Study On Molecular Mechanisms Of Glanzmann Thrombasthenia And Antithrombotic Mechanisms Of Holothurian Glycosaminoglycan Extracted From Sea Cucumber

Glanzmann thrombasthenia(GT)is a rare autosomal recessive bleeding disorder characterized by a life-long mucocutaneous bleeding tendency and absent or severely reduced platelet aggregation in response to the physiological agonists ADP,epinephrine,and collagen,but relatively normal aggregation in reponse to ristocetin.Known as Glanzmann thrombasthenia,It was first reported by Glanzmann in 1918.The disease is caused by either a lack or dysfunction of the platelet integrinαⅡbβ3,the glycoproteinⅡb/Ⅲa complex,which serves as a receptor for fibrinogen,von Willebrand factor,and other RGD-containing ligands and is essential for platelet aggregation.GT is not uncommon in China.Researches on the disease related mutations might not only be helpful to diagnosis,effective therapy and prepotency,but also provide clues for exploring the structure-function relationship of integrinαⅡbβ3.In this study,we worked on the molecular pathogenesis of four pedigree with Glanzmann thrombasthenia.In this study,all the four probands from four pedigrees suffered form recurrent bleeding episodes from early childhood showing epistaxis,gingival hemorrhage and menorrhagia in the female ones.Laboratory tests showed anormal platelet count and coagulation profiles,no clusters of normal platelets on the blood film,a prolonged bleeding time and severely reduced platelet aggregation in response to ADP,thrombin,collagen,adrenaline and arachidonic acid,but nearly normal platelet aggregation in response to ristocetin.Flow cytometry showed a strong reduction inαⅡb of proband 1,3, 4(＜5%of normal)classified as GT typeⅠGT.The proband 2 showed a moderate reduction inαⅡb classified as typeⅡGT.Genetic analysis showed that nonsense mutations C1750T(Arg584Stop,R584X)and 69-79 deletion mutation were identified in the proband 1;C1750T inherited from her father side and was inherited to her daughter,while her brother had heterozygous 69-79 deletion mutation,probably from her dead.mother;The proband 2 had homozygous A2334C(Gln747Pro,Q747P)missense mutation;Compound heterozygous mutations,T2255G(Leu721Arg,L721R)and C2671T (Gln860Stop,Q860X)were identified in the proband 3,the former being inherited from the maternal side and the latter the paternal side.the proband 4 was homozygous for C470A(Pro126His,P126H)missense mutation in herαⅡb gene,whose parents had a heterozygote C470A mutation.L721R, Q860X,P126H and 69-79 del mutations are novel discovered mutations. L721R,Q860X and P126H mutations were introduced independently by site-directed mutagenesis into a PCDM8Ⅱb mammalian expression plasmid. 293T cells cotransfected with cDNAs of mutatedαⅡbL721R,P126H and wild-typeβ3 expressed 2.1%and 3.6%respectivly of normal amount ofαⅡb on the cell surface as shown by FACS,in contrast to 31.9%of normal amount ofαⅡb on the cells cotransfected with cDNAs of mutatedαⅡbQ860X and wild-typeβ3.Western blot analysis of the cell lysates showed no detectable matureαⅡb in cells with P126H and L721R mutants,while a truncated form ofαⅡb was demonstrated in Q860X mutated GPⅡb/Ⅲa cells.Intracellular immunofluorescence studies demonstrated L721R,P126H and Q860X mutant pro-αⅡbβ3 complex and colocalized with an ER marker,but showed minimal-colocaliztion with an Golgi marker.In conclusion,The P126H mutation inαⅡbβ-propeller,L721R mutation inαⅡb calf-1 and Q860X mutation inαⅡb calf-1 domains causing typeⅠGT do not affect pro-αⅡbβ3 complex formation,but prevent transport of the pro-αⅡbβ3 complex from the endoplasmic reticulum to the Golgi to some extent,leading to intracellular retention and hindering its maturation and surface expression.L721R mutation possibly has dominant negative effect on expression of Q860X pro-αⅡbβ3 complex.Holothurian glycosaminoglycan(GAG),a glycosaminoglycan extracted from the body wall of sea cucumber is primarily composed of sulfated L-fucose.Previous clinical and experimental studies demonstrated that GAG has anticoagulant activities and antithrombotic actions in addition to reducing the blood viscosity and enhancing fat metabolism,while less bleeding than unfractionated heparin or low molecular weight heparin.Tissue factor pathway inhibitor(TFPI)is a Kunitz type serine protease inhibitor which inhibits the initial reaction of the tissue factor(TF)-mediated coagulation pathway. Heparin induces synthesis and secretion of TFPI from endothelial cells in vitro. No studies have not been reported about the effect of GAG on TFPI from endothelial cells.Thrombin activatable fibrinolysis inhibitor(TAFI)is a plasma metalloprotease that exhibits carboxypeptodase B-like activity.Upon activation by thrombin or plasmin,TAFI is converted to an enzyme(TAFIa) that inhibits fibrinolysis.Inhibition of TAFIa is a novel approach to arterial and venous thrombolysis.Effect of GAG on TAFI-dependent inhibition of fibrinolysis has not been reported.In order to further investigate the antithrombotic mechanisms of GAG,we studied the effctes of GAG on TFPI secretion and synthesis in endothelial cells in vitro,clot lysis and TAFIa in an in vitro thrombosis and thrombolysis model.Spontaneously transformed immortal endothelial cell line EA.hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin(UFH)for short-term incubation(15 min～2 h)and long-time incubation(6 h～48 h). Different doses of GAG were used to stimulate EA.hy926.Released free TFPI was determined by commercial ELISA kit.TFPI expression was investigated by Immunofluorescent method and TFPI mRNA level was analyzed by real-time PCR.In a 96-wells microtitre plate,pooled normal plasma to which different concentrations of GAG was added,was allowed to clot by addition of thrombin and calcium chloride,fibrinolysis was induced by addition of t-PA. Clotting times and clot lysis times were determined.TRR(TAFI-related retardation of clot lysis)was used to assess TAFI functional activity.GAG increased TFPI synthesis,expression and secretion by dose-dependent and time-course way.At low GAG concentrations,clot lysis times had been increased.At intermediate GAG concentrations,clot lysis times were significantly decreased compared to control values.TRR dose-dependently decreased on addition of GAG..In conclusion,GAG increases TFPI synthesis, expression and secretion of endothelial cells.GAG of intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.