Characteristics And Mechanism Of Anticoagulant And Antithrombotic Activity Of FG Oligosaccharides

Selective intrinsic coagulation factor inhibitors are the mainstream direction for developing anticoagulants with low bleeding tendency.The intrinsic tenase(FIXa-FVIIIa-PL-Ca²⁺complex,i Xase),the terminal rate-limiting enzyme of the intrinsic coagulation pathway,could be a promising target for novel anticoagulants.The depolymerized products of fucosylated glycosaminoglycan（FG）derived from sea cucumber have potent anti-i Xase activity,anticoagulation activity and the antithrombotic activity with a low bleeding risk.In this paper,using modern pharmacological techniques,the molecular mechanism of FG oligosaccharides acting on target protein,the characteristics of their anticoagulant and antithrombotic activity were systematically analyzed from the target protein,purified coagulation factor system,isolated plasma system and living animal level.The results showed that FG oligosaccharides selectively inhibited i Xase via binding to the exosite of FIXa,thereby inhibiting the intrinsic pathway and the amplification of coagulation triggered by limiting TF,and then inhibiting the pathological thrombosis.Whereas,they had a little effect on the physiological hemostasis.The main methods and results in this dissertation are listed as below.（1）The anticoagulant activity of FG oligosaccharides and their effects on the activity of coagulation factorsThe results about the clotting time of coagulation plasma showed that,FG oligosaccharides prolonged the activated partial thromboplastin time（APTT）in a chain length-dependent manner.And they had negligible effect on both prothrombin time（PT）and thrombin time（TT）.The effects on the activity of coagulation factors were analyzed using chromogenic substrate method.The results exhibited that FG oligosaccharides possessed potent activity for inhibition i Xase in a chain length-dependent manner.While they had no significant inhibitory activity for the extrinsic tenase（TF-FVIIa-PL-Ca2+complex,e Xase）,both Factor Xa（FXa）and thrombin（Active factor II,FIIa）in the presence of antithrombin（AT）.（2）The mechanistic study of i Xase inhibition by FG oligosaccharidesThe experimental results based on Bio-layer interferometry（BLI）technology exhibited that FG oligosaccharide could bind FIXa and zymogen FIX SP-EGF2 with high affinity,while the affinity to bind five FIX SP-EGF2 mutants decreased.Competitive BLI study indicated that FG oligosaccharides bound to an exosite on FIXa/FIX SP-EGF2 that overlapped with the binding sites for LMWH.These above results suggested that the binding to the exosite of FIXa underlay the molecular mechanism of i Xase inhibition by FG oligosaccharides,the importance of five amino acid sites in which was R233>R165>K230>K241>R170.The experimental results based on Fluorescence resonance energy transfer（FRET）technology showed that although FG oligosaccharide could inhibit the formation of functional i Xase complex,the binding to FIXa had no significant effect on the spatial distance from the active site of FIXa to PL in both FIXa-PL and FIXa-FVIIIa-PL-Ca²⁺complex.（3）The activity and mechanistic study of FG oligosaccharides inhibition of thrombin generationThe results of calibrated automated thrombogram（CAT）experiments displayed that FG oligosaccharides significantly inhibited FIIa generation depending on the intrinsic coagulation pathway（The inducers were limiting TF,APTT reagent and0.1%FIXa）in both human and rat plasma,while the effect on FIIa generation mainly depending on the extrinsic coagulation pathway（The inducer was massive TF）was relatively weak.（4）The antithrombotic activity of FG oligosaccharide and its effect on haemostatic functionIn a rat model of inferior caval vein thrombosis triggered by the combination of TF and ligation,FG oligosaccharide HS17 inhibited thrombosis triggered by limiting TF in a dose-dependent manner(ED₅₀,1.285 mg/kg sc).At five and ten times of ED₅₀doses,HS17 had little-to-no effect on thrombosis induced by massive TF,while LMWH remarkably inhibited thrombosis,suggesting that at a higher equivalent antithrombotic dose,HS17 should have less impact on the formation of physiological hemostatic plug initiated by massive TF than LMWH.（5）The study on the correlation between the inhibitory effect of FG oligosaccharide on thrombosis and its anticoagulant activity in vivoHS17 dose-dependently inhibited FIIa generation mediated by intrinsic pathway,and that its inhibitory activity was consistent with the antithrombotic activity at the corresponding dose.At five and ten times of ED₅₀ doses,HS17 had a little effect on both FIIa generation and thrombosis caused by massive TF,while LMWH significantly inhibited both FIIa generation and thrombosis under the same condition.Furthermore,HS17 dose-dependently prolonged APTT in rat plasma and had no effect on both PT and TT,whereas LMWH noticeably prolonged plasma APTT and TT at five and ten times of ED₅₀ doses.In summary,FG oligosaccharides possessed anticoagulant and antithrombotic activities depending on the inhibitory activity for i Xase,which clearly reflected the logical relationship among the anticoagulant target,anticoagulant activity and antithrombotic activity.These findings can provide sufficient and reliable basic data for the potential application of FG oligosaccharides,and provide new ideas and technical methods for the development of new i Xase inhibitors.