Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces

Abstract

<ns4:p><ns4:bold>Background:</ns4:bold> Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome.</ns4:p><ns4:p> <ns4:bold>Methods:</ns4:bold> We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin.</ns4:p><ns4:p> <ns4:bold>Conclusions:</ns4:bold> Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.</ns4:p>

Structural analysis of receptors and actin polarity in platelet protrusions

“…Moreover, microtubules are often found in the pseudopodia protrusions, in addition to the actin filaments. This observation is in agreement with a recent study that identified microtubules in over 30% of platelet protrusions (53).…”

“…Analysis of cryotomograms revealed densities attached to the extracellular phase of the plasma membrane ( Fig. 1 C , arrowheads, Inset ), while the cytoplasm contains cytoskeleton filaments (i.e., actin filaments and often microtubules), as previously described ( 52 , 53 ). Cryo-ET analysis of mouse platelets deficient in the α IIb β 3 integrin ( 54 ) shows a smooth membrane lacking extracellular densities ( SI Appendix , Fig.…”

Folylpolyglutamate synthetase mRNA G-quadruplexes regulate its cell protrusion localization and enhance a cancer cell invasive phenotype upon folate repletion

“…3 ), with no cells harboring localized mRNA after 2 h (Additional file 2 : Figure S3 A, C), VBT-treated cells retained the protrusion and the localized mRNA even after 3 h (Additional file 2 : Figure S3, E, G). Previous studies demonstrated that filopodia remain intact and motile after disruption of microtubule with VBT and/or NCZ [ 113 , 114 ]. Although many studies describe the transport of mRNA to cell protrusions, we could not find any publication describing the fate of mRNA after its local translation; does it undergo dispersion or degradation?…”

Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen

“…During the process of spreading, the actin cytoskeleton of platelets forms filamentous structures that associate with myosin to form actomyosin filaments that generate traction forces (4). This process is critical for stable adhesion (5)(6)(7), but there are differences in platelet spreading depending on the adhesive environment (8)(9)(10)(11).…”