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Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation., (© 2024. The Author(s).)

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

Background: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited., Objectives: The objective of this study was to profile the platelet proteomics signatures of IPDs., Methods: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development., Results: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins., Conclusions: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Hemostasis is a complex and tightly regulated system that attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis. Hemostasis reflects the subtle balance between procoagulant and anticoagulant factors in the pathways of primary hemostasis, secondary hemostasis, and fibrinolysis. The major components in this interplay include the vascular endothelium, platelets, coagulation factors, and fibrinolytic factors. After vessel wall injury, the subendothelium is exposed to the blood stream, followed by rapid activation of platelets via collagen binding and von Willebrand factor-mediated platelet adhesion to the damaged vessel wall through platelet glycoprotein receptor Ib/IX/V. Activated platelets change their shape, release bioactive molecules from their granules, and expose negatively charged phospholipids on their surface. For a proper function of this process, an adequate number of functional platelets are required. Subsequently, a rapid generation of sufficient amounts of thrombin begins; followed by activation of the coagulation system and its coagulation factors (secondary hemostasis), generating fibrin that consolidates the platelet plug. To maintain equilibrium between coagulation and anticoagulation, the naturally occurring anticoagulants such as protein C, protein S, and antithrombin keep this process in balance. Deficiencies (inherited or acquired) at any level of this fine-tuned system result in pathologic bleedings or increased hypercoagulability states leading to thrombosis. This review will focus on genetic diagnosis of inherited bleeding, thrombotic, and platelet disorders, discussing strengths and limitations of existing diagnostic settings and genetic tools and highlight some important considerations necessary for clinical application., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice., (© 2022 International Society on Thrombosis and Haemostasis.)

Platelets are generated and released into the bloodstream from their precursor cells, megakaryocytes that reside in the bone marrow. Though platelets have no nucleus or DNA, they contain a full transcriptome that, during platelet formation, is transported from the megakaryocyte to the platelet. It has been described that transcripts in platelets can be translated into proteins that influence platelet response. The platelet transcriptome is highly dynamic and has been extensively studied using microarrays and, more recently, RNA sequencing (RNA-seq) in relation to diverse conditions (inflammation, obesity, cancer, pathogens and others). In this review, we focus on bulk and single-cell RNA-seq studies that have aimed to characterize the coding transcriptome of healthy megakaryocytes and platelets in humans. It has been noted that bulk RNA-seq has limitations when studying in vitro-generated megakaryocyte cultures that are highly heterogeneous, while single-cell RNA-seq has not yet been applied to platelets due to their very limited RNA content. Next, we illustrate how these methods can be applied in the field of inherited platelet disorders for gene discovery and for unraveling novel disease mechanisms using RNA from platelets and megakaryocytes and rare disease bioinformatics. Next, future perspectives are discussed on how this field of coding transcriptomics can be integrated with other next-generation technologies to decipher unexplained inherited platelet disorders in a multiomics approach.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) form a clinical continuum associating progressive muscle weakness with additional multi-systemic anomalies of the bones, skin, spleen, and platelets. TAM/STRMK arises from excessive extracellular Ca 2+ entry due to gain-of-function mutations in the Ca 2+ sensor STIM1 or the Ca 2+ channel ORAI1. Currently, no treatment is available. Here we assessed the therapeutic potential of ORAI1 downregulation to anticipate and reverse disease development in a faithful mouse model carrying the most common TAM/STRMK mutation and recapitulating the main signs of the human disorder. To this aim, we crossed Stim1 R304W/+ mice with Orai1 +/- mice expressing 50% of ORAI1. Systematic phenotyping of the offspring revealed that the Stim1 R304W/+ Orai1 +/- mice were born with a normalized ratio and showed improved postnatal growth, bone architecture, and partly ameliorated muscle function and structure compared with their Stim1 R304W/+ littermates. We also produced AAV particles containing Orai1 -specific shRNAs, and intramuscular injections of Stim1 R304W/+ mice improved the skeletal muscle contraction and relaxation properties, while muscle histology remained unchanged. Altogether, we provide the proof-of-concept that Orai1 silencing partially prevents the development of the multi-systemic TAM/STRMK phenotype in mice, and we also established an approach to target Orai1 expression in postnatal tissues.

Objective: To analyze the molecular polymorphisms of CD36 among 58 blood donors with CD36 deficiency and compare with CD36 positive controls., Methods: A total of 58 donors with CD36 deficiency during a screening conducted in the laboratory from September 2019 to December 2020 were enrolled as the test group, including 39 males and 19 females, while 120 platelet donors with CD36 positive were randomly selected as the controls, including 76 males and 44 females. All of the subjects were Han nationality. The PCR-SBT method was used to detect coding region of CD36 gene, and molecular mutations were compared with those CD36 positive controls., Results: Among the 58 donors with CD36 deficiency, mutations appears in 32 individuals. The detection rate for type I was 71.43% (5/7), and type II was 51.92% (27/52), while among the 120 controls, mutations appears in 12 donors (10%). In the CD36 antigen-deficient donors, 16 variations were found, in which 329-330 del AC with the highest frequency accounted for 20.69%, followed by 1228-1239 del ATTGTGCCTATT(15.52%) and 1156 C>T(10.34%). Two variations, 198-205 del GATCTTTG and 220 C>T, led to premature termination of translation; four mutations, 329-330 del AC, 560 ins T, 1011-1049 39bp dupl and 1343-1344 ins TCTT, caused translation frame shift; 1228-1239 del ATTGTGCCTATT led to deletion of four amino acids (Ile-Val-Pro-Ile) at sites 410-413 of the peptide chain. The 1140 T>A and 1275 G>A were synonymous mutations, and the other 7 mutations resulted in the substitution of single nucleotide. The platelet expression in the donors of CD36 positive with 329-330 del AC or 1228-1239 del ATTGTGCCTATT mutation (heterozygote) was lower than those CD36 positive individuals without mutations (homozygote)., Conclusion: Multiple gene mutations in the CD36 coding region may cause CD36 deficiency, and the heterozygous individuals with mutations may lead to CD36 antigen reduction or deletion. Mutation is not detected in 44.83% of CD36 deficient individuals, there may be some other reasons for the CD36 antigen deficiency.

Cardiovascular complications remain the leading cause of morbidity and mortality in individuals with diabetes, driven by interlinked metabolic, inflammatory, and thrombotic changes. Hyperglycaemia, insulin resistance/deficiency, dyslipidaemia, and associated oxidative stress have been linked to abnormal platelet function leading to hyperactivity, and thus increasing vascular thrombotic risk. However, emerging evidence suggests platelets also contribute to low-grade inflammation and additionally possess the ability to interact with circulating immune cells, further driving vascular thrombo-inflammatory pathways. This narrative review highlights the role of platelets in inflammatory and immune processes beyond typical thrombotic effects and the impact these mechanisms have on cardiovascular disease in diabetes. We discuss pathways for platelet-induced inflammation and how platelet reprogramming in diabetes contributes to the high cardiovascular risk that characterises this population. Fully understanding the mechanistic pathways for platelet-induced vascular pathology will allow for the development of more effective management strategies that deal with the causes rather than the consequences of platelet function abnormalities in diabetes.

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2-3 min. In our cohort of healthy blood donors ( n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

Introduction/aims: Stromal interaction molecule 1 (STIM1) is a reticular Ca 2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1., Methods: We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel., Results: We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients., Discussion: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases., (© 2021 Wiley Periodicals LLC.)

Purpose of Review: Megakaryocytes are rare hematopoietic cells that play an instrumental role in hemostasis, and other important biological processes such as immunity and wound healing. With the advent of cell reprogramming technologies and advances in differentiation protocols, it is now possible to obtain megakaryocytes from any pluripotent stem cell (PSC) via hematopoietic induction. Here, we review recent advances in PSC-derived megakaryocyte (iMK) technology, focusing on platform validation, disease modeling and current limitations., Recent Findings: A comprehensive study confirmed that iMK can recapitulate many transcriptional and functional aspects of megakaryocyte and platelet biology, including variables associated with complex genetic traits such as sex and race. These findings were corroborated by several pathological models in which iMKs revealed molecular mechanisms behind inherited platelet disorders and assessed the efficacy of novel pharmacological interventions. However, current differentiation protocols generate primarily embryonic iMK, limiting the clinical and translational potential of this system., Summary: iMK are strong candidates to model pathologic mutations involved in platelet defects and develop innovative therapeutic strategies. Future efforts on generating definitive hematopoietic progenitors would improve current platelet generation protocols and expand our capacity to model neonatal and adult megakaryocyte disorders., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mutations in the transcription factors GATA binding factor 1 (GATA1), growth factor independence 1B (GFI1B), and Runt-related transcription factor 1 (RUNX1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction resulting in a grayish appearance in blood smears. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from 11 individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1Q154Rfs, or RUNX1TD2-6 and 28 healthy controls were examined by label-free quantitative mass spectrometry. In total, 2875 platelet proteins were reliably quantified. Clustering analysis of more than 300 differentially expressed proteins revealed profound differences between cases and controls. Among cases, 44 of 143 significantly downregulated proteins were assigned to platelet function, hemostasis, and granule biology, in line with platelet dysfunction and bleedings. Remarkably, none of these proteins were significantly diminished in all affected cases. Similarly, no proteins were commonly overrepresented in all affected cases compared with controls. These data indicate that the studied transcription factor mutations alter platelet proteomes in distinct largely nonoverlapping manners. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders., (© 2021 by The American Society of Hematology.)

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca 2+ homeostasis, and mutations in either gene result in excessive extracellular Ca 2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca 2+ levels, promote major nuclear import of the Ca 2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca 2+ entry, highlighting that TAM and STRMK involve a common pathomechanism., (© 2020 Japanese Society of Neuropathology.)

Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.

Cytoskeleton is composed of more than 100 proteins and represents a dynamic network of the cellular cytoplasm. Cytoskeletal functions include spatial organization of cellular components, structural connection of the cell with external environment, and biomechanical force generation. Cytoskeleton takes part, at different levels, in all phases of platelet biogenesis: megakaryocyte (MK) differentiation, MK maturation, and platelet formation. In addition, it also plays a major role in each stage of platelet function. Inherited platelet disorders (IPDs) are a group of rare diseases featured by low platelet count and/or impaired platelet function. Over the past decade, the investigation of platelet biomechanics has become a major and highly relevant theme of research due to its implications at every stage of development of human life. The initial use of diverse biophysical techniques (e.g., micropipette aspiration, atomic force and scanning ion conductance microscopy, real-time deformability cytometry) started unraveling biomechanical features of platelets that are expected to provide new explanations for physiological and pathological mechanisms. Although the impact of cytoskeletal alterations has been largely elucidated in various IPDs' pathogenesis, the understanding of their impact on biomechanical properties of platelets represents an unmet need. Regarding IPDs, improving biomechanical studies seems promising for diagnostic and prognostic implications. Potentially, these characteristics of platelets may also be used for the prediction of bleeding risk. This review addresses the current available methods for biophysical investigations of platelets and the possible implementations in the field of IPDs., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of TBXA2R and TUBB1 revealed heterozygosity for the novel TBXA2R*c.908T>C (p.L303P) mutation in D1 and D3. TUBB1 was either wild type (D2, D3) or heterozygous (D1, D4) for the common polymorphism TUBB1*c.920G>A (rs6070697; p.R307H). In conclusion, the bleeding phenotype of the index patient can be explained by a diminished platelet function caused by the TBXA2R*c.908T>C mutation inherited from the mother and a mild thrombocytopenia with unknown molecular basis that is inherited from the father.

Primary platelet secretion defects constitute a heterogeneous group of functional defects characterized by reduced platelet granule secretion upon stimulation by different agonists. The clinical and laboratory heterogeneity of primary platelet secretion defects warrants a tailored approach. We performed a pilot study in order to develop DNA sequence analysis pipelines for gene discovery and to create a list of candidate causal genes for platelet secretion defects. Whole-exome sequencing analysis of 14 unrelated Italian patients with primary secretion defects and 16 controls was performed on Illumina HiSeq. Variant prioritization was carried out using two filtering approaches: identification of rare, potentially damaging variants in platelet candidate genes or by selecting singletons. To corroborate the results, exome sequencing was applied in a family in which platelet secretion defects and a bleeding diathesis were present. Platelet candidate gene analysis revealed gene defects in 10/14 patients, which included ADRA2A , ARHGAP1 , DIAPH1 , EXOC1 , FCGR2A , ITPR1 , LTBP1 , PTPN7 , PTPN12 , PRKACG , PRKCD , RAP1GAP , STXBP5L , and VWF The analysis of singletons identified additional gene defects in PLG and PHACTR2 in two other patients. The family analysis confirmed a missense variant p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene as potentially causal. In summary, exome sequencing revealed potential causal variants in 12 of 14 patients with primary platelet secretion defects, highlighting the limitations of the genomic approaches for causal gene identification in this heterogeneous clinical and laboratory phenotype., (Copyright© 2019 Ferrata Storti Foundation.)

Purpose of Review: The increasing use of high throughput sequencing and genomic analysis has facilitated the discovery of new causes of inherited platelet disorders. Studies of these disorders and their respective mouse models have been central to understanding their biology, and also in revealing new aspects of platelet function and production. This review covers recent contributions to the identification of genes, proteins and variants associated with inherited platelet defects, and highlights how these studies have provided insights into platelet development and function., Recent Findings: Novel genes recently implicated in human platelet dysfunction include the galactose metabolism enzyme UDP-galactose-4-epimerase in macrothrombocytopenia, and erythropoietin-producing hepatoma-amplified sequence receptor transmembrane tyrosine kinase EPHB2 in a severe bleeding disorder with deficiencies in platelet agonist response and granule secretion. Recent studies of disease-associated variants established or clarified roles in platelet function and/or production for the membrane receptor G6b-B, the FYN-binding protein FYB1/ADAP, the RAS guanyl-releasing protein RASGRP2/CalDAG-GEFI and the receptor-like protein tyrosine phosphatase PTPRJ/CD148. Studies of genes associated with platelet disorders advanced understanding of the cellular roles of neurobeachin-like 2, as well as several genes influenced by the transcription regulator RUNT-related transcription factor 1 (RUNX1), including NOTCH4., Summary: The molecular bases of many hereditary platelet disorders have been elucidated by the application of recent advances in cell imaging and manipulation, genomics and protein function analysis. These techniques have also aided the detection of new disorders, and enabled studies of disease-associated genes and variants to enhance understanding of platelet development and function.

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1 -targets involved in platelet function by combining transcriptome analysis of patient and sh RUNX1 -transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (β)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2 , and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML., (Copyright© 2019 Ferrata Storti Foundation.)

Ca 2+ release-activated Ca 2+ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca 2+ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Essentials Platelets in trauma-induced coagulopathy (TIC) are impaired, but the mechanism is not known. We performed comprehensive longitudinal platelet function testing in trauma patient samples. Platelets in TIC are widely impaired early after injury, but platelet activatability is intact. This suggests a mechanism of transient platelet cytoskeletal/integrin dysfunction during TIC. SUMMARY: Background Trauma-induced coagulopathy (TIC) is a common and deadly bleeding disorder. Platelet dysfunction is present during TIC, but its mechanisms remain unclear. Platelets are currently thought to become "exhausted," a state in which they have released their granule contents and can no longer aggregate or contract. Methods This prospective observational cohort study tested the hypothesis that platelet exhaustion is present during TIC and characterized the early time course of platelet dysfunction. Blood was collected from 95 adult trauma patients at a Level I trauma center at time of Emergency Department arrival and several time points over 72 h. Platelet activation state and function were characterized using CD62P (P-selectin) and PAC-1 surface membrane staining, platelet function analyzer (PFA-100), aggregometry, viscoelastic platelet mapping, and, to test for exhaustion, their ability to express CD62P after ex vivo adenosine diphosphate (ADP) agonism. Platelet function was compared between patients with and without TIC, defined by prothrombin time ≥18 s. Results Platelets in TIC showed no initial increase in their level of surface activation markers or impairment of their capacity to express CD62P in response to ADP stimulation. However, TIC platelets were impaired in nearly all functional assays, spanning adhesion, aggregation, and contraction. These effects largely remained after controlling for platelet count and fibrinogen concentration and resolved after 8 h. Conclusion The TIC platelets exhibit early impairment of adhesion, aggregation, and contraction with retained alpha granule secretion ability, suggesting a specific mechanism of cytoskeletal or integrin dysfunction that is not a result of more general platelet exhaustion., (© 2019 International Society on Thrombosis and Haemostasis.)

Objectives: The bipolar affective disorder (BAD) pathophysiology is multifactorial and has not been fully clarified., Method: We measured selected mitochondrial parameters in peripheral blood components. The analyses were performed for patients suffering from a manic episode during remission and were compared to those performed for healthy controls. BAD was clinically evaluated using well-established diagnostic scales and questionnaires. Mitochondrial respiration was examined in intact and permeabilized blood platelets using high-resolution respirometry. The citrate synthase (CS) and electron transport system (ETS) complex (complex I, II, and IV) activities were examined in platelets., Results: The CS, complex II and complex IV activities were decreased in the BAD patients, complex I activity was increased, and the ratio of complex I to CS was significantly increased. In the intact platelets, respiration after complex I inhibition and residual oxygen consumption were decreased in the BAD patients compared to the healthy controls. In the permeabilized platelets, a decreased ETS capacity was found in the BAD patients. No significant differences were found between BAD patients in mania and remission., Conclusion: Increased complex I activity can be a compensatory mechanism for decreased CS and complex II and IV activities. We conclude that complex I and its abnormal activity contribute to defects in cellular energy metabolism during a manic episode and that the deficiency in the complex's functioning, but not the availability of oxidative phosphorylation substrates, seems to be responsible for the decreased ETS capacity in BAD patients. The observed parameters can be further evaluated as 'trait' markers of BAD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbβ3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2.

Calcium (Ca 2+ ) is a key regulator for a large number of cellular functions in all kinds of cells, and small disturbances of Ca 2+ homeostasis can severely compromise normal physiology in various tissues and organs. A major mechanism controlling Ca 2+ homeostasis is store-operated Ca 2+ entry (SOCE), which relies on the concerted action of the reticular Ca 2+ sensor STIM1 and the plasma membrane Ca 2+ channel ORAI1. Gain-of-function mutations in the respective genes induce excessive Ca 2+ entry, and cause tubular aggregate myopathy (TAM) and Stormorken syndrome. Both disorders are part of a clinical continuum and involve muscle weakness and additional variably pronounced features including miosis, thrombocytopenia, hyposplenism, ichthyosis, dyslexia, and short stature. Mutations in the reticular Ca 2+ buffer calsequestrin (CASQ1) have moreover been associated with the mild end of the TAM/Stormorken syndrome spectrum. Here we review the clinical and histological characteristics of both disorders, provide an overview on the genetic causes, and thereby focus on the pathomechanisms leading to muscle dysfunction and the multi-systemic phenotype of tubular aggregate myopathy and Stormorken syndrome., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

STIM1 and Orai1 are key components of the Ca 2+ -release activated Ca 2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca 2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.

Here, we describe a mother and son with a lifelong bleeding tendency and posttraumatic bleeding who were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study with a suspected platelet function disorder. However, despite a clinically significant bleeding score, both had normal platelet counts and normal platelet function. The patients' blood was analyzed by light transmission aggregometry and genotyping by whole exome sequencing, as outlined by the GAPP study. Approximately 25 000 genetic variants were found for each patient as a result of sequencing and were filtered using a specialized bioinformatics pipeline. A heterozygous variant displaying autosomal dominant inheritance (c.1611 C>A) was found in the gene THBD which encodes the glycoprotein thrombomodulin. This sequence change results in a stop codon (p.Cys537Stop) and truncation of the protein and has been previously described in two other families with bleeding events which suggests it may be a recurrent mutation. In summary, this study shows that patients with a suspected platelet disorder but who present with a normal pattern of platelet aggregation should be investigated for defects in nonplatelet genes.

Background: RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles., Methods and Results: We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (β-thromboglobulin) and AH-containing vesicles (β-glucuronidase, β-hexosaminidase, α-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 μmol L -1 ) were diminished. β-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production., Conclusions: These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations., (© 2017 John Wiley & Sons Ltd.)

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

Calcium (Ca 2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca 2+ levels regulates multiple cellular functions. Store-operated Ca 2+ entry (SOCE) is a major mechanism controlling Ca 2+ homeostasis, and is mediated by the concerted activity of the Ca 2+ sensor STIM1 and the Ca 2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca 2+ homeostasis and leading to muscle dysfunction., (© 2017 WILEY PERIODICALS, INC.)

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.

Evaluation of platelet function is important for understanding the physiology of hemostasis and thrombosis and is utilized in clinical practice to diagnose inherited and acquired platelet bleeding disorders. Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large number of platelets in whole blood and offers many advantages over other traditional methods. Attention to pre-analytical factors is required to ensure biologically valid and robust results.

Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction.

Recent years have seen increasing recognition of a subgroup of inherited platelet function disorders which are due to defects in transcription factors that are required to regulate megakaryopoiesis and platelet production. Thus, germline mutations in the genes encoding the haematopoietic transcription factors RUNX1, GATA-1, FLI1, GFI1b and ETV6 have been associated with both quantitative and qualitative platelet abnormalities, and variable bleeding symptoms in the affected patients. Some of the transcription factor defects are also associated with an increased predisposition to haematologic malignancies (RUNX1, ETV6), abnormal erythropoiesis (GATA-1, GFI1b, ETV6) and immune dysfunction (FLI1). The persistence of MYH10 expression in platelets is a surrogate marker for FLI1 and RUNX1 defects. Characterisation of the transcription factor defects that give rise to platelet function disorders, and of the genes that are differentially regulated as a result, are yielding insights into the roles of these genes in platelet formation and function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Platelet numbers are intricately regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. The growth factor thrombopoietin (TPO) drives platelet biogenesis by inducing megakaryocyte production. A recent study in mice identified a feedback mechanism by which clearance of aged, desialylated platelets stimulates TPO synthesis by hepatocytes. This new finding generated renewed interest in platelet clearance mechanisms. Here, different established and emerging mechanisms of platelet senescence and clearance will be reviewed with specific emphasis on the role of posttranslational modifications.

Unlabelled: Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet-binding sensor (PDI-sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s(-1) was correlated with the PDI-sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice., Summary: Background Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 μg mL(-1) corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s(-1) ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion PDI-sAb is a sensitive and real-time reporter of platelet- and vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients., (© 2016 International Society on Thrombosis and Haemostasis.)

In contrast to congenital platelet disorders, which are rare, acquired platelet dysfunctions are more common in clinical practice. Their main causes are medications and systemic/hematologic diseases. Typical clinical manifestations are mucosal bleeding, epistaxis, or superficial epidermal bleeding normally of modest entity. In most cases, the molecular mechanisms underlying impaired platelet function are not fully established, making it difficult to optimize patient care. We here provide a short overview of the various forms of acquired platelet disorders, with a particular focus on recent mechanistic studies on platelet dysfunction in von Willebrand disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Megakaryopoiesis is a complex, stepwise process that takes place largely in the bone marrow. At the apex of the hierarchy, hematopoietic stem cells undergo a number of lineage commitment decisions that ultimately lead to the production of polyploid megakaryocytes. On average, megakaryocytes release 10(11) platelets per day into the blood that repair vascular injuries and prevent excessive bleeding. This differentiation process is tightly controlled by exogenous and endogenous factors, which have been the topics of intense research in the hematopoietic field. Indeed, a skewing of megakaryocyte commitment and differentiation may entail the onset of myeloproliferative neoplasms and other preleukemic disorders together with acute megakaryoblastic leukemia, whereas quantitative or qualitative defects in platelet production can lead to inherited platelet disorders. The recent advent of next-generation sequencing has prompted mapping of the genomic landscape of these conditions to provide an accurate view of the underlying lesions. The aims of this review are to introduce the physiological pathways of megakaryopoiesis and to present landmark studies on acquired and inherited disorders that target them. These studies have not only introduced a new era in the fields of molecular medicine and targeted therapies but may also provide us with a better understanding of the mechanisms underlying normal megakaryopoiesis and thrombopoiesis that can inform efforts to create alternative sources of megakaryocytes and platelets., (© 2016 by The American Society of Hematology.)

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: The inherited platelet disorders have witnessed a surge in our understanding of molecular mechanisms of disease in the past few years due in large to part to the introduction of next-generation sequencing for discovery of novel genes. The purpose of this review is to update the reader on the novel discoveries with regard to the inherited platelet disorders, with a particular focus on describing the novel disorders described most recently., Recent Findings: The description of novel mechanisms of disease including mutations in PRKACG, in a family with severe macrothrombocytopenia, RUNX1 and FLI1 mutations in patients with inherited mild platelet function disorders and CalDAG-GEFI resulting in a severe platelet bleeding phenotype show that there is still much to be learned from studying families and molecular sequencing of patients with well phenotyped platelet disorders., Summary: The implications for clinical practice of the continually growing list of genes described in small numbers of families makes whole exome/genome tempting as an option for evaluation of patients, but use outside of the research setting still needs to be done with extreme caution as interpretation of variants is likely to require additional studies.

Background: The objective of this study was to investigate the change of platelet function and platelet mitochondrial membrane potential in contentious-flow left ventricular assist device (CF-LVAD)-implanted heart failure (HF) patients with or without systemic inflammatory response syndrome (SIRS)., Methods and Results: We recruited 31 CF-LVAD patients (16 SIRS and 15 non-SIRS) and 11 healthy volunteers as control. Pre- and post-implantation blood samples were collected. We used PFA-100 to test platelet functionality. Mitochondrial potential-sensitive dye was used to detect platelet dysfunction (mitochondrial membrane potential; ΔΨm) via flow cytometry. The percentage of depolarized-ΔΨm platelets was found to be a preexisting condition in all HF patients before CF-LVAD implantation compared with control subjects (10.3 ± 6.3% vs 2.8 ± 2.2%; P < .001). As evident from the PFA-100 test, the HF patients who developed SIRS after CF-LVAD implantation had significantly more qualitative platelet defects and thrombocytopathies compared with baseline. After implantation, the depolarized platelets in the SIRS patients increased by 2-fold compared with baseline (18.2 ± 8.4% vs 9.0 ± 6.6%; P < .01); whereas no change was noticed in the non-SIRS patients (10.9 ± 6.2% vs 11.7 ± 5.8%; P = .75)., Conclusions: We identified that platelet function and mitochondrial damage were enhanced in CF-LVAD patients with SIRS. Our findings suggest that depolarization of mitochondrial membrane potential is associated with SIRS after CF-LVAD implantation surgery., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS., (Published by Elsevier Inc.)

Defects of the platelet P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP) are associated with increased bleeding risk. The study of molecular abnormalities associated with inherited qualitative defects of the P2Y12R protein is useful to unravel structure-function relationships of the receptor. We describe the case of 2 brothers, sons of first cousins, with lifelong history of abnormal bleeding, associated with dysfunctional P2Y12R and a previously undescribed missense mutation in the encoding gene. ADP (4-20 µM)-induced aggregation of patients' platelets was markedly reduced and rapidly reversible. Other agonists induced borderline-normal aggregation. Inhibition of vasodilator-stimulated phosphoprotein phosphorylation and prostaglandin E1-induced increase in cyclic adenosine monophosphate (cAMP) by ADP was impaired, whereas inhibition of cAMP increase by epinephrine was normal. [(3)H]PSB-0413, a selective P2Y12R antagonist, bound to a normal number of binding sites; however, its affinity, and that of the agonists ADP and 2-methylthio-adenosine-5'-diphosphate, was reduced. Patients' DNA showed a homozygous c.847T>A substitution that changed the codon for His-187 to Gln (p.His187Gln). Crystallographic data and molecular modeling studies indicated that His187 in transmembrane 5 is important for agonist and nucleotide antagonist binding and located in a region undergoing conformational changes. These studies delineate a region of P2Y12R required for normal function after ADP binding.

The interaction between platelets and endothelium in vivo is a complex phenomenon. Our aim was to develop an in vitro system that mimics the in vivo environment and investigate platelet function in a common pathological condition. Human umbilical vein endothelial cells were used and platelets from 28 type 2 diabetes patients were studied under shear stress conditions. Mean coefficient of variation of platelet aggregation was 10% in dynamic conditions in the presence of endothelium. Endothelial cells increased the concentration of inductor needed to achieve 50% platelet aggregation to adenosine diphosphate from 2.6 ± 1.3 in static conditions to 3.7 ± 1.3 µM in dynamic conditions. A similar pattern was observed when collagen was used for platelet activation. Incubation of endothelium with a nitric oxide inhibitor abolished this effect, indicating platelet inhibitory effect of endothelial cells is nitric oxide mediated. Platelet reactivity of healthy controls was less influenced by the presence of endothelial cells and displayed reduced basal platelet reactivity compared with platelets from diabetes patients. We show that platelet aggregation in diabetes as commonly reported in vitro may not fully reflect the in vivo pathophysiological process. Future studies are warranted to investigate other pathological conditions and analyse the effects of antiplatelet agents using this system., (© The Author(s) 2014.)

Introduction: When measuring platelet aggregation using laser light scattering, small aggregates forming without the addition of agonists may be observed. This event is called 'spontaneous platelet aggregation (SPA)'. The platelet hyperactivity observed in arterial thrombotic diseases can be detected with relative ease by measuring SPA. Standardization is urgently needed because of differences between measurement conditions among various laboratories., Methods: We conducted a systematic study of factors that affect SPA measurement, compared SPA results to flow cytometry detection of surface antigens expressed on activated platelet membranes (P-selectin, activated glycoprotein IIb/IIIa), and determined conditions that yield stable measurements., Results and Conclusions: We evaluated results from 125 healthy volunteers and established conditions for a stable measurement of SPA. As the occurrence of SPA tended to increase with age, we determined conditions valid for subjects aged 20-60 years. Blood should be collected using a syringe, and the sample should be prepared after allowing the whole blood to rest for 30 min after collection. To isolate platelet-rich plasma, a 2-mL tube should be used and centrifuged at 150 g. The sample should be stored at room temperature, the platelet count of the sample should be (250 ± 10) × 10(9) /L, and the measurement should be completed within 90 min of blood collection., (© 2014 John Wiley & Sons Ltd.)

Stormorken syndrome is a rare autosomal dominant disorder characterized by a phenotype that includes miosis, thrombocytopenia/thrombocytopathy with bleeding time diathesis, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis. Using targeted sequencing and whole-exome sequencing, we identified the c.910C > T transition in a STIM1 allele (p.R304W) only in patients and not in their unaffected family members. STIM1 encodes stromal interaction molecule 1 protein (STIM1), which is a finely tuned endoplasmic reticulum Ca(2+) sensor. The effect of the mutation on the structure of STIM1 was investigated by molecular modeling, and its effect on function was explored by calcium imaging experiments. Results obtained from calcium imaging experiments using transfected cells together with fibroblasts from one patient are in agreement with impairment of calcium homeostasis. We show that the STIM1 p.R304W variant may affect the conformation of the inhibitory helix and unlock the inhibitory state of STIM1. The p.R304W mutation causes a gain of function effect associated with an increase in both resting Ca(2+) levels and store-operated calcium entry. Our study provides evidence that Stormorken syndrome may result from a single-gene defect, which is consistent with Mendelian-dominant inheritance., (© 2014 The Authors. *Human Mutation published by Wiley Periodicals, Inc.)