Purification And Characterization Of A Novel Fibrinolytic Enzyme From Cordyceps Militaris And Studies On Its Antithrombotic And Thrombolytic Activities

Cardiovascular diseases,including acute myocardial infarction,valvular heart disease, peripheral vascular disease,arrhythmias,intimidates mankind's life and health seriously. Heart diseases are responsible for 29%of the total mortality rate in the world,according to data provided by the World Health Organization(WHO) in 2000.The fibrinolytic agents available today for clinical use are thrombolytic drugs such as urokinase,the bacterial plasminogen activator streptokinase,and rt-PA;anticoagulants and Antithrombotic drugs.Despite their widespread use,all these agents have undesired side effects,exhibit low specificity for fibrin,and are also relatively expensive.Therefore,the searches for other fibrinolytic enzymes from various sources are being continued.The Cordyceps species,one of the famous Chinese traditional medicinal mushrooms, are entomogenous fungi belonging to Clavicipitaceae and Ascomycotina.In Chinese herbs Cordyceps species are generally called as "Dong-Chong-Xia-Cao",meaning "winter-worm and summer-grass".Recently,the fungus Cordyceps militaris,also called north cordyceps,has drawn attention as a traditional Chinese medicine due to its various biological and pharmacological activities.During the past several decades,many kinds of bioactive constituents from Cordyceps species have been isolated and characterized.These include cordycepin,ophicordin,polysaccharides and L-tryptophan.In addition,this northern Corydyceps showed several therapeutic effects,including immunoregulative, anticancer,antibacterial,and antifungal effects.But,considering the wide uses of the Cordyceps militaris,little information is available on the antithrombotic and thrombolytic activities.Therefore,in the present study,we purified and partially characterized the novel fibrinolytic enzyme from Cordyceps militaris for the first time,and investigated the antithrombotic and partial thrombolytic activities of CMase.The main results were given as follows:1.SN-18 with great thrombolytic activity was isolated and identified from 37 Cordyceps militaris around China.According to molecular analysis of ITS1-5.8S rDNA-ITS2 sequences,SN-18 has a high similarity with C.miitaris AY899258, Paecilomgces militaris DQ342250 and Paecilomgces militaris DQ342248.SN-18 could be determined to be a species of C.miitaris.The results of sequence analysis were in agreement with those of morphological features.2.Single factor experiment was used to optimize the suitable conditions and medium composition for the fibrinolytic activity of SN-18.The optimal temperature is 25℃. Medium volume of 30 mL in a 250 mL flask,160r/min shaking speed and inoculation of 10%wet mycelia were suitable for SN-18.The optimized medium compositions were 1% flour,5%Silkworm pupa powder.The activities of the fibrinolytic enzyme produced from this mediums were 32.34U/ml,2.31-fold against that from PDA.3.The fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized,which was designated C.militaris fibrinolytic enzyme(CMase).This extracellular enzyme from C.militaris was isolated by ammonium sulphate fraction,and purified to electrophoretic homogeneity using gel filtration chromatography.The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE.The optimum pH and temperature for the enzyme activity were pH 6.0 and 25℃,respectively. In the presence of metal ions such as Mg²⁺ and Fe²⁺ ions the activity of the enzyme increased,whereas EDTA and Cu²⁺ ion inhibited the enzyme activity.Interestingly the N-terminal amino acid sequences of the enzyme(Swiss prot accession no.P85318) is extremely similar to those of the trypsin proteinases from insects,and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom..4.Thrombolytic activities of CMase:The fibrinolysis activity was performed by degrading the fibrin directly,according to the experiment in vitro.The results of dissolving effects of CMase on the blood clot indicated that the blood clot in capillary was dissolved by 29.49%after treated in vitro for 3h at 37℃,while control group is 3.01%.After treated in vitro at 37℃,90r/min for 12h by 8ml CMase(24U/ml,12U/ml,6U/ml),Urokinase (12U/ml) and control,the blood clot in test tubes was significantly dissolved by 99%, 53.4%,14.2%and 8.6%respectively,but the control was 4.4%.5.The antithrombotic activities of CMase:In 8 min,it can dose-dependently inhibits platelet aggregation induced by adenosine diphosphate(ADP) in vivo and thrombus formation in Arterio-venous shunt model in vivo.ADP induced aggregation reached 9.21% and 7.66%for CMase(12,18U/kg),respectively,compared to 29.25%for control and 10.66%for Urokinase(500U/kg)(all p＜0.01),with 12U/kg having approximately the same effect as Urokinase(500U/kg).Compared to a control thrombus weight of 123.4 mg (SD 25.4),thrombus weight was 61.3(18.3) and 40.2(11.9) after treatment with CMase (12,18U/kg),and 61.8(32.2),59.3(26.2) after aspirin(i.g.0.4g/kg),Urokinase(250U/kg) (all p＜0.01),with 12U/kg having approximately the same effect as Urokinase(250U/kg).