Force-dependent von Willebrand factor activation and allosteric modulation by single-domain antibodies Open Access

Arce, Nicholas (Spring 2022)

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Over the course of several million years, animals have evolved to survive predation. The development of boney jaws and the ability to bite coincided with the ability to withstand and heal from an attack. While the flowing circulatory system of these animals was fundamental to transport oxygen and nutrients, the preservation of vessel integrity was also paramount. Hemostasis is the process of capturing platelets and activating coagulation factors to generate a clot. 

Primary hemostasis is carried out by two distinct interactions. Disruption or elimination of either interaction in humans by deleterious mutations or pharmacological inhibition will lead to bleeding. Subendothelial collagen contains binding sites for the A3 domain of the plasma glycoprotein, von Willebrand factor (VWF). VWF is a soluble, multimeric protein that can span enormous lengths. Its polymeric properties enable it to undergo extensional elongation after VWF has been immobilized or when it undergoes a mixture of shear and rotational forces. VWF A3 can bind to exposed collagen to serve as a long tether to capture platelets. The VWF A1 domain is responsible for binding to platelet membrane receptor glycoprotein (GP)Ibα. The binding of VWF to platelets in this manner not only physically sequesters platelets near the site of injury, but also stimulates platelets to engage other receptors to form a stable basis for coagulation to occur. The interactions of collagen with VWF and then VWF with GPIbα are the interactions which define primary hemostasis. 

The regulation of the GPIbα-VWF interaction has been the host of great debate over several decades. It postulated many years ago that force must be involved in the activation of VWF, as plasma VWF does not spontaneously bind to platelets. In this dissertation, I sought to elucidate the molecular mechanism that controls the force dependent activation of VWF. Using single-domain antibodies, I further probed VWF activation by modulating the functions of VWF both under flow and under static conditions. The research outlined in this dissertation demonstrates the importance of local autoinhibitory elements to the control of VWF activation and outlines an approach to target these elements to regulate VWF activity. 

Table of Contents

Abstract [4] 

Acknowledgements [6] 

Table of Contents [7] 

List of Figures and Tables [10] 

List of Abbreviations [13] 

Chapter 1 – General Introduction [14] 

1.1 Early history of von Willebrand factor [15] 

1.2 History of glycoprotein (GP)Ibα [16] 

1.3 Structure and organization of VWF [19] 

1.4 Von Willebrand’s Disease [23] 

1.5 Activation of VWF and the VWF-GPIbα interaction [25] 

1.6 Single-domain antibodies [36] 

1.7 Outstanding questions [42] 

Chapter 2 – Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module [44] 

2.1 Abstract [45] 

2.2 Introduction [46] 

2.3 Methods [48] 

2.4 Results [59] 

2.5 Discussion [79] 

2.6 Acknowledgements [85] 

2.7 Supplemental Information [85] 8 

Chapter 3 – Conservation and species-specific adaptation of force-dependent activation of von Willebrand factor [111] 

3.1 Abstract [112] 

3.2 Introduction [113] 

3.3 Methods [115] 

3.4 Results [120] 

3.5 Discussion [131] 

3.6 Acknowledgements [137] 

3.7 Supplemental Information [138] 

Chapter 4 – Nanobody activation of von Willebrand factor by disrupting the inhibition of the N-terminal autoinhibitory module [145] 

4.1 Abstract [146] 

4.2 Introduction [147] 

4.3 Methods [149] 

4.4 Results [158] 

4.5 Discussion [172] 

4.6 Acknowledgements [174] 

4.7 Supplemental Information [175] 

Chapter 5 – Mechanism-directed selection of allosteric inhibitors of a mechanosensory protein [187] 

5.1 Abstract [188] 

5.2 Introduction [189] 

5.3 Methods [191] 

5.4 Results [195] 

5.5 Discussion [203] 9 

5.6 Acknowledgements [206] 

5.7 Supplemental Information [207] 

Chapter 6 – General Discussion [213] 

6.1 Summary of Results and General Discussion [214] 

6.2 Limitations and Future Directions [220] 

6.3 Conclusion [223] 

References [224] 

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