neutralized by platelet factor 4. LMWHs have a longer half-life that is independent of dose, and thus produce more predictable results than those obtained with standard heparin. This explains why once-daily subcutaneous injection is effective for the prophylaxis or treatment of venous thrombosis. Second, for an equivalent antithrombotic effect, the LMWHs may produce less bleeding than standard heparin even in the perioperative period (29). Heparin0 ids
Two heparinoids are currently being investigated. These are dermatan sulfate and ORG 10172, which is a mixture of dermatan sulfate, chon droitin sulfate, and heparan sulfate. Dermatan sulfate catalyzes heparin cofactor II (30), which is a secondary inhibitor of thrombin. Since heparin cofactor II only inhibits thrombin, unlike antithrombin III, dermatan sulfate has minimal anti-factor Xa activity. In contrast, because ORG 10172 contains small amounts of heparan sulfate, this preparation has both antithrombin and anti-factor Xa activity. Limited information is available concerning the clinical effectiveness of these heparinoids. In the setting of cardiopulmonary bypass surgery, dermatan sulfate may be a useful alternative to heparin for patients with a history of heparin-associ ated thrombocytopenia or sensitivity to protamine.
ANTITHROMBIN III-INDEPENDENT INHIBITORS Several antithrombin III-independent inhibitors are now available. These include hirudin, hirudin fragments, argatroban, and the peptide chloro methyl ketone inhibitor, D-phe-pro-argCH2Cl (PPACK) and its deriva tives. Although all of these inhibitors bind directly to thrombin, they have different mechanisms of action, as is described below. The potential advantage of the antithrombin III-independent inhibitors is that, unlike heparin, these agents can reach and inactivate thrombin that is bound to fibrin (19). As a result, these inhibitors have proven to be more effective than heparin in experimental animal models of venous and arterial throm bosis (21, 22), and as adjuncts to tissue plasminogen activator-induced thrombolysis using a variety of model systems (31, 32). These observations illustrate the importance of thrombin inhibition in achieving an optimal antithrombotic effect. Hirudin and Its Derivatives
Hirudin is a potent and specific thrombin inhibitor initially isolated from the medicinal leech and now available through recombinant DNA tech-
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14
WEITZ & HIRSH
nology. It forms an essentially irreversible, stoichiometric complex with thrombin. Analysis of the crystal structure of the thrombin-hirudin com plex illustrates the extensive contact that hirudin makes with thrombin as it binds to both the active center and the substrate recognition site of the enzyme (33). Some of these contact points may be inaccessible when thrombin is bound to fibrin, a fact that would explain why hirudin is a slightly less effective inhibitor of clot-bound thrombin than some of the other antithrombin III-independent inhibitors ( 19). Hirugen is a synthetic dodecapeptide comprising residues 53 to 64 of the carboxy-terminal region of hirudin (34). Unlike hirudin, this peptide does not interact with the catalytic center of thrombin. Instead, hirugen binds to the nearby substrate recognition site and thereby blocks thrombin from interacting with its substrates. By adding D-phe-pro-arg-pro-(gIY)4 to the amino-terminal region, researchers have converted hirugen from a weak competitive inhibitor to a potent bivalent inhibitor known as hirulog (35). Like hirudin, this inhibitor blocks both the active center and the substrate recognition site. However, active site inhibition is transient because once complexed, thrombin can slowly cleave the pro-arg bond on the amino terminal extension and thereby convert hirulog to a hirugen like species. Argatroban
Argatroban, a synthetic argmme derivative, is a relatively weak com petitive inhibitor of the enzyme (36). Argatroban interacts with the active site of thrombin and has a half-life of only a few minutes. PPACK and Its Derivatives
The tripeptide chloromethyl ketone, PPACK, irreversibly inhibits throm bin by alkylating the active center histidine (37). Since thrombin binds to fibrin through a site distinct from its catalytic center, PPACK readily inhibits clot-bound thrombin (19). Recently, a PPACK derivative, D-phe pro-arg-borate, has been developed that is a more specific inhibitor of thrombin than the parent molecule (38).
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17. Walker, F. J., Esmon, C . T. 1979. The effects of phospholipid and factor V. on the inhibition of factor X. by anti thrombin III. Biochem. Biophys. Res. Commun. 90: 641--47 18. Hogg, P. J., Jackson, C. M. 1989. Fibrin monomer protects thrombin from inac tivation by heparin-antithrombin III: implications for heparin efficacy. Proc. Natl. Acad. Sci. USA 86: 3619-23 19. Weitz, J. I., Hudoba, M., Massel, D., Maraganore, J., Hirsh, J. 1990. Clot bound thrombin is protected from inhi bition by heparin-antithrombin III but is susceptible to inactivation by anti thrombin III-independent inhibitors. J. c/in. Invest. 86: 385-91 20. Bar-Shavit, R., Eldor, A., Vlodavsky, 1. 1989. Binding of thrombin to suben dothelial extracellular matrix: pro tection and expression of functional properties. J. Clin. Invest. 84: 1096-1104 21. Heras, M., Chesebro, 1. H., Penny, W. J., Bailey, K. R., Badimon, L., et al. 1989. Effects of thrombin inhibition on the development of acute platelet thrombus deposition during angioplasty in pigs: heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation 79: 657-65 22. Agnelli, G., Pascucci, c., Cosmi, B., Nenci, G. G. 1990. The comparative effects of recombinant hirudin (CGP 39393) and standard heparin on throm bus growth in rabbits. Thromb. Hae mostasis 63: 204--7 23. Andersson, L. 0., Barrowdiffe, T. W., Holmer, E., Johnson, E. A., Sims, G. E. 1976. Anticoagulant properties of hep arin fractionated by affinity chro matography on matrix-bound anti thrombin III and by gel filtration. Thromb. Res. 9: 575 24. Carter, C. J., Kelton, J. G., Hirsh, J., Cerskus, A. L., Santos, A. V., et al. 1982. between relationship the The hemorrhagic and antithrombotic prop erties of low molecular weight heparins and heparin. Blood 59: 1239 25. Casu, B., Oreste, P., Torri, G., Zoppetti, G., Choay, 1., et al. 1981. The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity con taining the minimal antithrombin 111binding sequence. Biochem. J. 197: 599609 26. Salzman, E. W., Rosenberg, R. D., Smith, M. H., Lindon, J. N., Favreau, L. 1980. Effect of heparin and heparin fractions on platelet aggregation. J. C/in. Invest. 65: 64--73 27. Blajchman, M. A., Young, E., Ofosu, F. A. 1989. Effects of unfractionated
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Fenton, J., Weitz, J. 1990. Effects of heparin and hirulog on t-PA induced thrombolysis in a rat model. Fibrinolysis 4: 9 33. Rydel, T. J., Ravichandran, K. G., Tulinsky, A., Bode, W., Huber, R., et al 1990. The structure of a complex of recombinant hirudin and human (i thrombin. Science 249: 277-80 34. Maraganore, J. M., Chao, B., Joseph, M. L., Jablonski, J., Ramachandron, K. L. 1989. Anticoagulant activity of syn thetic hirudin fragments. J. Bioi. Chem. 264: 8692-98 35. Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandron, K. L., Fenton, J. W. II. 1990. Design and char acterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin. Biochemistry 29: 7095-7101 36. Kikumoto, R., Tamao, Y., Tezuka, T., Tonomura, S., Hara, H., et al. 1984. Selective inhibition of thrombin by (2R,4R) - 4 - methyl- I[NZ- [(3- methyl1,2,3,4 - tetrahydro - 8 - quinolinyl) sul fonyl] - L - arginyl)] - 2 - piper idinecarboxylic acid. Biochemistry 23: 85-90 37. Kettner, C., Shaw, E. 1979. D-Phe-Pro ArgChzCI. A selective affinity label for thrombin. Thromb. Res. 14: 969-73 38. Kettner, C., Mersinger, L., Knabb, R. 1990.The selective inhibition of throm bin by peptides of boroarginine. J. Bioi. Chem.265: 18289-97 .
