a-uniform-shear-assay-for-human-platelet-cell-surface-receptors-via

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Children's Healthcare of Atlanta

Emory University School of Medicine

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets.

Hundreds of billions of platelets are cleared daily from circulation via efficient and highly regulated mechanisms. These mechanisms may be stimulated by exogenous reagents or environmental changes to accelerate platelet clearance, leading to thrombocytopenia. The interplay between antiapoptotic Bcl-x and proapoptotic molecules Bax and Bak sets an internal clock for the platelet lifespan, and BH3-only proteins, mitochondrial permeabilization, and phosphatidylserine (PS) exposure may also contribute to apoptosis-induced platelet clearance. Binding of plasma von Willebrand factor or antibodies to the ligand-binding domain of glycoprotein Ibα (GPIbα) on platelets can activate GPIb-IX in a shear-dependent manner by inducing unfolding of the mechanosensory domain therein, and trigger downstream signaling in the platelet including desialylation and PS exposure. Deglycosylated platelets are recognized by the Ashwell-Morell receptor and potentially other scavenger receptors, and are rapidly cleared by hepatocytes and/or macrophages. Inhibitors of platelet clearance pathways, including inhibitors of GPIbα shedding, neuraminidases, and platelet signaling, are efficacious at preserving the viability of platelets during storage and improving their recovery and survival in vivo. Overall, common mechanisms of platelet clearance have begun to emerge, suggesting potential strategies to extend the shelf-life of platelets stored at room temperature or to enable refrigerated storage.

Article	Year
Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets. Blood\| PubMed ID: 29203584	2018
Mechanisms of platelet clearance and translation to improve platelet storage. Blood\| PubMed ID: 29475962	2018

M. Edward Quach

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