Platelet Clearance and Mechanosensation via GPIb-IX-V: How Platelets Use the Force Público
Quach, Michael (Fall 2019)
Abstract
GPIb-IX-V is the primary mechanosensitive receptor complex on the platelet surface. All ligands targeting the complex bind to the GPIbα subunit and unfolding of a membrane proximal mechanosensory domain (MSD) in GPIbα leads to receptor activation. Despite this, expression of the other subunits of GPIb-IX-V (GPIbβ and GPIX) is required for expression on the platelet membrane. I present evidence that antibodies targeting GPIbβ modulate the activity of GPIb-IX-V, supporting a model wherein conformational changes in GPIbβ are part of the transduction pathway for mechanical signals through GPIb-IX-V. Many patients suffering from immune thrombocytopenia are resistant to first-line immunosuppressive treatments, a prognosis associated with autoantibodies targeting GPIb-IX-V. However, the mechanism of this resistance is unknown. Here, we present evidence that antibodies against the ligand binding domain (LBD) of GPIb-IX-V activate the receptor in a shear-dependent manner. Single-molecule optical tweezer experiments demonstrate that this effect depends on and antibody's unbinding force from the LBD. Antibodies with unbinding forces too low to unfold the MSD in GPIbα cannot activate the receptor, nor can they induce IVIG resistant ITP. Imaging flow cytometry and light transmission aggregometry reveal that activating antibodies can sustain crosslinking of platelets via GPIb-IX-V under shear, suggesting a mechanism by which anti-LBD antibodies exert force on GPIb-IX-V via crosslinking, activate GPIb-IX-V by unfolding its MSD, and induce Fc-independent platelet clearance.
Table of Contents
I. Introduction…………………………………………………………………………………… 1
A. Stop! In the Name of Blood: Circulation and Hemostasis [2]
B. If I Only Had a Brain: Platelets and Other Thrombocytes [18]
C. Symphonies of Destruction: Mechanisms of Platelet Clearance [29]
D. My Own Worst Enemy: Platelet Clearance in Immune Thrombocytopenia (ITP) [38]
E. The Force Theme: GPIb-IX-V and Platelet Mechanosensation [46]
F. No Line on the Horizon: What We Know and What We Hope to Learn [54]
II. A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-Plate Viscometry…………58
A. More Than a Feeling: Detecting the Effects of Force on Mechanoreceptors [60]
B. Protocol [65]
C. Notes on Performing This Assay [67]
III. Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets…………………… 69
A. Shadowboxin' - Fighting Immune Thrombocytopenia in the Dark [71]
B. Methods [75]
C. Results [78]
D. Living in the World Today: A Discussion of Results at Hand and Future Directions [95]
IV. Rethinking Ligands of GPIb-IX-V………………………………………………………… 98
A. Come Together: Divalent Ligands for GPIb-IX-V [99]
B. F(X)I Gon' Give it to Ya: The Curious Case of FXI and GPIb-IX-V [100]
C. Rules of Engagement: A MAb Against the Sialomucin Region Activates GPIb-IX-V [104]
D. Hold On (We're Going Home): Prototype of a Crosslinking Ligand [107]
V. Anti-GPIbb antibodies regulate activation of the platelet GPIb-IX-V complex……110
A. Dancing in the Dark: Pulling Back the Veil on GPIbb's Contribution to GPIb-IX-V Activation [111]
B. Methods [113]
C. Results [114]
D. Discussion [122]
VI. Conclusion and closing remarks …………………………………………………………………128
A. Back at One [129]
References [139]
Appendix [163]
Table of Abbreviations [165]
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