Sunday, November 24, 2024
11/24/2024
PROJECT SUMMARY/ABSTRACT In human bodies, bleeding is stopped when a clot is formed at the site of vascular damage. Under rapid flow conditions, the plasma protein von Willebrand factor (VWF) plays an indispensable role in capturing both platelets and collagen on damaged vessel walls, allowing the formation of platelet plugs. The adhesion between VWF and platelets is mediated by the interaction between the A1 domain of VWF and the Ib chain of the platelet receptor GPIb-IX complex. Gain-of-function mutations in A1 that enhance this interaction lead to type 2B von Willebrand disease (VWD). Targeting the A1GPIb-IX interaction has been an emerging strategy to treat or preempt bleeding and thrombotic disorders, though success in this area has been very limited. The lack of progress is due largely to the enigmatic nature of how exactly A1 remains inactive in blood circulation and how it is instantly activated to bind to GPIb-IX upon bleeding. Our recent identification of an autoinhibitory module (AIM), consisting of N- and C-terminal flanking regions on A1 and their O-linked glycans, is crucial for understanding A1 mechanoactivation during bleeding. In addition, AIM can be unfolded by a tensile pulling force of 8 to 20 pN. Based on these preliminary discoveries, we hypothesize that O-linked glycan structures, particularly sialic acids, further stabilize AIM and contribute to the mechanical regulation of A1GPIb-IX binding and that modulating AIM’s mechanical properties can be utilized to treat or preempt blood diseases. We propose to test this potentially paradigm-shifting hypothesis using state-of-the-art analytical biophysical tools, including single-molecule force spectroscopy, single-molecule fluorescence microscopy and all-atom molecular dynamics simulation. Three specific aims will be pursued to test the hypotheses. Aim 1 is to characterize the structure and biomechanical properties of AIM. Aim 2 is to determine how autoinhibition is regulated by O-linked glycosylation in AIM. And Aim 3 is to investigate the role of AIM in type 2B VWD and therapeutic applications. Completion of the proposed studies will identify the key molecular and biophysical mechanisms underlying how AIM mechanically regulates VWF function and platelet binding and will aid in devising novel therapeutic strategies for the prevention and treatment of human blood disease.
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