A novel mechanism of thrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency

Sphingomyelin synthase 1 (SMS1) contributes to the generation of membrane sphingomyelin (SM) and affects SM-mediated physiological functions. Here, we describe the hematologic phenotypes, such as reduced circulating platelets and dysfunctional hemostasis, in SMS1-deficient (SMS1-KO) mice. SMS1-KO mice display pathologic manifestations related to idiopathic thrombocytopenia (ITP), including relatively high amounts of peripheral blood reticulated platelets, enhanced megakaryopoiesis in the bone marrow and spleen, and splenomegaly. Deficiency of SMS1, but not SMS2, prevented SM production and enhanced phosphatidylserine (PS) externalization on the plasma membranes of platelets and megakaryocytes. Consequently, SMS1-KO platelets were excessively cleared by macrophages in the spleen. Multimer formation in the plasma membrane of TMEM16F, a known calcium (Ca2+)-activated nonselective ion channel and Ca2+-dependent PS scramblase, was enhanced; the result was PS externalization to outer leaflets through increased Ca2+ influx in immortalized mouse embryonic fibroblasts established from SMS1-KO mice (SMS1-KO tMEFs), as seen with SMS1-KO platelets. Thus, SMS1 deficiency changed the TMEM16F distribution on the membrane microdomain, regulating Ca2+ influx-dependent PS exposure. SMS1-KO tMEFs in which TMEM16F was knocked out by using the CRISPR/Cas9 system lacked both the Ca2+ influx and excess PS exposure seen in SMS1-KO tMEFs. Therefore, SM depletion on platelet membrane microdomains due to SMS1 deficiency enhanced PS externalization via a Ca2+ influx through TMEM16F activation, leading to elevated platelet clearance and causing hemostasis dysfunction through thrombocytopenia. Our current findings show that the SM-rich microdomain generated by SMS1 is a potent regulator of thrombocytopenia through TMEM16F, suggesting that its dysfunction may be a novel additional mechanism of ITP.

Meta-analysis of coagulation disbalances in COVID-19: 41 studies and 17601 patients

Introduction. Coagulation parameters are important determinants for COVID-19 infection. We conducted meta-analysis to assess the early hemostatic parameters in retrospective studies in association with severity of infection. Methods. Ovid, PubMed, Web of Sciences, and Google Scholar were searched for research articles that addressed clinical characteristics of COVID-19 patients and disease severity. Results were filtered using exclusion and inclusion criteria and then pooled into a meta-analysis to estimate the standardized mean difference with 95% CI for each of five coagulation parameters (D-dimers, fibrinogen, prothrombin time, platelets count, activated partial thromboplastin time). Two authors independently extracted data and assessed study quality. To explore the heterogeneity and robustness of our fundings, sensitivity and subgroup analyses were conducted. Publication bias was assessed with contour-enhanced funnel plots and Egger test by linear regression. Results. Overall, 41 original studies (17601 patients) on SARS-CoV2 were included. For the two groups of patients, stratified by severity, we identified that D-dimers, fibrinogen, activated partial thromboplastin time, and prothrombin time were significantly higher in the severe group (SMD 0.6985 with 95%CI [0.5155; 0.8815]); SMD 0.661with 95%CI [0.3387; 0.9833]; SMD 0.2683 with 95%CI [0.1357; 0.4009]; SMD 0.284 with 95%CI [0.1472; 0.4208]). In contrast, PLT was significantly lower in patients with more severe cases of COVID-19 (SMD -0.1684 with 95%CI [-0.2826; -0.0542]). Neither the analysis by the leave-one-out method nor the influence diagnostic have identified studies that solely cause significant change in the effect size estimates. Subgroup analysis showed no significant difference between articles originated from different countries but revealed that severity assessment criteria might have influence over estimated effect sizes for platelets and D-dimers. Contour-enhanced funnel plots and the Egger test for D-dimers and fibrinogen revealed significant asymmetry that might be a sign of publication bias. Conclusions. The standard coagulation laboratory parameters with exception of platelets counts are significantly elevated in patients with severe COVID-19. However, fibrinolysis shutdown requires evaluation outside conventional coagulation tests and analysis of additional specific markers related to clotting formation and PLT characteristics. We hypothesize that a proportion and parameters of immature reticulated platelets may serve as additional biomarkers for prediction of adverse events.

Impact of Reticulated Platelets on Platelet Reactivity in Neonates

Neonatal megakaryopoiesis and platelet turnover form a developmentally unique pattern by generating a pool of newly released reticulated platelets from the bone marrow into the circulation. Reticulated platelets are more reactive and hyperaggregable compared to mature platelets, due to their high residual mRNA content, large size, increased expression of platelet surface receptors, and degranulation. The proportion of reticulated platelets in neonates is higher compared to that in adults. Due to the emergence of an uninhibited platelet subpopulation, the newly formed reticulated platelet pool is inherently hyporesponsive to antiplatelets. An elevated population of reticulated platelets is often associated with increased platelet reactivity and is inversely related to high on-treatment platelet reactivity, which can contribute to ischemia. Measurements of the reticulated platelet subpopulation could be a useful indicator of increased tendency for platelet aggregation. Future research is anticipated to define the distinct functional properties of newly formed reticulated or immature platelets in neonates, as well as determine the impact of enhanced platelet turnover and high residual platelet reactivity on the response to antiplatelet agents.

Reticulated platelets – a new risk factor of atherothrombosis?

In this review we described the properties of reticulated platelets (RP) and showed how variations of their content might influence platelet activity, efficacy of antiplatelet drugs and the rate of thrombotic events in patients with cardiovascular diseases. RP represent a minor platelet fraction containing residual RNA from megakaryocytes. Platelets have no nucleus and do not synthesize RNA de novo, and RNA of megakaryocytic origin is destroyed during their circulation. That is why only recently produced “young” platelets contain RNA. In healthy donors RP are identified by staining with the RNA specific fluorescent dyes by flow cytofluorimetry or using standard protocols in modern flow haematological analyzers. RP content in blood reflects the level of thrombocytopoesis in the bone marrow. RP on average amounted from 3 to 10% of all platelets in the circulation depending on the method applied for their determination. RP absolute amount and/or their percentage is changed in haematological diseases associated with the alterations of megakaryocyte productive properties. RT measurements in patients with cardiovascular diseases have shown that their content is increased in acute coronary syndrome patients. RP are larger and functionally more active in comparison with not reticulated forms. They more frequently incorporate into the platelet aggregates and contain more intracellular granules. Increase of RT content in the circulation correlates with the increase of the average size and functional activity in the whole platelet population. High RP content in patients with cardiovascular diseases reduces antiaggregative effects of aspirin and P2Y12 APD receptor antagonists and increases the risk of atherothrombotic events.

Immature Platelet Fraction: Its Clinical Utility in Thrombocytopenia Patients

Objectives Etiology of thrombocytopenia is multifactorial and its pathogenesis should be distinguished for appropriate management. Newly formed immature platelets are called reticulated platelets (RPs) and can be estimated in peripheral blood using automated hematology analyzers, which express them as immature platelet fraction (IPF). In the present study we intend to assess and establish the clinical utility of IPF in differentiating the two major causes of thrombocytopenia—decreased production and increased destruction of platelets—along with determining its significance in monitoring patients with thrombocytopenia. Materials and Methods Sixty-one cases of thrombocytopenia and 101 healthy controls with normal platelet count were included in the study. IPF and all the other usual blood cell parameters were measured using a fully automated hematology analyzer. Based on the pathogenesis of thrombocytopenia, the cases were divided into groups and the difference in IPF value between the groups was evaluated. Results The reference range of IPF among healthy controls was estimated to be 0.7 to 5.7%. The mean IPF was significantly higher in patients with increased peripheral destruction of platelets (13.4%) as compared to patients with decreased production of platelets (4.6%). The optimal cutoff value of IPF for differentiating patients with increased peripheral destruction of platelets from patients with decreased production of platelets was 5.95% with a sensitivity of 88% and specificity of 75.9%. Conclusion Measurement of IPF is useful for detecting evidence of increased platelet production and helps in the initial evaluation of thrombocytopenia patients. It is a novel diagnostic method which can be used to differentiate patients with thrombocytopenia due to increased destruction of platelets from patients with thrombocytopenia due to bone marrow failure/suppression.

Reticulated Platelets—Which Functions Have Been Established by In Vivo and In Vitro Data?

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy. This review seeks to provide an overview of the key features of RP as well as preanalytical and analytical aspects that need to be considered when working with this platelet population.

FRAÇÃO DE PLAQUETAS IMATURAS (IPF): UMA REVISÃO DA LITERATURA

Introdução: A fração de plaquetas imaturas (IPF) é um parâmetro ainda pouco explorado na rotina clínica e sua determinação laboratorial através de citometria de fluxo tem sido pesquisado como um marcador de função medular. As plaquetas reticuladas (PR) representam plaquetas jovens recentemente liberadas pela medula óssea, e são caracterizadas pelo alto conteúdo de ácido ribonucleico (RNA) em seu citoplasma. Objetivo: O objetivo deste trabalho foi pesquisar a utilidade diagnóstica das PRs através do parâmetro IPF (fração de plaquetas imaturas) na rotina médica e suas limitações, além das perspectivas futuras para seu uso. Material e métodos: Foi realizada uma pesquisa bibliográfica em bases de dados confiáveis, Medline, PubMed (US. National Library of Medicine National Institutes of Health, USA), SciELO Brazil (Scientific Eletronic Library Online) e BIREME (Biblioteca Regional de Medicina, Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde) utilizando os descritores “Platelet índices”, “Immature platelet fraction” e “Reticulated platelets”. Resultados: As plaquetas reticuladas são um importante indicador da etiologia da trombocitopenia em diversos quadros clínicos, diferenciando os casos de consumo periférico plaquetário de uma falha na produção na medula óssea. As plaquetas imaturas permanecem na circulação por 24 a 36 horas, quando progressivamente têm seu RNA degradado e seu volume diminuído passando ao estado da plaqueta madura. Portanto, assim como os reticulócitos para os eritrócitos, a quantificação de plaquetas imaturas é capaz de trazer informações precoces em relação a produção de plaquetas na medula óssea. Além disto, alguns estudos apontam que o IPF pode ser útil no diagnóstico precoce de sepse bem como no acompanhamento de pacientes transplantados. Conclusão: Através desta revisão bibliográfica, conclui-se que a fração de plaquetas imaturas é um promissor parâmetro laboratorial para identificar a etiologia da trombocitopenia (consumo periférico ou falha medular). Embora novos estudos precisam aprofundar o conhecimento e a segurança do uso do IPF, este parâmetro mostra-se útil para o auxílio diagnostico e acompanhamento terapêutico de uma série de patologias associadas à trombocitopenia, como púrpura trombocitopênica, acompanhamento de pacientes transplantados e em pacientes com risco de sepse.

Correlation between Percentage of Reticulated Platelets and Heart Score in Patients with Suspected Non-ST Elevation Acute Coronary Syndromes

Thrombus formation in non-ST Elevation Acute Coronary Syndrome (NSTE-ACS) causes increased plateletconsumption, leading to a 20-fold increase of Reticulated Platelets (RP) release. Reticulated platelets have more granulesand proteins that make them quickly forming thrombus than mature platelets, potent to form bigger thrombus, andincrease the risk of Major Adverse Cardiac Events (MACE). HEART score is a risk stratification for possible NSTE-ACS, whichcan predict MACE. The study aimed to analyze the correlation between the percentage of reticulated platelets and HEARTscore. This research was a correlation observational cross-sectional study performed in Dr. Hasan Sadikin Hospital,Bandung, from August 2018 to May 2019. The subjects were patients suspected with NSTE-ACS by clinicians in theEmergency Department of Dr. Hasan Sadikin Hospital. These subjects were assessed for the HEART score andRP percentage. This study involved 52 subjects consisting of a higher number of males (76.9%) aged 45-64 years old (69.2%).HEART score stratification in this study was mostly high risk (69.2%), but none was low risk. Mean of platelet count, absolute3 3 RP, and RP percentage were 271±73 x103/mm , 9.3±4.3 x 103/mm , and 3.6±1.7%, respectively. The correlation testbetween RP percentage and HEART score with a 95% confidence interval using Spearman's correlation test showed asignificant positive correlation with moderate strength (p < 0.001 and r=0.475). The percentage of RP in this study was in thenormal range. However, there was a significant positive correlation with moderate strength between the percentage of RPand HEART scores in patients with suspected non-ST elevation acute coronary syndrome.