Studies On The Polypeptide Compositions And Anticoagulant Mechanism Of Leeches

In recent years,the health of the elderly is seriously threatened by cardiovascular diseases and thrombotic diseases which are staying at a high level.Anticoagulation therapy is the primary measures to deal with thromboembolism disease.The commonly used anticoagulant drugs such as heparin and warfarin,had deadly shortcomings like thrombocytopenia,bleeding,monitoring INR necessarily and so on.Exploiting thrombin specific inhibitors or coagulation factor specific inhibitors has been becoming a research direction for new anticoagulant drugs which can effectively avoid bleeding and thrombocytopenia side-effects when curing thromboembolism disease.Traditional Chinese medicine thinks that leeches have pharmacological effects of anticoagulant,antithrombotic and anti-hyperlipidemia,etc.Therefore leeches have been widely used in the treatment of high blood pressure and hemostasis swelling sores,but they have been unknown questions for years that what exactly the effective anticoagulants of dried leeches are and what their anticoagulant mechanisms are.Hirudin extracted from Hirudo nipponica Whitman saliva has been the strongest thrombin specific inhibitor so far and is the only leech extract that has been applied to clinical use,while other leech extracts are still under study.Among the common leech medicines,Hirudo nipponica Whitman and Poecilobdella manillensis lesson belong to Hirudinidae,a bloodsucking type,mainly take the blood of animals as food.Whitmania acranulate Whitman and Whitmania pigra Whitman leech belong to Haemopidae,a kind of non-bloodsucking leech,lived on subterranean insects and conches.At present,two strong anticoagulants have been extracted from leeches,and that is hirudin from H.nipponica Whitman and Bufrudin from P.manillensis lesson,It can be concluded that maybe there are unknown anticoagulants in W.acranulate Whitman and W.pigra Whitman since these two leeches not contain hirudin whereas they have antithrombotic and anticoagulation effects.In order to screen the better extraction methods in the present study,NP-40 lysis extraction,water extraction,salt precipitation-sephadex G-25 column extraction and acetone precipitation method were adopt in the assays to extract the total protein of leeches.The mass spectrometry named the Q-TOF high resolution system was applied to analysis the molecular weight of the total proteins from H.nipponica Whitman,P.manillensis lesson,W.acranulate Whitman and W.pigra Whitman.Anticoagulant exploration target of the leeches at the coagulation factor level can be done by running Native-PAGE electrophoresis which make protein separated and then detect the separated F?a?F?a?F?a through Western blot.The experimental results showed that:1)NP-40 lysis extraction was used for preparing electrophoresis samples for remaining the most anticoagulant activity;Water extraction was used for preparing ESI-MS samples.2)ESI HRMS calculated 11 proteins from H.nipponica Whitman saliva including hirudin whose molecular weght is 7349.5Da and 5 proteins from H.nipponica worms,their difference was obviously significant.Three proteins from P.manillensis lesson proved to 14997.5Da,15098 Da,15986.5Da weight and 4 proteins of W.pigra Whitman were analyzed whose MW respectively are 3999 Da,9397Da,12781.5Da,13727.5Da and 9397 Da,which was stable in quality and high in abundance.The proteins from W.acranulate Whitman with MW 7978.5Da,9396.5Da,10701.5Da,11181.5Da,12970 Da and 13715.0Da,while the protein with MW 9397 Da was also extracted from W.pigra Whitman,the same molecular weight polypeptide may have similar anticoagulant mechanism.3)Western blot detected the active peptides from H.nipponica Whitman,P.manillensis lesson,W.pigra Whitmanand W.acranulate Whitman that could bind to thrombin.It was also verified that the active anticoagulant from W.pigra Whitman could form a stable protein complex with thrombin at different proportions.The active peptides bound to coagulation factor F?a or F?a could be found in the total protein of W.pigra Whitman and W.acranulate Whitman.In order to exploit the anticoagulation targets of the active peptides from leeches,the Native-PAGE combined with western blot method was established to detect the complexes consisted of the active anticoagulant peptides and coagulation factors.This method extremely simplified the purification process of active peptides and is more flexible,sensitive,intuitively and easily controlled compared with the method of chromogenic substrate.The further study finds that W.pigra Whitman and W.acranulate Whitman contain specific coagulation factor F ? a or F ? a inhibitors,and the active anticoagulant from W.pigra Whitman could form a stable protein complexes with thrombin proportionably.These study provided data base and theoretical basis for the development of new specific thrombin or coagulation factor inhibitors.These studies provided modern scientifical basis for using leeches to cure thromboembolism diseases and lay the Data and theoretical foundation for the development of new specific thrombin or coagulation factor inhibitors.