The Research On The Pathogensis Of Von Willebrand Disease

Objective: Compared with endothelial progenitor cells, outgrowth endothelial cells(BOECs) from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells because of the latter’s limited life span and drift of phenotype, and become new tools for vascular abnormalities. Herein we aim to establish the protocol of BOECs, and then analyze the cell phenotype and function of BOECs. Methods: Mononuclear cells were collected from peripheral blood of healthy donor by gradient centrifugation, seeded on plates and cultivated in EGM-2 medium for 4 weeks. The morphological changes of cells, cell phenotype and von Willebrand factor(VWF) multimers were analyzed. The storage of VWF in BOECs and secretion of VWF strings of BOECs upon stimulation were observed by confocal fluorescence microscope. Results: After 4-week-culture in vitro, we found cell colonies and characteristic cobblestone-like morphology of BOECs in plates. For another three weeks of expansion, BOECs were trypsinized and detected by flow cytometry. BOECs expressed CD31, CD34, and EPCR, without the expression of CD14, CD45 and CD133. VWF in the supernatant of BOECs shared the same multimer pattern with that observed in normal plasma. VWF was detected in the cytoplasm of BOECs by confocal fluorescence microscope. The amount of VWF increased in cells and VWF strings formed on cells surface by the stimulation of phorbol-12-myristate-13-acetate(PMA). Conclusions: We first succeeded in establishing BOECs, and analyzing cell phenotype and function of BOECs. They are the native cell models for the pathogenesis of von Willebrand diseases(VWD), and could be the new tools of gene therapy for VWD patients.Objective: The quantitative deficit or qualitative defect of VWF results in the most prevalent inherited bleeding disease, von Willebrand disease. Although many VWF gene mutations have been registered in VWD database funded by International Thrombosis and Hemostasis Society, little is known about VWD patients from Asia, especially in China. The gene type and the relation between genotype and VWD diagnosis of Chinese patients are expected to further explore. Methods:We collected the clinical data and laboratory results of 38 VWD patients and their family members, and then analyzed the whole VWF gene sequence of the blood samples from these people. Results:The patients and their family members were divided into type 1(12 cases), type 2A(6 cases), type 3(13 cases) and healthy carriers(7 cases). VWF: RCo/VWF: Ag in type 2A was dramatically lower than those in type 1 and type 3. In our study, 30 VWF gene mutations were reported, including missense mutation, nonsense mutation, deletion and insertion, splice-site mutations, and compound mutations. Twenty of them were not reported in the literature. Conclusions: We reported twenty novel mutations of VWF gene identified in 38 VWD patients and their family members, which broadened the VWD database of Chinese patients. Meanwhile, VWF gene mutations played an important role in the diagnosis and subtype classification of VWD. We will further explore the pathological nature of these novel mutations in the VWD patients, which will provide the laboratorical data for the precision medicine of these patients.Objective: Von Willebrand factor propeptide(VWFpp), composed of D1 and D2 domains, is indispensable for the multimerization, intracellular trafficking, and secretion of the factor. While numerous mutations in mature von Willebrand factor have been identified in von Willebrand disease, few mutations in the propeptide have been reported, and the pathogenic nature of these mutations remains largely unknown. Methods: The multimer analysis, basal secretion and stimulated secretion by phorbol-12-myristate-13-acetate were analyzed in the transient transfection of wild type or mutant full-length von Willebrand factor. Endoplasmic reticulum retention was studied using confocal immunofluorescence and western blotting. The dimerization of truncated von Willebrand factor was examined to further elucidate the deficit in multimerization of full-length von Willebrand factor. Results: We identified three novel mutations(p.Gly39 Arg, p.Lys157 Glu, p.Cys379Gly) and one previously known mutation(p. Asp141Asn) in three unrelated patients with von Willebrand disease. Upon expression in the human embryonic kidney 293 cells, all the four mutations impaired multimerization of von Willebrand factor. Compared to the wild type D1D2D’D3, truncated fragments containing four mutations showed the reduction in dimerization of D’D3 domain. Furthermore, the basal and phorbol-12-myristate-13-acetate-induced secretions of VWF were reduced by all mutations. Immunofluorescence data showed mutant constructs led to the retention of von Willebrand factor in the endothelial reticulum. Conclusions: Missense mutations in the D1 domain of von Willebrand propeptide lead to the defects in multimerization, intracellular trafficking and secretion of von Willebrand factor, and consequently cause a bleeding tendency in von Willebrand disease patients. It will be the therapy target of the precision medicine for VWD patients carried with the mutations in VWFpp.