Integrin dynamics and platelet mechanosensing via surface receptors GPIbα and integrin αIIbβ3

platelet;molecular mechanotransduction;GPIbα;αIIbβ3;binding kinetics;signaling

Chen, Yunfeng ; Zhu, Cheng Mechanical Engineering McIntire, Larry Lam, Wilbur Li, Renhao Du, Xiaoping ; Zhu, Cheng

University:Georgia Institute of Technology

Department:Mechanical Engineering

关键词: platelet;    molecular mechanotransduction;    GPIbα;    αIIbβ3;    binding kinetics;    signaling;   

Others  :  https://smartech.gatech.edu/bitstream/1853/58596/1/CHEN-DISSERTATION-2016.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

In hemostasis and thrombosis, platelet adhesion and signaling play key roles. Two platelet receptors, glycoprotein Ibα (GPIbα) and integrin αIIbβ3, mediate the early and mid-stages of platelet adhesion in arterial environments. GPIbα is part of the GPIbα-V-IX complex that constitutes the receptor for von Willebrand factor (VWF). Its binding to VWF A1 domain enables rolling of platelets on the sites of vascular injury. αIIbβ3, upon activation, allows for platelet stable adhesion to the sub-endothelial surface and facilitates the platelets aggregation by cross-linking via soluble fibrinogen, fibronectin and VWF. GPIbα and αIIbβ3 has been reported to trigger outside-in mechano-activating signals upon ligand engagement in a sequential fashion, but exactly how the extracellular mechano-signals are transduced and translated to intracellular chemical signals remains unknown. In my PhD thesis research, I investigate the molecular dynamics of integrins, and study GPIbα and αIIbβ3 in the context of platelet adhesion and signal initiation. Using a cutting-edge technique, biomembrane force probe, I conduct single-molecule and single-cell level experiments to investigate the conformational and functional dynamics of integrin molecules under mechanical forces, and the relation between mechanical signals received by GPIbα and αIIbβ3 and platelet activation readouts. My research brought us new biomechanical insights on integrin dynamics and platelets adhesion and signaling: 1) Integrin αVβ3 undergoes bending and unbending conformational changes in a dynamic fashion. This process does not require energetic or signaling support from a cell. The probability, rate and speed of these bending and unbending conformational changes are regulated by mechanical force, ligand, integrin genotype, cation and purified/cellular environment. Force-enabled αVβ3 unbending does not reinforce the strength of its binding in the physiological Ca2+/Mg2+, but does in Mn2+.2) A single GPIbα-VWFA1 binding event is able to trigger platelet intracellular Ca2+ flux. A positive correlation was found between the binding duration (lifetime) and the intensity of the Ca2+ signal, so that the signaling is stronger triggered by WT VWFA1 at higher forces because of its catch-bond behavior, but weaker by a Von Willibrand disease (VWD) mutant R1450E which manifest a slip-bond. This suggests a new concept about VWD: signaling deficiency. Unfolding of two domains in the GPIbα structure, lucine-rich-repeat (LRRD) and mechanosensitive (MSD) domains, mediates the signal transduction in that, LRRD unfolding prolongs the binding lifetime and analogously enhances signaling intensity, whereas MSD unfolding digitally affects the fluxed Ca2+ type. Abolishment the binding of a cytoplasmic molecule 14-3-3ζ to GPIb complex depleted most of the α-type Ca2+. The strong correlation between mechanical force, structural conformational changes and molecular association of GPIbα, and the amplitude and type of the resulted Ca2+ signal suggested that GPIb together with its functionally adjacent molecules form a molecular mechanosensor.3) The mechano-signaling of GPIbα triggers an intermediate level of integrin αIIbβ3 activation, P-selectin expression and phosphatidylserine exposure as compared to ADP and thrombin, two soluble platelet activators, which suggests that GPIbα triggers platelets into an intermediate activation state. The intermediate up-regulation of αIIbβ3 is unstable and transient, but is important in that it opens the pathway of outside-in mechano-signaling, so that the αIIbβ3 can be further activated through repeated binding and pulling of ligand. This defines cooperation between integrin inside-out and outside-in mechano-signaling. On the other hand, ADP-activated αIIbβ3 can also be further activated via outside-in mechano-signaling to a hyperactive state. Overall, my study discovered a four-stage regulation of platelet αIIbβ3 by bidirectional signaling.Finally, I extended my research to tentatively investigate atherothrombosis in type I and II diabetes. I found that platelet hyperreactivity in diabetes is closely associated with enhanced adhesive capacity and exaggerated mechano-signaling of GPIbα and integrin αIIbβ3. These results imply the medical importance of my research, and reveal the advantages of single-cell biomechanics in disease studies and diagnostics.