BACKGROUND:Platelets are essential for hemostasis and thrombosis and play vital roles during metastatic cancer progression and infection. Hallmarks of platelet function are activation, cytoskeletal rearrangements, and the degranulation of their cellular contents upon stimulation. While α-granules and dense granules are the most studied platelet secretory granules, the dense tubular system (DTS) also functions as a secretory system for vascular thiol isomerases. However, how DTS cargo is packaged and transported from megakaryocytes (MKs) to platelets is poorly understood. OBJECTIVES:To underpin the mechanisms responsible for DTS cargo transport and leverage those for therapeutic protein packaging into platelets. METHODS:A retroviral expression system combined with immunofluorescence confocal microscopy was employed to track protein DTS cargo protein disulfide isomerase fused to enhanced green fluorescent protein (eGFP-PDI) during platelet production. Murine bone marrow transplantation models were used to determine the release of therapeutic proteins from platelets. RESULTS:We demonstrated that the endoplasmic reticulum retrieval motif Lys-Asp-Glu-Leu (KDEL) located at the C-terminus of protein disulfide isomerase was essential for the regular transport of eGFP-PDI-containing granules. eGFP-PDIΔKDEL, in which the retrieval signal was deleted, was aberrantly packaged, and its expression was upregulated within clathrin-coated endosomes. Finally, we found that ectopic transgenic proteins, such as tissue factor pathway inhibitor and interleukin 2, can be packaged into MKs and proplatelets by adding a KDEL retrieval sequence. CONCLUSION:Our data corroborate the DTS as a noncanonical secretory system in platelets and demonstrate that in vitro-generated MKs and platelets may be used as a delivery system for transgenic proteins during cellular therapy.

Megakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and lightsheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

Background: Despite the importance of platelets in thrombosis and hemostasis, the mechanism by which megakaryocytes (MKs) differentiate and initiate platelet release is incompletely understood. Although thrombopoietin (TPO) has been identified as the most important regulator of MK maturation, recent reports indicate that MK maturation and platelet biogenesis can occur independently of TPO. For instance, in inflammatory conditions, platelet counts can rapidly elevate within hours. We previous identified CCL5 [chemokine ligand 5 (CCL5, also known as RANTES)] as an additional regulator of MK differentiation and maturation during inflammation. In an in vitro model, we found that CCL5 enhanced proplatelet production in a CCR5-dependent manner. In addition, other work has shown that exposing isolated hematopoietic stem cells (HSCs) to CCL5 before transplantation can directly affect fate choices of them and their progenitor cells. Building upon these observations, we aimed to investigate whether CCL5 affects megakaryo- and thrombopoiesis using in vivo mouse models, and in a novel human bone marrow organoid. Results: To determine the effect of CCL5 under steady state, we administered acute CCL5 to mice and verified that cytokine levels were elevated in both plasma and bone marrow fluid 24 hours post administration. Quantification of MKs by flow cytometry revealed an increase in both MK progenitors [CD41+] and mature MKs [CD41+CD42d+] 24 hours after treatment with CCL5. Additionally, immunofluorescence imaging of femoral cryosections confirmed an increase in MK numbers, suggesting that acute exposure to CCL5 enhanced megakaryopoiesis. Expanding our analysis to HSPCs, we observed that CCL5 also induced the expansion of MK-biased CD41+ long-term (LT)-HSCs [Lin-cKithighSca-1+Flt3-CD48-CD150+CD41+] and MkPs [Lin-cKithighCD150+CD41+], 2.8- and 2.1-fold respectively, suggesting a potential non-canonical pathway triggered by CCL5. To elucidate the mechanism responsible for increased MK numbers, we aimed to determine the in vivo expression patterns of the CCL5 receptors CCR1, CCR3, and CCR5 with and without CCL5 treatment. While no differences were found in LT- and short-term (ST)-HSCs, CCR1 and CCR5 expression was increased in MkPs 24 hours after CCL5 administration, suggesting that CCL5 signaling through CCR1 and CCR5 represents a pathway by which CCL5 signals to drive megakaryopoiesis. To investigate the effect of CCL5 administration upon stress, i.e. during platelet recovery following anti-GPIba antibody-induced platelet depletion, mice were treated with either vehicle or CCL5 48 hours post platelet depletion. After 6 days, platelets counts were higher in CCL5-treated mice compared to the vehicle-treated group. Due to the faster recovery observed in CCL5-treated mice, we aimed to determine if the increase in platelet counts occurred via non-canonical pathways. Quantification of MKs by flow cytometry revealed an increase in MK-biased CD41+ LT-HSCs while we did not observe differences in multipotent progenitors (MPPs) compared to the vehicle-treated group, indicating that CCL5 facilitated the expansion of MKs in the bone marrow post depletion. To substantiate our findings in a human system, we utilized an iPCs-derived human bone marrow organoid, which recapitulates essential features of the adult human bone marrow and offers a platform to study cellular behavior within a bone marrow microenvironment. We treated bone marrow organoids with CCL5 for 24 hours and quantified populations including endothelial cells, HSPCs, erythroid cells and MKs using flow cytometry. Consistent with our observations in the murine model, the organoids exhibited 2-fold increase in MKs [CD41+]. Conclusions: Using murine and human models, we demonstrate a CCL5-induced expansion of MkPs, revealing a role for CCL5 in promoting megakaryopoiesis in the bone marrow. These data suggest that CCL5 may be one component of the inflammatory milieu that triggers elevated platelet counts in inflammatory thrombocytosis.

Introduction: We have recently shown that membrane fatty acids are key modulators of megakaryocyte differentiation and platelet formation. In obesity, patients have an altered plasma lipid composition, which coincides with enhanced platelet responsiveness to activation. However, the mechanism underlying this platelet phenotype remains unknown. Hypothesis: We hypothesized that the obese plasma lipidome alters the composition of the megakaryocyte membrane, which ultimately leads to the generation of hyper-reactive platelets. Aim: To investigate the impact of high fat diets with different fatty acid compositions on megakaryocyte development and platelet reactivity. Results: Mice were fed chow or high fat diets enriched in either saturated fatty acids (50% SFAs) or polyunsaturated fatty acids (50% PUFAs, omega-6-enriched). Neither high fat diet resulted in changes in megakaryocyte numbers, but both caused a significant increase in the size of bone marrow megakaryocytes, suggesting diet-derived lipid uptake into megakaryocytes. When examining platelet reactivity, we found that mice fed the SFA-enriched high fat diet exhibited increased expression of the active form of integrin αIIbβ3 (JON/A) in resting platelets and that both JON/A and P-selectin expression were significantly decreased after platelets were stimulated with ADP and the thromboxane A 2 analogue U46619. In contrast, platelets from mice fed the PUFA omega-6-enriched high fat diet had no differences in platelet reactivity compared to mice fed normal chow. Conclusions: Our data reveal that dietary fatty acid saturation status affects platelet reactivity, consistent with previous human studies showing variability in platelet reactivity in obese individuals. Further explorations will elucidate if plasma and/or platelet lipid content may be an indicator of risk for thrombosis or cardiovascular disease.

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts.

Platelet-derived extracellular vesicles (PEVs) were originally studied for their potential as regulators of coagulation, a function redundant with that of their parent cells. However, as the understanding of the diverse roles of platelets in hemostasis and disease has developed, so has the understanding of PEVs. In addition, the more recent revelation of constitutively released megakaryocyte-derived extracellular vesicles (MKEVs) in circulation provides an interesting counterpoint and avenue for investigation. In this review, we highlight the historical link of PEVs to thrombosis and hemostasis and provide critical updates. We also expand our discussion to encompass the roles that distinguish PEVs and MKEVs from their parent cells. Furthermore, the role of extracellular vesicles in disease pathology, both as biomarkers and as exacerbators, has been of great interest in recent years. We highlight some of the key roles that PEVs and MKEVs play in autoimmune blood cell disorders, liver pathology, and cardiovascular disease. We then look at the future of PEVs and MKEVs as candidates for novel therapeutics.

Valoracion del Jurado: El muro y la pergola. La solucion esencial de las casas gemelas en Arrabida es una delicada e inteligente adaptacion al terreno y al paisaje. Bebiendo de la tradicion mediterranea, consiguen una arquitectura que se hace naturaleza. Destaca la sensibilidad en la resolucion material, que le confiere un caracter telurico. Este proyecto se encuentra en Sesimbra, una pequena villa de pescadores al sur de Lisboa, en una parcela atipica: una parcela en pendiente, de grandes dimensiones, situada a 600 metros del mar y que se extiende desde la villa al castillo, delimitada por un pinar y una antigua via romana. El paisaje aqui es de una belleza salvaje, con una vista deslumbrante sobre el mar. Los olivos, las higueras y los pinos crecen sobre un suelo rocoso, arido, caracterizado por el color rojizo de la tierra. Los clientes, dos hermanas, pretendian transformar una pequena casa construida en los anos 60 en una casa de vacaciones, en la que puedan reunir a las familias y amigos en verano. Una casa de vacaciones debe evocar un universo propio. Can Lis de Jorn Utzon y la Casa Rotonda de Cini Boeri son dos ejemplos, casas esenciales, intimas y generosas, profundamente relacionadas con el sitio y la cultura donde se implantan. Casas en las que el tiempo pasa de forma diferente y ayudan a comprender fisicamente donde estamos. La primera decision fue dividir la casa en dos cuerpos, garantizando autonomia a cada familia. Como gemelos, comparten las mismas propiedades y dimensiones, pero cada uno se expresa de forma ligeramente diferente; uno mas alto, expuesto al paisaje, en busca del mar, otro mas recogido y horizontal, buscando una relacion mas intima con el terreno circundante. Dado que no era posible sobrepasar los 250m² de ocupacion debido a las restricciones legales, se introducen patios y se crea una pergola alrededor de los volumenes prolongando los espacios hacia el exterior, generando zonas de estar al aire libre con vistas al mar, cubiertas con vegetacion, duplicando el area de la casa. Encastrandose en el terreno, la casa surge como una prolongacion de la roca, invisible en la ladera. Para lograr este efecto se utiliza un hormigon con pigmentos de color castano, probados innumerables veces hasta que se encontro un color identico al de la tierra de alrededor. Su superficie, con los agregados del hormigon expuestos, es a la vez aspera y suave al tacto, como una piedra o la piel de un animal. Para obtener esta textura, todo el hormigon fue vertido al anochecer, utilizandose un proceso quimico para retrasar su fraguado, decapando la capa superficial a la madrugada siguiente.