Platelet extracellular vesicles and the secretory interactome join forces in health and disease.

Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells under various conditions. They are found in the extracellular milieu in all biological fluids. As the concentrations, contents, and origin of EVs can change during inflammation, the assessment of EVs can be used as a proxy of cellular activation. Here, we review the literature regarding EVs, more particularly those released by platelets and their mother cells, the megakaryocytes. Their cargo includes cytokines, growth factors, organelles (mitochondria and proteasomes), nucleic acids (messenger and non-coding RNA), transcription factors, and autoantigens. EVs may thus contribute to intercellular communication by facilitating exchange of material between cells. EVs also interact with other molecules secreted by cells. In autoimmune diseases, EVs are associated with antibodies secreted by B cells. By definition, EVs necessarily comprise a phospholipid moiety, which is thus the target of secreted phospholipases also abundantly expressed in the extracellular milieu. We discuss how platelet-derived EVs, which represent the majority of the circulating EVs, may contribute to immunity through the activity of their cargo or in combination with the secretory interactome.

Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Adipocyte Fatty Acid Transfer Supports Megakaryocyte Maturation.

Megakaryocytes (MKs) come from a complex process of hematopoietic progenitor maturation within the bone marrow that gives rise to de novo circulating platelets. Bone marrow microenvironment contains a large number of adipocytes with a still ill-defined role. This study aims to analyze the influence of adipocytes and increased medullar adiposity in megakaryopoiesis. An in vivo increased medullar adiposity in mice caused by high-fat-diet-induced obesity is associated to an enhanced MK maturation and proplatelet formation. In vitro co-culture of adipocytes with bone marrow hematopoietic progenitors shows that delipidation of adipocytes directly supports MK maturation by enhancing polyploidization, amplifying the demarcation membrane system, and accelerating proplatelet formation. This direct crosstalk between adipocytes and MKs occurs through adipocyte fatty acid transfer to MKs involving CD36 to reinforce megakaryocytic maturation. Thus, these findings unveil an influence of adiposity on MK homeostasis based on a dialogue between adipocytes and MKs.

Phosphatidylinositol 3-monophosphate: A novel actor in thrombopoiesis and thrombosis.

Phosphoinositides are lipid second messengers regulating in time and place the formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking, and cytoskeleton/membrane dynamics. One of these lipids, phosphatidylinositol 3 monophosphate (PtdIns3P), is present in small amounts in mammalian cells and is involved in the control of endocytic/endosomal trafficking and in autophagy. Its metabolism is finely regulated by specific kinases and phosphatases including class II phosphoinositide 3-kinases (PI3KC2s) and the class III PI3K, Vps34. Recently, PtdIns3P has emerged as an important regulator of megakaryocyte/platelet structure and functions. Here, we summarize the current knowledge in the role of different pools of PtdIns3P regulated by class II and III PI3Ks in platelet production and thrombosis. Potential new antithrombotic therapeutic perspectives based on the use of inhibitors targeting specifically PtdIns3P-metabolizing enzymes will also be discussed. Finally, we provide report of new research in this area presented at the International Society of Thrombosis and Haemostasis 2019 Annual Congress.

Phosphatidylinositol 3 monophosphate metabolizing enzymes in blood platelet production and in thrombosis.

Blood platelets, produced by the fragmentation of megakaryocytes, play a key role in hemostasis and thrombosis. Being implicated in atherothrombosis and other thromboembolic disorders, they represent a major therapeutic target for antithrombotic drug development. Several recent studies have highlighted an important role for the lipid phosphatidylinositol 3 monophosphate (PtdIns3P) in megakaryocytes and platelets. PtdIns3P, present in small amounts in mammalian cells, is involved in the control of endocytic trafficking and autophagy. Its metabolism is finely regulated by specific kinases and phosphatases. Class II (α, ß and γ) and III (Vps34) phosphoinositide-3-kinases (PI3Ks), INPP4 and Fig4 are involved in the production of PtdIns3P whereas PIKFyve, myotubularins (MTMs) and type II PIPK metabolize PtdIns3P. By regulating the turnover of different pools of PtdIns3P, class II (PI3KC2α) and class III (Vps34) PI3Ks have been recently involved in the regulation of platelet production and functions. These pools of PtdIns3P appear to modulate membrane organization and intracellular trafficking. Moreover, PIKFyve and INPP4 have been recently implicated in arterial thrombosis. In this review, we will discuss the role of PtdIns3P metabolizing enzymes in platelet production and function. Potential new anti-thrombotic therapeutic perspectives based on inhibitors targeting specifically PtdIns3P metabolizing enzymes will also be commented.

Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions.

Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbß3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.