Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator Simone M. Flight a,b , Paul P. Masci a,b , Martin F. Lavin c and Patrick J. Gaffney d In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 W 1.4 and 2.9 W 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 W 6.3 and 93.3 W 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 W 3 h) compared with the human (3.5 W 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis. Blood Coagul Fibrinolysis 17:417– 420 ß 2006 Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2006, 17:417–420 Keywords: fibrinolysis, plasminogen activation, porcine, thromboelastography a School of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, b School of Molecular and Microbial Sciences, University of Queensland, Brisbane, c Queensland Institute of Medical Research, Radiation Biology and Oncology, Royal Brisbane Hospital, Brisbane, Australia and d Academic Department of Surgery, St Thomas’ Hospital, London, UK Correspondence and requests for reprints to Simone Flight, R-Wing, School of Medicine, Princess Alexandra Hospital, Brisbane, QLD 4102, Australia Tel: +61 7 3240 2051; fax: +61 7 3240 5063; e-mail: sflight@soms.uq.edu.au Received 4 January 2006 Accepted 10 March 2006 Introduction Plasmin is one of the main enzymatic mediators of fibrinolysis [1,2], catalyzing the cleavage of insoluble fibrin and thereby accelerating the lysis of a thrombus. The proenzyme precursor of plasmin is plasminogen (Plg), a 90-kDa single-chain glycoprotein that is synthe- sized mainly in the liver and circulates in the plasma at a concentration of approximately 2 mmol/l [3]. Plasmin catalyzes the conversion of Glu-Plg to Lys-Plg through the cleavage of bonds at –K62, –R68 and –K77 [4–7] and to a greater extent when bound to cell surfaces [8], which is then converted to two-chain plasmin through the cleavage of the R561–V562 peptide bond. This conver- sion to Lys-Plg is a significant but not essential step in the activation of Glu-Plg in fibrinolysis. The two enzymes that cleave this bond under physiological conditions are tissue plasminogen activator (t-PA) and urokinase [9]. While urokinase activation of plasminogen requires no cofactors, activation with t-PA requires the presence of a surface and, under physiologic conditions, this surface is fibrin [10]. Pigs are frequently used to assess the therapeutic effects of compounds in clinical conditions that involve disrup- tion of the coagulation, fibrinolysis and the hemodynamic response. These involve injury [11–13], surgical pro- cedures [14–16] and chronic disease conditions such as arteriosclerosis [17]. Accordingly, it is imperative that the coagulation and fibrinolytic systems of the pig should approximately mimic those of the human. It has been reported [18] that pig plasminogen is resistant to acti- vation by urokinase and that this resistance is related to differing conformations between human and porcine plasminogen. Should it be confirmed that bleeding during surgery is mainly associated with plasmin- mediated degradation of clots and coagulation factors [1] then the value of the pig as a model of hemorrhage would need to be reassessed. This comparative study investigates the human recombinant t-PA (rt-PA)-stimu- lated lysis of porcine and human whole blood clots in the thrombelastograph and compares the relative effects of endogenous t-PA using the euglobulin clot lysis time (ECLT) assay for both human and porcine plasmas. Materials and methods Materials rt-PA was obtained from Boehringer Ingelheim (Ingel- heim, Germany) as Actilyse. Citrated porcine whole blood and plasma were obtained from the Herston Medical Research Centre, Brisbane, Australia. Citrated human blood and plasma was obtained from the Pathol- ogy unit at Princess Alexandra Hospital, Brisbane. Lysine-Sepharose affinity resin was purchased from Amersham Biosciences (Uppsala, Sweden). Bovine thrombin was purchased from Sigma Aldrich (St Louis, Missouri, USA). Short communication 417 0957-5235 ß 2006 Lippincott Williams & Wilkins