557 © 2002 Schattauer GmbH, Stuttgart Thromb Haemost 2002; 87: 557–62 Local proCPU (TAFI) Activation during Thrombolytic Treatment in a Dog Model of Coronary Artery Thrombosis can be Inhibited with a Direct, Small Molecule Thrombin Inhibitor (Melagatran) C. Mattsson 1 , J. A. Björkman 1 , T. Abrahamsson 1 , V. Nerme 1 , K. Schatteman 2 , J. Leurs 2 , S. Scharpé 2 , D. Hendriks 2 1 AstraZeneca, Mölndal, Sweden; 2 Laboratory of Medical Biochemistry, University of Antwerp, Belgium Keywords TAFI, thrombolysis, thrombin inhibitor, melagatran, CPU assay Summary To test the hypothesis that the direct thrombin inhibitor, melagatran is able to inhibit local pro-carboxypeptidase U (proCPU) activation that occurs during thrombolytic treatment, t-PA alone, or in combination with melagatran, was given to dogs with a coronary artery thrombosis. Blood samples from the great cardiac vein and aorta were collected at baseline, during thrombus formation, throughout the t-PA±melagatran infusion and during the patency period, for analysis of CPU activity using a novel assay. A higher CPU activity in venous compared to arterial blood (V-A difference) indicates CPU activation in coronary vessels. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Dogs (n = 26) were randomized to receive either 1) t-PA, 1 mg/kg as an intravenous 20-min infusion; 2) t-PA as in group 1,+ melagatran bolus, 0.3 mg/kg, followed by a 3-h infusion (0.15 mg/kg per h); 3) sham-operated but no coronary thrombus, and administered t-PA as for Group 1. All groups had similar baseline characteristics. Significant increases in CPU activity were observed in Groups 1 and 2 during thrombus formation, with V-A differences of 5.5 and 4.5 U/L, respectively. No significant V-A difference was observed in the sham-operated group. CPU activity increased in Group 1 during the t-PA infusion (V-A difference 15.9 U/L), whereas the V-A difference in Group 2 decreased to 2.6 U/L following melagatran treatment. These results demonstrate that melagatran attenuates generation of CPU in the coronary circulation. The mechanism is probably indirect, via inhibition of thrombin-mediated activation of proCPU. Introduction Carboxypeptidase U (CPU, EC 3.4.17.20) is a basic carboxypeptidase, which circulates in plasma as the zymogen proCPU (1, 2). This zymogen is also known as thrombin activatable fibrinolysis inhibitor (TAFI) or plasma pro-carboxypeptidase B (3). Upon activation by thrombin, the thrombin/thrombomodulin complex or plasmin, CPU is able to prolong the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on the thrombus (fibrin) surface, thereby reducing the number of plasminogen binding sites and subsequently the level of plasmin production (4). Clot lysis experiments in vitro have demonstrated that lysis time increases with increasing proCPU concentrations (3) and that inhibitors of CPU (5, 6) or inhibition of proCPU activation (5) facilitate clot lysis. Redlitz et al. (7) showed in a dog model of coronary artery thrombosis that thrombus formation and subsequent lysis with tissue type plasminogen activator (t-PA) led to the appearance of a carboxypeptidase activity in plasma. The impact of a CPU inhibitor on t-PA-induced thrombolysis in vivo has also been studied by other investigators (8, 9) who found a two- to three-fold potentiation of clot lysis and a shorter time to restore blood flow when combining t-PA with a CPU inhibitor. It has also been shown, both in porcine as well as in canine models of coronary artery thrombosis, that small, direct thrombin inhibitors are able to improve the thrombolytic efficacy of t-PA (10–12). This led us to the hypothesis that the pro-fibrinolytic effects of direct thrombin inhibitors may, at least partly, be due to an inhibition of thrombin- mediated activation of proCPU. The aim of the present study was to investigate the effects of melagatran, a reversible, direct thrombin inhibitor, on the generation of active CPU in coronary vessels using a dog model of coronary artery thrombosis. Methods Drugs t-PA (Actilyse ® ) was purchased from Boehringer Ingelheim (Germany) and dissolved in sterile water to a concentration of 1 mg/ml. Melagatran, a reversible inhibitor of thrombin [inhibition constant (K i ) 2 nmol/L] (13) with a molecular mass of 429.5 dalton, was synthesized by AstraZeneca (Södertälje, Sweden). PPACK and aprotinin were purchased from Alexis Biochemicals (Läufelfingen, Switzerland) and Sigma, (St. Louis, MO, USA), respectively. Correspondence to: Christer Mattsson, Department of Cell Biology and Bio- chemistry AstraZeneca R&D, S-431 83 Mölndal, Sweden – Tel.: +46 31 776 22 28; Fax: +46 31 776 37 36; E-mail: Christer.Mattsson@astrazeneca.com In Focus For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-05-06 | ID: 1001066444 | IP: 54.70.40.11