Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.
© 2024. The Author(s).

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit-megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.
Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.

We previously demonstrated that the percentage of blood eosinophils that are associated with platelets and thus positive for CD41 (integrin αIIb-subunit) correlates with and predicts peak eosinophil count (PEC) in biopsies of eosinophilic esophagitis (EoE) patients after treatment. Thus, flow cytometric determination of CD41+ eosinophils is a potential measure of EoE disease activity. Determinants of association of platelets with eosinophils and other leukocytes in EoE are largely unknown. The objectives of this study were to test the hypotheses that platelets associate with blood leukocytes other than eosinophils in EoE and that such associations also predict EoE activity. Whole blood flow cytometry was performed on samples from 25 subjects before and after two months of standard of care EoE treatment. CD41 positivity of cells within gates for eosinophils, neutrophils, monocytes, lymphocytes, and natural killer cells was compared. We found that percent CD41+ neutrophils, monocytes, and eosinophils correlated with one another such that principal component analysis of the five cell types identified "myeloid" and "lymphoid" factors. Percent CD41+ neutrophils or monocytes, or the myeloid factor, like CD41+ eosinophils, correlated with PEC after treatment, and CD41+ neutrophils or the myeloid factor predicted PEC < 6/high power field after treatment, albeit with lower area under the curve than for CD41+ eosinophils. We conclude that the processes driving platelets to associate with eosinophils in EoE also drive association of platelets with neutrophils and monocytes and that association of platelets with all three cell types is related to disease activity. Clinicaltrials.gov identifier: NCT02775045.

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.