Projects Overview
Overview
This Program Project Grant (PPG) on remaining critical issues concerning the molecular and biologic control of von Willebrand factor in von Willebrand disease. This PPG is comprised of 4 projects and 3 cores that are all focused on von Willebrand disease and mechanisms causing its dysfunction, increased clearance or increased synthesis, and the role carbohydrate modification plays in VWF biology. While this represents a new PPG application, this PPG makes use of the samples and biodata from the pre-existing PPG on historically diagnosed subjects and an R01 grant on new VWD subjects recruited prospectively. Project 1 aims to define longitudinal changes in VWF concentration and changes in semiquantitative bleeding assessment during follow-up intervals, the fidelity of the diagnosis of type 2 VWD, the frequency of antibodies to VWF in type 3 VWD, and the development of animal models of VWD and VWD variants. Subjects with low von Willebrand factor (LVWF), and type 1 or 3 VWD with no sequence variants will be studied in Core B with full genome sequencing. Abnormalities identified will be sought in their family members through Project 1 and the mechanism studied by Projects 1, 2, 3, or 4 based on the candidate pathway. Project 2 will define the role of carbohydrate modification of VWF through the determination of N- and O-glycan profiles, study the glycan differences in plasma, endothelial, and platelet VWF, and racial differences using MS-glycomic and glycoproteomic approaches. Model systems will be set up in mice to mirror the changes found in human mechanisms. Project 3 will examine the upstream regulatory control as a mechanism causing VWD. The cell biology of VWF will be studied using BOECs obtained from patients with VWD and used to investigate the role of promoters and enhancers. Genetically engineered mice will study the mechanisms by which clearance receptors alter VWF concentration. Project 4 will study the transcription regulation of VWF and the inhibition with microRNAs. This project will also examine the role of epigenetics in modulating plasma VWF and the effect of chronic inflammation on these mechanisms. There are three cores to support this PPG. Together, these studies will comprehensively study unique mechanisms that lead to reduced or abnormal VWF in VWD.
Leader: Dr. Robert Montgomery
Aim of Project 1 is to utilize the combined cohort to further characterize the longitudinal changes in VWF and bleeding symptoms, contrast type 2 with type 1 VWD through functional assays of VWF and comparison of representative animal models, and linkage of genetic variations detected by whole genome sequencing within the families of subjects with “yet identified” genetic variation. This Project addresses multiple clinical and basic questions about VWD and how assays and clinical scoring vary over time, to better elucidate the critical mechanisms in type 2 VWD, and contrast “affected” with “unaffected” family members to further understand VWF function and genetic mechanisms involving genetic changes outside of the VWF coding regions.
Leader: Drs. James O’Donnell and Sandra Haberichter
Aim of Project 2 is to define the quantitative and/or qualitative changes in VWF carbohydrate structure that lead to alteration of VWF synthesis or clearance that underlies type 1 von Willebrand disease (VWD) and Low VWF levels. This project will use lectin binding assays to characterize and quantify carbohydrate differences in the VWF of subjects that have been enrolled in this program. The specific glycan structures that cause these differences in lectin binding, and determine the biologic mechanisms that causes VWD or LVWF.
Leader: Drs. David Lillicrap and Paula James
Aim of Project 3 is to identify genetic variations outside of the von Willebrand factor gene that contribute significantly to quantitative pathologies of von Willebrand factor. This project will study the transcriptional regulation of VWF starting at the proximal promoter and the further upstream-enhancer on the 5’ end of the VWF gene. Several variants in syntaxin or its binding proteins have been shown to affect VWF levels in normal individuals but their role in VWD has not been optimally studied. Using BOECs from VWD patients or the development of engineered animals, the mechanisms behind these observations will be studied.
Leader: Dr. Christopher Ng
Aim of Project 4 is to identify specific transcriptional and epigenetic mechanisms in endothelial cells that are critical determinants of plasma and endothelial VWF levels. This project will study transcriptional regulation of VWF synthesis by miR-24 and TCF4 and determine other candidates through single cell RNA sequencing in BOECs. Epigenetics has not been extensively studied as a modulator of VWF, so the role of methylation and histone acetylation in regulating VWF in BOECs from older individuals might be explored as a reason for increased VWF levels with aging.