The Study On Fibrinolytic Mechanis M Of Indolone Compound From Marine Microbial Sources

2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8- tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid(FGFC1)is a natural product isolated from the metabolites of marine fungus Stachybotrys longispora 216, which belongs to the pyrano isoindole ketones alkaloid. The study found that in vitro fibrinolytic system, with the increase of FGFC1 concentration, FGFC1 obviously degradated fibrin; in vitro thrombolys is in rats model of pulmonary embolism, adding more than FGFC1 5mg/kg can cause pulmonary thrombosis completely dissolved. Adding of FGFC1 did not change blood fibrinogen in rats and the content of FDP,which confirmed that FGFC1 promoting thrombolys is was safety. With optimizing of FGFC1 products, the yield of fermentation can be greatly improved.So FGFC1 is an excellent potential thrombolytic drugs,studying on the mechanism of FGFC1 promoting thrombolysis has important significanceIn the present study, polyacrylamide gel electrophoresis(SDS-PAGE) and the chromogenic substrate method was used to study the role of FGFC1 in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator and plasminogen;circular dichroism spectroscopy(CD) was used to investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor;using the chromogenic substrate method studied on the kinetics of enzyme catalyzed reaction of pro-u PA activate plg.The above three methods were explored the mechanisms of FGFC1 to promote fibrinolys is.The first chapter summarized molecular structure and functional characteristics of components of the fibrinolytic system; elaborated the mechanism of CD and its application in the interaction between protein and small molecule compounds;summed up the relevant theoretical basis of enzyme kinetics and related methods to explore the mechanism of thrombolytic drugs.In the second chapter(SDS-PAGE)and the chromogenic substrate method was used to study the role of FGFC1 in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator and plasminogen. SDS-PAGE results showing FGFC1 promotes the degradation of FITC-fibrinogen and he chromogenic substrate results showing FGFC1 promotes the producing of the plm and u PA,these resultes indicated that FGFC1 accelerates the mutual activation reaction between single chain urokinase-type plasminogen activator and plasminogen.In the third chapter CD was used to investigate the influences of FGFC1 on spatial structure of the fibrinolytic activity factor. The results of far-ultraviolet CD spectra showed that in the concentration of 23 μmol·L-1 to 115 μmol·L-1 FGFC1 converted the α-helix to random coil for single-chain urokinase-type plasminogen activator;α-helix to random coil for urokinase; β-sheet to random coil for plasminogen;and β-sheet to random coil and β-turn for plasmin.The above far-ultraviolet CD spectra results revealed that changes in the secondary structure of these enzymes could be responsible for the increased molecular flexibility and more susceptible to react with substrate.The results of near-ultraviolet CD spectra showed that there was an increase in the ellipticity of the plasminogen with an enhancement of absorption about at 285 nm.The results of far and near-ultraviolet CD spectra exposed that FGFC1 slightly influenced the conformation of single chain urokinase-type plasminogen activator and streptokinase;and highly influenced the conformation of plasminogen and plasmin.The present study explained FGFC1 mainly reacts with plasminogen/plasmin than single-chain urokinase-type plasminogen activator as a result the FGFC1 could influence the fibrinolytic process.In the fourth chapter using the chromogenic substrate method studied on the kinetics of enzyme catalyzed reaction of pro-u PA activate plg. In the reaction system of pro-u PA and plg, with the increasing of concentration of FGFC1, the value of Kcat increases, 40μg·m L-1 FGFC1 increased the value of Kcat for 26.5 times, which meaned that FGFC1 significantly increased the maximum catalytic efficiency of pro-u PA. With the increasing of concentration of FGFC1, the value of kcat/km increases, 40μg·m L-1 FGFC1 increased the value of Kcat for 22.8 times, which means that FGFC1 significantly increased the overall catalytic activity of pro-u PA. With the increase of concentration of FGFC1,the value of Km increased slightly, indicating that FGFC1 weakly reduced binding activity of plg and pro-u PA.In plasminogen and pro-u PA reaction system, the active center of pro-u PA acted on Arg560-Val561 of plasminogen. Similar to activity of u PA, pro-u PA by endogenous active breaked Arg560-Val561 of plasminogen. FGFC1 accelerates the mutual activation reaction between pro-u PA and plg. From the fact that FGFC1 changed conformation of plg but FGFC1 increased the activity of plg,we can infer that FGFC1 changed the spatial structure of K5 of plg to make the Arg560-Val561 peptide of plg more consistent the activity center agent of pro-u PA.