Study On The Regulation Mechanism Of Activation Of Plasminogen And Promotion Of Fibrinolysis The By FGFC1

Fibrin was a main composition in thrombosis.Degradation of fibrin to dissolve thrombus was the key to the treatment of thrombotic cardiovascular and cerebrovascular diseases.Plasminogen was the zymogen form of plasmin.It was activated to plasmin by tissue-type plasminogen activator?t PA?or urokinase-type plasminogen activator?u PA?.Plasmin could degrade fibrin.Plasminogen/plasmin conversion was the core process of thrombolysis.FGFC1 was a rare marine-derived bisindole compound that could enhance fibrinolysis in vitro and vivo.FGFC1 promoted the production of plasmin to degrade of fibrin.FGFC1 could dissolve the pulmonary thrombus in rat and was a potential thrombolytic drug.The study showed that the target of FGFC1 was plasminogen,causing the changes in the secondary and tertiary structure of plasminogen and making plasminogen easily activated into plasmin.However,the mechanism of FGFC1 on plasminogen needed to be further researched.In this paper,through the study of the fibrinolytic characterization of FGFC1 and the analysis of the binding site and binding mode of FGFC1 and plasminogen,the preliminary activation mechanism of FGFC1 to plasminogen was analyzed.The change of the lysine residues of plasminogen by FGFC1 was explored via ¹⁹F NMR.The study could provide a theoretical basis for the clinical pharmacology of FGFC1 and the development of new thrombolytic drugs.The first chapter summarized the structure and function of the constituent factors of the fibrinolytic system.We summarized the structure and function of plasminogen,plasmin and plasminogen activator and the physiological effect of related activators and inhibitors.The lysine binding sites?LBS?of plasminogen were important targets in the process of activating and inhibiting plasminogen.Plasminogen in the blood circulation adopted a closed conformation.Plasminogen would take an open conformation and was more easily activated by the combination between LBS and thrombus.The activity of t PA and u PA would also be greatly improved.The Serine protease domain of t PA or u PA cleaved the peptide bond between plasminogen Arg⁵⁶¹and Val⁵⁶²,activating plasminogen to plasmin,which would rapidly degrade fibrin and dissolve the thrombus.In order to prevent the excessive production of plasmin and cause the risk of bleeding,plasminogen activator inhibitor 1?PAI-1?could inhibit the activity of t PA and u PA and?2-antiplasmin could also inhibited the activity of free plasmin.Therefore,the blood maintained balance in the rapid transformation of fibrinolysis and coagulation.The second chapter constructed the fibrinolytic system composed of plasminogen?plg?and single-chain urokinase-type plasminogen activator?pro-u PA?.The effect of pro-u PA and plg on the fibrinolytic activity of FGFC1 was researched.The fibrinolytic characterization of FGFC1 mediated by Glu-plg and Lys-plg was compared.Furthermore,we simulated the stability of FGFC1 fibrinolytic function under the influencing factors in vivo.The fibrinolytic activity of FGFC1 is expressed by the rate of urokinase production and was measured by the chromogenic substrate method.The results showed that 18 n M pro-u PA enhanced 7.7-fold the fibrinolytic activity and 80 n M plg enhanced 5.2-fold the fibrinolytic activity.The fibrinolytic activity first enhanced and then decreased with the increasing of the FGFC1 concentration.When FGFC1 concentration was 0.096 m M,the fibrinolytic activity was the strongest and enhanced by 2.2 times.When the concentration of FGFC1 was0.24 m M,the fibrinolytic activity was inhibited.The action mechanism of FGFC1 to plasminogen may include the formation of the Lys-plg conformation.The fibrinolytic activity of FGFC1 was only slightly reduced in the presence of[Na⁺]and glucose.It was inferred that the fibrinolytic activity of FGFC1 was stable in vivo.In chapter three,the binding sites and binding modes of FGFC1 to plasminogen were investigated by measuring the effect of plasminogen inhibitor 6-aminocaproic acid and tranexamic acid on fibrinolytic activity of FGFC1 and using the molecular docking of FGFC1 and 6-aminocaproic acid to KR1-5,respectively.The results showed that 6-aminocaproic acid and tranexamic acid significantly inhibited the fibrinolytic activity of FGFC1,indicating that lysine binding site?LBS?has an essential role in the process of FGFC1 binding to plasminogen.The docking showed that FGFC1 mainly bound to arginine,tyrosine,and tryptophan residues,which affected the function of LBS in plasminogen,further confirming that the action of FGFC1 on plasminogen is mainly due to the LBS.FGFC1 bound to the binding pocket of KR1-5.The main binding effect of FGFC1 on plasminogen was H-bonds.FGFC1 had the highest affinity to KR2.According to the fibrinolytic characterization of FGFC1 and the binding sites and binding modes of FGFC1 to plasminogen,it was preliminarily determined that FGFC1 activated plasminogen by LBS.This could lay a foundation for the analysis of activation mechanism of plasminogen by FGFC1.Chapter four explored the effect of FGFC1 on lysine residues of plasminogen via¹⁹F NMR.S-ethyl trifluorothioacetate could alkylate the lysine residues of plasminogen thus lysine residues were labeled.The change of fluorine signal indirectly reflected the change of the lysine residues.The results showed that the lysine residues were successfully labeled.There was no chemical shift,peak increasing or line broadening change,indicating that the labeled lysine residue was not affected or included in the process of FGFC1 binding to plasminogen.The fluorinated plasminogen still possessed active.The chemical modification will not denature plasminogen or cause major structural disturbances,indicating that the method of labeling plasminogen with S-ethyl trifluorothioacetate is feasible.The successful labeling of plasminogen provided a new perspective and method for studying the changes of amino acid residues in full-length plasminogen.The study preliminary explored the action mechanism of FGFC1 to plasminogen.The fibrinolytic characterization of FGFC1 indicated that relatively low FGFC1 concentration enhanced the fibrinolytic activity and relatively high FGFC1 concentration inhibited the fibrinolytic activity.At 0.096 m M,the fibrinolytic activity was the strongest and enhanced by 2.2 times.The action of FGFC1 to plasminogen may be divided into two aspects:activation and inhibition.The activation mechanism of FGFC1 to plasminogen may involve the formation of Lys-plasminogen-like form.Both experiments and docking results indicated that FGFC1 acted on plasminogen mainly by binding LBS.The results of ¹⁹F NMR showed that the labeled lysine residue was not affected or included in the process of FGFC1 binding to plasminogen.