A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant

Summary

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAKRGD- K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir.The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg.There were no observable changes in plasma fibrinogen concentration.In conclusion,our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats.Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGDK2- Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAKRGD- K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Keywords

Recombinant protein - antiplatelet activity - thrombolytic agent - arterial thrombosis - venous thrombosis

• References

• 1 Ueshima S, Matsuno H, Hayashi M. et al Function of tissue-type plasminogen activator releaser on vascular endothelial cells and thrombolysis in vivo. Thromb Haemost 2002; 87: 1069-1074.
• 2 Collen D, Lijnen HR. Thrombolytic agents. Thromb Haemost 2005; 93: 627-628.
• 3 Van de Werf FJ. The ideal fibrinolytic: can drug design improve clinical results?. Eur Heart J 1999; 20: 1452-1458.
• 4 Baruah DB, Dash RN, Chaudhari MR. et al Plasminogen activators: a comparison. Vascul Pharmacol 2006; 44: 1-9.
• 5 Vanderschueren S, Collen D, van de Werf F. A pilot study on bolus administration of recombinant staphylokinase for coronary artery thrombolysis. Thromb Haemost 1996; 76: 541-544.
• 6 Rozponczyk E, Szemraj J, Malinowski M. et al Staphylokinase-a specific plasminogen activator. Postepy Biochem 2006; 52: 80-86.
• 7 Moreadith RW, Collen D. Clinical development of PEGylated recombinant staphylokinase (PEG-Sak) for bolus thrombolytic treatment of patients with acute myocardial infarction. Adv Drug Deliv Rev 2003; 55: 1337-1345.
• 8 Armstrong PW, Burton J, Pakola S. et al CAPTORS II Investigators. Collaborative Angiographic Patency Trial Of Recombinant Staphylokinase (CAPTORS II). Am Heart J 2003; 146: 484-488.
• 9 Ueshima S, Matsuo O. Development of new fibrinolytic agents. Curr Pharm Des 2006; 12: 849-857.
• 10 Collen D, Sinnaeve P, Demarsin E. et al Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction. Circulation 2000; 102: 1766-1772.
• 11 Leadley Jr RJ , Kasiewski CJ, Bostwick JS. et al Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery. Thromb Haemost 1997; 78: 1278-1285.
• 12 Welsh RC, Armstrong PW. A marriage of enhancement: fibrinolysis and conjunctive therapy. Thromb Haemost 2004; 92: 1194-1200.
• 13 van Zyl WB, Pretorius GH, Lamprecht S. et al PLATSAK, a potent antithrombotic and fibrinolytic protein, inhibits arterial and venous thrombosis in a baboon model. Thromb Res 2000; 98: 435-443.
• 14 Lu HR, Wu Z, Pauwels P. et al Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog. J Am Coll Cardiol 1992; 19: 1350-1359.
• 15 Schneider J, Hauser R, Hennies HH. et al A novel chimaeric derivative of saruplase, rscu-PA-40 kDA/ Hir, binds to thrombin and exerts thrombus-specific fibrinolysis in arterial and venous thrombosis in dogs. Thromb Haemost 1997; 77: 535-539.
• 16 Szemraj J, Walkowiak B, Kawecka I. et al A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity–a staphylokinase variant. I. In vitro study. J Thromb Haemost 2005; 3: 2156-2165.
• 17 Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost 1987; 58: 1078-1084.
• 18 Schumacher WA, Steinbacher TE, Heran CL. et al Effects of antithrombotic drugs in a rat model of aspirin- insensitive arterial thrombosis. Thromb Haemost 1993; 69: 509-514.
• 19 Guarini S. A highly reproducible model of arterial thrombosis in rats. J Pharmacol Toxicol Methods 1996; 35: 101-105.
• 20 Kawasaki T, Sato K, Hirayama F. et al Effect of a synthetic factor Xa inhibitor, YM-60828, on blood vessel patency in combination with a thrombolytic agent and on blood loss from the operation site in a rat model of arterial thrombosis. Thromb Haemost 1998; 79: 859-864.
• 21 Barbanti M, Calanni F, Milani MR. et al Therapeutic effect of a low molecular weight dermatan sulphate (Desmin 370) in rat venous thrombosis–evidence for an anticoagulant–independent mechanism. Thromb Haemost 1993; 69: 147-151.
• 22 Dejana E, Villa S, de Gaetano G. Bleeding time in rats: A comparison of different experimental conditions. Thromb Haemost 1982; 48: 108-111.
• 23 Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 1990; 40: 198-201.
• 24 Hollander M, Wolfe DA. Nonparametric statistical methods. New York: Wiley; 1973: 115-123.
• 25 Yamamoto J, Kawano M, Hashimoto M. et al Adjuvant effect of antibodies against von Willebrand factor, fibrinogen, and fibronectin on staphylokinase-induced thrombolysis as measured using mural thrombi formed in rat mesenteric venules. Thromb Res 2000; 97: 327-333.
• 26 Dewerchin M, Vandamme AM, Holvoet P. et al Thrombolytic and pharmacokinetic properties of a recombinant chimeric plasminogen activator consisting of a fibrin fragment D-dimer specific humanized monoclonal antibody and a truncated single-chain urokinase. Thromb Haemost 1992; 68: 170-179.
• 27 Collen D, Dewerchin M, Rapold HJ. et al Thrombolytic and pharmacokinetic properties of a conjugate of recombinant single-chain urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin in a baboon venous thrombosis model. Circulation 1990; 82: 1744-1753.
• 28 Collen D, Stassen JM, Demarsin E. et al Pharmacokinetics and thrombolytic properties of chimaeric plasminogen activators consisting of the NH2 -terminal region of human tissue-type plasminogen activator and the COOH-terminal region of human single chain urokinase-type plasminogen activator. J Vasc Med Biol 1989; 1: 234-240.
• 29 Mogielnicki A, Chabielska E, Pawlak R. et al Angiotensin II enhances thrombosis development in renovascular hypertensive rats. Thromb Haemost 2005; 93: 1069-1076.
• 30 Jang IK, Gold HK, Ziskind AA. et al Differential sensitivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator. A possible explanation for resistance to coronary thrombolysis. Circulation 1989; 79: 920-928.
• 31 Yasuda T, Gold HK, Leinbach RC. et al Lysis of plasminogen activator-resistant platelet-rich coronary artery thrombus with combined bolus injection of recombinant tissue-type plasminogen activator and antiplatelet GPIIb/IIIa antibody. J Am Coll Cardiol 1990; 16: 1728-1735.