Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles.
ABSTRACT: Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.
Project description:Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin ?IIb?3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin ?IIb?3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin ?IIb?3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.
Project description:Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIb?) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41(+) cells in the lineage Sca1(+) cKit(+) (LSK) fraction (subCD41(+) LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41(+) LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3(+) CD41(-) LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41(+) LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41(+) LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41(+) CD42b(+) LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis.
Project description:BACKGROUND AND OBJECTIVES:Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS:The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS:Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION:This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.
Project description:BACKGROUND:Idiopathic pulmonary arterial hypertension (IPAH) is a devastating disease characterized by increased pulmonary vascular resistance, smooth muscle and endothelial cell proliferation, perivascular inflammatory infiltrates, and in situ thrombosis. Circulating intravascular ATP, ADP, AMP and adenosine activate purinergic cell signaling pathways and appear to induce many of the same pathologic processes that underlie IPAH. Extracellular dephosphorylation of ATP to ADP and AMP occurs primarily via CD39 (ENTPD1), an ectonucleotidase found on the surface of leukocytes, platelets, and endothelial cells. Microparticles are micron-sized phospholipid vesicles formed from the membranes of platelets and endothelial cells. OBJECTIVES:Studies here examine whether CD39 is an important microparticle surface nucleotidase, and whether patients with IPAH have altered microparticle-bound CD39 activity that may contribute to the pathophysiology of the disease. METHODOLOGY/PRINCIPAL FINDINGS:Kinetic parameters, inhibitor blocking experiments, and immunogold labeling with electron microscopy support the role of CD39 as a major nucleotidase on the surface of microparticles. Comparison of microparticle surface CD39 expression and nucleotidase activity in 10 patients with advanced IPAH and 10 healthy controls using flow cytometry and thin layer chromatograph demonstrate the following: 1) circulating platelet (CD39(+)CD31(+)CD42b(+)) and endothelial (CD39(+)CD31(+)CD42b(-)) microparticle subpopulations in patients with IPAH show increased CD39 expression; 2) microparticle ATPase and ADPase activity in patients with IPAH is increased. CONCLUSIONS/SIGNIFICANCE:We demonstrate for the first time increased CD39 expression and function on circulating microparticles in patients with IPAH. Further research is needed to elucidate whether these findings identify an important trigger for the development of the disease, or reflect a physiologic response to IPAH.
Project description:BACKGROUND:Microparticles are submicrometer vesicles that contain RNA and protein derived from their parent cells. Platelet and megakaryocyte microparticles represent 80% of circulating microparticles, and their numbers are elevated in diseases such as cancer and type 2 diabetes. The ability of microparticles to transport protein, lipid and RNA to target cells, as a means of transcellular communication, remains poorly understood. Determining the influence that microparticles have on circulating cells is essential for understanding their role in health and in disease. OBJECTIVES:To develop a novel approach to modify the composition of platelet microparticles, and understand how such changes impact their transcellular communication. METHODS:This novel model utilizes a lentiviral technology to alter the transcription factor peroxisome proliferator-activated receptor-? (PPAR?) content of megakaryoblastic cell lines and primary megakaryocytes, and also the protein composition of generated platelets and microparticles. The subsequent microparticles were isolated and added to target cells for assessment of uptake and resultant signaling events. RESULTS:We successfully engineered microparticles to contain green fluorescent protein and elevated levels of PPAR?. We found that these altered microparticles could be internalized by the monocytic cell line THP-1 and primary human microvascular endothelial cells. Importantly, microparticle-delivered PPAR? was shown to increase the expression of fatty acid-binding protein 4 (FABP4), which is a known PPAR? target gene in THP-1 cells. CONCLUSION:This proof-of-concept modification of megakaryocyte, platelet and microparticle composition and subsequent change in target cell physiology is an important new tool to address transcellular communication of microparticles.
Project description:Metabolic syndrome (MetS) is a common challenge in the world, and the platelet activation is enhanced in MetS patients. However, the fundamental mechanism that underlies platelet activation in MetS remains incompletely understood. Endothelial cells are damaged seriously in MetS patients, then they release more endothelial microparticles (EMPs). After all, whether the EMPs participate in platelet activation is still obscure. If they were, how did they work?We demonstrated that the levels of EMPs, PMPs (platelet derived microparticles) and microparticle-carried-PDI activity increased in MetS patients. IR endothelial cells released more EMPs, the EMP-PDI was more activated. EMPs can enhance the activation of CD62P, GPIIb/IIIa and platelet aggregation and this process can be partly inhibited by PDI inhibitor such as RL90 and rutin. Activated platelets stimulated by EMPs expressed more PDI on cytoplasm and released more PMPs.We obtained plasma from 23 MetS patients and 8 normal healthy controls. First we built insulin resistance (IR) model of human umbilical vein endothelial cells (HUVECs), and then we separated EMPs from HUVECs culture medium and used these EMPs to stimulate platelets. Levels of microparticles, P-selectin(CD62P), Glycoprotein IIb/IIIa (GPIIb/IIIa) were detected by flow cytometry and levels of EMPs were detected by enzyme-linked immunosorbent assay (ELISA). The protein disulfide isomerase (PDI) activity was detected by insulin transhydrogenase assay. Platelet aggregation was assessed by turbidimetry.EMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs.
Project description:Activated platelets form platelet-leukocyte aggregates in the circulation in inflammatory diseases. We investigated whether activated platelets in inflamed skin tissues are phagocytized and removed by neutrophils. To investigate the kinetics of platelets and neutrophils, we immunohistochemically examined the spatiotemporal distribution of them in a murine model of 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced dermatitis by using confocal and structured illumination microscopy. Four hours after elicitation, aggregates of CD41-positive platelets were adhered to CD31-positive endothelial cells within the vessels, and CD62P and PF4, markers of activated platelets, were expressed on platelet aggregates. At 8 hour post-elicitation, fragmented CD41-positive platelets were located both inside and outside vessels. Twenty-four hours after elicitation, the number of Ly-6G-positive neutrophils ingesting fragmented CD41-positive platelets outside vessels was increased, and CD62P and PF4 expression on the phagocytosed platelets was no longer observed. Disc-shaped CD41-positive platelets were not found outside vessels at any time during the experiment. Our data revealed that aggregates of activated platelets inside vessels were ingested and removed by neutrophils in the early stage of TNCB-induced dermatitis, suggesting that the process of removal of activated platelets by neutrophils may play an important role not only in the early phase of skin inflammation but also in other types of acute inflammation.
Project description:Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-? (PPAR?). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPAR? reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPAR? microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPAR? microparticles also changed monocyte mRNA levels of several genes including those under PPAR? control. Overall, the delivery of PPAR? from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.
Project description:BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis. METHODS: C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function. RESULTS: Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-? increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-?B and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin. CONCLUSIONS: Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.
Project description:BACKGROUND:Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS:We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS:Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS:These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.