MOLECULAR MECHANISMS OF VWF ALTERATION IN VITRO/VIVO

血管性血友病（VWD）是最常见的遗传性出血性疾病，其特征为

von Willebrand disease (VWD) is the most common inherited bleeding disorder and is characterized by decreased levels or defective function of von Willebrand factor (VWF). Novel mechanisms that have recently been identified in individuals with VWD include the intracellular retention of VWF, defective regulated storage and secretion, and increased VWF clearance from plasma. The molecular/structural requirements that regulate normal VWF function, clearance, and degradation in vivo are not completely understood, and we predict that a large-scale survey of patients will reveal unique mutations that delineate VWF domain structure-function relationships. Therefore, the goal of Project 2 is to define the mechanisms causing the clinical and laboratory phenotypes and genotypes in VWD patients identified in Projects 1, 3 and 4. This goal will be accomplished by completing the following two specific aims. Aim 1 will examine the effect of candidate type 1 and type 3 VWD mutations on the synthesis, intracellular processing, secretion, and clearance of VWF. Cell lines expressing altered forms of VWF will be examined for abnormalities in post-translational modification and secretion of VWF. We will examine the clearance of selected type 1 VWD variants from plasma using a murine model. In Aim 2, we will determine novel mechanisms of functional VWF variants. Much remains unknown regarding the mechanisms underlying type 2A VWD. We will examine type 2A variants to determine the relative abundance of the following mechanisms: intracellular retention and degradation, defective multimerization, or increased proteolysis by plasma proteases. The effects of type 2 A and selected type 1 VWF mutations on binding of platelets, FVIII, and collagen will also be determined. In all studies, the in vitro data set will be compared to patient data to determine genotype/phenotype correlation. As promoter and splice junction mutations are identified in Project 4 or modifying genes in Project 3, these alterations will be explored in murine models the further define their pathogenetic mechanisms. Through these studies we expect to identify unique phenotypes/genotypes in individual VWD patients that further define VWF structure/function relationships. The knowledge gained from this project will increase our understanding of VWD, and will lead to the development of more precise diagnostic techniques as well as aid the development of more effective treatment strategies.