| Intravascular thrombosis is one of the main causes of a wide variety ofcardiovascular diseases. The agents currently used in the clinics included tissue-typeplasminogen activator (t-PA), streptokinase, urokinase and their variants. Althoughthe thrombolytic agents effectively reduced the mortality of thromboembolicdiseases, they had such shortcomings as incomplete recanalization, delayedreperfusion time, reocclusion, bleeding and allergic reaction etc. Staphylokinase (Sak)is a promising thrombolytic agent. It is at least equipotent to t-PA for coronary arteryrecanalization, significantly more fibrin selective and easier to be expressed inEscherichia coli cytoplasm in high yield. It is understood that platelet aggregationplays an important role in the development of thrombus and depends on the presenceof membrane glycoprotein GPIIb/IIIa of platelet surface. Based on the tripeptidearginine-glycine-asparatic acid (RGD) sequence, many GPIIb/IIIa antagonists weredesigned and developed. In order to develop novel thrombolytic agent with fibrinolytic activity,antiplatelet activity and reduced immunogenicity, we designed rational Sak variantsusing structure-function information determined by computer modeling, based on thecrystal structure of staphylokinase monomer and the ternary complex ofmicroplasmin?staphylokianse?microplasmin. Thus RGD motif was grafted into theappropriate sites of staphylokinase molecule. The mutated genes were amplified byPCR on the template of pST-SAK and inserted into expression vector pLY-4. Therecombinant expression plasmids were transformed Escherichia coli and the variantswere expressed, purified and assayed for fibrinolytic activity, kinectics constants,antiplatelet activity and immunogenicity. Two variants, designated DGR and RL1,remarkably inhibited platelet aggregation and suffered no loss of fibrinolytic activity.Morever, the immunogenicity of these two variants was greatly reduced. A rapid, effective and simple strategy for pilot production of DGR wasestablished using selected strain with high expression level. Nearly 8 g wet bacteriawere harvested with low-density fermentation and over 60% expression level was 3DOCTORAL DISSERTATION ABSTRACTachieved. The protein of interest was then extracted by cell disruption, concentratedby ultrafiltration and purified by gel filtration and ion-exchange chromatography.The final product had a purity of over 97% and a specific activity of 11.5×104HU/mg. The yield of DGR reached 290 mg per liter culture. The molecular weight ofDGR was 15,455 and its isoelectric point was pH 6.6. The pharmacodynamics of DGR was evaluated for the thrombolytic efficacy,antithrombotic effect, antiplatelet aggregation activity and influences upon bloodcoagulation in vivo and compared with that of r-Sak. A rabbit thrombosis model wasestablished by producing a blood clot in an isolated femoral artery segment withendothelial damage and distal stenosis. 21 New Zealand white rabbits wererandomized into 4 groups treated with saline(n=3), r-Sak(0.3mg/kg, n=6), low doseof DGR (0.15 mg/kg, n=6) and high dose of DGR(0.3 mg/kg, n=6) respectively.Total dose was administrated by 4 repeated bolus injections via ear marginal vein,with a time interval of 10min, and the femoral blood flow was observed for 2 h.Arterial blood samples were collected at 0min, 60min and 120 min afteradministration. It was shown that 0.15mg/kg of DGR had the equipotentthrombolytic efficacy to that of 0.3 mg/kg of r-Sak while 0.3 mg/kg of DGRachieved shorter initial reperfusion time and longer total patency time. Reocclusionoccured in 50% of rabbits given wild type r-Sak(3/6) while no reocclusion wasobserved in 0.15 mg/kg or 0.30 mg/kg DGR groups. Platelet aggregation wasinhibited by 30%, 20% respctively at 60min and 120min in high-dose DGR groupand by 21%, 14% re... |
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