G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null-mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, while proplatelet release is hampered, but the underlying molecular mechanism remains unclear. Here, we report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis and osteosclerosis. As the mutation is based on a single nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller in size, displayed a less developed demarcation membrane system and reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1, and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.
Thromboembolic events are frequent and life-threating complications of COVID 19, but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct, but yet incompletely understood role. Several studies demonstrated altered platelet function in COVID 19 with in part controversial findings, while underlying disease-specific mechanisms remain ill-defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared to controls. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission on, platelets in COVID-19 failed to activate integrin GPIIb/IIIa in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole mount electron microscopy. In contrast, alpha-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of alpha-granule release was not observed in sepsis patients, despite a similar disease severity. We found overall unaltered thrombus formation in COVID 19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates thrombus formation was virtually abrogated in sepsis, while we detected overall normal-sized and stable thrombi in blood from COVID-19 patients. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers eptifibatide or tirofiban that had only a minor effect in control blood. We provide evidence that low dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.
Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.
Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, non-redundant functions in the megakaryocyte lineage.
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