Platelet actin nodules are podosome-like structures dependent on Wiskott–Aldrich syndrome protein and ARP2/3 complex

Abstract

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott–Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp−/− mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet–platelet interaction and their absence contributes to the bleeding diathesis of Wiskott–Aldrich syndrome.

Apolipoprotein A‐I Modulates Atherosclerosis Through Lymphatic Vessel‐Dependent Mechanisms in Mice

“…Furthermore, an assembling effect was observed, in which several platelets will aggregate together in an attempt to bring together several LECs (Figure 6A, right panel). Using the same experimental setting, quantification of pseudopodia‐shaped platelet adhesion to the lymphatic endothelium allowed us to observe a significant increase in the number of strongly adhered platelets45 per LECs when the latter were pretreated with apoA‐I (Figure 6B and 6C).…”

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice

“…Since platelets are significantly smaller than regular cells, we employed structured illumination microscopy (SIM) [35] to characterize the spatial organization of Pdlim7 with respect to other actin-associated proteins, α-actinin and vinculin (Figs 5 and 6). SIM provides a detailed image of protein localization to about ~110nm, which roughly doubles the lateral resolution of conventional light microscopy.…”

Integrin αIIbβ3 outside-in signaling

“…32,51,124 There have been contrasting reports regarding the role of talin in a IIb b 3 outside-in signaling, 125,126 although recent work suggests that, although not required for the initial outside-signaling responsible for platelet spreading, a later reassociation of talin with b 3 is required for clot retraction. 43,51 Upon stretchinduced changes in talin, binding sites for vinculin become exposed, and this crosslinker is important for consolidating the integrincytoskeletal linkage in nucleated cells.…”

“…32,120 However, although vinculin may support megakaryocyte membrane cytoskeletal integrity when force is applied to a IIb b 3 , assessment of vinculin-deficient platelets suggests it may not have a major role in platelet a IIb b 3 outside-in signaling, with mice only showing a mild hemostasis defect in a tailbleeding assay. 127 This is surprising given vinculin's abundance in platelets, 58,60,128 its presence in actin nodules, 124 and its importance for integrin-actin linkage in other cell types, 32 and may be due to robust compensatory mechanisms involving other proteins. 127 Similarly, a-actinin can consolidate adhesive complexes, 32,74 yet its role in a IIb b 3 outside-in signaling remains unclear.…”