Advances In Pathogenetic Understanding,Diagnosis And Therapy Of Von Willebrand Disease

Part One Clinical observation of desmopressin in the treatment of 15 patients with von Willebrand disease[Objective]Assess the significance of DDAVP use in the diagnosis and treatment of VWD.[Methods]An analysis of Hematology Division of First affiliated Hospital of Soochow University was conducted in the 15 VWD cases who treated with DDAVP from March 2016 to August 2018.Efficacy of DDAVP was monitored by observation of the plasma VWF antigen(VWF:Ag),VWF ristocetine cofactor activity(VWF:Rco),VWF collagen binding activity(VWF:CB),factor VIII procoagulant(FVIII:C),calculation of their ratios with VWF:Ag,namely VWF:Rco/VWF:Ag,VWF:CB/VWF:Ag and VWF multimer analysis before and 2h after DDAVP injection.The adverse events of DDAVP were observed.[Results]A total of 15 cases with VWD,7 males and 8 females with a median age of 23(6-46)years were enrolled.Among the patients above,7 of 9 type ? VWD patients achieve complete response(CR),1 type 2A VWD patient achieve CR,5 type 3 VWD patients achieve no response(NR).The VWF multimer analysis in 5 patients combined with other plasma VWF values were in accordance with the known diagnosis.[Conclusions]DDAVP is effective in most type 1 patients,and is ineffective in some type 2 and almost all type 3 patients.It is helpful for diagnosis and subsequent treatment planning.Part Two Construction and identification of recombinant mature von Willebrand factor plasmid[Objective]VWF is a large multimeric glycoprotein that mediates the attachment of platelets to damaged endothelium and also serves as the carrier protein for coagulation factor VIII(FVIII),protecting it from proteolytic degradation.To perform its role in hemostasis,VWF must be assembled into large multimers before secretion into the blood.VWF is comprised of several homologous domains(D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK),VWF propeptide contains two D domains(D1 and D2),while the mature VWF protein begins at the D' domain and continues to the CK domain.Until now,many mechanisms of VWFpp in the multimerization,transfer and storage of VWF are not well understood,this experiment intends to construct a mature VWF vector by homologous recombination method,which lays a molecular foundation for subsequent experimental research.[Methods]The total ORF of mature VWF gene was amplified from pSVHvWF1 plasmid,then the amplified fragments were clonded into the Linearized pSecTag2/Hygro B vector by the way of homologous recombination.The positive clones of eukaryotic expression vector of pSecTag2/Hygro B-MVWF were confirmed by colony PCR and sequencing.The correct cloning liquid was expanded and extracted,and a high-purity plasmid was obtained for subsequent experiments.Transient expression experiments were performed in HEK-293 cells transfected with wild type pSVHvWF1 plasmid and pSecTag2/Hygro B-MVWF vector,Cell transfection products were identified by ELISA and VWF multimer analysis.[Results]The recombinant vector pSecTag2/Hygro B-MVWF was successfully constructed and expressed in HEK-293 cells.Mature VWF fragments were detected by ELISA and VWF multimer analysis of the plasmid transfection products in HEK-293 cells.[Conclusions]We have successfully constructed a vector named pSecTag2/Hygro B-MVWF,which provided experiment basement for the follow study on the VWFpp.