Unraveling Autoinhibition and Activation Mechanisms of the von Willebrand Factor A1 Domain Restricted; Files Only

Abstract

The vascular system of an organism ensures proper nourishment of all tissues and organs in the body. Upon a breach in a blood vessel, various proteins and cells initiate a response to form a clot and repair the injured vessel in a process called hemostasis. Effective hemostasis is achieved by the mechanical properties of platelets and the enormous circulating glycoprotein von Willebrand factor (VWF) that is sensitive to changes in shear forces in flowing blood. VWF initiates hemostasis by activating platelets at the wounded vessel through the VWF A1 domain. Under normal conditions in the bloodstream, the A1 domain has low affinity for its cognate platelet receptor glycoprotein (GP)Ibα. How the A1 domain transitions from a low-affinity state to a high- affinity state that supports platelet binding under arterial shear is a long-standing question at the heart of many bleeding, clotting, and inflammatory diseases.

Recent studies of the A1 domain have demonstrated that the discontinuous glycosylated sequences at each termini form an autoinhibitory module, or AIM, that regulates A1 affinity. However, the role the AIM in various disease states is still poorly understood. Type 2B von Willebrand disease (VWD) is a genetic bleeding disorder in which mutations in the A1 domain spontaneously interact with platelets and initiate variable rates of platelet clearance, leading to heterogenous presentations of mild bleeding to fatal hemorrhaging, depending on the mutation. Using a variety of biochemical and biophysical approaches, I characterized representative type 2B VWD mutations to understand molecular signatures indicative of severe clinical phenotype. For the first time, I revealed structural differences underlying type 2B VWD severity and showed that all representative mutations reduce AIM-A1 thermal and mechanical stability. Next, assays with human platelets and additional mutagenesis studies revealed that charged regions of the A1 domain are critical for A1 and GPIbα complex formation as well as GPIbα-dependent platelet activation. Finally, studies of AIM-A1 conformational dynamics in various VWF fragments and purified plasma-derived VWF revealed that local AIM-A1 contacts largely recapitulate A1 dynamics in the multimeric glycoprotein. Collectively, this research provides critical structural details and mechanistic insights of VWF A1 domain activity. 

Table of Contents

Chapter I. Introduction.....................................................................................4

Circulation, Hemostasis, and Thrombosis {5} von Willebrand Factor and von Willebrand Disease {15} von Willebrand Factor Activation {23} Platelet Activation and Clearance {38} Refrain: Dissertation Goals and Major Questions {47}

Chapter II. Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain........................................................................49

Introduction {51} Materials and Methods {53} Results{58} Discussion {75}

Chapter III. Investigating factors that mediate A1-dependent platelet activation and allostery of the A1 domain..............................................................................82

Introduction {84} Materials and Methods {86} Results and Discussion {90}

Chapter IV. Conformational ensemble of plasma-derived von Willebrand factor A1 domain and autoinhibitory module largely recapitulates the recombinant fragment......112

Introduction {114} Materials and Methods {116} Results{120} Discussion {127}

Coda. Concluding remarks..............................................................................133

References...................................................................................................139

Appendices...................................................................................................159

Reported basal platelet counts of patients with indicated type 2B VWD mutation {160} Activating nanobody 6D12 unfolds autoinhibitory module of the A1 domain {162}

III. Conformational dynamics of plasma-derived von Willebrand factor A2 domain {164} 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, Biomechanics Chemistry, Biochemistry
Keyword	thrombocytopenia autoinhibition platelets hydrogen-deuterium exchange protein dynamics biochemistry blood von Willebrand factor protein stability von Willebrand disease
Committee Chair / Thesis Advisor	Renhao Li, PhD, Emory University
Committee Members	Lawrence Boise, PhD, Emory University Michael Koval, PhD, Emory University Laura Finzi, PhD, Emory University Christopher LaRock, PhD, Emory University

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