Platelet-like Particles Research Articles

Reinforcement of hydroxyapatite bone cement via thin glycerol-citrate polyester infiltration: Microstructural, mechanical and in-vitro evaluation

The development of calcium phosphate cements (CPCs) incorporating biopolymers, such as thermoplastic polylactides, is known as an efficient strategy to enhance the physico-chemical, biological, and mechanical properties of CPCs. However, the use of thermosetting polyol citrates as fillers in CPCs has rarely been reported. Hence, the main goal of the present work was to produce composite CPCs based on tetracalcium phosphate–monetite cement matrix (C) and glycerol-citrate polyester (GCA) and to thoroughly study the effect of GCA addition on microstructural, micro, and nano-mechanical as well as in-vitro cellular properties of final cement composites. The GCA polymer was synthesized by esterification of glycerol with citric acid and coated on the C cement powder matrix at a concentration of 2.5 wt% by infiltration in ethanol solution. Two systems were prepared, by drying the composite powder at 105 and 170 °C (denoted as GCA/C105 and GCA/C170), while the respective composite cements were produced by mixing the powders with the 2 wt% of NaH2PO4 liquid phase. The results showed that the GCA polyester had no significant effect on the hydrolysis and transformation of the original cement matrix, and the nanocrystalline hydroxyapatite (Hap) was found as the main hydration product in all types of cement. The cement setting time decreased slightly with the GCA addition. On the other hand, a significant increase in mechanical properties was reached by introducing the GCA polyester into the CPC matrix, achieving more than a 70 % increase in the compressive strength, and about 50 % and 20 % in micro and nanohardness values compared to that obtained for the original C cement sample. The nanoindentation analysis revealed that the improved compressive strength and hardness were due to the stronger boundaries between the platelet-like hydroxyapatite particles in GCA-doped cements and partially caused by the denser, less porous, and more compact microstructures. The results of in-vitro cytotoxicity testing revealed high proliferation activity of the osteoblastic cells in all cement extracts, providing useful information for designing novel composite cements with the potential to be applied as bone substitute materials.

Abstract 123: Hemostatic Fibrin Knob Triggered Microgel To Promote Clotting With Reduced Thrombotic Risks In Neonates

Introduction: Effective neonatal hemostatic therapies are vital for complex surgery-related life-threatening bleeding [1]. Neonatal hemostatic systems differ significantly from adults [2, 3]; however, current treatments for bleeding include adult blood products (ABP). Neonatal ABP use poses an elevated thrombotic risk [4, 5], necessitating alternative approaches. We propose hemostatic therapies leveraging age-dependent clotting differences. In adults, clot formation relies on fibrin(ogen) A:a knob-hole binding, while B:b binding dominates in neonates. Targeting B:b interactions with synthetic Bknob conjugated platelet-like particles (BK-PLP) could crosslink neonatal fibrinogen, catalyze clot formation, and enhance clot strength with lower thrombotic risk than ABP. To test this, we synthesized pNIPAm microgels and conjugated them to B-knob (AHRPYAAK), A-knob (GPRPFPAK), and non-binding control peptides (GPSPFPAK) to prepare BK-PLP, AK-PLP, and NB-PLP. Materials and Methods: We prepared BK-PLP, AK-PLP, and NB-PLP by conjugating microgels with mimetic peptides using EDC-Sulfo-NHS chemistry (Fig. 1A); Fibrin clot polymerization, stability, density, and stiffness, were analyzed via plate-based assays, confocal microscopy, and AFM. Results and Discussion: BK-PLPs significantly increased clot stability compared to AK-PLP, NB-PLP, or fibrin-only clots (Fig. 1B). Fiber density also increased with BK-PLPs, indicating a role in promoting denser fibrin clot structures (Fig. 1C). Clot mechanical properties assessment suggested that AKnob and NB-PLPs may disrupt clot networks, decreasing stiffness (Fig. 1D). Conclusion: Our study explored the effects of peptide conjugate particles on neonatal fibrin clots. BK-PLP incorporation increased clot stability, while AK-PLP and NB-PLP decreased clot stiffness, supporting further BK-PLP development for neonatal hemostasis.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Qing-Fei-Pai-Du Decoction ameliorated coagulopathy and thrombocytopenia in endotoxaemia rats and enhanced platelet production from megakaryocytes

BackgroundsThe Qing-Fei-Pai-Du Decoction (QFPDD) was reported to ameliorate the inflammatory coagulopathy and thrombocytopenia in COVID-19 patients. This work investigated the effects of QFPDD in endotoxaemia rats and then further studied its influence on platelet production using cultured megakaryocytes. MethodsThe coagulation indexes and platelet aggregation reactivity of LPS-induced endotoxemia rats with or without QFPDD treatment were analyzed. The platelet parameters, the expression of thrombopoietin (TPO) in the liver and the kidney, number of the megakaryocytes in bone marrow smear of rats were checked. The maturation and platelet production of human megakaryoblastic MEG-01 cells treated with QFPDD or isochlorogenic acid A (ISA), an active component of QFPDD, were observed. ResultsQFPDD improved the coagulation indexes of LPS-induced endotoxaemia rats and decreased the hyper-reactivity of platelets. QFPDD ameliorated LPS-induced decrease in platelet number, partially alleviated increase in liver TPO expression and exhibited no influence on increase in the number of bone marrow megakaryocytes. QFPDD promoted maturation and production of platelet-like particles of MEG-01 megakaryocytes. The effects of QFPDD on MEG-01 megakaryocytes might be related to activation of AKT pathway and up-regulation of related transcription factors. ISA also could induce the maturation of MEG-01 cells. ConclusionsQFPDD effectively ameliorated coagulopathy and thrombocytopenia in rats under inflammatory state. The mechanisms of QFPDD in ameliorating thrombocytopenia might be based on both decreasing effects on platelet depletion in coagulation and inducing effects on maturation and platelet production of megakaryocytes. ISA might be one of the components which contribute to the effects of QFPDD on megakaryocytes.

Murine Infusion Studies of Infused Human Megakaryocytes Support a “Catch-and-Release” Model of Thrombopoiesis

Introduction: Thrombopoiesis, the process by which megakaryocytes (Mks) release platelets, is still not well understood, including where it occurs and how a Mk releases its platelet content. The present study, based on infused human Mks (hMks) into immunocompromised mice, and measurements of the subsequent presence of nucleated Mks and anucleated cytoplasmic fragments in the lungs and in the peripheral blood were carried out to provide new insights into this process. Method: To gain a deeper understanding of the role of the lung in thrombopoiesis, we performed quantitative, temporal measurements of the size of human CD41 +CD42 + nucleated Mks and anucleated cytoplasmic fragments after tail-vein infusion of either mature (Day 12 of culture from CD34 + hematopoietic stem/progenitor cells (HSPCs)) hMks or immature (Day 7 of culture from CD34 + HSPCs) in NOD-scid gamma (NSG) mice. Data was collected from both the pulmonary bed by in situ microscopy ( Figure 1) and in the peripheral blood by flow cytometry ( Figure 2). To distinguish in the peripheral blood large cytoplasmic fragments from recirculating hMks, Hoechst-nuclear staining was done prior to flow cytometric analysis For size references, isolated donor platelets and input Hoechst-stained hMks, containing platelet-like particles (PLPs), were concurrently analyzed ( Figure 2, bottom left). Results: By pulmonary in situ microscopy, we found that infused mature hMks were entrapped on their first passage through the lungs. Over the subsequent hour, anucleate cytoplasmic fragments ( Figure 1, left) recycle to the lungs becoming increasingly smaller in size over the hour-long study. Infused immature hMks also shed cytoplasmic fragments, but these began much smaller in size though these, too, reduced in size over time ( Figure 1, right). In the peripheral blood, mature CD41 +CD42 + hMks made up <2% of all the circulating fragments ( Figure 2, top left). These hMks initially were comparable in size to the input hMks ( Figure 2, bottom), but decreased in size over time, presumably due to shedding of their cytoplasm. Already by 10 mins post-infusion, a dominant group of large-sized (L), anucleated CD41 +CD42 + cytoplasmic fragments were observed, slightly smaller than the input Hoechst-stained hMks. Their presence decreased, reaching near-zero in <20 hrs. In contrast, mid-sized (M), cytoplasmic fragments, comparable in size to donor platelets, increased in abundance over time. Small-sized (S), cytoplasmic fragments were only present at 10 mins and were largely the input PLPs. In contrast, up to 34% of the circulating CD41 +CD42 + fragments after infusing immature hMks were nucleated ( Figure 2, right), suggesting that unlike mature hMks that are efficiently entrapped by the murine lungs, many immature hMks were small enough to pass through the murine vascular beds and recycle multiple times without completely releasing their cytoplasm. Conclusions: In the murine lungs, mature hMks are efficiently entrapped and within minutes shed large cytoplasmic fragments. These cytoplasmic fragments then recycle back to the lung for further processing, resulting in a near complete release of platelet-size fragments by 90 mins. These findings are consistent with the recent observations of Zhao X, et al., using a distinct in vivo murine lung/heart system (PMC10329040) except that infused murine Mks recycled nucleated. Whether this difference is a true species-difference or related to hMks traversing the murine vasculature needs to be explored, but in aggregate, these two studies demonstrate that thrombopoiesis is a complex process that begins when whole Mks and/or large cytoplasmic fragments are released intravascular from the marrow, spleen and/or lung. Subsequently, the Mks and large cytoplasmic fragments are then converted into smaller fragments and finally platelets by repetitive cycles of “catch-and-release” in the pulmonary bed. The mechanistic basis controlling this process needs better elucidation, but at the same time, offers insights of potential clinical application.

Turbulence Enhances Fine-Tuning of Mitochondria Delivery in Megakaryocyte Maturation Contributing to Biogenesis of High Quality Platelets

Platelet (PLT) products have a short shelf life of 4-5 days but blood donations are limited. As a solution, we established induced pluripotent stem cell (iPSC)-derived immortalized megakaryocyte cell lines (imMKCLs) (Nakamura, Cell Stem Cell, 2014), which generated platelet-like particles (iPSC-PLTs) under static conditions, albeit with low yield and function. Subsequently, by developing turbulence-flow bioreactors, imMKCLs generated high yield of competent iPSC-PLTs (Ito, Cell, 2018). Meanwhile, it is still unclear the reason why turbulence stimulation into imMKCLs allow higher production efficiency and improved function. Herein we found turbulence increases CD42b (GPIbα) expression in iPSC-PLTs, which was inversely correlated with phosphatidylserine (PS) expression levels (as represented by Annexin V binding). Only CD42b + population of iPSC-PLTs responded to agonist stimulation, detected by P-selectin expression or PAC1 binding. We demonstrated functional CD42b + iPSC-PLTs are determined by mitochondria activity in iPSC-PLTs, but not in imMKCLs. Regarding CD42b expression, it is known that ADAM17 causes CD42b shedding upon exposure of phosphatidylserine on the cell surface (Sommer, Nature communications, 2016). Therefore, we examined whether mitochondria activity influence CD42b shedding. To test this, we added FCCP, a mitochondrial uncoupler depleting mitochondria membrane potential, in the absence or presence of shedding inhibitors in imMKCL culture. Depleting mitochondria activity decreased CD42b expression levels on iPSC-PLTs, independently of culture conditions. Pre-incubation with ADAM17 inhibitor or different type shedding inhibitors prior to FCCP treatment allowed to retain CD42b levels, suggesting that mitochondria function might regulate downstream ADAM17 activity or shedding mechanisms. This was confirmed by GPIX deficiency in imMKCLs, as no difference in mitochondria activity was found compared to the control. Differences in CD42b levels were not seen in MKs during maturation, independent of culture conditions, therefore we suspected turbulence promote intact mitochondria delivery in imMKCLs. To examine this hypothesis, we used Mdivi-1, which inhibit Drp1 activity and thereby suppress mitochondria fission, to mimic impaired mitochondria delivery. Administration of Mdivi-1 to turbulence-exposed imMKCLs decreased CD42b expression in subsequently generated iPSC-PLTs. Using confocal microscopy, we also visualized fused mitochondria in imMKCLs upon addition of Mdivi-1, strongly suggesting that negative CD42b expression in iPSC-PLTs is due to impaired mitochondria delivery. Next, to gain mechanistic insight at molecular levels, we differentiated imMKCLs in static or turbulence conditions and swapped the culture conditions on day 3 of the total 6 days of imMKCL maturation. It revealed that turbulence is required only after day 3 onwards, but not before. scRNA analysis and pharmacological examination results strongly suggested that increased HDAC5, 6, and 9 levels by turbulence stimulation are crucial for efficient mitochondria delivery into iPSC-PLTs. We conclude that turbulence increases ‘fine-tuning’ of mitochondria distribution in imMKCLs and efficient delivery to PLTs with relatively sufficient activity via an HDAC mediated mechanism, where mitochondria activity is required for inhibition of phosphatidylserine exposure on the cell surface and of ADAM17 activity.

Wound-triggered shape change microgels for the development of enhanced biomimetic function platelet-like particles.

Platelets play a pivotal role in hemostasis and wound healing and conditional shape change is an important component of platelet functionality. In normal circumstances, platelets travel through the circulatory system in an inactive rounded state, which enables platelets to easily move to vessel walls for attachment. When an injury occurs, platelets are prompted by molecules, such as thrombin, to shift into a stellate shape and increase exposure of fibrin-binding receptors. When active, platelets promote hemostasis and clot retraction, which enhances clot stability and promotes healing. However, in conditions where platelets are depleted or hyporeactive, these functions are diminished and lead to inhibited hemostasis and healing. To treat platelet depletion, our group developed platelet-like particles (PLPs) which consist of highly deformable microgels coupled to fibrin binding motif. However, first generation PLPs do not exhibit wound-triggered shape change like native platelets. Thus, the objective of these studies was to develop a PLP formulation that changes shape when prompted by thrombin. To create thrombin-sensitive PLPs (TS-PLPs), we incorporated a thrombin-cleavable peptide into the microgel body and then evaluated PLP properties before and after exposure to thrombin including morphology, size, and in vitro clot retraction. Once thrombin-prompted shape change ability was confirmed, the TS-PLPs were tested in vivo for hemostatic ability and subsequent wound healing outcomes in a murine liver trauma model. We found that TS-PLPs exhibit a wound-triggered shape change, induce significant clot retraction following exposure to thrombin and promote hemostasis and healing in vivo after trauma.

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

BackgroundGermline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. ObjectivesTo decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. MethodsPresuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. ResultsAnalysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core “ribosome biogenesis” pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. ConclusionThese findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

Structure and magnetic properties of Al3+ substituted M-type SrLaCo hexaferrite

M-type hexagonal ferrites (SrFe12O19) have attracted much attention as a widely used permanent magnet. Many studies have been done to improve permanent magnetic properties. In particular, the formula of La–Co co-replaced Sr–Fe has achieved higher performance. However, no one has studied the influence of a small amount of Al ions on the above formula. In this study, the Al3+ ion substituted M-type Sr0.7La0.3Fe11.75ￚxAlxCo0.25O19 (0 ​≤ ​x ​≤ ​0.12) hexagonal ferrites were synthesized by solid state reaction. The crystalline structure was investigated by using X-ray diffraction (XRD) and the Rietveld refining method. The morphological of powder and magnetically oriented bulk were observed by scanning electron microscopy (SEM), and magnetic properties were measured by physical property measurement system-vibrating sample magnetometer (PPMS-VSM) techniques. The results show that a single M-type strontium ferrite was obtained with the Aluminum (Al) content (x) from 0 to 0.08, and the impure phase appeared when x was 0.12. The lattice parameters a and c both decreased with the increasing Al3+ content. SEM images indicate the hexagonal platelet-like particles and the size of the magnetic powders about 2–5 ​μm, and the powders are easily arranged in one direction under a magnetic field. The saturation magnetization (Ms) of the hexaferrite decreased with the increase of x from 0 to 0.12, but coercivity (Hc) of the magnetic powders increased with Al3+ content (x) from 0 to 0.12.

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.

Histone deacetylation-regulated cell surface Siglec-7 expression promoted megakaryocytic maturation and enhanced platelet-like particle release

BackgroundFunctioning as important hematologic cells for hemostasis, wound healing and immune defense platelets are produced before being released into the blood by cytoplasmic fragmentation at the end of the megakaryocyte (MK) differentiation, during which the involvement of both apoptosis and autophagy has been reported. Inhibitory sialic acid–binding immunoglobulin-like lectin-7 gene (Siglec-7) can be expressed on platelets and induce apoptosis on activation for uncharacterized function. ObjectiveWe aimed to investigate the regulatory mechanism for Siglec-7 activation along MK differentiation and its physiologic role during the MK maturation and platelet formation. MethodsBy using 2 well-established MK differentiation models (HEL and K562) and human primary CD34+ cell, we examined the upregulations of transcript and protein levels of Siglec-7 during MK differentiation, and the effect of Siglec-7 surface presence on MK differentiation and platelet-like particles (PLPs) release. ResultsWe show that both transcripts and surface Siglec-7 were elevated during MK differentiation, and the histone deacetylase 1 (HDAC1) acted as a negative regulator for Siglec-7 activation. By increasing Siglec-7 surface expression, we found that increased presence of Siglec-7 not only enhanced MK maturation but also the release of PLPs by activating caspase 3-dependent signaling, as evidenced in the observation of more CD41, polyploidy, and platelet factor 4 transcript formations. ConclusionIn this study, we demonstrated that Siglec-7 activation was subjected to epigenetic regulation, and the resulting induced expression of surface Siglec-7 played an important regulatory role in promoting MK differentiation, maturation, and PLP formation.

Novel insights into mouse models of ectopic proplatelet release

AbstractMature 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 β1-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.

MALAT1 promotes platelet activity and thrombus formation through PI3k/Akt/GSK-3β signalling pathway

BackgroundIschaemic stroke and other cardiovascular illnesses are characterised by abnormalities in the processes of thrombosis and haemostasis, which rely on platelet activity. In platelets, a wide variety of microRNAs (long...

Dengue and Zika virus differential infection of human megakaryoblast MEG-01 reveals unique cellular markers

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48–72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%–25%) and 5% (3%–8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-β, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections.

Corrosion Resistance and Catalytic Activity toward the Oxygen Reduction Reaction of CoCrFexNi (0 ≤ x ≤ 0.7) Thin Films

Corrosion resistance and catalytic activity toward the oxygen reduction reaction (ORR) in an alkaline environment are two key properties for water recombination applications. In this work, CoCrFexNi (0 ≤ x ≤ 0.7) thin films were deposited by magnetron sputtering on polished steel substrates. The native passive layer was 2–4 nm thick and coherent to the columnar grains determined by transmission electron microscopy. The effect of Fe on the corrosion properties in 0.1 M NaCl and 1 M KOH and the catalytic activity of the films toward ORR were investigated. Electrochemical impedance spectroscopy and potentiodynamic polarization measurements indicate that CoCrFe0.7Ni and CoCrFe0.3Ni have the highest corrosion resistance of the studied films in NaCl and KOH, respectively. The high corrosion resistance of the CoCrFe0.7Ni film in NaCl was attributed to the smaller overall grain size, which leads to a more homogeneous film with a stronger passive layer. For CoCrFe0.3Ni in KOH, it was attributed to a lower Fe dissolution into the electrolyte and the build-up of a thick and protective hydroxide layer. Scanning Kelvin probe force microscopy showed no potential differences globally in any of the films, but locally, a potential gradient between the top of the columns and grain boundaries was observed. Corrosion of the films was likely initiated at the top of the columns where the potential was lowest. It was concluded that Fe is essential for the electrochemical activation of the surfaces and the catalytic activity toward ORR in an alkaline medium. The highest catalytic activity was recorded for high Fe-content films (x ≥ 0.5) and was attributed to the formation of platelet-like oxide particles on the film surface upon anodization. The study showed that the combination of corrosion resistance and catalytic activity toward ORR is possible for CoCrFexNi, making this material system a suitable candidate for water recombination in an alkaline environment.

Nd2O3, Cr2O3, and V2O3 Nanoparticles via Calcination: Synthesis, Characterization, Antimicrobial and Antioxidant Activities

Nd2O3, Cr2O3, and V2O3 nanoparticles were prepared by calcining the precursor materials that are novel mixed ligand complexes: [Nd(BDC)(ADMPY)(OAc)].H2O, [Cr(BDC)(ADM PY)Cl].H2O, and [V(BDC)(ADMPY)Cl].H2O, where BDC = 1,4-benzenedicarboxylic acid and ADMPY = 2-amino-4,6-dimethyl pyrimidine. The generated compounds were examined through several techniques such as elemental analysis (C.H.N), UV-Vis spectroscopy, thermal analysis (thermogravimetric, differential thermogravimetry, and differential thermal analysis), FT-IR spectra, X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM). The TEM micrographs showed that neodymium oxide nanoparticles assumed agglomerated platelet-like particles, with particle sizes around 30.16 nm, while chromium oxide NPs showed solid block material with compact density and fewer pores with nearly spherical shape and 56.12 nm size. The vanadium oxide NPs were an agglomeration of small spherical nanoparticles of 28.4 nm size. The antimicrobial properties of the samples were assessed using two strains of Gram-positive bacteria, two strains of Gram-negative bacteria, and one strain of yeast. The antimicrobial results demonstrated that a large spectrum of activity characterizes the tested compounds because they are active on Gram-positive and Gram-negative bacteria, especially on Gram-positive strains. The antioxidant activity of prepared compounds was assessed by scavenging free radicals of DPPH. Metal oxide NPs also showed promising results as antioxidants.

Expansion of Human Megakaryocyte-Lineage Progeny via Aryl Hydrocarbon Receptor Antagonism with CH223191.

Aryl hydrocarbon receptor (AhR) antagonism is known to expand human hematopoietic stem cells (HSCs). However, its regulatory effect on the lineage-skewed differentiation of HSCs has not been sufficiently studied. Here, we investigate the effect of the AhR-selective antagonist CH223191 on the regulation of HSC differentiation. Consistent with the well-known effects of AhR antagonists, CH223191 treatment increase phenotypic HSCs (Lin-CD34 + CD38-CD90 + CD45RA-) and preserves their functionality. On the other hand, CH223191 leads to an overall expansion of megakaryocyte (MK)-lineage populations, such as MK progenitors (MKps, CD34 + CD41 +), immature MKs (CD41 + CD42b-), and mature MKs (CD41 + CD42b +), and it also activates MK/platelet-associated signaling pathways. Furthermore, CH223191 expands MKps, mature MKs, and p-selectin (CD62p)-positive platelet-like particles in immune thrombocytopenia (ITP) patient bone marrow (BM). These results highlight the numerical expansion of human MK-lineage progeny through AhR antagonism with CH223191. This approach using CH2231291 may be applicable in the development of auxiliary treatment regimens for patients with abnormal thrombopoiesis.