Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots

Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis. Current clinically approved thrombolytic agents have significant drawbacks including reocclusion and bleeding complications. To address these problems, a staphylokinase-based thrombolytic agent equipped with antithrombotic activity from hirudin was engineered. Because the N termini for both staphylokinase and hirudin are required for their activities, a Y-shaped molecule is generated using engineered coiled-coil sequences as the heterodimerization domain. This agent, designated HE-SAKK, was produced and assembled from Bacillus subtilis via secretion using an optimized co-cultivation approach. After a simple in vitro treatment to reshuffle the disulfide bonds of hirudin, both staphylokinase and hirudin in HE-SAKK showed biological activities comparable with their parent molecules. This agent was capable of targeting thrombin-rich fibrin clots and inhibiting clot-bound thrombin activity. The time required for lysing 50% of fibrin clot in the absence or presence of fibrinogen was shortened 21 and 30%, respectively, with HE-SAKK in comparison with staphylokinase. In plasma clot studies, the HE-SAKK concentration required to achieve a comparable 50% clot lysis time was at least 12 times less than that of staphylokinase. Therefore, HE-SAKK is a promising thrombolytic agent with the capability to target thrombin-rich fibrin clots and to minimize clot reformation during fibrinolysis. Acute myocardial infarction (also known as heart attack) is a leading cause of death in the Western world. It is commonly caused by the formation of a pathologic clot (thrombus) at a critical position that results in obstructing the blood flow to heart tissues. The use of blood clot dissolving agents is one of the well established methods in treating patients with acute myocardial infarction (1The International Study GroupLancet. 1990; 336: 71-75Abstract PubMed Scopus (522) Google Scholar, 2Ohman E.M. Harrington R.A. Cannon C.P. Agnelli G. Cairns J.A. Kennedy J.W. Chest. 2001; 119: 253S-277SAbstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Results from several large scale clinical trials have firmly established the effectiveness of this approach in saving lives (1The International Study GroupLancet. 1990; 336: 71-75Abstract PubMed Scopus (522) Google Scholar, 2Ohman E.M. Harrington R.A. Cannon C.P. Agnelli G. Cairns J.A. Kennedy J.W. Chest. 2001; 119: 253S-277SAbstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 3The GUSTO InvestigatorsN. Engl. J. Med. 1993; 329: 673-682Crossref PubMed Scopus (3706) Google Scholar). Among the clinically approved blood clot dissolving agents including streptokinase, anisoylated plasminogen streptokinase activator complex (or anistreplase), tissue-specific plasminogen activator (tPA), 1The abbreviations used are: tPA, tissue-specific plasminogen activator; APSAC, anisoylated plasminogen streptokinase activator complex; HBS, HEPES-buffered saline; HE, hirudin-E coil; HE-SAKK, hirudin-E coil-staphylokinase-K coil heterodimer; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; SAK, staphylokinase; SAKK, staphylokinase-K coil; T 50%, time required for 50% clot lysis. and urokinase, tPA is the most commonly used blood clot dissolving agent in the Western world. Even though tPA is fibrin-specific, its short in vivo biological half-life and sensitivity to plasminogen activator inhibitors in circulation require the use of higher doses of tPA for effective clot lysis. At the pharmacological doses used, tPA exerts only partial fibrin specificity. This results in depletion of plasma proteins such as coagulation factors V and VIII, and to a certain degree, plasminogen and fibrinogen. Approximately 57% of the patients treated with tPA can restore their blood flow to an acceptable level within 90 min after receiving the treatment (2Ohman E.M. Harrington R.A. Cannon C.P. Agnelli G. Cairns J.A. Kennedy J.W. Chest. 2001; 119: 253S-277SAbstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Within this group, 10–30% of patients showed reocclusion shortly after clot dissolution. The reformed secondary clots are usually platelet-rich (4Jang I.K. Gold H.K. Ziskind A.A. Fallon J.T. Holt R.E. J.W. PubMed Scopus Google Scholar, Google Scholar, Gold H.K. I.K. Holt Fallon J.T. J. 1990; PubMed Scopus Google and to lysis by tPA Gold H.K. I.K. Holt Fallon J.T. J. 1990; PubMed Scopus Google Scholar). a significant of the patients from GUSTO InvestigatorsN. Engl. J. Med. 1993; 329: 673-682Crossref PubMed Scopus (3706) Google Scholar, International Study of GroupLancet. PubMed Scopus Google Scholar). To address these in the thrombolytic the of a to target thrombin-rich to the clot lysis At the the hirudin can minimize clot reformation during the of is a promising blood clot dissolving agent Med. PubMed Scopus Google Scholar). to can the fibrin of by a The complex can as the plasminogen activator to plasminogen to for clot lysis. comparable thrombolytic as tPA, several clinical trials that staphylokinase a fibrin in comparison to tPA PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). 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