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SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

ABSTRACT: BACKGROUND:Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES:Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS:Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS:Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS:These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.

SUBMITTER: Green HLH

PROVIDER: S-EPMC7186844 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

Green Hannah L H HLH Zuidscherwoude Malou M Alenazy Fawaz F Smith Christopher W CW Bender Markus M Thomas Steven G SG

Journal of thrombosis and haemostasis : JTH 20200217 4

<h4>Background</h4>Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail.<h4>Objectives</h4>Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains ...[more]

PMID: 31930764

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Project description:Essentials Platelets play an important role in pathogen recognition. Platelets contain several complement factors and can interact with E. coli. Platelet's complement protein C3 differs from plasmatic C3 in its electrophoretic mobility. Upon contact with bacteria, platelets are activated and can enhance complement activation.SummaryBackground The role of platelets in immune defense is increasingly being recognized. Platelets bind complement proteins from plasma, initiate complement activation, and interact with bacteria. However, the contribution of platelets to complement-mediated defense against bacterial infections is not known in detail. Objectives To assess platelet interactions with Escherichia coli strains, and evaluate the contributions of platelet complement proteins to host defense. Methods We studied the cell-cell interactions of a pathogenic and a non-pathogenic E. coli strain with platelet concentrates, washed platelets and manually isolated platelets by flow cytometry and ELISA. The presence of complement proteins and complement RNA in megakaryocytes and platelets was analyzed by PCR, RT-PCR, confocal microscopy, and western blotting. Results Incubation with E. coli leads to platelet activation, as indicated by the expression of CD62P and CD63 on the platelet surface. RNA and protein analyses show that megakaryocytes and platelets contain complement C3, and that platelet C3 migrates differently on polyacrylamide gels than plasmatic C3. Activation of platelets by bacteria leads to translocation of C3 to the cell surface. This translocation is not induced by thrombin receptor activating peptide or lipopolysaccharide. Interaction of platelets with E. coli occurs even in the absence of plasma proteins, and is independent of platelet toll-like receptor 4 and ?2b ?3 (glycoprotein IIbIIIa). Conclusion Platelets contain a specific form of C3. Importantly, they can modulate immune defense against bacteria by enhancing plasmatic complement activation.

Project description:BackgroundUpon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation.ResultsHere, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bβ3integrin signaling in D723H cells is sufficient to induce β1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and β1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of β1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation.ConclusionsWe characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.

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SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

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SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

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