Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2V617F mutation

Background: JAK2V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis. Objectives: Our main objective was to explore and compare platelet function depending on the MPN molecular marker. Methods: We analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels. Results: We observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN. Conclusions: These results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR

First description of an IgM monoclonal antibody causing αIIb β3 integrin activation and acquired Glanzmann thrombasthenia associated with macrothrombocytopenia

BACKGROUND: Acquired Glanzmann thrombasthenia (GT) is a bleeding disorder generally caused by anti-αIIb β3 autoantibodies. OBJECTIVES: We aimed to characterize the molecular mechanism leading to a progressive GT-like phenotype in a patient with chronic immune thrombocytopenia. PATIENT, METHODS AND RESULTS: The patient suffered from repeated episodes of gastrointestinal bleedings and further studies indicated a moderate platelet aggregation defect. Few months later, platelet function showed abolished aggregation using all agonists, but normal agglutination with ristocetin. No platelet-bound antibodies were detected, but the presence of large amounts of an IgM type antibody detected together with αIIb β3 in the patient's permeabilized platelets suggested that this IgM was an autoantibody causing the internalization of the complex. This was confirmed by the fact that the patient's IgM bound to normal platelets but not to platelets from GT type I patients. Moreover, patient's plasma activated αIIb β3 on controls' platelets as evidenced by increased PAC-1 binding. We also demonstrated that the patient's plasma triggered αIIb β3 outside-in signaling, as β3 Tyr773 and FAK were phosphorylated, and increased the rate of actin polymerization in resting platelets reflecting an impairment of cytoskeletal reorganization. As different signs of dysmegakariopoiesis were also observed in our patient, we evaluated the ability of its serum to impair proplatelets formation and showed that it significantly decreased the number of proplatelet-bearing megakaryocytes in controls' bone marrow stem cells culture as compared to normal serum. CONCLUSIONS: We present the case of a patient with a progressive and severely perturbed platelet function associated with the presence of an IgM activating autoantibody directed against αIIb β3 . This article is protected by copyright. All rights reserved

A mutation of the human EPHB2 gene leads to a major platelet functional defect.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans

Etude in vitro et ex-vivo des effets de concentres d'IgG a usage therapeutique sur les lymphocytes spleniques de malades atteints de thrombopenie autoimmune

SIGLECNRS T 56211 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Platelet δ-Storage Pool Disease: An Update

Platelet dense-granules are small organelles specific to the platelet lineage that contain small molecules (calcium, adenyl nucleotides, serotonin) and are essential for the activation of blood platelets prior to their aggregation in the event of a vascular injury. Delta-storage pool diseases (δ-SPDs) are platelet pathologies leading to hemorrhagic syndromes of variable severity and related to a qualitative (content) or quantitative (numerical) deficiency in dense-granules. These pathologies appear in a syndromic or non-syndromic form. The syndromic forms (Chediak–Higashi disease, Hermansky–Pudlak syndromes), whose causative genes are known, associate immune deficiencies and/or oculocutaneous albinism with a platelet function disorder (PFD). The non-syndromic forms correspond to an isolated PFD, but the genes responsible for the pathology are not yet known. The diagnosis of these pathologies is complex and poorly standardized. It is based on orientation tests performed by light transmission aggregometry or flow cytometry, which are supplemented by complementary tests based on the quantification of platelet dense-granules by electron microscopy using the whole platelet mount technique and the direct determination of granule contents (ADP/ATP and serotonin). The objective of this review is to present the state of our knowledge concerning platelet dense-granules and the tools available for the diagnosis of different forms of δ-SPD

Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail.We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation.Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C<A (TM1-536) mutant and 4 other TM mutants (TM1-515, TM1-525, TM1-533 and TM1-537) with a transmembrane domain having different lengths. The effect of shear stress, metalloprotease inhibitor, certain proteases and reducing agents were tested on TM shedding.Western blot and immunofluorescent analysis showed that TM1-536 was produced and a certain amount of TM1-536 was anchored on the cell membrane. A significantly higher levels of soluble TM was observed in the TM1-536 cell medium in comparison with TM-WT (56.3 +/- 5.2 vs 8.8 +/- 1.6 ng/mL, respectively, p = 0.001). The shedding of TM1-536 was 75% decreased in cells cultured in the presence of a metalloprotease inhibitor. No difference was observed between TM1-536 and TM-WT shedding after cell exposure to cathepsin G, elastase, several reducing agents and high shear stress (5000 s-1). Significantly higher levels of soluble TM were observed in the cell media of TM1-533, TM1-525, TM1-515 in comparison with TM-WT (p < 0.05).The mechanism responsible for TM shedding is complex and is not completely understood: higher sensitivity of the TM1-536 to the proteolysis by metalloproteases and a defect of synthesis due to the decreased size of the transmembrane domain might explain the high levels of soluble TM in plasma of the carriers

Cyclic thrombocytopenia related to erythropoietin-dependent anti-platelet anti-GPIV/IIIb antibody in hemodialysis

International audienceWe describe herein the case of a 65-year-old patient on chronic hemodialysis with a medical history of idiopathic thrombocytopenia who experienced numerous episodes of severe thrombocytopenia with no specific diagnosis. Further analysis of the evolution of the platelet count showed that cyclic thrombocytopenia occurred after each injection of recombinant erythropoietin (rHu-EPO). Exploration of the involved mechanisms revealed the presence of a rHu-EPO-dependent anti-GPIV/IIIb antibody associated with a significant increase in GPIV/IIIb expression on her platelets after the addition of rHu-EPO. EPO was discontinued and the patient was treated with roxadustat with favorable results on her hemoglobin and platelet counts

Atypical late diagnosis of Noonan syndrome revealed by bleedings due to platelet dysfunction.

International audienc