In normal platelets, four different types of granules have been recognized which differ in their content and their ultrastructural appearances. Porcine platelets differ from human platelets by their smaller size and the uneven size of their α-granules which are generally larger. It is interesting to note that vWf and thrombospondin, two α-granule components, are thought to be synthesized by megakaryocytes and that the EM studies support this hypothesis. Indeed, it has recently been demonstrated that circulating proteins can be removed from the plasma and then incorporated, via receptor coated vesicles, into the α-granules of megakaryocytes. Indeed, the study of porcine platelets has shown that the antigenic reactivity of vWf and the occurrence of tubular structures are both extensive. Immuno-EM is a simple and reliable technique for studying the localization and distribution of vWf within different cell types.

Abstract 3775 Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures: No relevant conflicts of interest to declare.

Multimerin 1 is a massive, soluble, disulfide-linked homopolymeric protein that is expressed in megakaryocytes, platelets and endothelial cells. Normally, multimerin 1 undergoes efficient sorting to secretion granules, and it is not detectable in plasma. Recently, multimerin 1 was designated as a member of the EMILIN protein family, a group of structurally similar, disulfide-linked multimeric proteins. Multimerin 1 has the structural features of an adhesive protein and it supports the adhesion of many different cell types in vitro, including activated platelets, neutrophils, and endothelial cells. Multimerin 1 also has the ability to self associate and form large, branching matrix fibers. In platelet alpha-granules, multimerin 1 functions as the binding protein for coagulation factor V, a key regulator of coagulation. This review summarizes the current knowledge on multimerin 1 including its orthologous genes, restricted pattern of expression, structure, biosynthesis and functions.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

The pathophysiology of microthrombocytopenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked thrombocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormalities are suggestive of intrinsic disturbances of production. In contrast to human patients, murine models of WASp deficiency exhibit only mild thrombocytopenia, and platelets are of normal size. Here, we have identified a critical role for WASp during murine platelet biogenesis. By electron microscopy, WASp-deficient MKs appeared to have shed platelets ectopically within the bone marrow space. WASp-deficient megakaryocytes (MKs) also displayed defects in response to fibrillar collagen I (CI) in vitro, the major matrix component of bone. These included a loss of normal CI receptor (alpha2beta1 integrin)-mediated inhibition of proplatelet formation, a marked abrogation of SDF-1-induced chemotactic migration of CD41+ MKs adherent to CI, and an almost complete lack of actin-rich podosomes, normally induced by interaction between CI and its receptors GPVI or alpha2beta1 integrin. These findings highlight the central and highly specialized role of WASp in MKs during platelet biogenesis, and may provide a mechanism for the mild thrombocytopenia observed in WASp-deficient mice. In addition, they suggest a novel explanation for some of the platelet abnormalities characteristic of patients with WAS.

Background and Aim: As platelets are able to endocytose human immunodeficiency virus (HIV), we have investigated the fate of lentiviruses when endocytosed by human platelets and megakaryocytes (MK), and have characterized a specific receptor directly involved in this function. Methods: Genetically modified (non-replicative) lentiviruses with an HIV envelope (HIV-e) or with a vesicular stomatitis virus protein G envelope (VSV-e) were alternatively used and their interaction with platelets and MK analyzed by electron microscopy (EM) and immunoEM. Results: When incubated with platelets, HIV-e and VSV-e lentiviruses were internalized in specific endocytic vesicles and trafficked to the surface connected canalicular system (SCCS). Double immunolabeling for the viral P24 core protein and alpha-granule markers showed that lentiviruses were degraded in the SCCS after contact with alpha-granule proteins. In culture MK, lentiviruses were found in endocytic vesicles and accumulated in acid phosphatase-containing multivesicular bodies (MVB). The expression of the pathogen receptor dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN) was then demonstrated in platelets by flow cytometry, immunoEM and Western blot. Anti-DC-SIGN antibodies decreased HIV-e lentivirus internalization by platelets, showing that the receptor is functional. Specific signals for DC-SIGN protein and mRNA were also found in MK. Conclusion: This study indicates that platelets and MK can internalize lentiviruses in a pathway, which either provide a shelter to lentiviral particles or alternatively disrupts viral integrity. The receptor DC-SIGN is involved in this function.

We report the case of a 60-year-old woman who was newly diagnosed for the gray platelet syndrome (GPS). This patient had long-term thrombocytopenia which had been initially misdiagnosed as idiopathic thrombocytopenic purpura (ITP). Blood smear displayed characteristic gray platelets, allowing the diagnosis to be made, which was confirmed by electron microscopy (EM). Polymorphonuclear neutrophils (PMN) appeared poorly granulated on the May-Grunwald-Giemsa-stained blood smear. Flow cytometry analysis of PMN demonstrated increased expression of CD35, CD11b and CD18 at resting PMN surface, without any changes after fMLP stimulation. Ultrastructural study retrieved a decreased number of myeloperoxidase (MPO)-negative secondary granules in PMN. Immunolabeling confirmed the presence of membrane proteins and the absence of soluble content in platelet and megakaryocyte (MK) alpha-granules, and the decrease of secondary granules and secretory vesicles in PMN. This new observation demonstrates that the impairment of the secretory compartment of PMN is definitely a hallmark of GPS, and that the detection of these subtle abnormalities should be searched with adequate and up-to-date technical approaches.

We report the unusual transformation of a case of Waldenström's macroglobulinemia (WM) into IgM multiple myeloma (MM). The initial clinical and biological presentation of the disease was typical smouldering WM, with lymphocytic infiltration of the bone marrow. Five years later, signs of transformation appeared: the patient presented with diffuse osteolytic bone lesions without organomegaly, and the bone marrow was infiltrated with characteristic malignant plasma cells. Electron microscopy (EM) examination showed that the endoplasmic reticulum (ER) of the dysmorphic plasma cells contained monoclonal IgM. Immunolabeling for calreticulin, a resident protein of the ER, demonstrated unequivocally that the characteristic intranuclear inclusions were indeed part of ER. Flow cytometry revealed an MM profile for the cellular proliferation. Molecular biology performed on the final marrow could only retrieve a single cellular clone. In conclusion, this is the first documented description of the transformation of typical WM into an aggressive form of MM.

Background: Leukosialin (CD43) membrane expression decreases during neutrophil apoptosis but CD43 is neither proteolysed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Methods: Membrane blebbing is a transient event, hardly appreciated during asynchronous spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15°C allowed to observe numerous blebbing neutrophils for a short 1-hour period at 37°C. Blebs detaching from the cell body were observed by time-lapse fluorescence microscopy and the release of bleb-derived vesicles was followed by flow cytometry. Results: CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Phosphatidylserine externalization required caspases and PKC but not the myosin light chain kinase MLCK. By contrast, bleb formation and release was caspase and PKC-independent but required an active MLCK, while CD43 down-regulation involved caspases but neither PKC nor MLCK. CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultra centrifugation of apoptotic cells supernatant allowed to recover <1 micron microparticles, which contained the entire CD43 molecule.These microparticles expressed neutrophil membrane markers such as CD11b, CD66b and CD63, together with CD43. Conclusion: Blebbing, phosphatidylserine externalization and CD43 down-regulation result from different signaling pathways and can occur independently from one another. CD43 downregulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degreesC made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degreesC. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 mum microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Background: Leukosialin (CD43) membrane expression decreases during neutrophil apoptosis but CD43 is neither proteolysed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Methods: Membrane blebbing is a transient event, hardly appreciated during asynchronous spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15°C allowed to observe numerous blebbing neutrophils for a short 1-hour period at 37°C. Blebs detaching from the cell body were observed by time-lapse fluorescence microscopy and the release of bleb-derived vesicles was followed by flow cytometry. Results: CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Phosphatidylserine externalization required caspases and PKC but not the myosin light chain kinase MLCK. By contrast, bleb formation and release was caspase and PKC-independent but required an active MLCK, while CD43 down-regulation involved caspases but neither PKC nor MLCK. CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultra centrifugation of apoptotic cells supernatant allowed to recover <1 micron microparticles, which contained the entire CD43 molecule.These microparticles expressed neutrophil membrane markers such as CD11b, CD66b and CD63, together with CD43. Conclusion: Blebbing, phosphatidylserine externalization and CD43 down-regulation result from different signaling pathways and can occur independently from one another. CD43 downregulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Hematopoietic zinc finger (HZF) null mice have features reminiscent of patients with gray platelet syndrome (GPS), a rare inherited bleeding disorder. This similarity has suggested that HZF deregulation might be involved in the human disease. The sequence of the eight exons of the HZF gene as well as the study of its expression in blood samples from five patients belonging to three different families did not reveal any modifications when compared with healthy donors. This study indicates that HZF is unlikely to be responsible for GPS.

Platelets exert their main function in hemostasis and thrombosis, but they also are able to internalise infectious particles such as bacteria and viruses [1, 2] as well as inert particles like latex beads [3, 4]. This was initially interpreted as a passive passage of the particles through the platelet surface connected canalicular system (SCCS), or as a spreading of platelets over the particle [5]. Indeed the internalized particle often remains connected to the extracellular medium. A work recently performed in our laboratory using S. aurei and HIV [1], confirmed anterior studies [2] showing that platelets were able to interact directly with these micro-organisms by engulfing them. Contrary to the opinion expressed by White in his recent publication in this journal [5], we thought that this characteristic could confer to platelets a role in the protection and defense of the organism against any invasion by micro-organisms. Alternatively, by including infectious agents, platelets could contribute to the transport and the dissemination of infection in the organism. In favour to the first hypothesis, platelets contain microbicidal substances: a bactericidal peptide (PMP) has been found in the rabbit platelet granules [6] and components similar to PMP called thrombocidin were shown in the -granules of human platelets. Moreover, -granules are the storage place of many chemokines which are -thromboglobulin, platelet factor 4, neutrophil activating peptide-2, macrophage inflammatory protein-1 , PDGF, TGF [7]. They allow the interaction of platelets with other cells of the immune system in order to accelerate the immunizing responses. Platelets are a significant source of the chemokines RANTES (regulate upon activation normal T-cell expressed and secreted) [7, 8] which block viral infection by their capacity to enter in competition with viral particles, by fixation on their receptors [9]. Engulfment of foreign particles by platelets has been compared to phagocytosis. The phenomenom of phagocytosis involves the ingestion of particles, whereas pinocytosis involves the internalization of micelles and soluble molecules. There are several steps of phagocytosis, among which are adherence, pseudopodium extension, phagosome and phagolysosome formation. In that sense, platelet endocytosis is related to phagocytosis. However, the comparison between the ingestion phenomenon of platelets and phagocytes stops here, since the fate of the endocytosed microorganisms remains to be determined. Indeed phagocytes are able to ingest, digest, and kill microorganisms. Platelets are able to ingest foreign particles, and this property has been clearly demonstrated. To do so, they can extend pseudopodia filled with microfilaments and devoid of granules and mitochondria, like phagocytes do. Figure 1A shows that endocytosis within platelets is not a passive entrance of the foreign particle into the opening of the SCCS, but corresponds to an active platelet phenomenon. The two last steps, digestion and killing, are under investigation, and we are currently working on this topic. Partial digestion is suggested by contact with toxic platelet secretion products: Lewis and Maldonado [10] demonstrated earlier that platelet lysosomes were released along the engulfed particles, and we have shown that -granules could follow the same route (Figure 1B,C): indeed, immunolabelling for the -granule protein, fibrinogen, definitely shows that they are able to fuse with the compartment where bacteria are trapped. Moreover, it was shown that the phagocytic vacuole of phagocytes has specific properties, and that these properties are transmitted to the limiting membrane of secretory organelles which align and make contact with each other. Thus, neutrophil granules form necklaces whose content seems to flow towards the phagosome. In a similar way, we could observe

Factor V is an essential coagulation cofactor that circulates in plasma and platelet alpha-granules where it is stored complexed to multimerin I (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN I complexes, we studied factorV in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factor V, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte alpha-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into alpha-granules and generate factorV-MMRN I complexes from endogenously and exogenously synthesized proteins.

SummaryMultimerin 1 (MMRN1) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family.In vivo, MMRN1 is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRN1 binding to activated platelets and endothelial cells, we investigated the identity of the major MMRN1 receptors on these cells using wild-type and RGE-forms of recombinant MMRN1. Ligand capture, cell adhesion, ELISA and flow cytometry analyses of platelet-MMRN1 binding, indicated that MMRN1 binds to integrins αIIbβ3 and αvβ3. Endothelial cell binding to MMRN1 was predominantly mediated by αvβ3 and did not require the MMRN1 RGD site or cellular activation. Like many other αvβ3 ligands, MMRN1 had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRN1 processing, indicated that MMRN1 adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRN1 attachment to cells and indicate that MMRN1 is a ligand for αIIbβ3 and αvβ3.

Background: Cellular prion protein (Pi-PC) is a naturally occurring protein in normal individuals which adopts an abnormal conformation, termed scrapie prion protein (PrPSc) that is associated with disease. There is great concern that clinically asymptomatic variant Creutzfeldt-Jacob disease (vCJD) may transmit PrPSc in blood transfusion products. PI-PC is widely expressed and has been found in human blood. The majority of cellular borne PrPC is associated with platelets (84%). Although PrPC rnRNA has been demonstrated in platelets, the quantity is unknown and may not reflect the total PrPC present. Objective: To investigate the expression of PrPC in the megakaryocyte lineage. Methods: The expression of PrPC was studied in CD34(+) cells, cultured nnegakaryocytes and platelets using electron microscopy; flow cytometry, semiquantitative RT-PCR and immunofluorescence confocal microscopy. Results and conclusions: The expression of PrPC appeared to increase with differentiation and polyploidization in the megakaryocyte lineage. PrPC was located within platelet a-granules and its source is likely to be from megakaryocyte precursors. If PrPSc has a similar distribution, these results have implications for the selection of blood donors and preparation of cell-depleted blood products.

Multimerin I (MMRNI) is a large, soluble, polymeric, factor V binding protein and member of the EMILIN protein family. In vivo, MMRNI is found in platelets, megakaryocytes, endothelium and extracellular matrix fibers, but not in plasma. To address the mechanism of MMRNI binding to activated platelets and endothelial cells,we investigated the identity of the major MMRNI receptors on these cells using wild-type and RGE-forms of recombinant MMRNI. Ligand capture,cell adhesion, ELISA and flow cytometry analyses of platelet-MMRNI binding, indicated that MMRNI binds to integrins alpha llb beta 3 and alpha v beta 3. Endothelial cell binding to MMRNI was predominantly mediated by alpha v beta 3 and did not require the MMRNI RGD site or cellular activation. Like many other alpha c beta 3 ligands, MMRNI had the ability to support adhesion of additional cell types, including stimulated neutrophils. Expression studies, using a cell line capable of endothelial-like MMRNI processing, indicated that MMRNI adhesion to cellular receptors enhanced its extracellular matrix fiber assembly. These studies implicate integrin-mediated binding in MMRNI attachment to cells and indicate that MMRNI is a ligand for alpha llb beta 3 and alpha v beta 3.