sign in sign up
<<
Home , Hirudin

JACC Vol. 22, No. 4 October 1993 :10`99-92 1(889

Editorial Comment bus formation after vascular injury such as that caused by angioplasty or plaque rupture . must also be con- Selective Thrombin Inhibitors: sidered an effector molecule . Inducible receptors for throm- bin have been demonstrated on endothelial and vascular "The Next Generation smooth muscle cells (3). The addition of thrombin in physi- of * ologic concentration to bovine aortic endothelial cells in culture significantly reduces endothelial cell proliferation EZRA DEUTSCH, MD, FACC, compared with controls . Vascular smooth muscle cell pro- liferation in cell culture is increased in response to addition A. KONETI RAO, MD,t of thrombin to the culture medium. Smooth muscle matrix ROBERT W. COLMAN, MDt production is altered by thrombin in this setting, with an Philadelphia, Pennsylvania increase in total protein and collagen content (4). Human umbilical vein endothelial cells in culture release a platelet-derived growth factorlike (PDGFc) molecule in Role of thrombin after vascular Injury . forma- response to physiologically relevant concentrations of tion plays a central role in the healing process that follows thrombin, mediated by intracellular conversion of PDGFc acute vascular injury . Thrombin is the critical enzyme in from an inactive to an active form (5). Cultured human early thrombus formation, cleaving fibrinopeptidt :s A and B microvascular renal endothelial cells exhibited increased from to yield insoluble , which effectively levels of a c-sis gene product for platelet-derived growth serves as the foundation of the evolving thrombus . Both free factor in response to thrombin exposure, with peak activity and fibrin-bound thrombin are able to convert fibrinogen to 4 h after exposure to thrombin . Receptor activation results in fibrin, allowing propagation of thrombus at the site of injury . an increase in messenger ribonucleic acid for the c-sis gene, Thrombin activates Factor XIII, which can then cross-link mediated by activation of protein kinase C (6) . Neither of fibrin, increasing its mechanical stability and resistance to these effects of thrombin on endothelial sells requires the lysis. Circulating platelets are recruited to the site of injury, proteolytic activity of the enzyme . adhere to thrombogenic substances such as subendothelial Human alpha thrombin has been shown to be a potent matrix proteins and contribute to clot formation. Thrombin mitogen for rat aortic smooth muscle cells in culture . How- then aggregates the platelets, further reinforcing the evolving ever, in this model the stimulus for proliferation was depen- thrombus . In addition to these prothrombotic effects, throm- dent on a functional proteolytic site (7). Thus, thrombin has bin initiates a series of reactions that activate endogenous direct effects on cell proliferation and influences the cellular systems . Thrombin generated at the site of synthetic mechanisms responsible for matrix protein and injury binds to thrombomodulin, an endothelial surface collagen production (4). Thrombin-mediated platelet-derived membrane protein, initiating activation of , which growth factor synthesis by endothelial cells further demon- in turn (in the presence of protein S) inactivates Factors Va strates the potential impact of this compound on the relative and Villa . Thrombin stimulates successive release of both proliferative response of endothelial and smooth muscle tissue (t-PA) and plasminogen activa- cells after an injury such as that created by balloon angio- tor inhibitor type I from endothelial cells, thus initiating plasty. endogeneous through plasmin generation from Thrombin has direct effects on white blood cells as well . plasminogen by t-PA with subsequent modulation through lnterleukin-1 release from macrophages can be induced by plasminogen activator inhibitor type 1 (1,2) . Thrombin there- thrombin (8). This cytokine stimulates smooth muscle cell fore plays a pivotal role in maintaining the intricate balance proliferation, and also induces endothelial expression of of initial prothrombotic reparative events and subsequent leukocyte adhesion molecules (9,10). Preliminary data from endogenous anticoagulant and thrombolytic pathways . our labotatory suggest that thrombin can potentiate neutro- The importance of thrombin lies not just in acute throm- phil degranulation in response to known agonists such as leukotriene B4 in vitro (Deutsch E, unpublished observa- tions). Thrombin can thus enhance recruitment of leuko- *Editorials published in Journal of the American College ofCardiology cytes to the site of injury and may contribute to local release reflect the views of the authors and do not necessarily represent the views of of leukotrienes and other vasoactive effectois and proteases JACC or the American College or Cardiology . From the Cardiology and Hematology Sections . Department of Internal such as elastase from adherent neutrophils . Medicine and the fSol Sherry Thrombosis Research Center, Temple Univer- Iiirudins versus heparlns as anticoagulants. is a . This work was sup- sity School of Medicine, Philadelphia, Pennsylvania selectOe thrombin inhibitor that binds directly, tightly and ported in part by Grant H L 47186 (Dr. Colman) and Grant H L 02658 (Dr. Rao : Academic Award in Vascular Diseases) from the National Heart . Lung, and rapidly to thrombin in a stoichiometric fashion. This 65- Blood Institute, National Institutes of Health, Bethesda, Maryland . amino acid polypeptide was isolated from the Hirudo . Address for correspondence : Ezra Deutsch . MD, Cardiology Section more than 30 years ago (I 1) . Subsequent de- Temple University Hospital, 9 Parkinson Pavilion, 3401 North Broad Street . medicina"is Philadelphia, Pennsylvania 19140. velopment of hirudin derivatives include recombinant

0735-1097/93156.00 01993 by the American College of Cardiology

JACC Vol. 22, No. 4 low DEUTSCH ET AL. EDITORIAL COMMENT October 1993 ;1099-92

desulfato-hirudin (r-hirudin, CGP 39393) and a 20-amino acid minimized or prevented by increasing systemic doses of synthetic peptide (hirulog) that binds both to the catalytic recombinant hirudin. The extent of thrombus formation at site of thrombin and to in exosite required for thrombin the site of arterial deendothelialization after balloon angio- binding to fibrinogen and thrombospondin (12). Hirudins plasty of the porcine carotid artery (24) or rabbit femoral offer several potential advantages as anticoagulants when artery (25) was reduced by intravenous r-hirudin in a dose- compared with . Hirudins neutralize thrombin di- dependent fashion . The magnitude of both platelet and fibrin rectly without the need for an intermediary molecule such as deposition at the angioplasty site in the porcine carotid ui. Although are inactivated by anti- artery was reduced by hirudin to a significantly greater heparin proteins such as platelet factor 4 (13), no similar degree than by heparin (24). Rethrombosis after balloon situation is observed with hirudins . Both recombinant hiru- arterial injury and thrombolysis was successfully inhibited din and hirulog can inhibit thrombin-mediated platelet acti- by subcutaneous hirudin in the rabbit jugular artery (25). The vation and generation of fibrin through cleavage of fibrino- addition of hirulog to t-PA resulted in enhanced thromboly- peptides A and B from fibrinogen in vitro and in in vivo sis and a reduction in rethrombosis in a canine model of models of thrombosis (1l). Although thrombin bound to coronary thrombosis (26) . Platelet and fibrin deposition at fibrin is resistant to inactivation by heparin-antithrombin III the site of stent deployment in the porcine coronary artery complex, inhibition of clot-bound thrombin is effectively was significantly reduced by r-hirudin relative to the levels achieved with recombinant hirudin (14). Thrombin-mediated achieved with heparin and dextran (27). R-hirudin has also platelet activation is not influenced by heparin (13) . How- been employed as an agent in cardiopulmo- ever, hirudin does inhibit thrombin-induced platelet activa- nary bypass in the dog (11). Effective anticoagulation was tion (11). These observations are relevant to clinical condi- achieved, paralleled by preservation of platelet count and tions such as rethrombosis after successful coronary fibrinogen levels . Hirudin also inhibits neutrophil degranu- thrombolysis or propagation of venous thrombi, conditions lation, as measured by elastase release, in a model of where fibrin-bound thrombin in an evolving clot is still able simulated cardiopulmonary bypass (Colman RW, unpub- to convert fibrinogen to fibrin and to activate circulating lished observations) . Thus, hirudin inhibits the prothrom- platelets in the face of ongoing anticoagulation with heparin. botic effects of free and surface-bound thrombin in experi- Preesut study. In this issue of the Journal, Verstraete et mental arterial and venous thrombosis and on synthetic al. (15) describe the biologic effects and pharmacokinetics of surfaces . recombinant desulfato-hirudin (COP 39393) when adminis- The potential impact of r-hirudin (CGP 39393) on inhibi- tered subcutaneously and intravenously in normal human tion of the cellular proliferative responses to thrombin is volunteers . This compound differs from the natural leech illustrated by a reduction in the magnitude of restenosis after hirudin only in the absence of a sulfato group on tyrosine 63 . balloon angioplasty in the femoral arteries of atherosclerotic The effects of single and multiple sequential doses of r- rabbits (28). Animals received either a heparin bolus or an hirudin on variables are presented, with parallel r-hirudin bolus immediately before angioplasty . A 2-h intra- measurements of plasma drug concentrations . Dose- venous infusion was continued in the r-hirudin cohort . dependent prolongation of thrombin time and activated Activated partial thromboplastin times were prolonged to 1 .5 partial thromboplastin time are observed . The magnitude of to 2 times control level in both treatment groups . Rabbits activated partial thr mboplastin time prolongation ranged receiving r-hirudin had significantly less angiographic resten- from 1.5 to 2.5 times baseline in the doses employed, a level osis at 28 days after the procedure, paralleled by a significant of anticoagulation useful in clinical practice, A peak effect on reduction in the extent of lumen cross-sectional area narrow- activated partial thromboplastin time was sustained for 3 to ing as deter-mined by planimetric analysis of histologic 6 h after subcutaneous administration. A linear relation was sections . This study demonstrates that, in this model, throm- observed between the extent of activated partial thrombo- bin generation at the time of angioplasty plays an important plastin time prolongation and plasma hirudin levels . There role in triggering the cellular events responsible for the was no evidence for cumulative effects on the magnitude or restenosis process. Thrombin inhibition with r-hirudin sig- time course of anticoagulation in volunteers who received nificantly reduced the magnitude of neointimal hyperplasia, multiple doses at intervals of every 8 or 12 h . No increase in and was not accompanied by hemorrhagic complications . bleeding time was observed, a not unexpected finding, Current clinical trials of hirudin . Several clinical trials of because hirudins have no direct effect on platelets and may hirudins are currently underway . A preliminary report com- not impair the establishment of primary hemostasis. These paring intravenous r-birudin (CGP 39393) with conventional and other studies (16-19) document the in vivo effects of anticoagulation with heparin during coronary angioplasty hirudins and constitute careful pharmacologic studies that examined the incidence of adverse events in a randomized define the appropriate doses for clinical use . cohort of patients (29). In comparison with patients treated Eitpethue t a studies of hh udin, Hirudins have been with heparin, patients who received r-hirudin had a lower shown to be effective in reducing the extent of vascular incidence of thrombotic events after angioplasty (acute thrombosis in a number of animal models. Experimental myocardial infarction, emergency coronary artery bypass venous thrombosis in the : rat (20.21) and rabbit (22,23) was surgery) . The relative risk of postprocedure ischemia, as

JACC Vol . 22, No. 4 DEUTSCH ET AL . 1091_ October 1993 :1099-92 EDITORIAL, COMMENT

nlvasured by ST segment changes on 24-hour Holier ambu- compared with a to i9% inc dence in historical control latory electrocardiographic monitoring, was significantly re- subjects (34). duced in the r-hirudin cohort . The investigators concluded Other thrombin inhibitors . In addition to hirudins, there are that hirudin was a safe and effective form of anticoagulation several other highly specific thrombin inhibitors under evalua- during coronary angioplasty and appeared to reduce the tion, including , a reversible competitive thrombin likelihood of recurrent thrmbotic events in the immediate inhibitor (35), argidipine (36), and D-phenylalanine-L-prolyl-L- postprocedure period . R-hirudin (CGP 39393) has also been arginyl-chloromethyl ketone (PPACK), an irreversible serine shown to be an effective anticoagulant for prophylaxis of protease inhibitor (37). In a recent clinical trial, intravenous thromboembolic complications in patients undergoing elec- argatroban was initially effective in the treatment of unstable tive hip surgery (30). A 10% incidence of embolic complica- angina (38). However, anginal symptoms reappeared in 9 of 43 tions was observed in 24 patients treated with 15 to 20 mg of patients, typically 4 to 8 hours after cessation of the argatroban r-hirudin subcutaneously two times daily . No increase in infusion, and may reflect renewed thrombin activity and throm- periprocedural bleeding was observed in these patients, who bus formation in parallel with diminishing plasma argatroban received the initial r-hirudin dose preoperatively. These levels. This observation serves to highlight the contribution of results compared favorably with those in control subjects thrombus formation as a final common event in acute ischemic treated with conventional or low molecular weight heparin . syndromes, and the initial efficacy of direct thrombin inhibition in the treatment of unstable angina . Hirulog has been employed as the sole anticoagulant in a series of 45 patients undergoing diagnostic coronary angiog- Conclusions. Hirudin and its derivatives show great promise as systemic anticoagulants . There would seem to be raphy (31). Two dosing regimens of hirulog were compared distinct advantages over heparin, which requires the pl :.rtic- with conventional anticoagulation with a 5,000 1' heparin ipation of antithrombin III and is rather ineffective in neu- intravenous bolus . Systemic anticoagulation with hirulog tralizing thrombin on cell surfaces . Nonetheless, hirudins, was rapidly achieved . Dose-dependent increases in activated like all global inhibitors of thrombin, have the potential to partial thromboplastin time, prolhrombin time and activated result in unwanted hemorrhage . The application of hirudins clotting time were observed, in a fashion similar to patients in clinical situations is thus tempered by the lack of an who received heparin . The mean half-life of this effect on effective antidote to rapidly terminate their action analogous activated partial thromboplastin time was 40 min . Fibrino- to the effect of prolamine on heparin . A number of potential peptide A generation was effectively suppressed, and there approaches to reverse the antithrombotic effect of hirudins was no evidence of major or minor hemorrhagic complica- need to be explored further. Acetylated (which . The tions during hirulog administration in these patients are not prothrombotic) bind to hirudins and thus inhibit their efficacy of hirulog as sole anticoagulant during coronary anticoagulant effects (39) . Recombinant Factor Vila will angioplasty was further evaluated in a multicenter study of generate new thrombin, which can then complex with circu- 208 patients (32). All patients received . Four dosing lating hirudin (1I). The development of monoclonal or regimens were employed, achieving decremental increases polyclonal antibodies to hirudins might provide a novel in activated clotting time . An 11% incidence of abrupt vessel approach to neutralize their anticoagulant effects . closure in the lower dose hirulog groups (median activated Available experimental data suggest that systemic or clotting time 268 and 290 s) was reduced to 3% in those site-specific delivery of hirudin compounds in the setting of patients receiving a higher dosing regimen (activated clotting plaque rupture or the period after angioplasty might favor- time 336 and 375 s, p = .03). Again, no hemorrhagic or ably influence the magnitude of long-term cellular prolifera- vascular complications were observed . Hirulog as a direct tive responses (24,28). The global inhibitory effects of hiru- thrombin inhibitor is well tolerated and in appropriate doses din, including thrombin action on platelets and other cells, may be a potent antithrombotic agent for use during coro- and blood coagulation via fibrin generation suggest that nary angioplasty . Hirulog has also been utilized as an specific thrombin inhibitors may be the next major advance anticoagulant for preventing venous thrombosis after hip in antithrombotic therapy . surgery (33). A dose of 1 .0 mglkg body weight was injected subcutaneously every 8 h for 3 days . No major bleeding was observed in 177 patients, who were followed up for 14 days . References The effects on coagulation variables observed were similar l. Colman RW, Marder Vi . Salzman EW, Hirsh J . Overview of hemostasis . . Hemostasis and to those reported for CGP 39393 by Verstraete et al. (15). In : Colman RW, Hirsh J, Marder Vi . Salzmtm EW. eds Thrombosis . Philadelphia: Lippincott, 1987:3-17 . The development of antibodies to hirulog was not observed 2. Wilcox JN. Thrombin and other potential mechanisms underlying resten- in these patients. Thus, it appears that there is no increased osis. Circulation 1991 ;84 :432-5 . incidence of risks or complications when using r-hirudin 3. Yu TH . Hung DT, Wheaton VI, Coujhlin SR . The molecular cloning of a mechanism of (COP 39393) or hirulog in lieu of heparin in all available functional thrombin receptor reveals a novel proleolytic receptor activation. Cell 1991 ;64 :1057-68. studies . Indeed, these preliminary studies suggest that anti- 4. Graham DJ . Alexander JJ . The effects of thrombin on bovine aortic coagulation with r-hirudin or hirulog during coronary angio- endothelial and smooth muscle cells . J Vasc Surg 19911;11 :307-I3 . . Thrombin induces plasty results in a reduction in abrupt vessel closure as 5. Harlan JM, Thompson PJ . Ross RR. Bowen-Pope DF

JACC Vol . 22, No. 4 DEUTSCH ET AL. 1'092 October 1993:1089-92 EDiTORJAL COMMENT

release of platelet-derived growth factor-like molecule(s) by cultured Effects of thrombin inhibition on the development of acute platelet- human endothelial cells. J Cell Biol 1986:103 :1129-33. thrombus deposition during angioplasty in pigs . Circulation 1989:79 :657- 6. Daniel TO, Gibbs VG. Milfay DF. Garovoy MR, Williams LT. Thrombin 65 . stimulates c-sis gene expression in microvascular endothelial cells . I Blot 25 . Kaiser B, Simon A, Markwardt F. Antithrombotic effects of recombinant Chem 1986:9579-82 . hirudin in experimental angioplasty and intravascular thrombolysis. 7. McNamara CA, Sarembock IJ . Gimple LW, Fenton3W 111, Coughlin SR, Thromb Haemost 1990.63 :11-7 . Owens GK . Thrombin stimulates proliferation of cultured rat aortic 26. Yao S-K, Ober J, Ferguson JJ, ct al . Combination of inhibition of smooth muscle cells by a proteotytically activated receptor . J Clin Invest thrombin and blockade of A2 synthetase and receptors 1993 ;93:948. enhances thrombolysis and delays reocclusion in canine coronary arter- 8. Jones A . Geczy CL . Thrombin and factor Xa enhance the production of ies . Circulation 1992:86 :19939. interleukin-1 . Immunology 1990:71 :236-41 . 27. Buchwald AB. Sandrock D, Unterberg C, et al . Platelet and fibrin 9. Libby P . Warner SJC, Friedman GB. IInterleukin-1 : a mitogen for human deposition on coronary stems in minipigs : effect of hirudin versus vascular smooth muscle cells that induces the release of growth factor heparin . J Am Coll Cardiol 199301 :249-34 . inhibitory pmstanolds . J Clin Invest 198&88&187-98. 28. Sarembock IJ . Gem SD . Gimple LW, Owen RM, Powers ER, Roberts 10, Dustin ML, Rothlein R, Bhan AK, DinarelloC4, Springer TA, Induction WC. Effectiveness of recombinant desulphatohirudin in reoucing resten- by IL-I and interferon gamma: tissue distribution, biochemistry . and osis after balloon angioplasty of atherosclerotic femoral arteries in rab- function of a natural adherence molecule (ICAM-I ). J Immunol 1986 :137 : bits. Circulation 1991 ;&4:232-43 . 245-54 . 29. van den Bos AA, Deckers JW, Heyndrickx GR . et al . PTCA with hirudin 11. Markwardt F . Hirudin and derivatives as anticoagulant agents . Thromb associated with less acute complications than with heparin (abstr). Haemost 1991 ;6ba4152, Circulation 1992 :861suppl I):1.372 . 12. Maraganore 3M, Boundon P, Jablonski J, Itamachandran KL . Fenton JW 30 . Eriksson BI, Kalebo P. Lindbratt S. Ekmnn S, Close P. Is direct thrombin Ill . Design and characterization of hiru :ogs : a Hovel class of bivalent inhibition with hirudin a safe prophylaxis of thromboembolic events after peptide inhibitors of thrombin . Biochemistry 1990 ;29:7095-101 . hip replacement? tabstr) . Circulation 1992 :86(suppl 1):1.410. 13. Hirsh J . Heparin. N Engl J Mod 1991 ;324:1365-74. 31 . Cannon CP . Marapnore JM . Loscalzo J . et al . Anticoagulant effects of 14. Weitz J! . Hudoba M, Mussel D. Maraganore JM, Hirsh J, Clot-bound hirulog, a novel thrombin inhibitor, in patients with coronary artery thrombin is protected from inhibition by heparin-antithrombin Ill but is disease . Am J Cardiol 1993;71 :778-82. . susceptible to inactivation by antithrotabin Ill-independent inhibitors inhibitor, J Clin Invest 19W,86:38341 . 32 . Bonau R. Jewitt D, Sigwart U. et al. Hinilog . a direct thrombin (nbstr). Circulation 1992: . Verstrnete M, Nurmobamed M. Kienast 1. et al. Biological effects of as anticoagulant during coronary angioplasty 15 86(suppl 11 :1 .649. recombinant hLrudin (CGP 39393) in human volunteers . J Am Coll Cardiol 1993 :22:1080-8. 33. Ginsberg JS. Hirsh J. Gent M . et al . A phase II study of hirulog in the 16 . Bichler J. Fichtl B . Siebeck M. Fritz H. Pharmacokinetics and phatma- prevention of venous thrombosis after major hip or knee surgery (absir). codynamics of hirrdin in man after single subcutaneous and intravenous Circulation 1992 :86(suppl l):1-409. bolus administration. Arzneim-Forsch 1988 :38:704-10. 34. Holmes DR, Holubkov R, Vlietstra RE, et al . Comparison of complica- 17, Markwardt F. Nowak 0 . Sturzebecher J . Griesshnch U, Walsmann P . dons of percutaneous transluminal coronary angioplasty from 1977 to Vogel 0 . Pharrnacokinetics and anticoagulant effect of hirudin in man . 1981 and from 1993 to 1986: The National Heart . Lung, and Blood Thromb Haemost 1984 ;32:160-3 . Institute's Percutaneous Transluminal Coronary Angioplasty Registry . 18, Matkwardt F, Nowak G, Sturzebecher J, Vogel G. Clin co-pharmacolog- J Am Call Cardiol 1988 ;12:1149-55 . ieal studies with recombinant hirudin. Thromb Res 1988 :52 :393-4110. 35, Jang IK. Gold HK . Leinbach RC. Fallon JT . Collen D . In vivo thrombin 19, Markwardt F. Nowak G, Sturzebecher J. Clinical pharmacology of inhibition enhances and sustains arterial recanalization with recombinant recombinant hirudin. Haemoslasis 1991 :211suppl l)ati33-6, tissue type plasminogen activator . Circ Res 1990;67:1552-61 . 20, Talbot M . Biology of recombinant hirudin (COP 39393) . a now prospect in 36 . Krupinski K, Breddin HK, Markwardt F . Haarmann W. Antithrombotic the treatment of thrombosis . Semin Thromb Hemost 1989:15:293-301 . effects of three thrombin inhibitors in a rat model of laser-induced 21 . Kaiser B, Markwardt F, AMithromb tie and haemorrhagic effects of thrombosis . Heemostasis 1989;19 :74-82. synthetic and naturally occurring thrombin inhibitors . Thromb Res 1956 : 37. Hansen SR. Harker LA . Interruption of acute platelet-dependent throm- 57;b13-20, bosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl 22, W.Jenp JM, Pilhnu R, Parsed J . Recombinant hirudin as antithrombotic chloromethyl ketone . Proc NatI Acad Sci U S A 198883:3184-8. agent, Drugs of the Future 1990-.15,267-80. 38. Gold HK. Toms FW, Garabedian HD, et al. Evidence for a rebound 23 . Walenp 3M . Pifarre R . Hoppensteedt D, Parsed J . Development of coagulation phenomenon after cessation of a 4 hour infusion of a specific recombinant hirudin as a therapeutic anticoagulant and antithrombotic thrombin inhibitor in patients with unstable angina pectoris . J Am Coll agent: some objective considerations. Semin Thromb Hemost 1989:15 : Cordial 1993 ;21 :1039-47. 316--33 . 39. Bnsggner E, Walsmann P. Markwatdt F. Neutralization of hirudin anti- 24. Reran M. Chesebro JH . Penny WJ, Bailey KR, Badimon L . Faster V . coagulation action by DIPthrombin. Pharmazic 1 1;36:860-I .