Morphometric analysis of spread platelets identifies integrin α_IIbβ₃-specific contractile phenotype

Abstract

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin αIIbβ3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner.The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of αIIbβ3 integrins. Cytoskeletal morphologies in vitro can thus inform about platelet adhesion receptor identity and functionality, and integrin αIIbβ3 mechanotransduction fundamentally determines the adoption of a highly contractile bipolar phenotype. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet contractility contributing to elusive bleeding phenotypes.

Platelets exploit fibrillar adhesions to assemble fibronectin matrix revealing new force-regulated thrombus remodeling mechanisms

“…Adhesion signaling of either α6β1 on laminin, α2β1 on Col4, or αIIbβ3 on Fn or fibrin is sufficient to drive αIIbβ3 to assemble Fn fibrils (Figures 1,2,6), in analogy to the pre-activation of α5β1 integrins by αvβ3 in fibroblasts Bharadwaj et al 2017), the initiation of α5β1-containing sliding fibrillar adhesions by α3β1 or α2β1 integrins on Matrigel (Lu et al 2020), and in accordance with previous observations (Olorundare et al 2001;Jaehyung Cho et al 2005;Mosher 2006b, 2006a;Jaehyung Cho and Mosher 2006). Adhesion signaling through αIIbβ3 is essential to develop full contractility (Figure 4), maximal stress fibers (Lickert et al 2018), as well as a 3D apical anchorage of Fn fibrils as seen in fibroblasts (Figure 1) and a similar mechanomolecular strain within fibrillar adhesions ( Figure 5). We for the first time derive a threshold of ~1 nN per cell-substrate adhesion to activate αIIbβ3 integrins to form 3D fibrils ( Figure 4).…”

“…As Fn fibrillogenesis is initiated by tensile forces that partially expose cryptic Fn-Fn assembly sites (Hocking, Smith, and McKeown-Longo 1996;Gretchen Baneyx and Vogel 1999) and an impaired cytoskeleton reduces Fn deposition by platelets (J. Cho and Mosher 2006b) and by other cells (G. Baneyx, Baugh, and Vogel 2002), we next analyzed the spatial organization of the actin cytoskeleton using dual color (2C) dSTORM. Confirming previous observations (Lickert et al 2018), we found that the majority of platelets on Fn coatings had formed a highly aligned 'bipolar' filamentous (F-)actin network with pronounced bundles traversing the cell (Figure 1m and Supplementary Figure S1). Longer Fn fibrils emanated parallel to these actin cables at adhesion sites situated at both ends and extended beyond the platelet edge ( Figure 1n).…”

“…Since platelets sense different ECM proteins after vascular injury, we asked whether the spatial architecture of de novo assembled Fn fibrils depends on the ECM proteins to which the platelets adhere. Fn fibrils were radially oriented on laminin (Ln), in agreement with previously reported Fn deposition patterns (J. Cho and Mosher 2006b), while the actin cytoskeleton showed a rather ring-like arrangement (Lickert et al 2018 Figure S5). No statistically significant differences were found for the tested laminin isoforms 111 or 521 (Supplementary Figure S5).…”

“…Talin unfolding is accompanied by an increasing spatial offset between integrins and vinculin (Case et al 2015;Liu et al 2015). In platelets spread on Fn, vinculin was strongly localized to peripheral adhesion sites and formed string-like patterns that were co-aligned with F-actin bundles (Lickert et al 2018). Fn fibrils overlapped with these elongated vinculin patterns and were tightly registered (Figure 5a,b), similar to fibrillar adhesions in fibroblasts .…”

“…Besides Fn, we selected fibrin as the major clot component since spreading on fibrin but not fibrinogen supports Fn matrix assembly by platelets (Jaehyung Cho et al 2005). Platelets on fibrin coatings showed the typical αIIbβ3-dependent contractile phenotype (Lickert et al 2018) and formed Fn fibrils mainly localized in the periphery of the cell ( Figure 6a). As on Fn coatings, Fn fibrils on fibrin were mainly pulled along the apical membrane ( Figure 6c) and only a moderate increase in fibril length and thickness were noted (Supplementary Figure S8).…”