Purification And Characterization Of NJP And Pharmacodynamics Study Of NJF

Thrombotic disease (TD) including thrombosis and thromboembolism. Thrombotic disease is the largest cause of mortality in human life; it not only ranks the highest incidence of various diseases, and has a high morbidity, mortality and recurrence rate. Currently, thrombolytic therapy is the the most effective method for clinical treatment for thrombotic disease. During the past decade, thrombolytic agents such as urokinase (UK), streptokinase (SK), staphylokinase (SaK), tissue-type plasminogen activator (t-PA). snake venom fibrinolytic enzymes (Batroxobin) and lumbrokinase (LK) have been widely used in the treatment of thrombosis. However, all these enzymes have undesired side effects, including need of large therapeutic doses, limited fibrin-specificity, re-occlusion and bleeding tendency. Therefore, the search for other thrombolytic agents with high specificity for fibrin, low bleeding tendency and relatively inexpensive from various nature sources are always the hot topic of the research aeras.Neanthes japonica (Izuka) is a marine invertebrates, which widely distributed in the Bohai Sea, Yellow Sea and the Yangtze River Estuary, it also specific to the Pacific coast of Japan. Nowadays, N. japonica is usually used as high-quality fish foods and marine fishing bait. A new serine protease named Neanthes japonica fibrinolytic enzyme (NJF) has been isolated from N. japonica by our laboratory. During the purification steps, we found another fraction named N. japonica protease (NJP) with specific fibrinolytic activity and characteristics, which might be developed into a novel thrombolytic agent, to achieve their most economic and social values. This thesis consist of two parts, the first part includes the first three chapters, which are about the process establishment of the separation, purification, the measurements of molecular characteristics and enzymatic properties of N. japonica protease (NJP); The second part includes the fourth chapter, which is about the in vivo pharmacodynamics experiments of NJF. Now the main results are presented as follows:1. By combination of crude enzyme extraction, ammonium sulfate fractionation, Phenyl Sepharose hydrophobic chromatography, anion-exchange chromatography and gel filtration, we obtained a single chain protein with high purity, named N. japonica protease (NJP). NJP was purified 1556-fold with a final yield of 13% after five purification steps. When S-2238 was used as the specific substrate, the specific enzyme activity of NJP was 10113.9 U/mg according to chromogenic substrate assay.2. The molecular weight of NJP was about 28.6-33.5 kDa according to MALDI-TOF MS and SDS-PAGE, which indicated that NJP was a single chain protein; the isoelectric point of NJP determined by two-dimensional electrophoresis (2-DE) was 9.2.3. We have obtained 3 peptides sequences of 28 amino acids by MALDI-TOF/TOF MS and De Novo Sequencing. The three peptides were presented as follows:VTVVQYR; STNASSGYLNLR and VYLLDTGLR. The homology with the amino acid sequences of NJP were searched through the non-redundant protein sequences (nr) and Swiss-Prot databases by using NCBI blastp program, the results indicated that NJP is a novel discovered protease;This protein sequence data will appear in the UniProt Knowledgebase under the accession number(s) P86834, EC 3.4.21.4. Azocasein was used as the substrate in the assay of the effects of temperature and pH on the protease activity of NJP. The optimum temperature was at 40℃, the enzyme activity was stable between 30℃and 60℃; The optimum pH was found to be 9.0, NJP showed at least 80% of the maximum activity over a pH range from pH 7.0 to 11.0, which indicated that NJP is an alkaline protease.5. The protease activity of NJP was completely inhibited by Hg2+ion according to azocasein asssy, and slightly inhibited by Co2+and Mg2+ions. However, other metal ions had no obvious effects on the inhibition or activation of the enzyme activity. 6. The protease activity of NJP was completely inhibited by the typical serine protease inhibitor PMSF using azocasein as a substrate, while other selected protease inhibitors were not effective or had weak inhibitory effects on NJP. These results suggest that NJF is a unique serine protease.7. The fibrinolytic activity was assayed using the fibrin plate method, the results indicated that NJP could directly degrade fibrin, it had none kinase activity and it was not a plasminogen activator; NJP had stronger fibrinolytic activity compared with UK, the fibrinolytic activity of NJP was about 12000 U/mg proteins.8. The fibrinogenolytic activity of NJP was analyzed by SDS-PAGE. The results indicated that the hydrolytic pattern of NJP on fibrinogen started from the Aa-chain, followed by the Bβ-chain, and the y-chain at last.9. NJP showed a higher degree of specificity for the substrate S-2238 for thrombin according to chromogenic substrates methods, the amidolytic activity was 10.11+2.7 mmol/min/mg; these above results suggest that the newly discovered NJP is an alkaline thrombin-like serine protease.10. The fibrinolytic activity of NJF was assayed using the fibrin plate method. The fibrinolytic activity of NJF was about 30000 U/mg proteins when UK was used as a control. We defined the fibrinolytic activity of NJF as 10 BU/mg.11. In this study, the rat middle cerebral artery occlusion (MCAO) model was successfully produced by intraluminal suture method.12. According to the results of neurological deficit scores and TTC stained, we can came to the conclusions that intravenous treatment of NJF could dose dependently reduce the cerebral infarction with significantly decreased the neurological deficit scores.13. Ischemia/reperfusion induced cerebral edema was reduced in a dose dependent manner after intravenous treatment of NJF.14. Intravenous treatment of NJF could significantly reduce the MDA levels and increase the SOD activities by attenuating lipid peroxidation and increasing endogenous antioxidation.15. Intravenous treatment with NJF at 5 BU/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema, lipid peroxidation and antioxidation; NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia; The neuroprotection mechanism shown by NJF may be attributed to inhibition of lipid peroxidation, increase endogenous antioxidant defense enzymes.In conclusion, we have purified and characterized a novel protease (NJP) from Neanthes japonica (Izuka) which was clearly different from the previous isolated NJF and other fibrinolytic enzymes. NJP is an alkaline thrombin-like serine protease with much higher fibrinolytic activity. It can directly degrade fibrin distinct from the plasminogen activators. Taking account of these results, NJP might be a potential candidate for the thrombosis prevention and thrombolytic therapy; meanwhile, this work is the first evidence that NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia, which will provide a basis for further researches about pharmacodynamics and clinical trials of NJF application.