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Fibronectin assembly of platelets on adhesion proteins of the provisional clot and the basement membrane. (a) 3D dSTORM image of pFn647-enriched Fn fibril assembly by a single platelet seeded on fibrin (Fb, binds αIIbβ3). See also Supplementary Movie S1. Scale bar 2 µm. (b) 3D dSTORM image of pFn647-enriched Fn fibril assembly by a single platelet seeded on collagen type IV (Col-4, binds α2β1). Scale bar 2 µm. (c) Systematic comparison of fibril start-end height between Fn (blue), Fb (blue), Col-4 (magenta) and Ln-111 (magenta). Data were pooled in addition to Figure 2 from two healthy human donors (31-33 years) and were compared with a non-parametric Kruskal-Wallis rank test with post-hoc Dunn test to make (multiple) comparisons. (d) Integrin mechanosensing of ECM proteins triggers dimensionality of the first deposited ECM network. Platelets spread on Fn and Fb (right) generate high traction forces (red arrows and dark red-yellow) and Fn fibrils are aligned to polarized actin bundles (top view). Mechanosignaling (black arrow) through αIIbβ3 (blue) instructs Fn fibril anchorage along the apical membrane of platelets and the mechanomolecular strain induces a spatial offset between vinculin (green) and Fn (see inset). On Ln or Col4 (middle), platelets contract less strong (yellow) and
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Upon vascular injury, platelets are crucial for thrombus formation and contraction, but do they directly initiate early tissue repair processes? Using 3D super-resolution microscopy, micropost traction force microscopy, and specific integrin or myosin IIa inhibitors, we discovered here that platelets form fibrillar adhesions. They assemble fibronec...
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... the architecture on laminin with respect to their anchorage beneath the cell (Figure 6c), length and thickness (Supplementary Figure S8). Exemplary line profiles (dashed) of the localization density along the line in (b) for the vinculin stain (green) and the pFn stain (blue). ...
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... is thus remarkable that platelets not only orchestrate thrombus formation and contraction, but also direct the initial tissue repair process by assembling Fn nanofibrils (Figures 1,2,6). This Our findings taken together with existing literature can be summarized in a new mechanoregulated thrombus formation model (Figure 6 d,e): we now propose the existence of at least three different platelet phenotypes, defined by either low, medium or high contractility. Each subtype serves contractile-specific functions, including their ability to sense their ECM environment and to respond by either being procoagulant or assembling de novo ECM. ...
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... our study investigated fundamental mechanisms of fibrillar adhesion formation by using isolated platelets, in order to specify the phenomenon, the integrins involved and how fibronectin fibrillogenesis is regulated by platelet contractility, we can only speculate here how the three platelet phenotypes might impact blood clot formation, contraction and the subsequent invasion of cells. Our mechanistic sketch (Figure 6e) though is in agreement with our previous observations of Fn fibrillogenesis and fibroblast invasion made on 2 and 24 hours old blood clots that had formed on titanium surfaces ( Burkhardt et al. 2016). ...
Citations
... When the platelets are exposed to ECM, they are activated by interaction with collagen and laminin, and also with fibrinogen in vascular events [48]. The activation of the platelets upon contact with ECM proteins occurs via their adhesion receptors, especially when the endothelial barrier is disrupted [49]. PolyP in the circulating blood or plasma has a half-life of~1.5-2 ...
Inorganic polyphosphates (polyP) are of increasing medical interest due to their unprecedented ability to exhibit both morphogenetic and ATP-delivering properties. However, these polymers are only physiologically active in the coacervate state, but not as amorphous nanoparticles (NP), the storage form of the polymer. Little is known about the mechanism of formation and interconversion of these two distinct polyP phases in the presence of metal ions. Based on in silico simulation studies, showing a differential clustering of polyP and calcium ions, the pH-dependent NP and coacervate formation of polyP was examined experimentally. Turbidimetric studies showed that Ca-polyP coacervate formation at pH 7 is a slow process compared to NP formation at pH 10. In FTIR spectra, the asymmetric stretching vibration signal of the internal (PO2)⁻ units, which is present in the Ca-polyP coacervate formed at pH 7, disappears in the NP formed at pH 10 using the conventional method (dropping of a CaCl2 solution into a Na-polyP solution). Surprisingly, when reversing the procedure, adding Na-polyP to CaCl2, a coacervate is obtained at both pH 7 and pH 10, as confirmed by SEM and FTIR analyses. The (PO2)⁻ signal also disappears when Ca-polyP-NP are exposed to peptides, leading to the transformation of the NP into the coacervate phase. From these results, a mechanistic model of pH-dependent coacervate and NP formation is proposed that considers not only electrostatic ion-ion but also ion-dipole interactions. Functional studies revealed a delayed polyP release kinetics for Ca-polyP-NP embedded in a hydrogel due to NP/coacervate conversion. Human A549 epithelial cells grown on the coacervate show increased proliferation and ATP production compared to cells cultured on particulate polyP. Ca-polyP NP taken up by endocytosis undergo intracellular coacervate transformation. Understanding the differential expression of the two polyP phases is of functional importance for the potential therapeutic application of this physiological, regeneratively active polymer.
... Currently, a wide array of biophysical methods is available for the investigation of platelet biomechanics. They include micropipette aspiration [15][16][17] , atomic force microscopy [18][19][20] , scanning ion conductance microscopy 21,22 , traction force microscopy 23,24 , including flexible micropost arrays [25][26][27] . Although these methods have proven valuable in advancing our insights into platelet biomechanics, these are technically demanding, labor-intensive, and mostly limited to analysis of adherent platelets 28 . ...
Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.
... 44,45 A wide variety of biophysical techniques are currently used to assess cytoskeleton-dependent contractile force generation at piconewton (pN) and biomechanical properties of platelets at nanometer (nm) regimes. [46][47][48][49] In this review we are focusing on micropipette aspiration, atomic force microscopy (AFM), scanning ion conductance microscopy (SICM), and real-time deformability cytometry (RT-DC) that offer varying degree of sensitivities and throughputs to investigate platelet biomechanics up to single platelets (►Table 1). ...
Cytoskeleton is composed of more than 100 proteins and represents a dynamic network of the cellular cytoplasm. Cytoskeletal functions include spatial organization of cellular components, structural connection of the cell with external environment, and biomechanical force generation. Cytoskeleton takes part, at different levels, in all phases of platelet biogenesis: megakaryocyte (MK) differentiation, MK maturation, and platelet formation. In addition, it also plays a major role in each stage of platelet function. Inherited platelet disorders (IPDs) are a group of rare diseases featured by low platelet count and/or impaired platelet function. Over the past decade, the investigation of platelet biomechanics has become a major and highly relevant theme of research due to its implications at every stage of development of human life. The initial use of diverse biophysical techniques (e.g., micropipette aspiration, atomic force and scanning ion conductance microscopy, real-time deformability cytometry) started unraveling biomechanical features of platelets that are expected to provide new explanations for physiological and pathological mechanisms. Although the impact of cytoskeletal alterations has been largely elucidated in various IPDs' pathogenesis, the understanding of their impact on biomechanical properties of platelets represents an unmet need. Regarding IPDs, improving biomechanical studies seems promising for diagnostic and prognostic implications. Potentially, these characteristics of platelets may also be used for the prediction of bleeding risk. This review addresses the current available methods for biophysical investigations of platelets and the possible implementations in the field of IPDs.
... Lastly, these studies alert us to how little we know about the function of CTL2 in platelets. Even the well-studied platelet integrin continues to surprise us with new facets of its versatile functionality, including its recently revealed role in orchestrating the assembly of new ECM [19], thereby constantly reshaping our understanding of platelet functions in and beyond thrombosis. ...
Nanoparticles of a particular, evolutionarily old inorganic polymer found across the biological kingdoms have attracted increasing interest in recent years not only because of their crucial role in metabolism but also their potential medical applicability: it is inorganic polyphosphate (polyP). This ubiquitous linear polymer is composed of 10–1000 phosphate residues linked by high‐energy anhydride bonds. PolyP causes induction of gene activity, provides phosphate for bone mineralization, and serves as an energy supplier through enzymatic cleavage of its acid anhydride bonds and subsequent ATP formation. The biomedical breakthrough of polyP came with the development of a successful fabrication process, in depot form, as Ca‐ or Mg‐polyP nanoparticles, or as the directly effective polymer, as soluble Na‐polyP, for regenerative repair and healing processes, especially in tissue areas with insufficient blood supply. Physiologically, the platelets are the main vehicles for polyP nanoparticles in the circulating blood. To be biomedically active, these particles undergo coacervation. This review provides an overview of the properties of polyP and polyP nanoparticles for applications in the regeneration and repair of bone, cartilage, and skin. In addition to studies on animal models, the first successful proof‐of‐concept studies on humans for the healing of chronic wounds are outlined.
