Background: Human platelet FcγRIIA recognizes IgG immune complexes with high affinity. This specificity plays a central role in heparin-induced thrombocytopenia and during interaction of platelets with IgG opsonized bacteria.

Aims: To quantitatively assess the lateral mobility dynamics of FcγRIIA on human platelets and its regulation.

Methods: Total internal reflection fluorescence microscopy (TIRFm) was used for imaging and tracking of single FcγRIIA on human platelets by photoluminescent quantum dot (QD) nanoparticles (5nm diameter) conjugated to monoclonal anti-FcγRIIA Fab (Figure 1a). Quantification of FcγRIIA mobility dynamics (e.g. single receptor trajectories, diffusion coefficients, and track length) on live spreading platelet filopodia, lamellipodia and platelet body was performed. To assess the role of FcγRIIA mobility for sensing of IgG opsonized particles by platelets, we used micropatterned arrays coated with aggregated IgG, or IgG coated bacteria-mimetic beads, or IgG opsonized live bacteria. The relevance of platelet cytoskeletal integrity was assessed by blocking cytoskeletal functions with cytochalasin D and blebbistatin.

Results: We report for the first time diffusion dynamics of single FcγRIIA molecules on human platelets (Fig. 1 A). The single FcγRIIA molecules moved longer distances on filopodia (Mean ± SD 1.53 µm 0.98)) and lamellipodia (1.22 ± 0.96) compared to the FcγRIIA molecules on platelet body (0.55 ± 0.51; P=0.0001) (Figure 1 B). Disruption of F-actin assembly using cytoclalasin D and blebbistatin decreased the diffusion and track length of single FcγRIIA dynamics in the platelet membrane. Cytoskeletal integrity was indispensable for FcγRIIA mediated ligand sensing, platelet adhesion, spreading and activation on aggregated IgG, IgG coated bacteria-mimetic beads, and IgG opsonized live bacteria.

Conclusions: Continuous actin-based “treadmilling” mechanism controls the dynamics of FcγRIIA lateral mobility on the platelets. This facilitates rapid ligand engagement by FcγRIIA and may have broad implications for the response of platelets to opsonized bacteria and IgG coated autologous cells, e.g. in immune thrombocytopenia or the anti-phospholipid syndrome.

[Lateral Mobility Dynamics of Single FcγRIIA on Human Platelets]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/platelets-as-immune-cells-lateral-mobility-dynamics-of-fc%ce%b3riia-on-human-platelets-is-directly-linked-to-integrity-of-platelet-cytoskeleton/