ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

SARS-CoV-2 Omicron variant infection affects blood platelets, a comparative analysis with Delta variant.

Introduction: In November 2021, the SARS-CoV-2 Omicron variant of concern has emerged and is currently dominating the COVID-19 pandemic over the world. Omicron displays a number of mutations, particularly in the spike protein, leading to specific characteristics including a higher potential for transmission. Although Omicron has caused a significant number of deaths worldwide, it generally induces less severe clinical signs compared to earlier variants. As its impact on blood platelets remains unknown, we investigated platelet behavior in severe patients infected with Omicron in comparison to Delta. Methods: Clinical and biological characteristics of severe COVID-19 patients infected with the Omicron (n=9) or Delta (n=11) variants were analyzed. Using complementary methods such as flow cytometry, confocal imaging and electron microscopy, we examined platelet activation, responsiveness and phenotype, presence of virus in platelets and induction of selective autophagy. We also explored the direct effect of spike proteins from the Omicron or Delta variants on healthy platelet signaling. Results: Severe Omicron variant infection resulted in platelet activation and partial desensitization, presence of the virus in platelets and selective autophagy response. The intraplatelet processing of Omicron viral cargo was different from Delta as evidenced by the distribution of spike protein-positive structures near the plasma membrane and the colocalization of spike and Rab7. Moreover, spike proteins from the Omicron or Delta variants alone activated signaling pathways in healthy platelets including phosphorylation of AKT, p38MAPK, LIMK and SPL76 with different kinetics. Discussion: Although SARS-CoV-2 Omicron has different biological characteristics compared to prior variants, it leads to platelet activation and desensitization as previously observed with the Delta variant. Omicron is also found in platelets from severe patients where it induces selective autophagy, but the mechanisms of intraplatelet processing of Omicron cargo, as part of the innate response, differs from Delta, suggesting that mutations on spike protein modify virus to platelet interactions.

Multicolor flow cytometry in clinical samples for platelet signaling assessment.

Background: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. Objectives: To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). Methods: By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI-related disease with granule secretion defect). Results: The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. Conclusion: In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.

Immunohistologic Features of Cerebral Venous Thrombosis Due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

OBJECTIVES: Vaccine-induced immune thrombotic thrombocytopenia (VITT), a recently described entity characterized by thrombosis at unusual locations such as cerebral venous sinus and splanchnic vein, has been rarely described after adenoviral-encoded COVID-19 vaccines. In this study, we report the immunohistological correlates in 3 fatal cases of cerebral venous thrombosis related to VITT analyzed at an academic medical center. METHODS: Detailed neuropathologic studies were performed in 3 cases of cerebral venous thrombosis related to VITT after adenoviral COVID-19 vaccination. RESULTS: Autopsy revealed extensive cerebral vein thrombosis in all 3 cases. Polarized thrombi were observed with a high density of neutrophils in the core and a low density in the tail. Endothelial cells adjacent to the thrombus were largely destroyed. Markers of neutrophil extracellular trap and complement activation were present at the border and within the cerebral vein thrombi. SARS-CoV-2 spike protein was detected within the thrombus and in the adjacent vessel wall. DISCUSSION: Data indicate that neutrophils and complement activation associated with antispike immunity triggered by the vaccine is probably involved in the disease process.

What can we learn from the platelet lipidome?

Besides their proteome, platelets use, in all responses to the environmental cues, a huge and diverse family of hydrophobic and amphipathic small molecules involved in structural, metabolic and signaling functions; the lipids. Studying how platelet lipidome changes modulate platelet function is an old story constantly renewed through the impressive technical advances allowing the discovery of new lipids, functions and metabolic pathways. Technical progress in analytical lipidomic profiling by top-of-the-line approaches such as nuclear magnetic resonance and gas chromatography or liquid chromatography coupled to mass spectrometry enables either large-scale analysis of lipids or targeted lipidomics. With the support of bioinformatics tools and databases, it is now possible to investigate thousands of lipids over a concentration range of several orders of magnitude. The lipidomic landscape of platelets is considered a treasure trove, not only able to expand our knowledge of platelet biology and pathologies but also to bring diagnostic and therapeutic opportunities. The aim of this commentary article is to summarize the advances in the field and to highlight what lipidomics can tell us about platelet biology and pathophysiology.

What is the context? Lipids are a huge and diverse family of molecules strongly involved in biological membranes organization and dynamics, signal transduction, cell metabolism and intercellular communication.Earlier seminal works using conventional lipid biochemistry methods have shown the essential role of certain classes of lipids in platelet biology and platelet-related pathologiesWhat is new? The important development of modern lipidomic analyses using mass-spectrometry now provides opportunities to investigate the entire platelet lipidome in different conditions.The application of lipidomic approaches to analyze large-scale lipid species allows platelet clinical lipidomics development.What is the impact? Study of the lipidomic landscape of platelets will expand our knowledge of platelet biology and should bring new diagnosis and therapeutic opportunities.Evaluating the functional and clinical significance of the data generated by modern platelet lipidomics appears as a vast and exciting challenge.

Platelet P2Y1 receptor exhibits constitutive G protein signaling and ß-arrestin 2 recruitment.

BACKGROUND: Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step. RESULTS: Here, we investigated the pharmacology of P2Y1-R signaling through Gq and ß-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of ß-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. CONCLUSIONS: This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.

HAT-field: a cheap, robust and quantitative Point-of-care serological test for Covid-19.

The haemagglutination test (HAT)-field protocol described here is an optimization of the recently published HAT, for the detection of antibodies directed against the receptor binding domain (RBD) of the SARS-Cov-2 virus. HAT and HAT-field are both based on haemagglutination triggered by a single reagent, the IH4-RBD recombinant protein. A sample of IH4-RBD sufficient for several thousand tests or a plasmid encoding IH4-RBD can be obtained from the authors of our first paper. Using titration of IH4-RBD, HAT-field now allows a quantitative assessment of antibody levels in a single step, using a few microliters of whole blood, such as can be obtained by finger prick, and requires only very simple disposable equipment. Because it is based on a single soluble reagent, the test can be adapted very simply and rapidly to detect antibodies against variants of the SARS-CoV-2, or conceivably against different pathogens. HAT-field appears well suited to provide quantitative assessments of the serological protection of populations as well as individuals, and given its very low cost, the stability of the IH4-RBD reagent in the adapted buffer and the simplicity of the procedure, could be deployed pretty much anywhere, including in the poorest countries and the most remote corners of the globe.

Platelet activation and partial desensitization are associated with viral xenophagy in patients with severe COVID-19.

Mild thrombocytopenia, changes in platelet gene expression, enhanced platelet functionality, and presence of platelet-rich thrombi in the lung have been associated with thromboinflammatory complications of patients with COVID-19. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gets internalized by platelets and directly alters their behavior and function in infected patients remains elusive. Here, we investigated platelet parameters and the presence of viral material in platelets from a prospective cohort of 29 patients with severe COVID-19 admitted to an intensive care unit. A combination of specific assays, tandem mass spectrometry, and flow cytometry indicated high levels of protein and lipid platelet activation markers in the plasma from patients with severe COVID-19 associated with an increase of proinflammatory cytokines and leukocyte-platelets interactions. Platelets were partly desensitized, as shown by a significant reduction of αIIbß3 activation and granule secretion in response to stimulation and a decrease of surface GPVI, whereas plasma from patients with severe COVID-19 potentiated washed healthy platelet aggregation response. Transmission electron microscopy indicated the presence of SARS-CoV-2 particles in a significant fraction of platelets as confirmed by immunogold labeling and immunofluorescence imaging of Spike and nucleocapsid proteins. Compared with platelets from healthy donors or patients with bacterial sepsis, platelets from patients with severe COVID-19 exhibited enlarged intracellular vesicles and autophagolysosomes. They had large LC3-positive structures and increased levels of LC3II with a co-localization of LC3 and Spike, suggesting that platelets can digest SARS-CoV-2 material by xenophagy in critically ill patients. Altogether, these data show that during severe COVID-19, platelets get activated, become partly desensitized, and develop a selective autophagy response.

Platelet Versus Megakaryocyte: Who Is the Real Bandleader of Thromboinflammation in Sepsis?

Platelets are mainly known for their key role in hemostasis and thrombosis. However, studies over the last two decades have shown their strong implication in mechanisms associated with inflammation, thrombosis, and the immune system in various neoplastic, inflammatory, autoimmune, and infectious diseases. During sepsis, platelets amplify the recruitment and activation of innate immune cells at the site of infection and contribute to the elimination of pathogens. In certain conditions, these mechanisms can lead to thromboinflammation resulting in severe organ dysfunction. Here, we discuss the interactions of platelets with leukocytes, neutrophil extracellular traps (NETs), and endothelial cells during sepsis. The intrinsic properties of platelets that generate an inflammatory signal through the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome are discussed. As an example of immunothrombosis, the implication of platelets in vaccine-induced immune thrombotic thrombocytopenia is documented. Finally, we discuss the role of megakaryocytes (MKs) in thromboinflammation and their adaptive responses.

Association between megakaryocyte abnormalities on bone marrow smear and response to thrombopoietin receptor agonists in adult patients with primary immune thrombocytopenia.

Impaired platelet production is a mechanism of immune thrombocytopenia (ITP). Morphological abnormalities of megakaryocytes (MKs) are sometimes observed in this disease. Two studies have suggested an association between MK abnormalities and response to corticosteroids in primary ITP, but none have investigated this association for thrombopoietin-receptor agonists (TPO-RAs). This was the aim of this study. The source of population was the French CARMEN registry with prospective follow-up of adult patients with incident ITP. We included patients with primary ITP, treated by TPO-RA and with a bone marrow smear before initiating TPO-RA. MK abnormalities were categorized by the presence of dysplasia and by the stage of maturation. Among 451 patients screened, 38 were included in the analysis. There was no difference in the median percentage of dysplastic MKs between responders to TPO-RA (4.0%, 95% confidence interval - CI: 2.3-6.4) and non-responders (4.5%, 95% CI: 0.7-7.1). There was a slightly higher proportion of granular MKs (4.5%, 95% CI: 3-6) and basophilic MKs (30.1%, 95% CI: 21.9-39.1) in non-responders compared to responders (granular: 2.0%, 95% CI: 0-4.1; basophilic: 21.3%, 95% CI: 11.4-40.7). In conclusion, MK abnormalities were not associated with response achievement in ITP patients treated with TPO-RA in this series of 38 patients.

Phosphoinositide 3-kinases in platelets, thrombosis and therapeutics.

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.

[Digital morphology analyzers in hematology: a French survey]. / État des lieux des pratiques d'utilisation du microscope automatisé en hématologie en France.

Digital morphology hematology analyzers are becoming more prevalent in laboratories Aims: investigate practices and assess the benefits and limits of digital automated microscopy in hematology. METHODS: questionnaire sent by e-mail in 2018 to French public and private laboratories. RESULTS: out of 118 responses (56 private, 62 public), 117 participants had a CellaVision® microscope, 1 had a West Medica®. Practices were sometimes different, especially in the choice of smears to be digitized or for quality controls (16.1% had internal quality controls, 48.3% external quality controls); 62.1% never used the red blood cell (RBC) characterization tool; the number of cells counted varied from 100 to 400. The study reported a high rate of agreement for these benefits: traceability (95.7%), staff training (94.1%), eye strain (91.4%), risk of error (87.2%), time saving (83.6%). Among the disadvantages, apart from the inadequate search for platelets clumps (93.2%), the agreement rates were often lower: adaptation to digital images (61.2%), difficult assessment of atypical morphologies (49.6%) or RBC morphology (49.6%). CONCLUSION: despite well-established benefits, standardization of practices and technical improvement are still needed.

Effects of novel Btk and Syk inhibitors on platelet functions alone and in combination in vitro and in vivo.

BACKGROUND: Inhibitors of tyrosine kinases downstream of the B-cell receptor, such as Bruton's tyrosine kinase (Btk) or Spleen tyrosine kinase (Syk), used alone or in combination are new therapeutic options in the treatment of B-cell malignancies. A challenge in the development of second-generation Btk inhibitors is to limit their side effects such as the increased bleeding risk. Considering the pivotal role of Syk in immunoreceptor tyrosine-based activation motif mediated platelet signaling, the impact of inhibiting this kinase on platelet functions is also worth analyzing. OBJECTIVES: We investigated the effect of a novel Btk inhibitor, tirabrutinib, and a Syk inhibitor, entospletinib, alone and in combination on platelet signaling and functions in vitro and ex vivo. METHODS: Platelet aggregation, secretion, and signaling responses as well as thrombus growth under flow were analyzed in the presence of the inhibitors alone or in combination in vitro, at clinically relevant doses, and ex vivo in patients treated with these inhibitors in the context of a phase I trial. RESULTS: Although tirabrutinib alone had modest effects on platelet activation in vitro and ex vivo, entospletinib alone efficiently inhibited washed platelet aggregation in response to collagen. However, entospletinib weakly affected platelet activation in platelet-rich plasma, in whole blood and ex vivo. Importantly, the combination of tirabrutinib and entospletinib induced a significant decrease in platelet response to collagen in vitro and ex vivo correlating with mild bleedings reported in some of the treated patients. CONCLUSION: These new results should contribute to improve the safety of these targeted therapies.

Coagulation and heparin requirements during ablation in patients under oral anticoagulant drugs.

BACKGROUND: Anticoagulation during catheter ablation should be closely monitored with activated clotting time (ACT). However vitamin K antagonists (VKA) or direct oral anticoagulant drugs (DOAC) may act differently on ACT and on heparin needs. The aim of this study was to compare ACT and heparin requirements during catheter ablation under various oral anticoagulant drugs and in controls. METHODS: Sixty consecutive patients referred for ablation were retrospectively included: group I (n = 15, VKA), group 2 (n = 15, uninterrupted rivaroxaban), group 3 (n = 15, uninterrupted apixaban), and group 4 (n = 15, controls). Heparin requirements and ACT were compared throughout the procedure. RESULTS: Heparin requirements during the procedure were significantly lower in patients under VKA compared to DOAC, but similar between DOAC patients and controls.Activated clotting time values were significantly higher in patients under VKA compared to DOAC and similar in DOAC patients versus controls. Furthermore, anticoagulation control as evaluated by the number/proportion of ACT> 300 as well as the time passed over 300 seconds was significantly better in patients under VKA versus DOAC, without significant differences between DOAC and controls. Finally, the number of patients/ACT with excessive ACT values was significantly higher in VKA versus DOAC patients versus controls.There was no significant difference between rivaroxaban and apixaban for ACT or heparin dosing throughout the procedure. CONCLUSION: Vitamin K antagonists allowed less heparin requirement despite reaching higher ACT values and more efficient anticoagulation control (with more excessive values) compared to patients under DOAC therapy and to controls. There was no difference in heparin requirements or ACT between DOAC patients and controls.

Thromboembolic events and Covid-19.

The novel Corona virus infection (Covid-19) first identified in China in December 2019 has rapidly progressed in pandemic leading to significant mortality and unprecedented challenge for healthcare systems. Although the clinical spectrum of Covid-19 is variable, acute respiratory failure and systemic coagulopathy are common in severe Covid-19 patients. Lung is an important target of the SARS-CoV-2 virus causing eventually acute respiratory distress syndrome associated to a thromboinflammatory state. The cytokinic storm, thromboinflammation and pulmonary tropism are the bedrock of tissue lesions responsible for acute respiratory failure and for prolonged infection that may lead to multiple organ failure and death. The thrombogenicity of this infectious disease is illustrated by the high frequency of thromboembolic events observed even in Covid-19 patients treated with anticoagulation. Increased D-Dimers, a biomarker reflecting activation of hemostasis and fibrinolysis, and low platelet count (thrombocytopenia) are associated with higher mortality in Covid-19 patients. In this review, we will summarize our current knowledge on the thromboembolic manifestations, the disturbed hemostatic parameters, and the thromboinflammatory conditions associated to Covid-19 and we will discuss the modalities of anticoagulant treatment or other potential antithrombotic options.

Platelet function defects and sertraline-induced bleeding: a case report.

A 71-year-old man is admitted for nose bleeds recurring for several days. His medical background shows in particular major depression for which he has been receiving sertraline for several years. The workup shows anemia, and no anomalies on head and neck CT angiography. However, further explorations suggest an acquired thrombopathy that could have contributed to the bleeding. During sertraline exposure, platelet functional exploration and platelet secretion were abnormal. Sertraline is often used as first-line treatment of depression. Pharmacological data and spontaneous notifications suggest increased potential risk with sertraline. It appears necessary to pay attention to bleeding with sertraline use.

Systematic Screening for Deep Vein Thrombosis in Critically Ill Inpatients With COVID-19: Impact on the Incidence of Venous Thromboembolism.

Background: Several studies suggest an increased incidence of thrombosis in COVID-19 patients. However, evidence on how to prevent and even treat it is scarce. The aim of this study was to compare the cumulative incidence of venous thromboembolism (VTE) of two different methods for lower extremity deep vein thrombosis (LE-DVT) diagnosis: systematic vs. clinically guided complete compression venous ultrasonography (CCUS). We conducted a monocentric, prospective, open-label, non-randomized study. All consecutive patients admitted in three intensive care units (ICUs) of University Hospital of Toulouse for COVID-19 pneumonia were included: one performed systematic screening for LE-DVT, the others did not. The primary outcome was the 21-day cumulative incidence of VTE. The secondary end points were the 21-day cumulative incidences of major bleeding and death. Results: Among the 78 patients included, 27 (34.6%) underwent systematic screening for DVT 7 ± 2 days after ICU admission. Thirty-two patients (41.0%) were diagnosed with VTE, with a 21-day cumulative incidence of 42.3% (95% CI, 31.4-55.2), without difference between screened and non-screened patients (hazard ratio 1.45, 95% CI, 0.72-2.93). In the screened group, the frequency of isolated DVT was higher (25.9 vs. 5.9%, p-value = 0.027), but the frequency of pulmonary embolism was not reduced (25.9 vs. 29.4%, p-value = 0.745). The 21-day cumulative incidences of major bleeding and death were 9.6% (95% CI, 4.7-19.2) and 10.3% (95% CI, 5.0-20.8), respectively, without difference between the two groups. Conclusions: A systematic screening for DVT in patients hospitalized in ICU was not associated with a higher diagnosis of VTE or a reduced diagnosis of PE.