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In vitro effect of melagatran and lepirudin on clot-bound thrombin
Thrombosis Research 110 (2003) 249 – 252
Brief Communication
In vitro effect of melagatran and lepirudin on clot-bound thrombin Jamal Lebrazi, Ismail Elalamy, Meyer Michel Samama * Department of Biological Hematology, Hoˆtel-Dieu Hospital, 1 Place du Parvis Notre-Dame, 75004 Paris, France Received 5 May 2003; received in revised form 11 June 2003; accepted 16 June 2003
Keywords: Clot-bound thrombin; Procoagulant activity; Direct thrombin inhibitors; Washed clots; Melagatran; Lepirudin
1. Introduction Thrombin converts fibrinogen into fibrin monomers by cleaving fibrinopeptides A and B from aminoterminal regions of Aa and Bh chains of fibrinogen, respectively [1]. During this reaction, thrombin is adsorbed to fibrin through the fibrin(ogen)-binding site located within the E domain [2,3]. The fibrin-bound thrombin retains its enzymatic activity and has the potential to induce thrombosis extension [4]. Given this central role of thrombin, most treatment strategies for acute coronary syndromes are aimed at inhibiting its generation or blocking its activity [5 –7]. In vitro studies have shown that fibrin-bound thrombin is relatively protected from inactivation by heparin-antithrombin and this inactivation requires ligation of both exosites 1 and 2 of thrombin [8,9]. These findings could explain why heparin is limited in its ability to inhibit the extension of venous thrombosis and to prevent rethrombosis after successful thrombolysis [3]. Thus, direct thrombin inhibitors may represent alternatives to the treatment with heparin. At present, most clinical benefits of recombinant (r)hirudin have been demonstrated in the management of patients with heparin-induced thrombocytopenia. For this reason, lepirudin (a form of r-hirudin) has recently been approved for anticoagulation in patients with heparin-induced thrombocytopenia and associated thromboembolic disease [10,11].
Abbreviations: PPP, platelet-poor plasma; CBT, clot-bound thrombin; FPT, fluid-phase thrombin; OD, optical density; DTI, direct thrombin inhibitor. * Corresponding author. Tel.: +33-1-42-34-82-66; fax: +33-1-42-3482-54. E-mail address: [email protected] (M.M. Samama). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00345-1
The development of reversible noncovalent direct thrombin inhibitors (DTI), such as inogatran and melagatran, has resulted in safer, more specific and predictable anticoagulant treatment. Melagatran is a new synthetic competitive and rapid thrombin inhibitor with a molecular mass of 429 Da. Melagatran is a dipeptide analogue which acts as fibrinopeptide A [12,13] and can be administered subcutaneously, and a prodrug form of melagatran, ximelagatran, is at present the only oral thrombin inhibitor available [5– 7,14]. Clinical trials have shown that melagatran was well tolerated with no clinically significant bleeding problems, suggesting that melagatran could be given to patients suffering from DVT [6,15]. The aim of our study was to evaluate and to compare the in vitro inhibitory effect of melagatran, the active form of the oral DTI ximelagatran [5,7 – 9], and lepirudin [11] on clot-associated thrombin activity by using standard washed clots prepared from human platelet-poor plasma (PPP) and a specific chromogenic substrate for thrombin measurement.
2. Materials and methods The clot-bound thrombin (CBT) activity was studied as previously described [16]. Aliquots (300 Al) of fresh human platelet-poor plasma (PPP) were clotted at 37 jC by 300 Al of calcium chloride (12.5 mmol/l, final concentration), in a glass tube and incubated at 37 jC for 10 min after clotting. The coagulation reaction was stopped by transferring the tubes in a water bath at 0 jC; clots were then removed, blotted on filter paper, washed twice (15 min) at 0 jC in 1 ml of EDTA – Tris buffer (Na2EDTA2H2O 7.5 mmol/l, NaCl 175 mmol/l, pH 8.4) containing 1% bovine albumin (Sigma, France) and again blotted on filter paper. Clots were incubated for 30 min at 37 jC with melagatran (H319/68,
250
J. Lebrazi et al. / Thrombosis Research 110 (2003) 249–252 Table 2 Residual activity of clot-bound thrombin after 1 h of incubation at room temperature with melagatran or lepirudin
Fig. 1. Standard amidolytic activity of increasing concentration of free human a-thrombin in buffer solution. Results are expressed in OD at 405 nm (mean F S.E.M.; n = 11).
Concentration (Amol/l)
Clot + melagatran (NIH/ml)
Clot + lepirudin (NIH/ml)
0 0.25 0.5 1 5 20
0.45 F 0.05 0.29 F 0.01 0.29 F 0.01 0.24 F 0.02 0.18 F 0.01 0.19 F 0.02
0.45 F 0.05 0.31 F 0.02 0.27 F 0.02 0.23 F 0.03 0.18 F 0.001 0.14 F 0.02
Results are expressed in NIH/ml (mean F S.E.M.; n = 11).
dose-dependent manner with the increase of free thrombin (Fig. 1). AstraZeneca, Sweden) or lepirudin (Refludan, Hœchst Marion Roussel, Deutschland) at different concentrations (0– 20 Amol/l). The different clots were then washed and introduced in each microtitre plate wells containing 100 Al of EDTA –Tris buffer, 1% bovine albumin; then, the microtitre plate was placed in a cold bath at 0 jC. The clot enzymatic activity was determined after addition of 100 Al of CBS 3447 (0.75 mmol/l), a specific substrate for thrombin (Diagnostica Stago, Asnie`res, France). After incubation for 20 min at 37 jC, the enzymatic reaction was stopped by addition of 50 Al of pure acetic acid; 200Al aliquots of each solution were placed in additional microtitre plate wells for optical density (OD) determinations at 405 nm.
3.2. Effect of melagatran and lepirudin on CBT The inhibition of CBT was assessed in clots prepared as indicated in Materials and methods and incubated with melagatran or lepirudin at various concentrations (0, 0.25,0.5, 1, 5 and 20 Amol/l) at room temperature for 1 h. After washing, they were blotted and incubated with the specific substrate for thrombin at 37 jC for 20 min. The reaction was stopped with acetic acid and the OD was determined at 405 nm (mean F SEM; n = 11) (Table 1). The amidolytic activity of CBT decreased in the clots incubated with melagatran or lepirudin with a residual activity of the clots (Table 2). The IC50 was near to 1 Amol/l.
3. Results
3.3. Effect of melagatran and lepirudin on free thrombin
3.1. Amidolytic activity of free thrombin at different concentrations in buffer
The inhibitory activity of increasing concentrations of melagatran or lepirudin against free thrombin was determined by incubating thrombin with melagatran or lepirudin. Free a-thrombin 0.45 NIH/ml, an equivalent activity of clotbound thrombin, was incubated for 1 h at room temperature with melagatran or lepirudin and the activity was measured with the chromogenic substrate for thrombin. IC50 values
The OD was measured in each solution of free human athrombin at different concentrations in buffer after 20 min of incubation at 37 jC in the presence of CBS 3447, a specific substrate for thrombin. The OD increases significantly in a
Table 1 The amidolytic activity of clots incubated 1 h with melagatran or lepirudin at different concentrations Concentration (Amol/l)
Clot + melagatran (OD F S.E.M.) (I%)*
Clot + lepirudin (OD F S.E.M. ) (I%)*
0 0.25 0.5 1 5 20
0.311 F 0.049 ( 0.202 F 0.057 ( 0.200 F 0.048 ( 0.169 F 0.061 ( 0.127 F 0.036 ( 0.138 F 0.035 (
0.311 F 0.049 ( 0.215 F 0.064 ( 0.190 F 0.070 ( 0.162 F 0.060 ( 0.129 F 0.002 ( 0.100 F 0.033 (
0) 35) 36) 46) 59) 56)
0) 31) 39) 48) 59) 68)
Results are expressed in OD at 405 nm (mean F S.E.M.; n = 11). * Inhibition in percentage of the control.
p
NS NS NS NS 0.09
Table 3 Free thrombin (0.45 NIH/ml) was incubated for 1 h at room temperature with melagatran or lepirudin, and the activity was measured in OD with the specific substrate for thrombin (mean F S.E.M.; n = 3) Antithrombin drug (Amol/l)
FPT + melagatran (OD) (I%)*
FPT + lepirudin (OD) (I%)*
0 0.25 0.5 1.25 5 20
0.310 ( 0.230 ( 0.150 ( 0.068 ( 0.023 ( 0.011 (
0.310 0.190 0.065 0.008 0.004 0.004
0) 26) 51) 78) 93) 96)
* Inhibition in percentage of the control.
( ( ( ( ( (
0) 39) 79) 97) 99) 99)
J. Lebrazi et al. / Thrombosis Research 110 (2003) 249–252
were approximately 0.5 and 0.3 Amol/l for melagatran and lepirudin, respectively (Table 3).
4. Discussion In our system, we have shown that the activity of clotbound thrombin measured with a chromogenic substrate for thrombin is equivalent to 0.45 F 0.05 NIH/ml. This value was obtained by extrapolation of the results on a standard curve corresponding to the measurement of increasing units of free human thrombin in buffer (Fig. 1). The CBT activity decreased significantly in a dose-dependent manner when clots were incubated with melagatran or lepirudin (Tables 1 and 2). In these conditions, we have a residual activity of 0.14 – 0.19 NIH/ml after 1-h incubation in the presence of 20 Amol/l melagatran or lepirudin. This result suggests the possibility that this activity is unspecific (not related to thrombin activity), because, if the small substrate penetrates into the clot and is cleaved by active thrombin, melagatran can also penetrate the clot; with such a high concentration, all thrombin activity should be inhibited. In the same conditions, we have studied the effects of melagatran and lepirudin on fluid-phase thrombin (FPT) in solution at 0.45 NIH/ml. We obtained an IC50 of approximately 0.5 Amol/l for melagatran and 0.34 Amol/l for lepirudin (Table 3). The IC50 for CBT were about 1 Amol/l for both melagatran and lepirudin. These values are higher than the therapeutic plasma concentration, which ranges between 0.05 and 0.5 Amol/l for melagatran [15] and between 0.02 and 0.10 Amol/l for lepirudin [10]. The results obtained for the IC50 were 1.8 – 5.8 and 10 –50 times higher than the therapeutic concentrations of melagatran and lepirudin, respectively. The ratio of the IC50 values for CBT versus FPT (IC50CBT/IC50FPT) were 2 and 2.9 for melagatran and lepirudin, respectively, suggesting that the activity of melagatran on FPT is 2 times higher than the activity on CBT and 2.9 times higher than the activity on CBT for lepirudin. In other experiments without the washing procedure, we have obtained an IC50 of 0.21 and 1 Amol/ l for melagatran and lepirudin, respectively [17]. This difference suggests that the washing procedure can induce some loss of the effect of melagatran. Melagatran, as lepirudin, is a direct, competitive, reversible and noncovalent thrombin inhibitor that does not require endogenous cofactors such as antithrombin or other cofactor for its antithrombin effect [8,12,14,15,18]. It binds rapidly and reversibly to both clotbound and soluble thrombin. In summary, inhibition of thrombin seems important, since thrombin is continuously generated in growing clots, and slow binding inhibition of thrombin seems to be associated with steep dose –response curves. The results of our present study suggest that melagatran could inhibit thrombus extension and improve prevention and treatment of venous thromboembolic episodes.
251
Acknowledgements We thank Dr. Christer Mattsson (Department of Cell Biology and Biochemistry, AstraZeneca R&D, Mo¨lndal, Sweden) for his generous gift of melagatran. This work has been supported by grants from AstraZeneca, Sweden.
References [1] Blomba¨ck B, Hessel B, Hogg D, Therkildsen L. A two-step fibrinogen – fibrin transition in blood coagulation. Nature 1978;275: 501 – 5. [2] Hsieh K. Thrombin interaction with fibrin polymerization sites. Thromb Res 1997;86:301 – 16. [3] Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-inedependent inhibitors. Circulation 1998;97:544 – 52. [4] Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990;86:385 – 91. [5] Hauptman J. Pharmacokinetics of an emerging new class of anticoagulant – antithrombotic drugs. A review of small-molecule thrombin inhibitors. Eur J Clin Pharmacol 2002;57:751 – 8. [6] Eriksson H, Ogren M, Eriksson UG, Kalebo P, Ahnfelt L, Bjorkstrom S, et al. Prophylaxis of venous thromboembolism with subcutaneous melagatran in total hip or total knee replacement. Results from phase II studies. Thromb Res 2002;105:371 – 8. [7] Hopfner R. Ximelagatran. Curr Opin Investig Drugs 2002;3: 246 – 51. [8] Ofosu F, Agnelli G, Hirsh J. New thrombin inhibitors: mode of action, experimental findings and clinical potential. Recent Adv Haematol 1992;6:45 – 72. [9] Becker DL, Fredenburgh JC, Stafford AR, Weitz JI. Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparincatalyzed inhibition by antithrombin and heparin cofactor II. J Biol Chem 1999;274:6226 – 33. [10] Challapalli R, Lefkovits J, Topol EJ. Clinical trials of recombinant hirudin in acute coronary syndromes. Coron Artery Dis 1996;7: 429 – 37. [11] Mudaliar JH, Liem TK, Nichols WK, Spadone DP, Silver D. Lepirudin is a safe and effective anticoagulant for patients with heparin-associated antiplatelet antibodies. J Vasc Surg 2001;34: 17 – 20. [12] Gustafsson D, Antonsson T, Bylund R, Eriksson U, Gyzander E, Nilsson I, et al. Effects of melagatran, a new low-molecular-weight thrombin inhibitor, on thrombin and fibrinolytic enzymes. Thromb Haemost 1998;79:110 – 8. [13] Mattsson C, Menschiek-Lundin A, Wahlander K, Lindahl TL. Effect of melagatran on prothrombin time assays depends on the sensitivity of the thromboplastin and the final dilution of the plasma sample. Thromb Haemost 2001;86:611 – 5. [14] Sarich TC, Eriksson UG, Mattsson C, Wolzt M, Frison L, Fager G, et al. Inhibition of thrombin generation by the oral direct thrombin inhibitor ximelagatran in shed blood from healthy male subjects. Thromb Haemost 2002;87:300 – 5. [15] Eriksson H, Eriksson UG, Frison L, Hansson PO, Held P, Holmstrom M, et al. Pharmacokinetics and pharmacodynamics of melagatran, a novel synthetic LMW thrombin inhibitor, in patients with acute DVT. Thromb Haemost 1999;81:358 – 63. [16] Meddahi S, Bara L, Fessi H, Samama MM. Evaluation of human clot associated procoagulant activities in the presence of DX9065a,
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r-Hirudin, Enoxaparin and heparin by a chromogenic technique, F1 + 2 and TAT measurements. Thromb Haemost, Suppl 1997; 1 – 844:PS 1204. [17] Lebrazi J, Elalamy I, Samama MM. Inhibitory effect of melagatran and hirudin on clot bound thrombin. 18th Congress of Interna-
tional Society on Thromb and Haemost, Paris; 2001. abstract 1090, CD3576. [18] Bates SM, Weitz JI. The mechanism of action of thrombin inhibitors. J Invasive Cardiol 2000;12:27 – 32.
Brief Communication
In vitro effect of melagatran and lepirudin on clot-bound thrombin Jamal Lebrazi, Ismail Elalamy, Meyer Michel Samama * Department of Biological Hematology, Hoˆtel-Dieu Hospital, 1 Place du Parvis Notre-Dame, 75004 Paris, France Received 5 May 2003; received in revised form 11 June 2003; accepted 16 June 2003
Keywords: Clot-bound thrombin; Procoagulant activity; Direct thrombin inhibitors; Washed clots; Melagatran; Lepirudin
1. Introduction Thrombin converts fibrinogen into fibrin monomers by cleaving fibrinopeptides A and B from aminoterminal regions of Aa and Bh chains of fibrinogen, respectively [1]. During this reaction, thrombin is adsorbed to fibrin through the fibrin(ogen)-binding site located within the E domain [2,3]. The fibrin-bound thrombin retains its enzymatic activity and has the potential to induce thrombosis extension [4]. Given this central role of thrombin, most treatment strategies for acute coronary syndromes are aimed at inhibiting its generation or blocking its activity [5 –7]. In vitro studies have shown that fibrin-bound thrombin is relatively protected from inactivation by heparin-antithrombin and this inactivation requires ligation of both exosites 1 and 2 of thrombin [8,9]. These findings could explain why heparin is limited in its ability to inhibit the extension of venous thrombosis and to prevent rethrombosis after successful thrombolysis [3]. Thus, direct thrombin inhibitors may represent alternatives to the treatment with heparin. At present, most clinical benefits of recombinant (r)hirudin have been demonstrated in the management of patients with heparin-induced thrombocytopenia. For this reason, lepirudin (a form of r-hirudin) has recently been approved for anticoagulation in patients with heparin-induced thrombocytopenia and associated thromboembolic disease [10,11].
Abbreviations: PPP, platelet-poor plasma; CBT, clot-bound thrombin; FPT, fluid-phase thrombin; OD, optical density; DTI, direct thrombin inhibitor. * Corresponding author. Tel.: +33-1-42-34-82-66; fax: +33-1-42-3482-54. E-mail address: [email protected] (M.M. Samama). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00345-1
The development of reversible noncovalent direct thrombin inhibitors (DTI), such as inogatran and melagatran, has resulted in safer, more specific and predictable anticoagulant treatment. Melagatran is a new synthetic competitive and rapid thrombin inhibitor with a molecular mass of 429 Da. Melagatran is a dipeptide analogue which acts as fibrinopeptide A [12,13] and can be administered subcutaneously, and a prodrug form of melagatran, ximelagatran, is at present the only oral thrombin inhibitor available [5– 7,14]. Clinical trials have shown that melagatran was well tolerated with no clinically significant bleeding problems, suggesting that melagatran could be given to patients suffering from DVT [6,15]. The aim of our study was to evaluate and to compare the in vitro inhibitory effect of melagatran, the active form of the oral DTI ximelagatran [5,7 – 9], and lepirudin [11] on clot-associated thrombin activity by using standard washed clots prepared from human platelet-poor plasma (PPP) and a specific chromogenic substrate for thrombin measurement.
2. Materials and methods The clot-bound thrombin (CBT) activity was studied as previously described [16]. Aliquots (300 Al) of fresh human platelet-poor plasma (PPP) were clotted at 37 jC by 300 Al of calcium chloride (12.5 mmol/l, final concentration), in a glass tube and incubated at 37 jC for 10 min after clotting. The coagulation reaction was stopped by transferring the tubes in a water bath at 0 jC; clots were then removed, blotted on filter paper, washed twice (15 min) at 0 jC in 1 ml of EDTA – Tris buffer (Na2EDTA2H2O 7.5 mmol/l, NaCl 175 mmol/l, pH 8.4) containing 1% bovine albumin (Sigma, France) and again blotted on filter paper. Clots were incubated for 30 min at 37 jC with melagatran (H319/68,
250
J. Lebrazi et al. / Thrombosis Research 110 (2003) 249–252 Table 2 Residual activity of clot-bound thrombin after 1 h of incubation at room temperature with melagatran or lepirudin
Fig. 1. Standard amidolytic activity of increasing concentration of free human a-thrombin in buffer solution. Results are expressed in OD at 405 nm (mean F S.E.M.; n = 11).
Concentration (Amol/l)
Clot + melagatran (NIH/ml)
Clot + lepirudin (NIH/ml)
0 0.25 0.5 1 5 20
0.45 F 0.05 0.29 F 0.01 0.29 F 0.01 0.24 F 0.02 0.18 F 0.01 0.19 F 0.02
0.45 F 0.05 0.31 F 0.02 0.27 F 0.02 0.23 F 0.03 0.18 F 0.001 0.14 F 0.02
Results are expressed in NIH/ml (mean F S.E.M.; n = 11).
dose-dependent manner with the increase of free thrombin (Fig. 1). AstraZeneca, Sweden) or lepirudin (Refludan, Hœchst Marion Roussel, Deutschland) at different concentrations (0– 20 Amol/l). The different clots were then washed and introduced in each microtitre plate wells containing 100 Al of EDTA –Tris buffer, 1% bovine albumin; then, the microtitre plate was placed in a cold bath at 0 jC. The clot enzymatic activity was determined after addition of 100 Al of CBS 3447 (0.75 mmol/l), a specific substrate for thrombin (Diagnostica Stago, Asnie`res, France). After incubation for 20 min at 37 jC, the enzymatic reaction was stopped by addition of 50 Al of pure acetic acid; 200Al aliquots of each solution were placed in additional microtitre plate wells for optical density (OD) determinations at 405 nm.
3.2. Effect of melagatran and lepirudin on CBT The inhibition of CBT was assessed in clots prepared as indicated in Materials and methods and incubated with melagatran or lepirudin at various concentrations (0, 0.25,0.5, 1, 5 and 20 Amol/l) at room temperature for 1 h. After washing, they were blotted and incubated with the specific substrate for thrombin at 37 jC for 20 min. The reaction was stopped with acetic acid and the OD was determined at 405 nm (mean F SEM; n = 11) (Table 1). The amidolytic activity of CBT decreased in the clots incubated with melagatran or lepirudin with a residual activity of the clots (Table 2). The IC50 was near to 1 Amol/l.
3. Results
3.3. Effect of melagatran and lepirudin on free thrombin
3.1. Amidolytic activity of free thrombin at different concentrations in buffer
The inhibitory activity of increasing concentrations of melagatran or lepirudin against free thrombin was determined by incubating thrombin with melagatran or lepirudin. Free a-thrombin 0.45 NIH/ml, an equivalent activity of clotbound thrombin, was incubated for 1 h at room temperature with melagatran or lepirudin and the activity was measured with the chromogenic substrate for thrombin. IC50 values
The OD was measured in each solution of free human athrombin at different concentrations in buffer after 20 min of incubation at 37 jC in the presence of CBS 3447, a specific substrate for thrombin. The OD increases significantly in a
Table 1 The amidolytic activity of clots incubated 1 h with melagatran or lepirudin at different concentrations Concentration (Amol/l)
Clot + melagatran (OD F S.E.M.) (I%)*
Clot + lepirudin (OD F S.E.M. ) (I%)*
0 0.25 0.5 1 5 20
0.311 F 0.049 ( 0.202 F 0.057 ( 0.200 F 0.048 ( 0.169 F 0.061 ( 0.127 F 0.036 ( 0.138 F 0.035 (
0.311 F 0.049 ( 0.215 F 0.064 ( 0.190 F 0.070 ( 0.162 F 0.060 ( 0.129 F 0.002 ( 0.100 F 0.033 (
0) 35) 36) 46) 59) 56)
0) 31) 39) 48) 59) 68)
Results are expressed in OD at 405 nm (mean F S.E.M.; n = 11). * Inhibition in percentage of the control.
p
NS NS NS NS 0.09
Table 3 Free thrombin (0.45 NIH/ml) was incubated for 1 h at room temperature with melagatran or lepirudin, and the activity was measured in OD with the specific substrate for thrombin (mean F S.E.M.; n = 3) Antithrombin drug (Amol/l)
FPT + melagatran (OD) (I%)*
FPT + lepirudin (OD) (I%)*
0 0.25 0.5 1.25 5 20
0.310 ( 0.230 ( 0.150 ( 0.068 ( 0.023 ( 0.011 (
0.310 0.190 0.065 0.008 0.004 0.004
0) 26) 51) 78) 93) 96)
* Inhibition in percentage of the control.
( ( ( ( ( (
0) 39) 79) 97) 99) 99)
J. Lebrazi et al. / Thrombosis Research 110 (2003) 249–252
were approximately 0.5 and 0.3 Amol/l for melagatran and lepirudin, respectively (Table 3).
4. Discussion In our system, we have shown that the activity of clotbound thrombin measured with a chromogenic substrate for thrombin is equivalent to 0.45 F 0.05 NIH/ml. This value was obtained by extrapolation of the results on a standard curve corresponding to the measurement of increasing units of free human thrombin in buffer (Fig. 1). The CBT activity decreased significantly in a dose-dependent manner when clots were incubated with melagatran or lepirudin (Tables 1 and 2). In these conditions, we have a residual activity of 0.14 – 0.19 NIH/ml after 1-h incubation in the presence of 20 Amol/l melagatran or lepirudin. This result suggests the possibility that this activity is unspecific (not related to thrombin activity), because, if the small substrate penetrates into the clot and is cleaved by active thrombin, melagatran can also penetrate the clot; with such a high concentration, all thrombin activity should be inhibited. In the same conditions, we have studied the effects of melagatran and lepirudin on fluid-phase thrombin (FPT) in solution at 0.45 NIH/ml. We obtained an IC50 of approximately 0.5 Amol/l for melagatran and 0.34 Amol/l for lepirudin (Table 3). The IC50 for CBT were about 1 Amol/l for both melagatran and lepirudin. These values are higher than the therapeutic plasma concentration, which ranges between 0.05 and 0.5 Amol/l for melagatran [15] and between 0.02 and 0.10 Amol/l for lepirudin [10]. The results obtained for the IC50 were 1.8 – 5.8 and 10 –50 times higher than the therapeutic concentrations of melagatran and lepirudin, respectively. The ratio of the IC50 values for CBT versus FPT (IC50CBT/IC50FPT) were 2 and 2.9 for melagatran and lepirudin, respectively, suggesting that the activity of melagatran on FPT is 2 times higher than the activity on CBT and 2.9 times higher than the activity on CBT for lepirudin. In other experiments without the washing procedure, we have obtained an IC50 of 0.21 and 1 Amol/ l for melagatran and lepirudin, respectively [17]. This difference suggests that the washing procedure can induce some loss of the effect of melagatran. Melagatran, as lepirudin, is a direct, competitive, reversible and noncovalent thrombin inhibitor that does not require endogenous cofactors such as antithrombin or other cofactor for its antithrombin effect [8,12,14,15,18]. It binds rapidly and reversibly to both clotbound and soluble thrombin. In summary, inhibition of thrombin seems important, since thrombin is continuously generated in growing clots, and slow binding inhibition of thrombin seems to be associated with steep dose –response curves. The results of our present study suggest that melagatran could inhibit thrombus extension and improve prevention and treatment of venous thromboembolic episodes.
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Acknowledgements We thank Dr. Christer Mattsson (Department of Cell Biology and Biochemistry, AstraZeneca R&D, Mo¨lndal, Sweden) for his generous gift of melagatran. This work has been supported by grants from AstraZeneca, Sweden.
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