| Thrombosis are major causes of morbidity and mortality,and the incidence of thromboembolic diseases(TD)increases as a population ages.China is moving to an aged country,and cardiovascular diseases will become a heavy burden on society.During the last few decades,various therapeutic treatments of thromboembolism complications,such as anti-platelet,anti-clotting,and other anti-thrombosis agents emerged with the continuous increase of TD incidence.However,more or less side effects are present in each of them.Therefore,the pursuit of high quality thrombolytic agent will never be stopped.Data from experimental models using mice and clinical studies have shown that coagulation factors XI or XII are important for thrombosis,while having minor or no apparent roles in processes that terminate blood loss.Pharmacological inhibition of FXII or FXI may offer the exciting possibility of anticoagulation therapies with minimal or no bleeding risk.Hameadipsa sylvestris is a blood-feeding annelid animal.In order to successfully take blood,blood-feeding animals evolved antihemostatic mechanisms to counteract their hosts.H,sylvestris provides a very good natural source for anticoagulant agents prospecting.By Sephadex G-50 gel filtration and reverse-phase high performance liquid chromatography(RP-HPLC),a peptide named as sylvestin,which could prolong the coagulation of plasma,was purified from homogenate of H.sylvestris.Its partial N-terminal amino acid sequence was determined by automated Edman degradation.Specific primers were designed according to the amino sequences determined by Edman degradation and cDNA sequence coding the peptide was cloned from cDNA library of the head of H.sylvestris.The mature peptide is composed of 43 amino acid residues and has six cysteines.Its molecular weight is 4795.4 Da by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS),which is 6 Da more than that of the natural peptide,indicating that it forms three pairs of disulfide bond.BLAST analysis revealed that sylvestin contained a canonical Kazal motif(C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C),suggesting that it is a novel Kazal-type protein.In order to investigate the structure-function relationship of the peptide,it was synthesized by solid-phase chemistry method.RP-HPLC and MALDI-TOF/TOF MS were used to monitor the oxidative refolding process of synthetic linear peptides.The activity of the synthetic peptide was confirmed to be the same as the natural peptide.By recalcification time assay,APTT(activated partial thromboplastin time)and PT(prothrombin time)assay and chromogenic substrate assays,the peptide was determined to be a novel FXIIa inhibitor.Kinetic studies including Dixon plot and Lineweaver-burk plot showed that sylvestin is a competitive inhibitor of FXIIa with a Ki of 2.98 μM.We also studied the anti-thrombosis activity in vivo by carrageenan-induced mouse tail thrombosis model.Tail-thrombus of the sylvestin treated mice is significantly shorter that of the saline group and the effect was dose-depended.2 mg/kg and 4 mg/kg of sylvestin reduced the thrombus by about 31.2%and 51.8%within 48 h,respectively.This work not only contributes to the blood-feeding mechanism of H.sylvestris,but also is helpful for the further study of the structure-function relationship of this antithromboic peptide. |
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