Effects of argatroban and heparin on thrombus formation and tissue plasminogen activator-induced thrombolysis in a microvascular thrombosis model

Thrombosis Research 109 (2003) 55 – 64

Regular Article

Effects of argatroban and heparin on thrombus formation and tissue plasminogen activator-induced thrombolysis in a microvascular thrombosis model Keizo Yamada, Hajime Tsuji *, Shinzo Kimura, Hisato Kato, Shingo Yano, Naoki Ukimura, Yuka Yamada, Katsumi Nakagawa, Masao Nakagawa Second Department of Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho Hirokoji Kawaramachi, Kamigyo-ku, Kyoto 602-8566, Japan Received 10 July 2002; received in revised form 14 November 2002; accepted 4 February 2003

Abstract Effects of (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidine-carboxylic acid monohydrate (argatroban) and unfractionated heparin (UFH) were compared with respect to thrombus formation and tissue-type plasminogen activator (t-PA)-induced thrombolysis in a microvasculature thrombosis model. The antithrombotic activities of anticoagulants were evaluated with respect to the time required for the initiation of thrombus formation (Ti) and the time required for the thrombus to stop blood flow (Ts). The effects of anticoagulants administered with t-PA were evaluated by percent stenosis of the vessel and percent area of the thrombus. Argatroban (1 – 3 mg/kg/bolus) significantly prolonged Ti and Ts in a dose-dependent fashion compared to control. Argatroban (3 mg/kg/bolus) significantly prolonged both the Ti and Ts more effectively than UFH (100 anti-XaU (a-XaU)/kg/bolus), despite equivalent prolongation of the activated partial thromboplastin time (aPTT). Higher doses of UFH (300 – 500 a-XaU/kg) were required to significantly prolong Ti and Ts, but at these doses, UFH caused over-prolongation of aPTT ( > 180 s), which might consequently cause bleeding complications. Argatroban (0.1 – 0.3 mg/kg/h) significantly accelerated thrombolysis by t-PA in both a dose- and time-dependent fashion. Although argatroban (0.1 – 0.2 mg/kg/h) did not significantly prolong the aPTT and bleeding time (BT) as compared with control, it significantly accelerated thrombolysis by t-PA at these doses of lower bleeding risk. Argatroban (0.3 mg/kg/h) significantly enhanced thrombolysis by t-PA, while UFH (12.5 anti-XaU/kg/h) attenuated it again, despite equivalent prolongation of the aPTT and BT. We conclude that argatroban seems to be a more efficient and safer anticoagulant than UFH for the prevention of thrombus formation and acceleration of tPA-induced thrombolysis. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: Thrombosis models; Argatroban; Unfractionated heparin (UFH); Tissue-type plasminogen activator (t-PA); Thrombus formation; Thrombolysis

1. Introduction

Abbreviations: UFH, unfractionated heparin; t-PA, tissue-type plasminogen activator; anti-XaU, a-XaU; Ti, the time required for the initiation of thrombus formation; Ts, the time required for the thrombus to stop blood flow; TIMI, thrombolysis in myocardial infarction; AT, antithrombin; MINT Trial, Myocardial Infarction with Novastan and TPA Trial; aPTT, activated partial thromboplastin time; BT, bleeding time; H1 – 4, the height of thrombus per hour; D0, the original vessel diameter; A1 – 4, the percent area of the thrombus per hour after t-PA infusion; A0, the original thrombus area; S.E., standard error; ANOVA, analysis of variance; GUSTO Trial, Global Utilization of Streptokinase and TPA for Occluded Coronary Arteries Trial; FPA, fibrinopeptide A; TAFI, thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin; TF, tissue factor. * Corresponding author. Tel.: +81-75-251-5511; fax: +81-75-251-5514. E-mail address: [email protected] (H. Tsuji).

Thrombolytic therapy with tissue-type plasminogen activator (t-PA) at present is limited by the relatively high incidence of reocclusion and resistance to reperfusion despite therapeutic heparinization [1]. There is a clinical need for more potent and less toxic agents because the optimal reperfusion rate [thrombolysis in myocardial infarction (TIMI) grade 3 flow] was only 54%, the time delay to reperfusion was 45– 120 min, the reocclusion rate after initial successful reperfusion was 5– 10%, and intracranial bleeding occurs in 0.9% of patients [2]. It has been suggested that thrombin bound to a clot (or fibrin) may play a critical role in resistance to thrombolysis and subsequent reocclusion [3]. Thrombin binds to fibrin via

0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00105-1

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Fig. 1. Schema for analysis of the thrombolytic process. Percent area of thrombus was calculated as the ratio between the area of thrombus per hour after t-PA infusion (A1 – 4) and the original area (A0). Percent stenosis of the lumen by thrombus was calculated as the ratio between the height of thrombus per hour (H1 – 4) and the original vessel diameter (D0).

the anion binding exosite, and clot-bound thrombin retains amidolytic activity. Clot-bound thrombin can lead to continued incorporation of fibrin into the thrombus and further growth of established thrombi despite lytic therapy. Unfractionated heparin (UFH) has been shown to be relatively ineffective for the inhibition of clot-bound thrombin because it is an indirect thrombin inhibitor requiring antithrombin (AT) and the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin [4,5]. However, clot-bound thrombin is not resistant to inhibition by AT-independent inhibitors such as argatroban, which is a direct thrombin inhibitor [6,7]. (2R,4R)-4-Methyl-1-[N2-(3methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]2-piperidine-carboxylic acid monohydrate (argatroban) is a

small molecule, synthetic direct thrombin inhibitor derived from L-arginine, which reversibly inhibits the active site of thrombin [8]. Argatroban is a highly selective thrombin inhibitor, which has no inhibitory activity against other serine proteases except at concentrations in excess of 100 times those required for thrombin inhibition [2,8,9]. Argatroban effectively inhibits all the physiologic ability of thrombin to cleave fibrinogen, factor XIII, and protein C, and to initiate platelet aggregation [10]. Structural studies have shown that argatroban binds tightly to thrombin by inserting dual hydrophobic constituents on its arginine backbone into clefts on thrombin that are close to its active site [11,12]. Thus, steric hindrance blocks thrombin’s physiologic substrates from access to its catalytic pocket [12,13]. Experimental thrombosis models have shown that argatroban enhances reperfusion by exogenous plasminogen activators [14] as compared with UFH. Argatroban enhances t-PA-mediated coronary reperfusion after acute myocardial infarction as documented in the Myocardial Infarction with Novastan and TPA (MINT) study [2]. Selective inhibition of thrombin seems to be an effective approach for the prevention of thrombus formation [15,16] and the acceleration of thrombolysis [14,17,18]. The aim of the present study was to evaluate the effect of argatroban as compared with UFH on thrombus formation and t-PA-induced thrombolysis in a microvascular thrombosis model.

Fig. 2. The time course of thrombus formation. Five minutes after the injection of fluorescent sodium (30 mg/kg) through the right subclavian artery, the venule measuring 40 Am in diameter was irradiated by filtered light (diameter, 100 Am; intensity, 10 mW/mm2; wave length, 400 – 500 nm) at the center of the venule until the developing thrombus stenosed the luminal area. The panels represent the time course of thrombus formation before (upper left), 1 min (upper right; Ti), 5 min (lower left), and 7 min (lower right; Ts) after starting irradiation. Ti=the time required for the initiation of thrombus formation, Ts=the time required for the thrombus to stop blood flow.

K. Yamada et al. / Thrombosis Research 109 (2003) 55–64

2. Materials and methods 2.1. Preparation of hamster cheek pouch Male golden hamsters with a body weight of 120 –130 g were anesthetized with pentobarbital sodium (100 mg/kg ip). Tracheotomy was performed to maintain adequate respiration. The right subclavian artery was catheterized with polyethylene tubing (PE10). The tubing was passed up to the orifice of the right common carotid artery. Placement of the tubing in this manner allowed the injection of pharmaceutical agents directly into the microvasculature of the right cheek pouch. In experiments of thrombolysis by t-PA with anticoagulants, the right femoral vein was also cannulated to allow drug administration. The microvasculature of the right cheek pouch was prepared for the experiment on a microscopic stage according to the method of Shanberge et al. [19]. The surface of the right cheek pouch was kept moist by the flow (1 ml/min) of warm (37 jC) bicarbonate buffer (131.9 M NaCl, 4.7 M KCl, 2.0 M CaCl2, 1.2 M MgSO4, 18.0 M NaHCO3, pH 7.4). The microscopic image was transmitted to a color monitor thorough a high-gain video camera (Flovel, Tokyo, Japan) and was recorded on videotape. Oral premedication including aspirin was not performed in these experiments. Maintenance of animals and experimental procedures were carried out in accordance with the guidelines of the Japanese Council on Animal Care. The experiments were approved by the Kyoto Prefectural University of Medicine Animal Care Committee. 2.2. Effects of anticoagulants on thrombus formation Five minutes after the injection of fluorescent sodium (30 mg/kg) and anticoagulants through the right subclavian artery, a selected venule measuring 40 Am in diameter was irradiated by filtered light (diameter, 100 Am; intensity, 10 mW/mm2; wave length, 400 – 500 nm) at the center of the venule until the developing thrombus stenosed the luminal area according to the method of Takada et al. [20,21]. The antithrombotic activities of argatroban (Mitsubishi Chemical Ind., Tokyo, Japan, 1, 2, 3 mg/kg bolus) and unfractionated heparin (100, 300, 500 anti-XaU (a-XaU)/kg bolus: Kabi Pharmacia, Uppsala, Sweden) were evaluated with respect to the time required for the initiation of thrombus formation (Ti: s) and the time to stop blood flow by thrombus (Ts: s) according to the method of Takada et al [20]. Control animals received a bolus injection of saline. Blood samples were obtained through the right atrium of the hamster 5 min after the injection of each medication. Measurement of activated partial thromboplastin time (aPTT) was performed on plasma using Platelin plus activator (Organon Teknika, North Carolina, USA).

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Bleeding time (BT) was measured on the shaved left medial hind leg before sampling the blood. Blood flowing from the incision was blotted with a filter disc at 30-s intervals without touching the injured site, and the time between incision and cessation of bleeding was determined as the bleeding time. 2.3. Effects of anticoagulants on t-PA-induced thrombolysis Five minutes after the injection of fluorescent sodium (50 mg/kg) through the right subclavian artery, a selected venule measuring 50 –70 Am in diameter was partially irradiated by filtered light (diameter, 100 Am; intensity, 20 mW/mm2; wave length, 400– 500 nm) until the developing thrombus stenosed 99% of the luminal area according to the method of Yoneda et al. [22]. Fifteen minutes after thrombus formation, t-PA (TD-2061; Toyobo, Tokyo, Japan, 72
104 IU/kg/ h) was administered continuously through the right subclavian artery in combination with either argatroban (0.05, 0.1, 0.2, and 0.3 mg/kg/h) or UFH (12.5 a-XaU/kg/h) through the right femoral vein. Control animals received a femoral infusion of saline. The recorded configuration of the thrombus was transferred to a computed image analyzer (XL-500; AVIONICS, Tokyo, Japan). The percent stenosis of the vascular lumen by the thrombus was calculated as the ratio between the height of thrombus per hour (H1 – 4) and

Table 1 Effects of argatroban and UFH on aPTT and thrombus formation Compound Dose

aPTT (s)

Saline

25.6F0.4

Argatroban 1 mg/kg 2 mg/kg 3 mg/kg

41.9F1.1** 79.3F1.8** 452.4F12.8* 51.1F1.0** 87.4F1.3** 509.4F11.2** 73.8F3.3**,*** 100.7F3.7**,z 602.6F26.8**,z

UFH

100 77.3F5.1** a-XaU/kg 300 >180** a-XaU/kg 500 >180** a-XaU/kg

Ti (s) 66.8F2.9

Ts (s) 405.9F9.8

69.1F2.0

417.6F12.3

85.0F4.3**

518.6F13.3**

116.7F3.7**

693.9F14.3**

Argatroban significantly prolonged Ti and Ts dose-dependently as compared with control. UFH given in doses of 300 and 500 a-XaU/kg significantly prolonged Ti and Ts dose-dependently as compared with control. However, the conventional therapeutic dose of UFH (100 a-XaU/ kg) did not prolong Ti and Ts significantly as compared with control. Argatroban significantly prolonged the aPTT dose-dependently as compared with control. UFH prolonged the aPTT at a dose of 100 a-XaU/kg by three times as much as control. UFH prolonged the aPTT to >180 s at doses of 300 and 500 a-XaU/kg. Argatroban (3 mg/kg) significantly inhibited Ti and Ts as compared with UFH (100 a-XaU/kg), despite equivalent prolongation of the aPTT. ns=not significant, anti-XaU=a-XaU. n=10. * P<0.05 vs. saline-treated group. ** P<0.01 vs. saline-treated group. *** ns vs. UFH (100 a-XaU/kg)-treated group. z P<0.01 vs. UFH (100 a-XaU/kg)-treated group.

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the original vessel diameter (D0). The percent area of the thrombus was calculated as the ratio between the area of thrombus per hour after t-PA infusion (A1 – 4) and the

original thrombus area (A0) (Fig. 1). The area of thrombus was measured by tracing the gray scale image with a cursor (planimetry).

Fig. 3. The time course of thrombus formation (A) and thrombolytic process (B) by t-PA. (A) The process of thrombus formation. Irradiation (diameter, 100 Am; intensity, 20 mW/mm2; wave length, 400 – 500 nm) was started on half of the venule (diameter, 70 Am) 5 min after injection of fluorescent sodium (50 mg/ kg). Two minutes later, initiation of thrombus was observed. Five-minute irradiation increased the size of thrombus to a semicircular shape. The panels represent the time course of thrombus formation immediately (upper left), 2 min (upper right), and 4 min (lower left) after starting irradiation. The lower right panel shows the completed thrombus with 99% stenosis 7 min after starting irradiation. (B) The process of thrombolysis by t-PA (72
104 IU/kg/h) before (upper left), 1 h (upper right), 2 h (lower left), and 4 h (lower right) after t-PA infusion. The mural thrombus showed intermittent reocclusion until 1 h after the infusion. At 2 h after infusion, thrombolysis had progressed and the thrombus occupied only 60% of the vascular lumen; at 4 h, very little thrombus remained.

K. Yamada et al. / Thrombosis Research 109 (2003) 55–64

2.4. Statistical analysis All values are expressed as meanFstandard error (S.E.) of two independent experiments. Statistical analysis was

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performed by analysis of variance (ANOVA), and Fisher’s protected least significant difference was used for individual comparisons. Values of p<0.05 were considered significant.

Fig. 4. Effect of argatroban on t-PA-induced thrombolysis. Argatroban (0.1 to 0.3 mg/kg/hr) in combination with t-PA significantly accelerated the decrease in percent stenosis and percent area of thrombus as compared with the t-PA-treated group. (n) control; ( ) t-PA (72
104 IU/kg/hr); t-PA+argatroban ( ) 0:05; ( ) 0.1; ( ) 0.2; ( ) 0.3 mg/kg/hr. *P<0.05, **P<0.01, t-PA: tissue-type plasminogen activator. n=4.

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3. Results 3.1. Effects of argatroban and UFH on inhibition of thrombus formation Representative figures of thrombus in order to assess Ti and Ts as described in Section 2 are shown in Fig. 2. Each panel shows the venule and thrombus before (upper left), 1 min (upper right), 5 min (lower left), and 7 min (lower right) after the beginning of irradiation. Thrombus began to be produced from the vessel wall by 1 min of irradiation (Ti). At 5 min, growth of thrombus was observed at the center of

the lumen, and at 7 min, the developing thrombus stenosed the luminal area (Ts). As shown in Table 1, argatroban significantly prolonged Ti and Ts in a dose-dependent fashion compared to control. However, conventional therapeutic doses of UFH (100 a-XaU/kg) did not show any significant prolongation, although UFH showed equivalent prolongation of the aPTT compared to the highest doses of argatroban. Higher doses of UFH (300 and 500 a-XaU/ kg) were required to significantly prolong Ti and Ts, but at these doses, UFH caused over-prolongation of the aPTT (>180 s).

Fig. 5. Effect of UFH on t-PA-induced thrombolysis. UFH in combination with t-PA delayed the decrease in percent stenosis and percent area of thrombus as compared with the t-PA treated group. (n) control; ( ) t-PA (72
104 IU/kg/hr); ( ) t-PA+UFH (12.5 a-XaU/kg/hr). *P<0.05. anti-XaU: a-XaU. n=4. *P<0.05, t-PA: tissue-type plasminogen activator, UFH: unfractionated heparin, anti-XaU: a-XaU.

K. Yamada et al. / Thrombosis Research 109 (2003) 55–64

3.2. Effects of argatroban and UFH on t-PA-induced thrombolysis Fig. 3A shows the representative thrombus produced to assess the thrombolytic processes. Each panel represents the time course of thrombus formation immediately (upper left), 2 min (upper right), and 4 min (lower left) after the beginning of irradiation. The lower right panel shows the final thrombus with 99% stenosis by 7 min after irradiation. The process of thrombolysis by t-PA (72
104 IU/kg/h) is shown in Fig. 3B: Each panel represents those prior to t-PA infusion (upper left), 1 h (upper right), 2 h (lower left), and 4 h (lower right) after t-PA infusion. Intermittent reocclusion was observed only until 1 h after infusion of t-PA. At 4 h, thrombolysis had markedly progressed and very little of the mural thrombus remained. Argatroban (0.1 – 0.3 mg/kg/h) significantly accelerated the decrease in both percent stenosis and percent area of thrombus induced by t-PA in both a dose- and timedependent fashion (Fig. 4). In the argatroban-treated group, a decrease in intermittent reocclusion was observed in the first hour after infusion of t-PA as compared to the t-PA-treated group. By contrast, a subtherapeutic dose of UFH (12.5 a-XaU/kg/h) attenuated the thrombolytic effects of t-PA (Fig. 5). In the UFH-treated group, an increase in intermittent reocclusion was observed in the hour after infusion of t-PA as compared to the t-PA-treated group. The effects of argatroban and UFH on aPTT and BT are summarized in Table 2 together with the data of tPA-induced thrombolysis. Although argatroban had a tendency to prolong the aPTT and BT, these were significantly greater than control only at the maximal dosage (0.3 mg/kg/h). Moreover, since prolongation of the BT by argatroban was not enhanced by t-PA, it can be speculated that argatroban will not promote hemorrhagic complications during thrombolytic therapy with t-PA. Argatroban (0.3 mg/kg/h) significantly accelerated the decrease in percent area of thrombus, while UFH (12.5 anti-XaU/kg/

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h) attenuated it again, despite equivalent prolongation of the aPTT and BT.

4. Discussion In this experimental model, a constant size of thrombus was produced in the microvasculature with excellent reproducibility by irradiation with filtered light and intravascular administration of fluorescent sodium [22]. Although the exact mechanism is not known, oxygen radicals were suggested to cause the injury to the endothelium and lead to thrombus formation [20]. By electron-microscopic observation, thrombus was composed of mostly spheroidal degranulated or pseudopod-formed platelets, endothelial cell membrane was partly ruptured, and basement membrane was exposed [20,21]. Thrombus formation in the microvessel was dependent on concentration of fluorescent dye and the light intensity [20,21]. So the irradiation was changed to make the suitable thrombus for each experiment. In an experiment like this, typically, the thrombus formed will dislodge and emboli will flow downstream, but the thrombus formed did not dislodge in the control situation. Sato [23] studied the differences in the growth pattern of the thrombus volume in venules and arterioles by using the filtered light and fluorescent dye in mesenteric microvessels of the rat. In arterioles, irregular growth curves of the thrombus volume and occasional detachment of the thrombus were seen. However, in venules, the smooth growth of the thrombus was seen. This result may suggest that flow condition could influence thrombus formation/embolization. Because of the above reason, we used venules instead of arterioles. Studies in healthy volunteers have demonstrated that argatroban (1 – 30 Ag/kg/min), with or without a bolus dose, increase the ratios of coagulation parameters (aPTT) in a similar, dose- and concentration-dependent manner with a predictable temporal response and a plateau between 1 and

Table 2 Effects of argatroban and UFH on aPTT, BT, and t-PA-induced thrombolysis Compound

Dose

Saline

t-PA ()

t-PA (+)

% Area of thrombus 1 h after t-PA infusion

aPTT (s)

BT (s)

BT (s)

28.8F0.4

204F6

210F13.4

93.2F2.5

Argatroban

0.1 mg/kg/h 0.2 mg/kg/h 0.3 mg/kg/h

30.7F0.8 31.6F0.4 34.6F2.1*,***

216F11 228F12 265F9**,***

222F7.3 235F9.2 270F7.8**

59.6F4.0** 52.5F6.9** 37.9F16.9**,z

UFH

12.5 a-XaU/kg/h

33.3F1.2**

252F7**

ND

79.4F9.5

Argatroban (0.1 – 0.3 mg/kg/h) prolonged the aPTT and BT dose-dependently and significantly at 0.3 mg/kg/h. Argatroban (0.1 – 0.3 mg/kg/h) with t-PA did not prolong BT more than with argatroban alone. Argatroban (0.3 mg/kg/h) significantly enhanced thrombolysis by t-PA, while UFH (12.5 anti-XaU/kg/h) attenuated it again, despite equivalent prolongation of the aPTT and BT. ns=not significant; ND=not determined; anti-XaU=a-XaU. n=4. * P<0.05 vs. saline-treated group. ** P<0.01 vs. saline-treated group. *** ns vs. UFH (100 a-XaU/kg)-treated group. z P<0.01 vs. UFH-treated group.

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3 h [24]. By other studies in hamsters, a strong linear correlation was observed between the antithrombotic effect of argatroban (1– 3 mg/kg/bolus) and prolongation of the aPTT [25]. These data suggest that aPTT could be useful to monitor therapeutic plasma levels of these thrombin inhibitors, and aPTT was used to compare the effects of anticoagulants in our study. Since thrombin is the key player in the formation of fibrin and in platelet aggregation during thrombus formation, it is essential to inhibit the action of thrombin to prevent and treat thrombotic disease. However, there is experimental evidence that thrombin bound to fibrin in vitro and in vivo is resistant to the heparin – AT complex [4,25]. Moreover, heparin is reported to be inhibited by fibrin monomer [26] and by platelet factor 4 [4,27] and thrombospondin [28]. Since heparin is also reported to activate platelets [29] and to be ineffective at inhibiting meizothrombin [30], platelet-rich thrombus is quite resistant to heparin. UFH also has the devastating side effect of the thrombocytopenia/thrombotic syndrome [31]. Thus, direct and specific inhibitors of thrombin have been developed as more potent and safer alternatives to heparin. The first specific thrombin inhibitor was argatroban, which is a synthesized arginine derivative with a molecular weight of 527 Da. With its high affinity to the reactive site of thrombin, argatroban is able to inhibit the action of thrombin even in the presence of fibrin binding. The present study was conducted to prove the superiority of the specific thrombin inhibitor over heparin at preventing thrombus formation and in preventing reocclusion during thrombolytic therapy. In the present study, although argatroban (3 mg/kg) and UFH (100 a-XaU/kg) showed equivalent effects on prolongation of the aPTT, the former significantly prolonged Ti and Ts while the latter did not. A much higher dose of UFH (500 a-XaU/kg) was necessary to cause similar effects on the Ti and Ts as compared to argatroban (3 mg/kg). However, at this high UFH dose, prolongation of the aPTT was beyond the acceptable range, which might consequently cause bleeding complications. Because of its relatively large molecules, UFH is poorly accessible to the thrombin molecules bound to fibrin. The reactive site of fibrin-bound thrombin is accessible to molecules of low molecular weight [18]. Our findings confirm that platelet-rich thrombus is resistant to UFH [16,32], and results indicate that although both argatroban and UFH inhibit platelet-rich thrombus formation, the antithrombotic effect of argatroban seems to be much stronger than UFH, correlating with prolongation of the aPTT without increased risk of hemorrhagic complications. Anticoagulants are thought to enhance the effects of thrombolysis by prevention of new fibrin formation and its incorporation into thrombus during fibrinolytic treatment [17]. Cercek et al. [33] showed that ancrod, a defibrinating agent, enhanced thrombolysis by streptokinase and urokinase by depleting fibrinogen and preventing new fibrin

formation. UFH is widely used to enhance the thrombolysis mediated by t-PA. However, reports indicate that it also interferes with the fibrinolytic system [5,6,17]. In order to achieve satisfactory acceleration of t-PA-induced thrombolysis, lower molecular weight and selective thrombin inhibitors are required. In our thrombosis model, Yoneda et al. [22,34] studied the differences according to dose of t-PA (Control: saline; Group I: 36
104; Group II: 72
104; and Group III: 108
104 U/kg/h). In comparison with controls, only Group II showed significant acceleration of thrombolysis without a significant reduction in fibrinolytic parameters. We therefore used 72
104 U/kg/h of t-PA for this experiment. In the clinical study of the Organization to Assess Strategies for Ischemic Syndromes (OASIS-2), a direct thrombin inhibitor, r-hirudin was superior to UFH in preventing cardiovascular death, myocardial infarction, and refractory angina [35]. Major bleeding episodes were more frequent in the hirudin group than in the heparin group (59 vs. 34; p=0.01), but there was no excess in life-threatening episode or stroke. In OASIS-2 study, early-onset thrombocytopenia may contribute to the increased risk of bleeding observed with hirudin [36]. In the MINT study, argatroban, as compared with heparin, appears to enhance reperfusion with t-PA in patient with AMI. Although not statistically significant, major and minor bleeding was lower in highdose argatroban patients as compared with heparin patients: 59.6% vs. 77.5% ( p=0.07). Other reports indicate that major bleeding occurred in 2.2% of argatroban-treated patients compared with 3.1% reported historically for UFH use during percutaneous coronary intervention [37]. Argatroban unlike hirudin forms fully reversible complexes with thrombin [37], and this fact may be related to a lower bleeding risk. In the MINT study, relatively low doses of argatroban were selected, which showed prolongation of the aPTT to 1.5 and 2.0 times baseline, similar to the Global Utilization of Streptokinase and TPA for Occluded Coronary Arteries (GUSTO) Trial IIa [38] and TIMI9A Trial [39]. In previous studies, there were suggestions that a low dose of direct thrombin inhibitor may be more effective than a high dose—the so-called ‘‘thrombin paradox’’ [40]. This fact can be explained by preservation of the thrombomodulin (TM) and protein C pathway. For these reasons, we chose relatively low doses of argatroban that prolonged the aPTT to 1.2 times baseline. The comparative doses of anticoagulants were chosen on the basis of a pilot experience, which showed prolongation of aPTT to about 1.2 times the base line. No bolus injection of anticoagulant was used, and a very low dose was infused because bleeding tendency occurred by using much more dose of anticoagulant or by adding bolus injection of anticoagulant especially around 4 h after the anticoagulant infusion. In our study, argatroban (0.1 – 0.3 mg/kg/h) significantly and dose-dependently enhanced the thrombolysis mediated by t-PA. Although argatroban (0.1 – 0.2 mg/kg/h) had no significant prolongation of the aPTT and BT as compared with control, arga-

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troban significantly enhanced the thrombolysis mediated by t-PA even at these doses of lower bleeding risk. Argatroban at 0.3 mg/kg/h significantly accelerated both the decrease in percent stenosis and percent area of thrombus as compared with the t-PA-treated group, while UFH at a dose of 12.5 aXaU/kg/h attenuated both. In the t-PA-treated group, the thrombolysis progressed with repeating reocclusion after reflow only until 1 h after infusion of t-PA, and persistent patency was observed thereafter. It is supposed that argatroban would decease this intermittent reocclusion by preventing new fibrin formation on the surface of thrombus, and UFH would increase the intermittent reocclusion by failing to prevent it compared with the t-PA-treated group. UFH was thus much less effective at inhibiting thrombin bound to fibrin. In vitro studies with 0.2– 0.4 U/ml of UFH, the therapeutic range, showed complete inhibition of fibrinopeptide A (FPA) release to free thrombin, while the same concentration of UFH produced only 20– 40% inhibition of clot-bound FPA generation [4]. In addition, a 20-fold higher concentration of UFH was required to produce equivalent inhibition of clot-bound FPA generation. In an in vitro thrombosis model, UFH (1 U/ml), when added prior to thrombus formation, considerably delayed t-PA-induced thrombolysis by inhibiting the binding of t-PA (17%, p<0.02) and plasminogen (88%, p<0.0005) to activated platelets [41]. Thus, UFH interferes with the fibrinolytic system by competing with fibrin for the active site on plasminogen and/or t-PA at the surface of activated platelets [42]. In an in vivo study, enhancement of saruplase-induced thrombolysis by UFH could be achieved only by a dose of UFH that prolonged the aPTT by 5-fold, while a lower dose of UFH with a 2.5-fold aPTT prolongation did not influence the thrombolysis [16]. In our experiments, a subtherapeutic dose of UFH is used, and this may have influenced the results. Our findings agree with those reports using lower doses. In humans, such a high dose would be considered outside the accepted therapeutic range in association with thrombolytic therapy. Moreover, UFH delayed the thrombolysis mediated by t-PA, whereas the synthetic thrombin inhibitors, argatroban significantly enhanced thrombolysis. There are other reports showing the superiority of direct thrombin inhibitors over UFH. Inhibition of thrombin by UFH depends largely upon enhancement of AT. The higher levels of thrombin during thrombus formation and thrombolysis are thought to deplete AT [17], under these circumstances, direct thrombin inhibitors would be more effective than UFH. Argatroban inhibits clot-incorporated thrombin effectively because it is more accessible to the clot-fluid interphase as a result of its low molecular weight and direct interaction with the active site of thrombin [43]. In vitro study demonstrated that argatroban dose-dependently accelerate fibrinolysis by the inhibition of thrombin-activatable fibrinolysis inhibitor (TAFI) activation in the presence of TM [44]. In this study, it is indicated that not only TM concentration but also tissue factor (TF) concentration is important for the inhibition of TAFI activation by argatro-

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ban. Plasma clot generated in the presence of argatroban is lysed more easily than one generated in the absence of argatroban because fibrin generated in the presence of argatroban contains more monomeric a-chains and decreased a-chain crosslinkage [14]. Argatroban also decreases the binding of a2-plasmin inhibitor to fibrin by inhibiting the generation of factor XIIIa, further promoting thrombolysis [14,45]. We conclude that argatroban seems to be a more efficient and safer anticoagulant than UFH for the prevention of thrombus formation and acceleration of t-PA-induced thrombolysis.

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