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In our laboratory, we study thrombopoiesis and hemostasis using zebrafish as a model organism to unravel the mechanisms of differentiation and development of thrombocytes. We have shown in our earlier work that thrombocytes are functional equivalents of platelets and have transcriptional machinery similar to megakaryocytes. We recently found evidence that hox genes play a role in their development. We used piggyback gene knockdown and thrombocyte quantification assays to understand the influence of these ancient developmental regulators on thrombopoiesis. In this chapter, we describe methods used to discover these hox genes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5'-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups., (© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

This comprehensive review examines the role of fish thrombocytes, cells considered functionally analogous to platelets in terms of coagulation, but which differ in their origin and morphology. Despite the evolutionary distance between teleosts and mammals, genomic studies reveal conserved patterns in blood coagulation, although there are exceptions such as the absence of factors belonging to the contact system. Beyond coagulation, fish thrombocytes have important immunological functions. These cells express both proinflammatory genes and genes involved in antigen presentation, suggesting a role in both innate and adaptive immune responses. Moreover, having demonstrated their phagocytic abilities, crucial in the fight against pathogenic microorganisms, underscores their multifaceted involvement in immunity. Finally, the need for further research on the functions of these cells is highlighted, in order to better understand their involvement in maintaining the health of aquaculture fish. The use of standardized and automated methods for the analysis of these activities is advocated, emphaiszing their potential to facilitate the early detection of stress or infection, thus minimizing the economic losses that these adverse situations can generate in the field of aquaculture., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Acute exacerbations (AE) are severe complications of chronic obstructive pulmonary disease (COPD); however, the need for biomarkers which predict them is still unmet. High platelet count (PLC) and platelet-to-lymphocyte ratio (PLR) are associated with higher mortality in patients with COPD. We investigated if PLC and PLR at the onset of a severe AE could predict the time of the next relapse., Methods: In a prospective observational cohort study, data of 152 patients hospitalized with AECOPD were collected, and patients were divided into PLC-low (<239 ​× ​10 9 /L, n ​= ​51), PLC-medium (239-297 ​× ​10 9 /L, n ​= ​51) and PLC-high (>297 ​× ​10 9 /L, n ​= ​50) or PLR-low (<147, N ​= ​51), PLR-medium (147-295, n ​= ​51) and PLR high (>295, n ​= ​50) groups based on PLC and PLR tertiles using admission laboratory results. Clinical characteristics and the time to the next severe or moderate AE within 52 weeks were compared among subgroups using log-rank test., Results: PLC and PLR tertiles did not differ in clinical characteristics or the time till the next AE (p ​> ​0.05). PLC and PLR showed a direct weak correlation to neutrophil count (Pearson r ​= ​0.26, p ​< ​0.01 and r ​= ​0.20, p ​= ​0.01) and PLC also demonstrated a weak relationship to white blood cell counts (Pearson r ​= ​0.29, p ​< ​0.001). However, PLR presented an inverse relationship to monocyte and eosinophil counts (r ​= ​-0.32, p ​< ​0.001 and r ​= ​-0.17, p ​= ​0.03)., Conclusion: PLC and PLR do not predict the time till the next relapse; however, they may reflect on neutrophilic inflammatory response during an exacerbation of COPD., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024. Published by Elsevier B.V.)

Surgical-induced hemostasis is a critical step in the closure of incisions, which is frequently achieved via electrocauterization and subsequent tissue necrotization. The latter is associated with postoperative complications. Recent in vivo work suggested reactive species-producing gas plasma technology as a pro-homeostatic agent acting via platelet activation. However, it remained elusive how platelet activation is linked to lipid and protein oxidation and the reactive species compositions. A direct relation between the reactive species composition and platelet activation was revealed by assessing the production of several reactive species and by using antioxidants. In addition, platelet lipidome and proteome analysis identified significantly regulated key lipids in the platelet activation pathway, such as diacylglycerols and phosphatidylinositol as well as oxylipins like thromboxanes. Lipid oxidation products mainly derived from phosphatidylethanolamine and phosphatidylserine species were observed at modest levels. In addition, oxidative post-translational modifications were identified on key proteins of the hemostasis machinery. This study provides new insights into oxidation-induced platelet activation in general and suggests a potential role of those processes in gas plasma-mediated hemostasis in particular., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: The study aimed to determine a simple diagnostic test that could predict the risk of anastomotic leakage in early postoperative period., Methods: A single-center, retrospective study was conducted. The electronic medical records of patients who underwent resection for rectal tumor between January 1, 2016, and December 31, 2021, in University Hospital Olomouc, were reviewed. The data included risk factors for leakage and laboratory parameters commonly obtained., Results: The decrease in platelets was significant as for the possibility of being a marker of anastomotic leakage; OR = 0.980 (p = 0.036). A decrease of 34 or higher predicts leakage with a sensitivity of 45 % (95 % CI: 23.1-68.5 %) and specificity of 81.1 % (95 % CI: 75.2-86.1 %). Postoperative leukocyte blood level (OR = 1.134; p = 0.019) and leukocyte level on postoperative day 1 (OR = 1.184; p = 0.023) were significant predictors for leakage. WBC values ≥ 8.8 predict leakage with a sensitivity of 70.0 % (95 % CI: 45.7-88.1 %) and specificity of 55.3 % (95 % CI: 48.4-62.0 %).  Hemoglobin blood level ≤ 79.5 predicts leakage with a sensitivity of 70.0 % (95 % CI: 45.7-88.1 %) and specificity of 62.2 % (95 % CI: 55.5-68.7 %)., Conclusion: Despite the fact that the specificity and sensitivity of the followed parameters are low, they could serve as markers useful for early diagnosis or suspicion for leakage (Tab. 5, Fig. 3, Ref. 14).

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga -/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga +/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα Δ 19-56 . This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα +/ Δ 19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga -/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα +/ Δ 19-56 . We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y 1 and P2Y 12 receptors might ameliorate the septic response to HlyA-producing E. coli . The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y 1 and P2Y 12 antagonists. Our data show that the P2Y 1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y 1 , by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y 12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y 1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y 1 receptor antagonists.

In vitro pharmacodynamic models are used to optimize in vivo dosing regimens in antimicrobial drug development. One limiting factor of such models is the lack of host factors such as corpuscular blood components as erythrocytes which have already been shown to impact activity of antibiotics and/or growth of the pathogen. However, the impact of thrombocytes has not previously been investigated. We set out to investigate if the addition of thrombocytes (set to physiological concentrations in blood of healthy human, i.e., 5 × 10 5 thrombocytes/μL standard growth media Mueller Hinton Broth, MHB) has an influence on bacterial growth and on the efficacy of antibiotics against Gram+ and Gram- bacteria. Growth assays and time-killing-curves (TKC) were performed with ATCC-strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in triplicate over 24 h. The same approach was followed for 5 clinical isolates of Escherichia coli. Meropenem, ciprofloxacin, and tigecycline were tested as representatives of broad-spectrum antibiotics, and concentrations several-fold above and below the minimal inhibitory concentration (MIC) were simulated. No significant impact of thrombocytes was found on bacterial growth or antimicrobial stability for the investigated agents. Bacteria reduced thrombocyte content to different degree, indicating direct interaction of pathogens and thrombocytes. Impact on bacterial killing was observed but was not fully reproducible when thrombocytes from different donors where used. While interaction of bacteria and thrombocytes was evident in the present study, interaction between antibiotic activity and thrombocytes seems unlikely. Whether variability was caused by different thrombocyte concentrates needs further investigation.

Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially bacteraemia. The number of sepsis cases has been steadily increasing over the last decades, and there are still no specific, molecular supportive therapies for sepsis to supplement antibiotic treatment. P2X 1 receptors are expressed by a number of immune cells including thrombocytes, which presently have been established as an important player in the acute immune response to bacterial infections. P2X 1 receptor-deficient mice have been shown to be relatively protected against urosepsis, with markedly reduced levels of circulating proinflammatory cytokines and intravascular coagulation. However, here we show that continuous intravenous infusion with P2X 1 receptor antagonist markedly accelerates development of a septic response to induced bacteraemia with uropathogenic E. coli. Mice exposed to the P2X 1 receptor antagonists die very early with haematuria, substantially elevated plasma levels of proinflammatory cytokines, massive intravascular coagulation and a concomitant reduction in circulating thrombocytes. Interestingly, infusion of P2X 1 receptor antagonists causes a marked acute reduction in circulating thrombocytes and a higher number of bacteria in the blood. These data support the notion that the number of functional thrombocytes is important for the acute defence against bacteria in the circulation and that the P2X 1 receptor potentially could be essential for this response.

Human studies have shown an association between certain platelet indices and active inflammatory bowel disease when compared to healthy controls. The objectives of this retrospective study were to determine if any platelet indices differ between dogs with chronic enteropathies and healthy age- and sex-matched control dogs and are predictive of the severity of chronic enteropathy based on canine chronic enteropathy clinical activity index (CCECAI) scores. Medical records for 22 chronic enteropathy-positive dogs and 22 healthy control dogs were reviewed for historical and physical examination findings, platelet indices, and histologic diagnoses of chronic enteropathy. Platelet indices were compared between the groups, and an association between platelet indices and CCECAI scores in dogs with chronic enteropathy was investigated. Chronic diarrhea and weight loss were common clinical signs associated with chronic enteropathy. Lymphoplasmacytic enteritis was the most common histologic diagnosis. Only one platelet index, platelet component distribution width (P =  0.045), was found to be significantly different between the groups. For predicting severity of disease, determined by CCECAI score, statistically significant differences in indices associated with higher scores were platelet count (P = 0.024) and platelet dry mass distribution width (P =  0.036). Only platelet component distribution width showed potential in characterizing dogs with chronic enteropathy compared to normal dogs. Elevated platelet count and decreased platelet dry mass distribution width had a significant effect on total CCECAI scores. These findings suggest further investigation into the utility of platelet indices as predictors of disease presence and severity in dogs with chronic enteropathy is warranted., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

Recently, ketamine has been investigated as a potential antidepressant option for treatment resistant depression. Unlike traditional drugs, it yields immediate effects, most likely via increased glutamatergic transmission and synaptic plasticity. However, ketamine administration in humans is systemic and its long-term impact on blood parameters has not yet been described in clinical studies. Here we investigated potential sustained effects of ketamine administration (0.5 mg/kg ketamine racemate) on hematological and biochemical values in plasma and serum in a randomized double-blinded study. 80 healthy young participants were included and whole blood samples were collected 5 days before, and 14 days after the infusion. To assess the group effect, repeated measure analyses of co-variance (rmANCOVA) were conducted for the following blood parameters: levels of sodium, potassium, calcium, hemoglobin and number of erythrocytes, lymphocytes, and thrombocytes. RmANCOVA revealed a significant time by treatment effect on thrombocyte levels (F 1, 74 = 13.54, p < 0.001, eta = 0.155), driven by an increase in the ketamine group (paired t-test, t = -3.51, df = 38, p = 0.001). Specificity of thrombocyte effect was confirmed by logistic regression, and in addition, no other coagulation parameters showed significant interaction. Moreover, the relative increase in the ketamine group was stable across sexes and not predicted by age, BMI, smoking, alcohol or drug use, and contraception. Our results describe aftereffects of sub-anesthetic ketamine administration on blood coagulation parameters, which should be considered especially when targeting psychiatric populations with relevant clinical comorbidities., (Copyright © 2018 Elsevier B.V. and ECNP. All rights reserved.)

The historical concentration of thrombocytes is an average of three subsequent concentrations of thrombocytes before donation (during two years) in blood of donors. The evaluation was applied to characteristics of historical concentration of thrombocytes concerning concentration of thrombocytes in blood of 99 regular donors before every donation of apheresis thrombocytes. It is established that less than 20% dispersion between historical concentration of thrombocytes and value obtained in day of donation is observed in 96% of regular donors. The average concentration of thrombocytes before three subsequent donations of apparatus apheresis in males is statistically higher than in females: 235,2±6,7 and 222,1±11,0 (× 109 cells/l) correspondingly (p < 0,05). The concentration of thrombocytes has no relationship with intervals between donations. There is no statistically significant differences between historical concentration of thrombocytes and concentration of thrombocytes before fourth donation and in general and between separate donations both in males and females. It is appropriate to analyze quality of concentrates of thrombocytes received through program accounting historical concentration of thrombocytes., Competing Interests: The authors declare no conflict of interest.

Background and Objective: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed., Material and Methods: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and β-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS., Results: Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes., Conclusion: Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Platelet preparations can stimulate the healing process and have mitogenic properties. We hypothesized that collagen barrier membranes (CBM), clinically used in guided bone regeneration and guided tissue regeneration, can serve as carriers for platelet secretome., Methods: Secretome was generated from washed platelets and unwashed platelets (washed/unwashed PSEC) and lyophilized onto CBM. Overall appearance of CBM was evaluated by scanning electron microscopy. The impact of PSEC on cell attachment was measured based on fluorescence microscopy with DiI-labeled cells. To assess the release kinetics, supernatants of CBM were collected and medium was replaced at hour 1-48. The mitogenic effect was evaluated with periodontal fibroblasts. Furthermore, the release of total protein, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF) β1 was measured., Results: CBM overall appearance and cell attachment was not modulated by PSEC. Supernatants taken after one hour induced a mitogenic response in fibroblasts and showed the highest levels of total protein, TGFβ1 and PDGF-BB. These effects decreased rapidly in subsequent supernatants. While supernatants of CBM loaded with unwashed PSEC induced a stronger mitogenic response than supernatants of CBM loaded with washed PSEC this difference between the PSEC preparations was not observed when cells were seeded on 48-hours-washed CBM., Conclusions: CBM release platelet-derived factors in continuously declining release kinetics.

Toxoplasma (T.) gondii is able to infect various cell types in different hosts. The replication of this parasite within different peripheral mononuclear blood cell populations in chicken has not yet been fully understood. Aim of the present study was to investigate the impact of chicken erythrocytes and thrombocytes as potential host cells for T. gondii. Cultures of primary avian erythrocytes and thrombocytes were inoculated with tachyzoites of T. gondii type II strain ME49. Parasite replication was detected by a quantitative real-time PCR at different times postinoculation until 24 or 48 h, respectively, displaying long-term investigations for the chosen cultures. The parasite replication curve showed a continuous decrease of parasite stages in erythrocytes and thrombocytes. Observations by light microscopy showed massive destruction for both cell populations. Few macrophages in between the infected thrombocytes were viable during the investigation period and showed internalised tachyzoites by confocal laser scanning microscopy. These findings show that T. gondii is not capable of replication in chicken erythrocytes and thrombocytes; therefore, both cannot be considered as potential host cells. In further consequence, monocyte-derived macrophages seem to be the key to the dissemination mechanisms for T. gondii in chicken.

Platelets display a number of properties besides the crucial function of repairing a damaged vascular endothelium and stopping bleeding. Platelets constitutively express molecules that are classically acknowledged to function in primary haemostasis. Platelets specialize in pro-inflammatory activities, and can secrete a large number of molecules, many of which display biological response modifier functions. Recently, platelets expressing receptors for infectious and possibly noninfectious danger signals were shown to be involved in pathophysiological reactions including an immune-inflammatory response. In sepsis, platelets play a key role in immunothrombosis, participate in the formation of NETs (neutrophil extracellular trap) resulting in the trapping and killing of pathogens and are one of the main factors influencing mortality.

Background: The storage time of platelet products negatively affects bacterial safety and platelet function. However, low maximum storage time increases outdating of valuable products. Thus, to quantify the effect of platelet storage time on platelet measurements after platelet transfusion, a systematic review and meta-analyses were performed., Methods: Reports and meeting abstracts of randomized trials and observational studies, performed in humans, reporting platelet measurements after transfusion of platelet products of different storage times, were selected until February 2016. Meta-analyses were performed for four different storage time contrasts, each answering a different question. Random-effects models were used to account for substantial heterogeneity and the weighted mean differences calculated., Results: Our search strategy yielded 4234 studies of which 46 papers satisfied the inclusion criteria. As judged by the 1-h corrected count increment, transfusions of fresher platelets compared to stored platelets showed better increment. The weighed mean difference varied from 2·11 (95%CI: 1·51-2·71) to 2·68 (95%CI: 1·92-3·45). For the 24-h corrected count increment, the weighted mean difference varied from 1·36 (95%CI: 0·12-2·60) to 1·68 (95%CI: 1·07-2·28) depending on the contrast. Recovery and survival of old platelets as percentage of fresh platelets were 81% and 73% for the original definition contrast. For the extended storage contrast, recovery and survival were 75% and 68%., Conclusions: Fresh platelets were superior to old platelets for all platelet measurements and for all storage time contrasts meta-analysed., (© 2016 International Society of Blood Transfusion.)

Raising ostriches became an important economic activity after their products became commodities. The health of farm animals is of paramount importance, so assessing basic immunological responses is necessary to better understand health problems. We developed a method to obtain ostrich thrombocytes and macrophages. The thrombocytes died by apoptosis after 48 h in culture, and the macrophages expanded in size and increased the number of acidic compartments. Macrophages were activated by chicken interferon-γ, producing high levels of nitric oxide. Toxoplasma gondii was able to infect these macrophages, and activation controlled parasitic reproduction. T. gondii, however, persisted in these cells, and infection reduced the production of nitric oxide. These results are important for the future assessment of the basic cellular and immunobiology of ostriches and demonstrate T. gondii suppression of nitric oxide production., (© 2016 Poultry Science Association Inc.)

The hematologic system performs a number of essential functions, including oxygen transport, the execution of the immune response against tumor cells and invading pathogens, and hemostasis (blood clotting). These roles are performed by erythrocytes (red blood cells), leukocytes (white blood cells), and thrombocytes (platelets), respectively. Critically, circadian rhythms are evident in the function of all 3 cell types. In this review, we describe these oscillations, explore their mechanistic bases, and highlight their key implications. Since erythrocytes are anucleate, circadian rhythms in these cells testify to the existence of a nontranscriptional circadian clock. From a clinical perspective, leukocyte rhythms could underlie daily variation in the severity of allergic reactions, the symptoms of chronic inflammatory diseases, and the body's response to infection, while the rhythmic properties of thrombocytes may explain daily fluctuations in the incidence of heart attack and stroke. Consequently, the efficacy of treatments for these conditions is likely to depend on the timing of their administration. Last, we outline preliminary evidence that circadian disruption in the hematologic system could contribute to the deleterious effects of poor diet, shift work, and alcohol abuse on human health., (© 2015 The Author(s).)

Invasive aspergillosis is characterized by vascular invasion and thrombosis. In order to determine the antifungal activity of human platelets, hyphal elongation and metabolic activity of a clinical A. fumigatus isolate were measured. Genome-wide identification of differentially expressed genes in A. fumigatus was performed after exposure to platelets for 15, 30, 60 and 180 min. Data were analyzed by gene ontology annotation as well as functional categories (FunCat) and KEGG enrichment analyses. Platelets attenuated hyphal elongation and viability of A. fumigatus and in total 584 differentially expressed genes were identified, many of which were associated with regulation of biological processes, stress response, transport and metabolism. FunCat and KEGG enrichment analyses showed stress response and metabolic adaptation to be increased in response to platelets. Our findings demonstrate that A. fumigatus displayed a specific transcriptional response when exposed to platelets, thus reflecting their antifungal activities., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish., (Copyright © 2014. Published by Elsevier Inc.)

Vascular endothelial growth factor C (Vegfc) is crucial for lymphatic and blood vessel development, yet its cellular sources and specific functions in heart development remain unclear. To address this, we created a vegfc reporter and an inducible overexpression line in zebrafish. We found vegfc expression in large coronary arteries, circulating thrombocytes, cardiac adipocytes, and outflow tract smooth muscle cells. Notably, although coronary lymphangiogenesis aligns with Vegfc-expressing arteries in juveniles, it occurs only after coronary artery formation. Vegfc overexpression induced ectopic lymphatics on the ventricular surface prior to arterial formation, indicating that Vegfc abundance, rather than arterial presence, drives lymphatic development. However, this overexpression did not affect coronary artery coverage, suggesting a specific role for Vegfc in lymphatic, rather than arterial, development. Thrombocytes emerged as the initial Vegfc source during inflammation following heart injuries, transitioning to endocardial and myocardial expression during regeneration. Lower Vegfc levels in an amputation model corresponded with a lack of lymphatic expansion. Importantly, Vegfc overexpression enhanced lymphatic expansion and promoted scar resolution without affecting cardiomyocyte proliferation, highlighting its role in regulating lymphangiogenesis and promoting heart regeneration. [ABSTRACT FROM AUTHOR]

Introduction: Studies assessing the impact of preoperative and first-day postoperative values of leukocytes, thrombocytes, and platelet/leukocyte ratio (PLR) after radical cystectomy (RC) are sparse. We aimed to assess the impact of these factors on long-term survival after RC. Methods: An analysis of patients undergoing open RC from 2004 to 2023 at our center was performed. Leukocytosis was defined as ≥8,000 leukocytes/μL and thrombocytosis as ≥400,000 thrombocytes/μL. Similarly, the cutoff for PLR was set at 28. A multivariable Cox regression analysis was performed to assess the role of leukocytosis, thrombocytosis, and PLR on long-term survival after RC. For all analyses, hazard ratios (HRs) with the corresponding 95% confidence intervals (CIs) were estimated. Results: A total of 1,817 patients with a median age of 70 years (interquartile range [IQR]: 62–77) were included. Overall, 804 (44%), 175 (10%), and 1,296 (71%) patients presented with leukocytosis, thrombocytosis, and PLR ≥28 preoperatively. Accordingly, 1,414 (78%), 37 (2%), and 249 (14%) patients presented with leukocytosis, thrombocytosis, and PLR ≥28 on the first day after RC. At a median follow-up of 26 months (IQR: 8–68) after RC, 896 (49%) patients died. In the multivariate Cox regression analysis after adjusting for major perioperative risk factors, only preoperative leukocytosis (HR: 1.3, 95% CI: 1.1–1.6, p = 0.01), as well as both preoperative and first-day thrombocytosis (HR: 2.1, 95% CI: 1.5–2.9, and HR: 2.8, 95% CI: 1.6–5.1, p < 0.001, accordingly) were associated with worse overall survival. Conclusion: PLR should not be used as a prognostic marker for survival after RC. On the contrary, preoperative leukocytosis, as well as preoperative and first-day thrombocytosis should raise awareness among clinicians performing RC since they were independently associated with worse survival after RC. [ABSTRACT FROM AUTHOR]

Thrombocytes play an essential role in hemostasis and thrombosis. Moreover, the controlled activation of thrombocytes is required in reproduction and fertility. The platelet-activating factor and the controlled activation of platelets have important roles in folliculogenesis, ovulation, placental development, implantation and embryo development. Activated platelets accumulate in the follicular vessels surrounding the follicle and, due to its released soluble molecules (factors, mediators, chemokines, cytokines, neurotransmitters), locally increase oocyte maturation and hormone secretion. Furthermore, activated platelets are involved in the pathogenesis of ovarian hyperstimulation syndrome (OHSS) and preeclampsia. Low-dose aspirin can prevent OHSS during ovulation induction, while intrauterine or intraovarian administration of platelet-rich plasma (PRP) increases the endometrium thickness and receptivity as well as oocyte maturation. Activated thrombocytes rapidly release the contents of intracellular granules and have multiple adhesion molecules and receptors on their surface. Considering the numerous homeostatic endocrine functions of thrombocytes, it is reasonable to suppose a platelet-associated regulatory system (PARS) in reproduction. Although we are far from a complete understanding of the regulatory processes, the results of PARS research and the therapeutic application of aspirin and PRP during in vitro fertilization are promising. [ABSTRACT FROM AUTHOR]

Background: Phenobarbital (PB) is used as a first‐line treatment for recurrent epileptic seizures in cats. While hematologic abnormalities are well‐known side effects of antiepileptic therapy with PB in humans and dogs, little is known about such alterations in cats. Objectives: The aim of this retrospective study was to investigate the prevalence and clinical relevance of cytopenia during PB treatment in cats. Methods: In this single‐center, retrospective clinical study, 69 cats—with suspected idiopathic epilepsy admitted to the Small Animal Clinic of the University of Veterinary Medicine in Vienna (VMU)—were included. A complete blood count for each patient was performed, and changes in hematocrit, leukocytes, neutrophils, and thrombocytes were documented and graded. Results: Fifty‐three out of 69 cats (76.8%) showed cytopenias with a reduction of at least one cell fraction during PB treatment. The most frequent change was neutropenia (60%), followed by leukopenia (49.3%), thrombocytopenia (24.1%), and anemia (20.3%). Most of the changes were mild or moderate; only one patient (1.5%) showed severe leukopenia and neutropenia, and one was a life‐threatening neutropenia (1.5%) with a serum PB concentration within or even below the therapeutic range. These patients did not present with clinical symptoms other than those related to epileptic episodes. Cats who received combination therapy showed lower hematocrits than those who received monotherapy. A tendency for leukocytes and neutrophils to decrease during PB treatment was also seen. Conclusions: Blood cytopenias may frequently occur in cats on chronic PB therapy, even when serum drug levels are within the therapeutic range. However, clinical signs are typically mild to moderate and rarely severe. [ABSTRACT FROM AUTHOR]

Patients with severe infection have an increased risk of cardiovascular events. A possible underlying mechanism is inflammation-induced platelet aggregation. We investigated whether hyperaggregation occurs during infection, and whether aspirin inhibits this. In this multicentre, open-label, randomised controlled trial, patients hospitalised due to acute infection were randomised to receive 10 days of aspirin treatment (80 mg 1dd or 40 mg 2dd) or no intervention (1:1:1 allocation). Measurements were performed during infection (T1; days 1–3), after intervention (T2; day 14) and without infection (T3; day > 90). The primary endpoint was platelet aggregation measured by the Platelet Function Analyzer® closure time (CT), and the secondary outcomes were serum and plasma thromboxane B2 (sTxB2 and pTxB2). Fifty-four patients (28 females) were included between January 2018 and December 2020. CT was 18% (95%CI 6;32) higher at T3 compared with T1 in the control group (n = 16), whereas sTxB2 and pTxB2 did not differ. Aspirin prolonged CT with 100% (95%CI 77; 127) from T1 to T2 in the intervention group (n = 38), while it increased with only 12% (95%CI 1;25) in controls. sTxB2 decreased with 95% (95%CI − 97; − 92) from T1 to T2, while it increased in the control group. pTxB2 was not affected compared with controls. Platelet aggregation is increased during severe infection, and this can be inhibited by aspirin. Optimisation of the treatment regimen may further diminish the persisting pTxB2 levels that point towards remaining platelet activity. This trial was registered on 13 April 2017 at EudraCT (2016-004303-32). [ABSTRACT FROM AUTHOR]

Aim: Recurrent pregnancy loss (RPL) is known to be associated with increased thrombophilia and oxidative toxicity. However, the mechanism of thrombophilia apoptosis and oxidative toxicity is still unclear. In addition, the treatment of heparin induced regulator roles on intracellular free Ca2+ ([Ca2+]i) and cytosolic reactive oxygen species (cytROS) concentrations in several diseases. TRPM2 and TRPV1 channels are activated by different stimuli, including oxidative toxicity. The aim of this study was to investigate the effects of low molecular weight heparin (LMWH) via modulation of TRPM2 and TRPV1 on calcium signaling, oxidative toxicity, and apoptosis in the thrombocytes of RPL patients. Study Design: Thrombocyte and plasma samples collected from 10 patients with RPL and 10 healthy controls were used in the current study. Main Findings: The [Ca2+]i concentration, cytROS (DCFH‐DA), mitochondrial membrane potential (JC‐1), apoptosis, caspase‐3, and caspase‐9 levels were high in the plasma and thrombocytes of RPL patients, although they were diminished by the treatments of LMWH, TRPM2 (N‐(p‐amylcinnamoyl)anthranilic acid) and TRPV1 (capsazepine) channel blockers. Conclusions: The current study results suggest that the treatment of LMWH is useful against apoptotic cell death and oxidative toxicity in the thrombocytes of patients with RPL, which seem to be dependent on increased levels of [Ca2+]i concentration via the activation of TRPM2 and TRPV1. [ABSTRACT FROM AUTHOR]

Pegagan embun (Hydrocotyle sibthorpioides Lam.) has been studied as an immunostimulant, increasing macrophage cell activity and phagocytosis capacity. Based on that circumstance, the study aims to prove the immunostimulating effect by measuring the activity of Hydrocotyle sibthorpioides Lam. extract in the capsule for the complete blood profile of healthy volunteers. The number of volunteers used was twenty people, and the volunteers were divided into two groups. The first group was given a placebo capsule without Hydrocotyle sibthorpioides Lam. extract. The second group was given a capsule with Hydrocotyle sibthorpioides Lam. extract with a dose of 67 mg, which was taken once a day for three days. Blood sampling was obtained before and after taking the capsule preparation. Observation of the complete blood profile was conducted by investigating changes in blood parameters such as haemoglobin levels, number of erythrocytes, number of leukocytes, hematocrit values, number of thrombocytes and the percentage of leukocyte types (banded neutrophils, segmented neutrophils, eosinophils, basophils, monocytes, and lymphocytes). The second group given Hydrocotyle sibthorpioides Lam. extract showed a significant effect on the increase in haemoglobin levels, number of thrombocytes and hematocrit values (p<0.05). The percentage of leukocyte type values showed that lymphocytes increased significantly (p<0.05). In contrast, the segmented neutrophil increased but did not show a significant difference with the percentage of banded neutrophils, eosinophils, basophils and monocytes (p>0.05). There was a nonsignificant result in all parameters for the first group, which was administrated with a placebo capsule without Hydrocotyle sibthorpioides Lam. extract.v [ABSTRACT FROM AUTHOR]

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2- related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2- related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis. [ABSTRACT FROM AUTHOR]

In humans, splenectomy is performed to treat many clinical disorders, including immune thrombocytopenia. However, the incidence of splenectomies for immune thrombocytopenia as a therapeutic has significantly declined over the past decade due to the availability of new therapies. Infection and sepsis as a result of splenectomies are well documented, but other long-term effects are not well characterized. Evidence suggests that persons who have had a prior splenectomy may be at an increased risk of vascular conditions. Also, elevated levels of cell-derived microparticles appear to contribute to an increased risk of thrombosis and cardiovascular disease. However, in vivo studies on the increased levels of microparticles following splenectomy are limited. In order to understand the effects of splenectomies, we developed a protocol for splenectomy in adult zebrafish. After anesthesia, the spleen was removed under a stereomicroscope after making an incision on the ventral side of the fish. The spleen was removed by pulling with forceps. The incision was closed by Vetbond tissue glue. Blood collected from both splenectomized zebrafish and those that underwent sham surgeries was immunolabeled with polyclonal antisera against αIIb, followed by flow cytometry. We observed elevated levels of thrombocytes and their microparticles in splenectomized zebrafish. Finally, by injecting αIIb antibody intravenously into zebrafish, we found the thrombocyte counts decreased, suggesting the fish developed immune thrombocytopenia like conditions, which were then reversed by splenectomy. In summary, the model developed here should be useful to study molecular changes due to splenectomy. Also, the zebrafish will be useful in modeling treatment of immune thrombocytopenia like conditions. [ABSTRACT FROM AUTHOR]

Thrombocytes circulate in the blood of nonmammalian vertebrates and are involved in hemostasis; however, many detailed characteristics of thrombocytes remain unclear. Recently, we established an amphibian thrombocyte cell line. Here, we report the finding that thrombocytes produce integrin alpha IIb (CD41)‐positive extracellular vesicles (EVs), which include microRNAs (miRs). Flow cytometric analysis showed the expression of CD41+ and phosphatidylserine on the surface of EVs. Nanotracking analysis showed that these CD41+ EVs were approximately 100 nm in diameter. As CD41+ EVs were also observed from African clawed frogs, the production of CD41+ EVs might be common to amphibians. Microarray analysis showed that the CD41+ EVs contain many kinds of miRs. These CD41+ EVs were phagocytosed by endothelial cells and macrophages. qPCR analysis showed that many angiogenesis‐related genes were up‐regulated in CD41+ EV‐treated endothelial cells. Over‐expression of some miRs in the CD41+ EVs increased the proliferation of endothelial cells. These results indicated that thrombocytes produced CD41+ EVs, including miRs, that were received by endothelial cells to induce the expression of angiogenesis‐related genes. These results indicated that the CD41+ EVs produced from thrombocytes act as signaling molecules to repair damaged blood vessels. [ABSTRACT FROM AUTHOR]

Background: Thrombocytopenia is a common condition in hospitalised critically ill children and most platelet transfusions are given as prophylaxis to non‐bleeding children prior to invasive procedures such as central venous catheterisation and lumbar puncture. Platelet transfusion may reduce bleeding complications but have also been associated with potential adverse effects and variation in clinical practice exist. To direct future research, we aim to assess the current evidence regarding prophylactic platelet transfusion prior to procedures in hospitalised thrombocytopenic children. Methods: We will conduct a scoping review of all study designs in accordance with the Preferred Reporting Items for Systematic and Meta‐Analyses (PRISMA) statement. We will include studies on hospitalised children undergoing invasive procedures where the prevalence of thrombocytopenia and the predefined outcome measures, including bleeding, transfusion‐related adverse events and use of blood products and life support are reported. We will provide descriptive analyses of the included studies/trials and the quality of evidence will be assessed according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. Conclusion: The outlined scoping review will provide an overview on the benefits and harms of prophylactic platelet transfusion prior to invasive procedures in thrombocytopenic hospitalised children. [ABSTRACT FROM AUTHOR]

Thrombocytes are blood cells that protect fish by aggregation to hinder hemorrhages and play a role on pathogen phagocytosis. Moreover, there is a lack of information regarding to the population of thrombocytes and its relationship with fish ploidy. The goal of this work was to determine the thrombocyte numbers and its aggregation capacity in adult diploid and triploid rainbow trouts Oncorhynchus mykiss by flow cytometry screening using monoclonal antibody and magnetic separation. As expected, thrombocytes were in similar proportions between ploidies with 15.26 ± 6.77% in diploid and 13.11 ± 6.37% in triploid blood samples; however, with thrombocytes of triploid trouts showing less aggregation activity when exposed to collagen (85.59 ± 3.36% vs. 95.51 ± 1.25%). In conclusion, triploid thrombocytes have lower aggregation capacity than the diploid ones. This response is possibly related to the deficiency of receptors on the surface of thrombocytes of triploid trouts, which requires further studies for confirmation. [ABSTRACT FROM AUTHOR]

Background: The examination of a peripheral blood smear is mandatory in case of unexplained thrombocytopenia or thrombocytosis. First, the number of platelets should be estimated in order to confirm the platelet count determined by the haematology analyser, and to rule out causes of spuriously low or elevated platelet counts. Second, the size and morphological features of the platelets, which may provide information on the underlying cause of the low or enhanced platelet count, have to be assessed. Content: This review summarizes the physiological and pathological features of platelet size and morphology, circulating megakaryocytes, micromegakaryocytes and megakaryoblasts, and provides an overview of current guidelines on the reporting of platelet morphology. Summary: In the diagnostic work-up of a patient with thrombocytopenia, the size of the platelets is of diagnostic relevance. Thrombocytopenia with small platelets is suggestive of a defect in platelet production, whereas the presence of large platelets is more likely to be associated with enhanced platelet turnover or hereditary thrombocytopenias. Morphological platelet abnormalities may affect the granulation and the shape and are frequently associated with abnormalities of platelet size. Platelet anomalies can be found in various haematologic disorders, such as myelodysplastic syndromes, myeloproliferative neoplasms, acute megakaryoblastic leukaemia or hereditary thrombocytopenias. [ABSTRACT FROM AUTHOR]

Objective. To study the platelet antiaggregant activity of new 11-amino acid derivatives of erythropoietin, containing tripeptide motifs. Materials and methods. The platelet aggregation degree was determined using the platelet-rich plasma of male Wistar rats. Formation of platelet aggregates results in an increase in light transmission through the sample; the kinetics of the responses and maximal aggregation provide a quantitative assessment of platelet aggregation The degree of platelet aggregation is estimated by the magnitude of the amplitude of aggregatograms, and the time of onset of light transmission level as well the time of onset of 85% of light transmission. Results. When blood was incubated with the studied peptides P-αB1, P-αB3, and P-αB4, a pronounced antiplatelet effect was observed. This is evidenced by a decrease in the maximum light transmittance of the plasma to 28.4±1.00%, 29.7±1.13% and 30.1±0.97%, respectively, and a delay in its onset to 134.4±2,90, 135.8±3.72 and 132.0 ± 3.59 seconds, respectively. In this case, a shift in the plasma light transmission curve to the right was observed which characterizes the platelet aggregation process. Conclusion. The addition of P-αB1, P-αB3, and P-αB4 produces antiplatelet activity, which is reflected by the shifting to the right of the platelet aggregation curve. The data obtained indicate a prolongation of platelet aggregation time and a decrease in its degree. [ABSTRACT FROM AUTHOR]

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Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b , hoxb2a , hoxc5a , hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b , hoxb2a , hoxc5a , and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a , and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b , as a negative regulator for thrombocyte development. [ABSTRACT FROM AUTHOR]

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their nonneutropenic parents. Materials and Methods: Fifteen patients with SCN and 21 nonneutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescenceassociated β-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents. Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95- mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/ thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate. Conclusion: In cases of SCN, patients' pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect. [ABSTRACT FROM AUTHOR]

Objective: There are currently 2 representative diagnostic criteria for essential thrombocythemia (ET), the 2014 British Committee for Standards in Hematology Guidelines (BCSH) criteria and the 2016 World Health Organization (WHO) criteria. We compare and discuss the advantages and disadvantages of the 2 criteria. Method: We applied the 2 criteria to 403 patients with thrombocytosis and suspected myeloproliferative neoplasms (MPN) and compared patient populations. Results: The BCSH criteria diagnosed ET in 279 patients (BCSH-ET) whereas the WHO criteria diagnosed ET in 203 patients (WHO-ET). There were 83 patients diagnosable only by the BCSH criteria (BCSH-only-ET), of which under the WHO classification, 69 patients fell under the category of MPN, unclassifiable (MPN-u), 12 patients were PMF, prefibrotic/early stage (pre-PMF), and 2 patients were polycythemia vera. Patient characteristics such as age, hemoglobin, hematocrit, platelet counts, lactate dehydrogenase levels, JAK2V617F allele burdens, prevalence of myelofibrosis and splenomegaly, and frequencies of thrombotic events and treatment did not differ between WHO-ET and BCSH-only-ET, but BCSH-only-ET patients showed higher WBC counts and higher JAK2V617F mutation frequencies. Conclusion: The BCSH criteria diagnosed ET in a broader range of patients encompassing a significant number of patients who would otherwise be diagnosed as pre-PMF or MPN-u. [ABSTRACT FROM AUTHOR]

Objectives To evaluate the efficacy of a salvage treatment with rituximab (RTX) in adults with primary immune thrombocytopenia (ITP), in terms of short-term response and long-term response (LTR, i.e., probability to achieve and maintain response) and to identify biological and clinical predictors of response. Methods We retrospectively evaluated the outcome of patients with primary ITP treated with standard dosage RTX (375 mg/m2 × 4) as salvage therapy in five Italian centers. One hundred and three patients, median age of 46 yr, were included. The median period of observation was 59 months. Results Response (R) and complete response (CR) were documented in 57 (55%) and 37 (36%) patients, respectively. Patients younger than 40 yr had a higher probability to achieve CR ( P = 0.025). Younger women (age < 40 yr) had a significantly higher probability to achieve R and CR ( P = 0.039 and P = 0.009, respectively). The estimated LTR rate was 36% and 31% after 48 and 72 months, respectively; female sex ( P = 0.033) and younger age ( P = 0.021) were associated with better LTR. Younger women had the highest LTR rate ( P = 0.006). Response duration was associated with the obtainment of CR after RTX (CR vs. partial response, P = 0.002). Conclusions The effect of RTX salvage treatment appears higher in younger women, with LTR rate possibly approaching that of splenectomy. [ABSTRACT FROM AUTHOR]

Balance of components' activity of thrombocyte-vessel hemostasis is the basis of the whole hemostasis process functioning in conditions of 'in vivo' and, thus, optimum of fl uidic blood features and adequacy of blood supply of all the tissues of an animal during whole its life. The aim is – to fi nd out physiological peculiarities of thrombocyte-vessel hemostasis of healthy cows during the normal process of pregnancy. The investigation was fulfilled with the help of 47 healthy cows of black-variegated breed which were examined and investigated 7 times during pregnancy: on the day of insemination, on the 45th, 90th, 135th, 180th, 230th and 280th day of pregnancy. The received results of state evaluation of thrombocyte-vessel hemostasis of cows after successful insemination testify to the existence of physiological regularity in the dynamics of its separate components' activity. It was found that during pregnancy the cows were noted to have weakening of thrombocyte hemostasis component's activity and strengthening of vessel hemostasis component. It provides them with necessary hemostatic and fl uidic blood features, thus promoting the formation of optimal conditions for placenta blood supply and anabolism in fetus' tissues. [ABSTRACT FROM AUTHOR]

Equilibria between the membranes of erythrocytes as well as thrombocytes and solution ions in fatal accidental hypothermia were analyzed using a theoretical four-equilibria model. The model was developed to determinate parameters characterizing cell membrane-surrounding ion interactions: the total surface concentrations of both acidic and basic groups C A, C B, and association constants K AH, K BOH. Knowledge of these parameters was necessary to calculate the theoretical values of surface charge density. The model was validated by curve-fitting the experimental data points to simulated data generated by the model. The experimental and theoretical surface charge density values agree at pH 2-8, at higher pH the deviation was observed. [ABSTRACT FROM AUTHOR]

Background & Aims Different mechanisms including portal hypertension and hypersplenism have been involved in the development of thrombocytopenia in cirrhosis. However, the relative contribution of each one is unknown. The aim was to evaluate simultaneously different mechanisms that determine platelet count in cirrhosis. Methods Cross-sectional study including cirrhotics ( n = 120) with hepatic venous pressure gradient ( HVPG) measurement. Samples were obtained from peripheral (P) veins to evaluate thrombopoietin ( TPO), stem cell factor, hepatocyte growth factor ( HGF), tumour necrosis factor, interleukin-(IL6) and (IL11) and from hepatic (H) veins to evaluate TPO. A subgroup ( n = 72) had spleen volume estimation. H and P- TPO were also measured in non-cirrhotic patients ( n = 15). Results Patients (Child A: 55, B: 43, C: 22) had a median platelet count of 81 000/mm3 ( IQR 60 500, 110 750), which correlated with spleen volume ( r = −0.38, P < 0.001). Platelets were associated also to HVPG ( r = −0.47, P = 0.004) and P- TPO ( r = 0.31, P = 0.050) only in compensated patients. H- TPO decreased, and the proportion of patients with P-TPO > H-TPO increased, with the presence and the severity of liver disease. H-TPO was correlated with liver function (bilirubin r = −0.350, P < 0.001 and international normalized ratio r = −0.227, P = 0.011). Patients with H-TPO < P-TPO had higher levels of IL-11 and HGF. Conclusion Platelet count in cirrhosis is associated mainly to spleen volume, although portal hypertension as estimated by HVPG and liver function plays a significant role in compensated patients. H- TPO and the proportion of patients with P-TPO > H- TPO were associated to the presence and severity of liver disease. [ABSTRACT FROM AUTHOR]

Purpose: Aspirin is an antiplatelet drug which is commonly used in secondary prevention in ischemic heart disease and cerebrovascular events, and in newly diagnosed myocardial infarction. The aim of the present study was to examine effect of aspirin on the level of selected sphingolipid intermediates in plasma, erythrocytes and platelets. Material and Method: Forty two healthy volunteers participated in the study. They were divided into two groups. In one group aspirin was given orally, daily, for one week in a dose of 75mg (n=25). The subjects from the second group received one 300mg dose of the drug (n=17). In both groups the blood was taken 4h after the last dose of aspirin. The following sphingolipid intermediates were quantified using high-pressure liquid chromatography: sphinganine, sphingosine, sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and ceramide. Results: It was found that lower dose of aspirin increased the level of S1P and ceramide in erythrocytes (by 23 and 37%, respectively) having no effect on plasma and platelet sphingolipid levels. Higher dose of the drug reduced S1P and SA1P concentration in the plasma (by 16 and 10%, respectively). Conclusion: We conclude that aspirin interferes with sphingolipid metabolism in blood and that this effect depends on a dose of the drug. Since S1P is a potent cardioprotectant, the reduction in its plasma concentration after the loading dose of aspirin could be undesired side effect of the drug. [ABSTRACT FROM AUTHOR]

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo. [ABSTRACT FROM AUTHOR]