Normal Platelet Numbers Research Articles

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Thrombotic thrombocytopenic purpura in a patient on long-term alpha-interferon therapy for essential thrombocythemia: a case report

BackgroundThrombotic thrombocytopenic purpura (TTP) is rare and severe thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and renal dysfunction. In contrast, essential thrombocythemia (ET) is a myeloproliferative disease associated with an abnormal increase in platelet numbers. Previous studies reported several cases of the development of ET in patients with TTP. However, the case of an ET patient complicated with TTP has not been previously reported. In this case study, we present a patient with TTP who was previously diagnosed with ET. Therefore, to the best of our knowledge, this is the first report of TTP in ET.Case presentationA 31-year-old Chinese female who was previously diagnosed with ET presented with anemia and renal dysfunction. The patient had been on long-term treatment with hydroxyurea, aspirin, and alpha interferon (INF-α) for ten years. The diagnosis of TTP was confirmed by clinical features, schistocytes noted on the peripheral blood smear, and lower ADAMTS13 activity (8.5%), together with the renal biopsy results. INF-α was discontinued, and the patient was then treated with plasma exchange and corticosteroids. After one year of follow-up, the patient had a normal hemoglobin level and platelet numbers, and her ADAMTS13 activity had improved. However, the patient’s renal function remains impaired.ConclusionsWe report a case of an ET patient complicated with TTP that was possibly due to INF-α, highlighting the potential complications associated with long-term ET therapy. The case also highlights the importance of considering TTP in patients with pre-existing ET who present with anemia and renal dysfunction, extending the spectrum of known studies.

Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis. IL6 and TNF expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.

VASCULAR-PLATELET HEMOSTASIS OF INJURED PATIENTS: PROSPECTIVE OBSERVATIONAL STUDY.

The aim: We study vascular-platelet hemostasis peculiarities in patients with severe trauma. Materials and methods: We included 50 patients, who were divided into control (n=15) and study (n=35) groups. The control group included patients without traumatic injuries, study group - patients with severe trauma. The study group was divided into the I subgroup (patients received 1 g tranexamic acid IV at the prehospital stage), and the II subgroup (1 g tranexamic acid IV after hospital admission). Results: The main changes in the I subgroup started on the 3rd day, while in the II subgroup - on the 1st day. Patients of both subgroups on the 1st and 3rd days had a normal number of platelets in venous blood, however, on the 3rd day, there was a decreasing level of discocytes whereas the level of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased in comparison with the control group (p<0.05). Conclusions: The changes in vascular-platelet hemostasis in patients appeared in the I subgroup on the 3rd day, while in the II subgroup - on the 1st day. For the I subgroup was the decreasing level of discocytes, whereas the level of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased. For the II subgroup on the 1st day, there was an increasing sum of active forms of platelets, on the 3rd day - the level of discocytes was decreased, and levels of discoechinocytes, spherocytes, spheroechinocytes, and the sum of active forms of platelets were increased.

Oculocutaneous albinism and bleeding diathesis due to a novel deletion in the HPS3 gene.

Hermansky–Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism (OCA) and bleeding diathesis. To date, 11 HPS types have been reported (HPS-1 to HPS-11), each defined by disease-causing variants in specific genes. Variants in the HPS1 gene were found in approximately 15% of HPS patients, most of whom harbor the Puerto Rican founder mutation. In this study, we report six affected individuals from three nonconsanguineous families of Ashkenazi Jewish descent, who presented with OCA and multiple ecchymoses and had normal platelet number and size. Linkage analysis indicated complete segregation to HPS3. Sequencing of the whole coding region and the intron boundaries of HPS3 revealed a heterozygous c.1163+1G>A variant in all six patients. Long-range PCR amplification revealed that all affected individuals also carry a 14,761bp deletion that includes the 5′UTR and exon 1 of HPS3, encompassing regions with long interspersed nuclear elements. The frequency of the c.1163+1G>A splice site variant was found to be 1:200 in the Ashkenazi Jewish population, whereas the large deletion was not detected in 300 Ashkenazi Jewish controls. These results present a novel HPS3 deletion mutation and suggest that the prevalence of HPS-3 in Ashkenazi Jews is more common than previously thought.

Cerebral vasculature exhibits dose-dependent sensitivity to thrombocytopenia that is limited to fetal/neonatal life

AbstractWhether increasing platelet counts in fetal and neonatal alloimmune thrombocytopenia (FNAIT) is effective at preventing intracerebral hemorrhage (ICH) has been a subject of debate. The crux of the matter has been whether thrombocytopenia is the major driver of ICH in diseases such as FNAIT. We recently demonstrated in mice that severe thrombocytopenia was sufficient to drive ICH in utero and in early neonatal life. It remains unclear what degree of thrombocytopenia is required to drive ICH and for how long after birth thrombocytopenia can cause ICH. By inducing a thrombocytopenic range, we demonstrate that there is a large buffer zone of mild thrombocytopenia that does not result in ICH, that ICH becomes probabilistic at 40% of the normal platelet number, and that ICH becomes fully penetrant below 10% of the normal platelet number. We also demonstrate that although the neonatal mouse is susceptible to thrombocytopenia-induced ICH, this sensitivity is rapidly lost between postnatal days 7 and 14. These findings provide important insights into the risk of in utero ICH with varying degrees of thrombocytopenia and into defining the developmental high-risk period for thrombocytopenia-driven ICH in a mouse model of FNAIT.

Trauma-induced coagulopathy: prospective observation study

Coagulopathy remains the leading cause of illness and death in people with severe trauma.&#x0D; The aim was to study the indicators of vascular-platelet hemostasis in severe trauma and to establish changes in the main pathophysiological mechanisms of primary hemostasis that occur in patients with trauma-induced coagulopathy, compared with almost healthy individuals of the same age.&#x0D; Materials and methods. The study included 44 patients aged 19 to 55 years (36.1 [28.7; 43.2] years). The control group included 20 patients of the therapeutic department without preconditions for changes in the hemostasis system, and the main group - 24 patients with diagnosis of “severe trauma” who were treated in the anesthesiology and intensive care department.&#x0D; Results and discussion. We studied indicators of intravascular platelet activation. Patients had a normal number of platelets in the venous blood, but the level of spherocytes and spheroechinocytes increased. On the 3rd day after the injury, number of platelets in the venous blood was normal, however the level of discocytes decreased, and the level of discochinocytes, spheroechinocytes and the sum of active forms of platelets increased. On the fifth day, the level of discochinocytes and active forms of platelets, significantly higher (p&lt;0.05) among patients of the main group, and spherocytes, spheroechinocytes and platelets involved in the aggregates, were significantly lower than in the control group of patients (p&lt;0.05).&#x0D; Conclusions. Indicators of vascular-platelet hemostasis in patients with polytrauma had significant differences from those of the control group. This may be evidence of activation of the vascular-platelet system of the hemostasis system and may be a prerequisite for late thrombotic complications in patients with polytrauma

Изменения функционального состава тромбоцитов у пациентов с политравмой

Акутальность. Политравма остается главной причиной глобальной заболеваемости и смертности и является причиной более 10 % смертей. Цель: изучить данные литературы об изменениях показателей сосудисто-тромбоцитарного гемостаза у пациентов с политравмой, исследовать динамику морфофункционального состояния тромбоцитов, проанализировать изменения показателей внутрисосудистой активации тромбоцитов у пациента с политравмой. Результаты. Для нормального свертывания крови нужны как минимум 4 компонента — кровеносные сосуды, тромбоциты, способность крови к свертыванию и фибринолиз. Определение таких компонентов, как показатели внутрисосудистой активации тромбоцитов, может стать важным шагом оценки нарушений тромбоцитарного звена гемостаза у пациентов с политравмой. Сосудисто-тромбоцитарный гемостаз начинается рефлекторным первичным спазмом артериол, после чего возникает вторичный спазм артериол, дальше образуется первичная тромбоцитарная пробка (адгезия и агрегация) и, соответственно, консолидация тромба, в результате чего формируется окончательный тромбоцитарный тромб. Еще до контакта тромбоцитов с обнаженным коллагеном происходит первичная активация тромбоцитов. Сначала меняется форма интактных тромбоцитов с дискоидной формы к активированым клеткам — дискоэхиноцитам, сфероцитам и/или сфероэхиноцитам. Нами установлено, что на 3-и сутки после травмы при нормальном количестве тромбоцитов в венозной крови уменьшается количество интактных тромбоцитов — дискоцитов, растет количество активных форм тромбоцитов — дискоэхиноцитов и сфероэхиноцитов, соответственно, растет общая сумма активных форм тромбоцитов. Нормальный уровень тромбоцитов у пациентов с политравмой может замаскировать выраженность коагулопатии, а проведение исследований показателей внутрисосудистой активации тромбоцитов может стать диагностическим компонентом сосудисто-тромбоцитарного звена гемостаза у пациентов с политравмой. Выводы. У пациентов с коагулопатией вследствие политравмы на 3 сутки наблюдаются изменения показателей внутрисосудистой активации тромбоцитов и агрегации тромбоцитов, индуцированной адреналином и АДФ.

SARS-CoV-2 Protease-Dependent Human Cell Infection Induces Immunological Response Imbalance and Neutrophils “Proteolytic Storm”. Proteolysis Inhibitors can Prevent and Treat Covid-19

Based on Chinese CDCP report on COVID-19, 14% of patients presented severe disease and 5% critical conditions. The average case-fatality rate was 2.3%, but mortality was as high as 49% in patients with critical illness. Serious life threatening thromboembolic complications have been found in 71•4% of non survivors and micro/macro angiopathic coagulopathy has been found, at autopsy also, with highly increased neutrophil number, fibrinogen, concentrations of D-dimer and FDPs and NETs, ATIII decrease and normal number of platelets. A cytokine storm and interaction between inflammation and coagulation has been advocated as explanation of hypercoagulability. It has been shown that SARS-CoV-2 infection of alveolar cells is driven by the S-protein by engaging ACE2 and TMPRSS2 cell receptors. whose activation depends on the activity of various host proteases. Full inhibition of SARS-CoV-2 entry was observed when serine proteases inhibitor camostat mesylate was coupled with Cathepsin B/L inhibitor E-64d. In addition multiple proteases are involved in host immune response against viral invasion and immunopathology related to imbalanced immune activation. In this paper it’s hypothesised that the severity of Covid-19 is induced by recruitment of innate responder neutrophils, which release proteases and NETs inducing endothelial damage and imbalance of the four major proteolytic cascades (coagulation, complement, fibrinolysis and kallikrein) with prevalence of activators over inhibitors and consequent thrombotic complications. Platelets adhesion to damaged endothelium and vWFVIII multimers presence, due to loss of ADAMTS13, contributes to hypercoagulability state. Human plasma or serine protease inhibitors like aprotinin can help to control neutrophil induced “proteolytic storm”. The goal of this paper is to support the view that, in SARS-CoV-2 infection, proteases have a key role and exceeding imbalanced neutrophil innate “unfriendly fire” response can be identified as the trigger of a “proteolytic storm”, responsible for subsequent well known hypercoagulation and “cytokine storm” and human plasma, in adequate volumes, together with serine proteases inhibitors can be an effective therapeutic strategy.

Assessment of Platelet Numbers and Prevalence of Thrombocytopenia among Children with Severe Malaria at a Tertiary Teaching Hospital

Background: Thrombocytopenia plays an important role in the diagnosis of malaria in most countries because of the rapid decrease of platelets during malaria onset and also in severe forms of the disease. The study sought to assess platelet numbers and determine the prevalence of malarial thrombocytopenia among children with febrile malaria at the Korle-Bu Teaching Hospital (KBTH). Methodology: It was a cross-sectional study carried out in the Department of Child Health and the Polyclinic of KBTH from April to July 2018. A total of 100 children aged from 1 to 12 years who tested malaria positive for both thick and thin blood film and Rapid Diagnostic Tests (RDTs) were recruited into the study. Venous blood sample was obtained from the children and analyzed for platelet count using a haematology auto analyzer. Parasite speciation and quantification was used to classify the disease into mild, moderate and severe, and platelet numbers were also categorized into low, normal and high. Data obtained was then analyzed for prevalence of malarial thrombocytopenia in severe malaria. Results: The total studied participants were 100, 61 (61%) were males whiles 39 (39%) were females. Both severe malaria and thrombocytopenia were mostly seen in children less than 5 years of age and the males recorded more thrombocytopenia (66%) and normal platelet numbers 34 (64.2%) than the females. There was a significant higher percentage of malaria children with thrombocytosis being females (64%) as compared to those being males (36%). 53 (53%) children had normal platelet numbers, 33 (33%) had thrombocytopenia, and 14 (14%) had thrombocytosis. The prevalence of uncomplicated malaria and complicated malaria were 80% and 20% respectively. The overall prevalence of thrombocytopenia was 33%, and the prevalence of thrombocytopenia in uncomplicated and severe malaria was 18.8% and 90% respectively. The prevalence of malaria types among children with thrombocytopenia was assessed. Out of the 33 (33%) patients who had thrombocytopenia, 15 (45.5%) of them had uncomplicated malaria and 18 (54.5%) of them had complicated or severe malaria. No child was found to have thrombocytosis during complicated malaria. Conclusion: The study indicates a significant higher prevalence of thrombocytopenia in severe malaria among children at the Korle-Bu Teaching Hospital, and this could be used as a useful indicator to investigate malaria or as a diagnostic clue for malaria in children.

SARS-CoV-2 Infection is Protease-Dependent and Induces Neutrophils “Proteolytic Storm” Triggering Clinical Worsening and Viral Sepsis. Proteolysis and Inhibitors of Neutrophil Release Can Prevent and Treat Covid-19

Based on Chinese CDCP report on COVID-19, 14% of patients presented severe disease and 5% critical conditions. The average case-fatality rate was 2.3%, but mortality was as high as 49% in patients with critical illness. Serious life threatening thromboembolic complications have been found in 71.4% of non-survivors and micro/macro angiopathic coagulopathy has been found, at autopsy also, with highly increased neutrophil number, fibrinogen, concentrations of D-dimer and FDPs and NETs, ATIII decrease and normal number of platelets. A cytokine storm and interaction between inflammation and coagulation has been advocated as explanation of hypercoagulability. It has been shown that SARS-CoV-2 infection of alveolar cells is driven by the S-protein by engaging ACE2 and TMPRSS2 cell receptors. Whose activation depends on the activity of various host proteases. Full inhibition of SARS-CoV-2 entry was observed when serine proteases inhibitor camostat mesylate was coupled with Cathepsin B/L inhibitor E-64d. In addition multiple proteases are involved in host immune response against viral invasion and immunopathology related to imbalanced immune activation. In this paper it’s hypothesized that the severity of Covid-19 is induced by recruitment of innate responder neutrophils, which release proteases and NETs inducing endothelial damage and imbalance of the four major proteolytic cascades (coagulation, complement, fibrinolysis and kallikrein) with prevalence of activators over inhibitors and consequent thrombotic complications. Platelets adhesion to damaged endothelium and vWFVIII multimers presence, due to loss of ADAMTS13, contributes to hypercoagulability state. Human plasma or serine protease inhibitors like aprotinin can help to control neutrophil induced “proteolytic storm”. The goal of this paper is to support the view that, in SARS-CoV-2 infection, proteases have a key role and exceeding imbalanced neutrophil innate “unfriendly fire” response can be identified as the trigger of a “proteolytic storm”, responsible for subsequent well known hyper coagulation and “cytokine storm” and human plasma, in adequate volumes, together with serine proteases inhibitors can be an effective therapeutic strategy.

Strategies for Analysis of Novel Molecular Variants in the RUNX1 Gene As a Cause of Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML)

Introduction: Runx 1 is a key transcription factor (TF) in hematopoiesis. Germline mutations in RUNX1 (≈40 described) are associated with FPD/AML, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction and a high risk (40% before 35 yr) of developing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Overestimating the pathogenicity of new molecular variants detected by high throughput sequencing (HTS) in the diagnosis of inherited thrombocytopenias (IT), even affecting well established candidate genes, could be more negative for the management and quality of life of patients than lacking such diagnosis (Lentaigne, Blood 2016). This is especially relevant for variants in TF such as Runx1, due to the risk of developing myeloid malignancies. Objective: To evaluate the pathogenicity of new variants in RUNX1 identified in patients enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with Congenital Platelet Disorders" (TPC-GT-PH-SETH). Methods: Three unrelated women aged 27 (P1), 46 (P2) and 50 (P3) years, were included in TPC-GT-PH-SETH because of their history of mild thrombocytopenia (≈105pl/uL) with normal volume (MPV) and moderate bleeding. A daughter and a brother of P2 died after developing MDS and LAM, respectively, and a sister of P3 had breast cancer. Only P1 and P2 had previously showed moderate functional platelet abnormalities (reduced aggregation, P-selectin secretion and number of δ-granules). By HTS (Bastida, Haematologica 2018) we identified new heterozygous variants in RUNX1: P1, de novo c.802C&lt;T [p.Gln.268*]; P2, c.586A&gt;G [p.Thr196Ala], segregated with thrombocytopenia and neoplasia in the family; P3, c.476A&gt;G; [p.Asn159Ser], no segregation with thrombocytopenia, with 4 of 6 carriers in the family having normal platelet numbers. For this study, we obtained blood from patients and three controls, and isolated ultrapure platelets by filtration+immunoselection. Platelet RNA was analyzed using Clariom-D Array (≈540 000 transcripts) (Caparrós-Pérez, PLoS One 2017). In P1 we isolated CD34+ cells from blood, differentiated them in vitro to megakaryocytes (Mks), and evaluated the formation of proplatelets. Results: Analysis of transcriptomes showed that when considering the 13 genes well recognized as targets of RUNX1 (including MYL9, MYH9, ALOX12), the percentage of those with altered expression in patients, vs. controls, was 69.2% and 61.5% in P1 and P2, respectively, and only 7.7% in P3. Previously, the transcriptome of a single patient with RUNX1 variant (83aa deletion) (Sun, JTH 2007) has reported the infraexpression of 100 genes. Our cases showed low expression of 74.7%, 67.7% and 7.1% (P1, P2 and P3, respectively) of those genes. Principal component analysis of the array intensity signals, grouped together controls and the P3 case, while the P1 and P2 cases were clustered together. Additionally, in vitro culture of CD34-Mks from patient P1 showed reduced pro-platelet formation. Conclusion: Platelet phenotyping, family segregation studies, platelet transcriptome and in vitro pro-platelet formation, support the pathogenicity of Runx 1 variants p.Gln268* and p.Thr196Ala, but not of p.Asn159Ser. Platelet transcriptome analysis is a useful tool in the pathogenic characterization of new molecular variants affecting TF genes in patients with IT, such as RUNX1 and can help to identify new target genes of these TF. Disclosures No relevant conflicts of interest to declare.

Complication hémorragique inattendue pour un cancer bronchique sous double immunothérapie

Specific immune-related adverse events in lung cancer treatment are rare and it is important that they are identified as they may have important adverse consequences. We report such a case here. A Caucasian female diagnosed with KRAS mutant advanced adenocarcinoma of the lung was enrolled in a phase Ib trial assessing the combination of an anti cytotoxic T-lymphocyte- associated protein 4 antibody and a programmed death-Ligand 1 inhibitor. For several years, she had also been taking warfarin for recurrent pulmonary embolism. At day 15 of treatment, she presented with grade 1 haematomas and signs of grade 2 hyperthyroidism. Blood tests revealed a normal number of platelets but an INR increased to 6.5. Thyroid function tests and auto antibodies confirmed the presence of an autoimmune thyroitidis. The study treatment was then stopped and the patient received 1mg/kg of prednisone and 40mg of propranolol. At day 28, the thyroid function and symptoms were normalized. No direct interactions exist between immunotherapy and vitamin K antagonists (VKA) but hyperthyroidism, through pharmacokinetic and metabolic mechanisms, can boost VKA plasma levels and increase INR, leading to hemorrhagic complications. This case emphasizes that special consideration should be given to patients with VKA treatment planned to receive immunotherapy.

Determination of platelet-derived growth factors in platelet unenriched plasma

The authors of this study proposed to use for clinical purposes not enriched with platelets plasma, as a source of autologous growth factors. The aim of the study was to determine the content of growth factors in plasma with a normal number of platelets. Materials and methods. Peripheral blood samples of 10 healthy volunteers (6 men and 4 women), aged 30 to 40, were analyzed. To obtain plasma, the blood was taken into MeaPlasma vacuum tubes. Anticoagulant was fractionated sodium heparinate. The number of platelets was measured with the Biol ALFT-2 230 LA. The concentration of platelet growth factors was determined by ELISA using Bio-Ocean LLC diagnostic kits (USA). Results. Not enriched with platelets plasma were obtained (mean platelet count in the samples was 280,000/μl). The level of TGF-β1 appeared to be elevated in women only in 1 case of 4. In men, no significant changes in the content of TGF-β1 were detected. Both in men and in women, there was an increased content of platelet growth factors PDGF-AA, VEGF, IGF-1. Significant changes in the content of EGF have not been established. Conclusions. The data obtained are preliminary and require further confirmation. Not enriched with platelets plasma obtained by the new method can be effective due to a moderate accumulation in it of the most important platelet growth factors for skin regeneration.

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia

AbstractThe role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

Lysyl oxidase is associated with increased thrombosis and platelet reactivity

AbstractLysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Loxtg/tg) were generated. Pf4-Loxtg/tg mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Loxtg/tg platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Loxtg/tg platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Treatment Experience of Acute Lymphoblastic Leukemia with Hgm/LAL07 Regimen at Hospital General De Mexico

IntroductionAcute lymphoblastic leukemia is a lymphoproliferative malignancy characterized by an uncontrolled growth of lymphoid progenitors of B or T lineage, who evade apoptosis and displace normal hematopoiesis. Based on the pediatric pre-induction steroid, the protocol assessed the impact ALL0288 GIMEMA 7 days pre-induction with steroids, complete remissions (CR). Similar to this study, our institutional protocol also considers the pre-induction with steroids, but unlike the Italian protocol fewer cases steroid responders and a lower rate of complete remissions were recorded. A 36 month follow-up overall survival was 32% The main objective of this study was to evaluate the results at 5 years of follow institutional protocol based on a pre-treatment with steroids HGMLAL07.Patients.We included patients diagnosed with acute lymphoblastic leukemia under morphological criteria French-American-British (FAB) and corroborated by flow cytometry. The criteria for complete remission was seen at 4 weeks of treatment (<5% blasts in bone marrow with normal number of leukocytes and platelets). Prophylaxis central nervous system was carried out by weekly and then monthly lumbar punctures to the maintenance stage. If express the BCR-ABL oncogene was added to treatment Imatinib 400mg PO every 24 hours you first 14 days of each cycle.Materials and methods.Study design. Retrospective cohort study of adult patients treated with institutional protocol HGMLAL07 carriers de novo acute lymphoid leukemia in the period from 2007 to 2015 in the Department of Hematology, General Hospital of Mexico. We excluded patients treated with another induction therapy and those of mixed lineage leukemia.General treatment. It was considered relapse if they had at any time monitoring the presence of more than 10% blasts in bone marrow or isolation in the cerebrospinal fluid. If u have HLA-matched donor, he referred to the area of stem cell transplantation.Statistic analysis. SPSS statistical software version 20.0 was used. Chi-Square test considered significant at p <0.05 (95% CI) was used to test hypothesis testing. Survival analysis was performed using the Kaplan-Meyer test for comparing groups for overall survival and disease-free survival test was used log-rank2. The COX regression model was used for the risk function between different risk variablesResults. Of the 262 patients with LLA de novo, 255 patients met the inclusion criteria and were treated with institutional protocol HGMLAL07, 52.9% were male gender (n = 135) and 47.1% (n = 120) Gender female. The mean age was 31 years (range 16- 80 years), the average for older female compared with male (34 versus 29years, p = 0.001, 95% CI). The average of leukocytes at diagnosis was 56.1 x 10 (9) / L. Phenotypically, most were classified leukemia B-cell (95.3%, n = 243) and the remaining T lineage (4.7%, n = 12). Only 3.1% of cases expressed the oncogene BCRABL1 (n = 8). Finally concluding that 62.7% of cases were classified as high risk (n = 160) and 37.3% (n = 95) and normal risk.Response to induction therapy.Of the 255 patients who started the protocol remission induction in 1.6% of confirmed with CNS infiltration diagnosis (n = 4). The complete remission rate was around 82.7% (n = 211), registering an induction mortality 3.9% (n = 10). A total of 34 patients were considered refractory leukemia (n = 34), requiring a second line treatment. Among the variables that showed the impact of the failure to initial treatment (refractory or death) were the type of risk at diagnosis (p = 0.020) and white blood cell count> 30 x 10 (9) / L (p = 0.001). Mean neutrophil recovery was 28 days and the platelet recovery was at 32 days. The main cause of death was infectious processes, followed by central nervous system bleeding.Postremisión treatment and outcomeOf the total patients, 51% (n = 130) presented relapse, the main site bone marrow, followed by infiltration of the central nervous systemPrognostic factors that impacted on survivalThe median overall survival (OS) was 1053 days, with survival at 5 years of follow-up of 29%, overall survival at 5 years was influenced by the type of risk (p = 0.020, 95% CI). None of the risk factors impact on survival at one year. The disease-free survival was 11% at 5 years of follow-up, within the variables that impacted on the SLE, a leukocyte count> 30 (9) / L and age> 35 years directly impacted prognosis (p = 0.006 and p = 0.030, 95% CI respectively). DisclosuresNo relevant conflicts of interest to declare.

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis

AbstractAutophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.

Abnormal cytoplasmic extensions associated with active αIIbβ3 are probably the cause for macrothrombocytopenia in Glanzmann thrombasthenia-like syndrome.

Mutations in the ITGA2B or ITGB3 genes that encode for the αIIbβ3 platelet integrin usually cause Glanzmann thrombasthenia, a severe autosomal recessive bleeding disorder characterized by absence of platelet aggregation, but normal platelet number and size. Several rare mutations cause a Glanzmann-like syndrome which manifests macrothrombocytopenia and usually displays autosomal dominant inheritance. The exact mechanism causing Glanzmann-like syndrome is unknown. One typical example of Glanzmann-like mutations causes deletion of 40 amino acids (p.647-686) in the β3 β-tail domain (βTD_del) that was found in the heterozygous state in Italian and Japanese families. A second example is a missense mutation, C560R, located in the epidermal growth factor-like domain, found in the homozygous state in a French patient. Both mutations cause constitutive activation of αIIbβ3, but differ in their surface expression. In the current study, we generated cultured cells expressing β3-βTD_del or β3-C560R mutations along with wild-type αIIb, and examined the cells' ability to create tubulin-dependent protrusions compared to cells expressing wild-type αIIbβ3. Unlike cells expressing wild-type αIIbβ3, cells harboring each of the mutations exhibited abnormal cytoplasmic extensions on immobilized fibrinogen or Von Willebrand factor, which resembled extensions formed in megakaryocyte leading to proplatelets. Moreover, we showed that formation of abnormal extensions occurred also in wild-type αIIbβ3 cells when activated by activating antibody. These results suggest that the active conformation of αIIbβ3 can induce cytoskeletal rearrangements that lead to impaired proplatelet formation.