Covalent Linkage of Streptokinase to Recombinant Hirudin: A Novel Thrombolytic Agent with Antithrombotic Properties

Summary

In a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SK) was covalently bound to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to ¹²⁵I-rHir was accomplished via heterobifunctional crosslinkers in an average molar ratio of 1:1. The ¹²⁵I-rHir-SK complex was purified from starting components by anion exchange and gel filtration chromatography. The major band containing covalently bound ¹²⁵I-rHir had a molecular weight of 53 kDa as determined by SDS-PAGE and autoradiography. Biologic activity of each component was then assayed utilizing the chromogenic substrate for each compound. Complex bound ¹²⁵I-rHir exhibited a 1.2 fold decrease in thrombin inhibition when compared to concentrations of, ²⁵I-rHir greater than 3.13 nM. Complex bound ⁱ²⁵I-SK, replacing the ¹²⁵I label on rHir, displayed a 7.9-fold loss in plasminogen activation when compared to ^l25I-SK. These chromogenic assay results were not adversely altered in the presence of the converse compound’s substrate. The ¹²⁵I-SK-rHir complex (examined at various concentrations) also demonstrated a 0. 17- to 17-fold greater affinity for thrombin immobilized onto Sepha- rose beads as compared to ¹²⁵I-SK. These findings indicate the rHir-SK complex maintained both thrombolytic and antithrombin properties while also obtaining affinity for immobilized thrombin.

• References

• 1 Haber E, Quertermous T, Matsueda GR, Runge MS. Innovative approaches to plasminogen therapy. Science 1989; 243: 51-6
• 2 National Heart and Lung Cooperative Study Group Urokinase-streptokinase embolism trial: phase II results. JAMA 1974; 229: 1606-13
• 3 Elliot MS, Immelman EJ, Jeffery P, Benatar SR, Funston MR, Smith JA, Shepstone BJ, Ferguson AD, Jacobs P, Walker W, Louw JH. A comparative randomized trial of heparin versus streptokinase in the treatment of acute proximal venous thrombosis: An interim report of a prospective trial. Br J Surg 1979; 66: 838-43
• 4 Bode C, Runge M, Schonermark S, Eberle T, Newell JB, Kubler W, Haber E. Conjugation to antifibrin Fab’ enhances fibrinolytic potency of singlechain urokinase plasminogen activator. Circulation 1990; 81 (06) 1974-1980
• 5 Smith RAG, Dupe RJ, English PD, Green J. Fibrinolysis with acvlenzymes: a new approach to thrombolytic therapy. Nature 1981; 290: 505-8
• 6 Collen D, Dewerchin M, Rapold H, Lijnen H, Stassen J. 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 (05) 1744-53
• 7 Summaria L. The plasmin b-chain streptokinase complex. In: Thrombolytic Therapy Comerota AJ. (ed). Orlando FL: Grune and Stratton; 1988
• 8 Third International Study of Infarct Survival, ISIS 3 a randomised comparison of streptokinase versus tissue plasminogen activator versus anistre- plase and of aspirin plus heparin versus aspirin alone among 41,299 cases of suspected acute myocardial infarction. Lancet 1993; 339: 753-70
• 9 Lowry OH, Rosebrough NT, Farr AT, Randall RJ. Protein measurement with the folm phenol reagent. J Biol Chem 1951; 193: 265-75
• 10 Laemmli UK. Cleavage of structural proteins during the assemhly of the head of bacteriophage T4. Nature 1970; 227: 680-5
• 11 Bode C, Matsueda G, Hui K, Haber E. Antibody-directed urokinase: a specific fibrinolytic agent. Science 1985; 229: 765-7
• 12 Rutherford JD, Braunwald E. Thrombolytic therapy in acute myocardial infarction. Chest 1990; 97 (04) 136-45
• 13 White HD, Norris RM, Brown MA, Takayama M, Maslowski A, Bass NM, Ormiston JA, Whitlock T. Effect of intravenous streptokinase on left ventricular function and early survival after acute myocardial infarction. N Engl J Med 1987; 317 (14) 850-5
• 14 Francis CW, Marder VJ. Fibrinolytic therapy for venous thrombosis. Prog CardioDis 1991; 34 (03) 193-204
• 15 Molina JE, Hunter DW, Yedlicka JW, Cerra FB. Thrombolytic therapy for postoperative pulmonary embolism. Am J Surg 1992; 163: 375-80
• 16 Thrombolysis in Myocardial Infarction Study Group (TIMI Trials). The thrombolysis in myocardial infarction (TIMI) trial. N Engl J Med 1985; 312: 932-6
• 17 Thrombolysis and Angioplasty in Acute Myocardial Infarction Study Group (TAMI-2 Trials). Coronary thrombolysis with combined infusion of recombinant tissue-type plasminogen activator and urokinase in patients with acute myocardial infarction. Circulation 1988; 77: 1100-7
• 18 Graor R, Risius B, Denny K, Young JR, Beven EG, Hertzer NR, Ruschaupt III WF, O'Hara PJ, Geisinger MA, Zelch MG. Local thrombolysis in the treatment of thrombosed arteries bypass grafts and arteriovenous fistulas. J Vase Surg 1985 2 (03) 406-14
• 19 Sussman B, Dardik H, Ibrahim I, Mendes D, Kahn M. Improved patient selection for enzymatic lysis of peripheral arterial and graft occlusions. Am J Surg 1984; 148: 244-8
• 20 The Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico II (GISSI-2): a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet 1990; 336: 65-71
• 21 Sherry S, Marder VJ. Streptokinase and recombinant tissue plasminogen activator (rt-PA) are equally effective in treating acute myocardial infarction. Ann Intern Med 1991; 114 (05) 417-23
• 22 Topol EJ. Which thrombolytic agent should one choose?. Prog Cardiov Dis 1991; 34 (03) 165-78
• 23 The Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico II (GISSI-2). and The International Study Group (ISG). The risk of stroke with acute myocardial infarction after thrombolytic and antithrombotic treatment-GISSI-2 study. N Engl J Med 1992; 327 (01) 1-6
• 24 Intravenous Streptokinase in Acute Myocardial Infarction Study Group (ISAM Study Group). A prospective trial of intravenous streptokinase in acute myocardial infarction (ISAM). N Engl J Med 1986; 314 (23) 1465-71
• 25 Quinones-Baldrich WJ. Thrombolytic therapy for vascular diseases. In: Vascular Surgery: A Comprehensive Review Moore WS. (ed). W. B. Saunders Company Philadelphia; 1991: 237-61
• 26 Rosenchein U, Lenz R, Radnay J, Tovim TB, Rozenszajn LA. Streptokinase immunogenicity in thrombolytic therapy for acute myocardial infarction. Israel J Med Sci 1991; 27 (10) 541-5
• 27 Vaughan DE, Houtte EV, Declerck PJ, Collen D. Streptokinase-induced platelet aggregation. Circulation 1991; 84 (01) 84-91
• 28 Fenton JW. Regulation of thrombin generation and functions. Sem Thromb Hemost 1988; 14: 234-40
• 29 Hogg PJ, Jackson CM. Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: Implications for heparin efficacy. Proc Natl Acad Sci USA 1989; 86: 3619-23
• 30 Gulba D, Barthels M, Westhoff-Bleck M, Jost S, Rafflenbeul W, Daniel WG, Hecker H, Lichtlen PR. Increased thrombin levels during thrombolytic therapy in acute myocardial infarction. Circulation 1991; 83 (03) 937-44
• 31 Anderson H, Willerson J. Thrombolysis in acute myocardial infarction. N Engl J Med 1993; 329 (10) 703-9
• 32 Walz DA, Anderson GF, Fenton JW. Responses of aortic smooth muscle to thrombin and thrombin analogues. Ann NY Acad Sci 1986; 485: 323-34
• 33 Bar-Shavit R, Kahn A, Wilner G, Fenton J. Monocyte chemo-taxis: stimulation by specific exosite region in thrombin. Science 1983; 220: 728-31
• 34 Bizios R, Lai L, Fenton J, Malik A. Thrombin-induced chemotaxis and aggregation of neutrophils. J Cell Physiol 1986; 128 (03) 485-90
• 35 Kaplan JE, Moon DG, Weston LK, Minnear FL, Vecchio PJD, Shepard JM, Fenton JW. Platelets adhere to thrombin-treated endothelial cells in vitro. Am J Physiol 1989; 257: H423-H433
• 36 Tollefsen DM, Feagler JR, Majerus PW. The binding of thrombin to the surface of human platelets. J Biol Chem 1974; 249: 2646-51
• 37 The GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med 1993; 329 (10) 673-82
• 38 Dewerchin M, Lijnen H, Stassen J, De Cock F, Quertermous T, Ginsberg MH, Plow EF, Collen D. Effect of chemical conjugation of recombinant single-chain urokinase-type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo. Blood 1991; 78 (04) 1005-18
• 39 Markwardt F. Pharmacology of hirudin: One hundred years after the first report of the anticoagulant agent in medicinal leeches. Biochim Acta 1985; 44: 1007-13
• 40 Obberghen-Schilling EV, Perez-Rodriguez R, Pouyssegur J. Hirudin a probe to analyze the growth-promoting activity of thrombin in fibroblasts; reevaluation of the temporal action of competence factors. Biochem Biophys Res Comm 1982; 106: 79-86
• 41 Fenton JW, Bing DH. Thrombin active-site regions. Sem Thromb Hemost 1986; 12 (03) 200-8
• 42 Weitz J, Hudoba M, Massel D, Maganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin Ill-independent inhibitors. J Clin Invest 1990; 86: 385-91
• 43 Ito RK, Phaneuf MD, LoGerfo FW. Thrombin inhibition by covalently bound hirudin. Blood Coagulation and Fibrinolysis 1991; 2: 77-81
• 44 Rydel T, Ravichandran K, Tulinsky A, Bode W, Huber R, Roitsch C, Fenton JW. The structure of a complex of recombinant hirudin and human alpha thrombin. Science 1990; 249: 277-80