Platelets can bind and phagocytose infectious microorganisms and so enable their transport for a prolonged time. To investigate the subcellular events of these interactions, platelets were incubated either with Staphylococcus aureus or with HIV and analyzed by electron microscopy (EM) and immuno-EM. HIV and bacteria internalization occurred exclusively within platelets showing morphological evidence of activation. Platelet activation enhanced the degree of bacterial internalization. Immunolabeling revealed that the engulfing vacuoles and the open canalicular system (OCS) were composed of distinct antigens. The engulfing vacuoles eventually became the site of prominent alpha-granule release. In platelets incubated with HIV, characteristic endocytic vacuoles were identified close to the plasma membrane, tightly surrounding 1 or 2 HIV particles. Virus particles were also located within the OCS. Immunogold labeling for the viral core protein p24 confirmed the presence of HIV within platelets. Finally, examination of platelets from a patient with acquired immunodeficiency syndrome and high viremia suggested that HIV endocytosis may also occur in vivo.

CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction.Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actindisrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction. ( IntroductionThe vascular endothelium forms a continuous monolayer on the inner surface of the vessel wall and plays a pivotal role in regulating blood flow, vascular permeability, thrombogenesis, and hematogenous metastasis. 1 Positioned at the interface between blood and tissues, quiescent endothelial cells (ECs) generate an antithrombotic surface equipped to respond quickly to biologic needs. 2 The endothelial monolayer requires highly effective intercellular junctions that control the contact between adjacent cells and the trafficking of circulating blood cells. 3,4 At least 2 types of cell-cell junctional structures have been identified in the endothelium: adherens junctions (AJs) and tight junctions (TJs). These play a central part in the control of paracellular permeability and maintenance of cell polarity. [5][6][7] The junctions are tightly regulated structures composed of several adhesion molecules interacting with cytoskeletal proteins. Among the adhesive molecules, the endothelium-specific cadherin 5 or vascular endothelial cadherin (VEcadherin) 8,9 is localized in AJs, whereas the junctional adhesion molecule (JAM) 10 was reported to be present in TJs. Other molecules, such as platelet endothelial cell adhesion molecule 1 (PECAM-1)/CD31, are not restricted to one type of junctional structure, and their specific localization appears to be important to their vascular functions. 11,12 The S-Endo 1-associated antigen (CD146), also referred to as MelCAM or MUC18, 13 is a transmembrane glycoprotein that is constitutively expressed in the whole human endothelium, irrespective of its anatomical site or vessel caliber. 14,15 CD146 expression is not restricted to ECs; it has also been observed on several other cell types, including melanoma cells, 13 smooth muscle cells, and follicular dendritic cells. 14 Using optical microscopy, we previously showed that,...

The origin of platelet a-granule fibrinogen (Fg), whether from endogeneous synthesis or exogeneous derivation, remains unknown. Although Fg biosynthesis by megakaryocytes (MK) has been suggested, recent studies have demonstrated that certain a-granular proteins originate primarily from plasma. To study the origin of a-granule Fg, platelet-associated Fg was measured by ELISA and Western blotting, and localized by immunofluorescence and immunoelectron microscopy in a patient with symptomatic congenital afibrinogenemia before and after replacement therapy with cryoprecipitate. a-Granule Fg was detected in the majority of platelets as early as 24 h postinfusion, suggesting that direct platelet uptake was occurring. Platelet Fg reached a maximum value of 42.5% of normal values at 3 d postinfusion and was localized in the a-granules, while plasma levels followed a typical half-life profile. Significant a-granule Fg was still detectable at 13 d postinfusion, with plasma Fg virtually absent. Studies on cultured CFU-MKs from the patient also confirmed that MKs can incorporate exogeneous Fg into a-granules. These results indicate that platelet a-granule Fg can be derived from the circulating plasma pool and that Fg uptake can occur in both platelets and MKs.

The site and mechanism of platelet production by bone marrow megakaryocytes (MKs) has been the subject of extensive studies, but is still a matter of controversy. However, the recent discovery of the Mpl ligand (Mpl-l), also called megakaryocyte growth and development factor (MGDF ) or thrombopoietin, has resulted in considerable progress in the understanding of the maturation of the MK lineage. To better understand the mechanism of platelet production, we examined the late stage of MK maturation by electron microscopy in cells cultured in the presence of Mpl-l. Human bone marrow CD34+CD38+ cells, which contain late MK progenitors, were purified by flow cytometry and cultured in a serum-free liquid medium containing recombinant human Mpl-l (MGDF 10 ng/mL) for 7 days. In this system, the majority of cultured cells were large MKs with lobulated polyploid nuclei. The MKs displayed a smooth surface with harmonious cytoplasmic maturation and abundant, regularly distributed demarcation membranes and α-granules, and even some dense granules. Interestingly, approximately 30% of the MKs observed displayed morphologic evidence of platelet production: at optical microscopy, MKs formed long filamentous cytoplasmic extensions (proplatelets) that fragmented into platelet-sized particles. Moreover, flow cytometric analysis of this cultured cell population showed GPIIb-positive particles of the size of platelets. Electron microscopic observation showed that MKs producing platelets displayed thin pseudopods on the surface, and that the channels of the demarcation membrane system were dilated, allowing long strands of cytoplasm to extend from the cell periphery. These cytoplasmic strands displayed beading with constrictions separating platelet-sized segments; the more distal to the cell core, the smaller the fragments were. They eventually detached from the cell core into the culture medium either occasionally still elongated or, more often, separated into individual platelets. Parallel longitudinal and perpendicular microtubules were visualized in the constricted regions of these cytoplasmic strips; immunogold study of tubulin localization confirmed this subcellular distribution. On both sides of the constricted areas, vacuoles were noted, the fusion of which might have led to the detachment of individual platelets. Finally, in close proximity to the platelet-forming MKs, numerous microparticles were shed. Although some of these particles might correspond to transverse sections of pseudopods, this did not seem to be the case, since they were rarely seen around thrombin-stimulated MKs with surfaces bristled by numerous pseudopods. Flow cytometry showed that apart from shed cytoplasmic fragments of platelet size, numerous smaller particles strongly labeled for CD41 were also released by mature MKs. In conclusion, this study describes the ultrastructure of human platelet production in cultured MKs, involving the formation of proplatelets and the shedding of microparticles.