Chemical Structures And Anticoagulant Activities Of Polysaccharides From Holothuria Leucospilota

In recent years,the incidence of thrombotic diseases is increasing,and the affected population is getting younger,which cause a serious health hazard.Thrombotic disease can be treated with antithrombotic drugs,these drugs mainly include three categories:anticoagulant drugs,antiplatelet drugs and thrombolytic drugs.Among them,anticoagulant drugs can disrupt coagulation factors,which mainly include heparins,vitamin K antagonists,direct thrombin inhibitors and synthesized small molecule as coagulation factor inhibitors,etc.Currently,the antithrombotics may result in the side effect of hemorrhage in varying degrees.Recent researches showed that the intrinsic coagulation pathway is closely associated with the pathological thrombosis,while it is not required for the physiological hemostasis.Therefore,the antithrombotic drugs with low bleeding tendency may be developed by selectively targeting this pathway.Fucosylated glycosaminoglycan（FG）is a distinct structurally complex glycosaminoglycan derivate.Native FG has various pharmacological activities,it has potent inhibition of i Xase,and also has side effects such as factor XII（FXII）activation and platelet aggregation.The depolymerized FGs with certain molecular weight keep the potent anticoagulant and antithrombotic activities with lower bleeding tendency.In this research,chemicalβ-elimination depolymerization with unique glycosidic-bond-selectivity was applied on FG from sea cucumber Holothuria leucospilota,the definite structure were clarified on the basis of the high-regular oligosaccharides obtained from depolymerized FGs with“bottom up”strategy.Additionally,the anticoagulant activities of native FG and the pure oligosaccharides were systematically studied.In addition,the anticoagulant structure-activity relationship of HLFG was elucidated.The methods and results of this dissertation are listed as below.（1）Extraction,purification and physicochemical properties analysis of polysaccharides from Holothuria leucospilota:Herein,HLFG、HLFS、HLNG from Holothuria leucospilota were extracted and purification by enzymatic-alkaline hydrolysis,grading-enthanol precipitation and strong ion exchange chromatography.The physicochemical properties including molecular weight,monosaccharide composition,IR and the ratio of sulfate to carboxyl group were analyzed.The HPGPC showed that these polysaccharides had good homogeneity,and the purity is more than 95%.（2）Structural analysis of HLFG:The depolymerized product of HLFG（d HLFG）was prepared by hydrogen peroxide,and its structure was studied by 1D/2D NMR spectra analysis.According to the 1D/2D NMR spectra of the native（HLFG）and its depolymerized products,the chemical structure of HLFG was deduced.The backbone sequence of HLFG was(-4-D-Glc A-β1,3-D-Gal NAc_4S6S-β1-),and the branches L-Fuc linked to Glc A viaα1,3 glycosidic bonds,but its side chains may be 2,4-sulfated,3,4-sulfated and 4-sulfated.The result of 1D/2D NMR spectra analysis indicated that disaccharide branches probably exist.Branches of L-Fuc:F1,F2 were prepared by mild acid hydrolysis,and their structures were analyzed by 1D/2D NMR,the existence of disaccharide branches in native HLFG was proved again.（3）Structure characterization of a series of oligosaccharides of HLFG:HLFG was depolymerized by glycosidic-selectiveβ-eliminative method.A series of oligosaccharides HL3,HL5,HL8 were purified from the depolymerized products（d′HLFG）by GPC.According to the HPGPC and NMR analysis,the structure of HL3 was L-Fuc _XS4S-（α1-3）-L-Δ^4,5Glc A-（α1-3）-D-Gal NAc_4S6S-ol（R=2 or 3）.The structure of HL5 was L-Fuc _R-（α1-3）-D-Δ^4,5Glc A-（α1-3）-L-Gal NAc_4S6S-β-（1-4）-[L-Fuc _R-（α1-3）]-D-Glc A-ol（R=2S4S,3S4S or 4S）。（4）Anticoagulant activities of HLFG,HLFS,d HLFG and oligosaccharides:The results of APTT,TT and PT showed that HLFG and HLFS had anticoagulant effect by inhibiting intrinsic coagulation pathway,and also on the common coagulation.The anticoagulant activity of HLFG was stronger than HLFS.For HLFG,the activity decreased with the molecular weight,while HLNG had no obvious anticoagulant activity.Particularly,HLFG and its depolymerized products displayed potent anti-i Xase activity,and their activity decreased with the reduction of the chain length.Additionally,native HLFG showed significant factor XII activation and human platelets aggregation,while its low-molecular-weight products（d HLFG-1 and d HLFG-2）did not produce these activities,indicating FXII and platelet activation by FG may be dependent on their molecular weight.