Differential Role of the RAC1-Binding Proteins FAM49b (CYRI-B) and CYFIP1 in Platelets.

Platelet function at vascular injury sites is tightly regulated through the actin cytoskeleton. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex (WRC) activates lamellipodia formation via ARP2/3, initiated by GTP-bound RAC1 interacting with the WRC subunit CYFIP1. The protein FAM49b (Family of Unknown Function 49b), also known as CYRI-B (CYFIP-Related RAC Interactor B), has been found to interact with activated RAC1, leading to the negative regulation of the WRC in mammalian cells. To investigate the role of FAM49b in platelet function, we studied platelet-specific Fam49b-/--, Cyfip1-/--, and Cyfip1/Fam49b-/--mice. Platelet counts and activation of Fam49b-/- mice were comparable to those of control mice. On fully fibrinogen-coated surfaces, Fam49b-/--platelets spread faster with an increased mean projected cell area than control platelets, whereas Cyfip1/Fam49b-/--platelets did not form lamellipodia, phenocopying the Cyfip1-/--platelets. However, Fam49b-/--platelets often assumed a polarized shape and were more prone to migrate on fibrinogen-coated surfaces. On 2D structured micropatterns, however, Fam49b-/--platelets displayed reduced spreading, whereas spreading of Cyfip1-/-- and Cyfip1/Fam49b-/--platelets was enhanced. In summary, FAM49b contributes to the regulation of morphology and migration of spread platelets, but to exert its inhibitory effect on actin polymerization, the functional WAVE complex must be present.

Thrombopoietin levels in sepsis and septic shock - a systematic review and meta-analysis.

OBJECTIVES: Sepsis is a life-threatening condition implicating an inadequate activation of the immune system. Platelets act as modulators and contributors to immune processes. Indeed, altered platelet turnover, thrombotic events, and changes in thrombopoietin levels in systemic inflammation have been reported, but thrombopoietin-levels in sepsis and septic-shock have not yet been systematically evaluated. We therefore performed a meta-analysis of thrombopoietin (TPO)-levels in patients with sepsis. METHODS: Two independent reviewers screened records and full-text articles for inclusion. Scientific databases were searched for studies examining thrombopoietin levels in adult sepsis and septic-shock patients until August 1st 2022. RESULTS: Of 95 items screened, six studies met the inclusion criteria, including 598 subjects. Both sepsis and severe sepsis were associated with increased levels of thrombopoietin (sepsis vs. control: standardized mean difference 3.06, 95 % CI 1.35-4.77; Z=3.50, p=0.0005) (sepsis vs. severe sepsis: standardized mean difference -1.67, 95 % CI -2.46 to -0.88; Z=4.14, p<0.0001). TPO-levels did not show significant differences between severe sepsis and septic shock patients but differed between sepsis and inflammation-associated non-septic controls. Overall, high heterogeneity and low sample size could be noted. CONCLUSIONS: Concluding, increased levels of thrombopoietin appear to be present both in sepsis and severe sepsis with high heterogeneity but thrombopoietin does not allow to differentiate between severe sepsis and septic-shock. TPO may potentially serve to differentiate sepsis from non-septic trauma and/or tissue damage related (systemic) inflammation. Usage of different assays and high heterogeneity demand standardization of methods and further large multicenter trials.

The WASH-complex subunit Strumpellin regulates integrin αIIbß3 trafficking in murine platelets.

The platelet specific integrin αIIbß3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbß3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbß3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbß3 surface expression. While exposure of the internal αIIbß3 pool after platelet activation was unaffected, the uptake of the αIIbß3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbß3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbß3 trafficking in murine platelets.

Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration.

Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIbα, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.

Oculocerebrorenal syndrome of Lowe protein controls cytoskeletal reorganisation during human platelet spreading.

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

Absence of early platelet increment in healthy mice during decitabine treatment.

Treatment of myelodysplastic syndromes includes the administration of the hypomethylating agent decitabine. An early platelet response in decitabine-treated myelodysplastic syndrome patients is a predictor of overall survival. The effect of decitabine on megakaryocytes and the bone marrow, however, is understudied. We show that an early platelet increment was not detectable in healthy mice during decitabine treatment. Analyses of bone marrow sections revealed vessels with dilated lumina, decreased cellularity, but increased number of red blood cells and the presence of (pro)platelet-like particles. Taken together, decitabine treatment of healthy mice does not induce an early platelet increment, but affects the bone marrow.

Novel insights into mouse models of ectopic proplatelet release.

Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished ß1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease.

MYH9-related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9-related disease, and treatment with tranexamic acid can improve the hemostatic function.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation.

The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

Short-range organization and photophysical properties of CdSe quantum dots coupled with aryleneethynylenes.

Hybrid organic-inorganic nanomaterials composed of organic semiconductors and inorganic quantum dots (QDs) are promising candidates for opto-electronic devices in a sustainable internet of things. Especially their ability to combine the advantages of both compounds in one material with new functionality, the energy-efficient production possibility and the applicability in thin films with little resource consumption are key benefits of these materials. However, a major challenge one is facing for these hybrid materials is the lack of a detailed understanding of the organic-inorganic interface which hampers the widespread application in devices. We advance the understanding of this interface by studying the short-range organization and binding motif of aryleneethynylenes coupled to CdSe QDs as an example system with various experimental methods. Clear evidence for an incorporation of the organic ligands in between the inorganic QDs is found, and polarization-modulation infrared reflection-absorption spectroscopy is shown to be a powerful technique to directly detect the binding in such hybrid thin-film systems. A monodentate binding and a connection of neighboring QDs by the aryleneethynylene molecules is identified. Using steady-state and time resolved spectroscopy, we further investigated the photophysics of these hybrid systems. Different passivation capabilities resulting in different decay dynamics of the QDs turned out to be the main influence of the ligands on the photophysics.

Cyclophilin A Is Not Acetylated at Lysine-82 and Lysine-125 in Resting and Stimulated Platelets.

Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed-neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping.

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.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Thymosin ß4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets.

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin ß4 (encoded by Tmsb4x) is one of the two main G-actin-sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin ß4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. Megakaryocyte numbers in the bone marrow and spleen were unaltered, however, Tmsb4x KO megakaryocytes showed defective proplatelet formation in vitro and in vivo. Thymosin ß4-deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin ß4 for actin dynamics during platelet biogenesis, platelet activation downstream of glycoprotein VI and thrombus stability.

Platelet Shape Changes during Thrombus Formation: Role of Actin-Based Protrusions.

Platelet activation and aggregation are essential to limit blood loss at sites of vascular injury but may also lead to occlusion of diseased vessels. The platelet cytoskeleton is a critical component for proper hemostatic function. Platelets change their shape after activation and their contractile machinery mediates thrombus stabilization and clot retraction. In vitro studies have shown that platelets, which come into contact with proteins such as fibrinogen, spread and first form filopodia and then lamellipodia, the latter being plate-like protrusions with branched actin filaments. However, the role of platelet lamellipodia in hemostasis and thrombus formation has been unclear until recently. This short review will briefly summarize the recent findings on the contribution of the actin cytoskeleton and lamellipodial structures to platelet function.