The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34+ cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl-/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [125I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.

BACKGROUND:Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood.OBJECTIVES/PATIENTS:To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls.METHODS:Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy.RESULTS AND CONCLUSIONS:We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34+ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Abstract S78454 (also called PCI-24781) is an orally bioavailable, hydroxamate-based pan-HDACi currently being tested in clinical trials in the US and EU. Like many other HDACi, this drug induces a reversible thrombocytopenia. This thrombocytopenia, which was associated with a decrease in either GATA1 transcriptional activity or the expression of proteins of the Rho GTPase family (RhoA, Cdc42 or Rac), remains poorly understood. Given that the S78454 plasma level is above 100nM in treated patients, we performed a dose-response analysis (from 10 to 100 nM) of the drug effects on CFU-MK growth and cell proliferation. CFU-MKs were generated by ex vivo culture of CD34-positive cells and their differentiation was dose-dependently decreased after either a 24-hour treatment or a continuous exposure to S78454. This effect was associated with a dose-dependent decrease in MK proliferation. When added at day 8 of the culture, at a time-point when MK have nearly finished their endomitotic process and are entering in the cytoplasmic maturation step, S78454 induced a dose-dependent decrease (ten- to- two fold at 100nM and 20nM, respectively) in proplatelet formation, even when secondarily removed from the culture medium. The decreased proliferation was associated with an increased apoptosis, as demonstrated by the presence of a sub-G1 peak after propidium iodide staining, Annexin V binding, and caspase-3 proteolysis. We did not observed any blockade in the TPO/MPL/JAK2 signaling since ERK, Stat3 and Stat5 phosphorylation remained unaffected. Interestingly, MK exposed to S78454 displayed an increase in γH2AX foci formation and a decrease in Rad51 expression, a major mediator of homologous recombination, indicating a defective repair of DNA double-strand breaks. Consequently, P53 became phosphorylated in MKs, and the expression of its target genes BAX and P21 was increased. Altogether, these results suggest that S78454-induced thrombocytopenia may be mediated by induction of apoptosis in MK due to DNA-double-strand breaks and p53 activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2022. doi:1538-7445.AM2012-2022

Abstract Abstract 4826 MYH9-related disorders (MYH9-RD) are characterized by an autosomal-dominant macrothrombocytopenia associated or not with glomerular impairment, hearing loss and/or cataracts. It is caused by mutations of the MYH9 gene (encoding for the nonmuscle myosin heavy chain IIA), present at the heterozygous state in patients. In knockout animal models, proplatelet formation (PPF) was found to be increased in certain conditions, but decreased in others (Chen et al, Blood 2007, Eckly et al, Blood 2007 and Thrombosis and Haemostasis 2010) and samples with heterozygosity did not show differences with the wild type. More recently, in a mouse model with a mutated MYH9 gene, mice with the mutation showed a phenotype similar to the one found in MYH9-RD, affecting multiple organs and, interestingly, mice heterozygous for the mutation showed an abnormal phenotype as well, although attenuated (Anderson et al, ASH Meeting 2010, Abstract 2527). However, opposite to the initial reports in mouse models with a knockout, proplatelet formation was found to be impaired in 4 patients (Pecci et al, Thrombosis and Haemostasis 2009). Therefore the question for the exact mechanism for thrombocytopenia still stands, so does the question whether the mutated protein causes disease through a dominant negative effect versus a functional haploinsufficiency. To address these questions, we performed studies on patient samples on one hand and seeked to knock down the gene expression in human megakaryocytes (MKs) on the other hand. To study PPF in these patients, we analyzed MK differentiation in CD34+ cells from 9 patients with mutations in exons encoding for the motor domain (2 patients) or for the coiled coil domain of myosin IIA (7 patients). Compared with cultured MKs derived from healthy donors, a 2 to 5 –fold decrease in PPF was observed in the patients (4.9 ± 1.2% for patients vs. 15.1 ± 1.8% for control) and proplatelet area was decreased in patients as well (threefold decrease, 6156 μm2 for patients versus 18064 μm2 for control). In confocal microscopy with immunofluorescence staining, we observed disorganization of the granules, abnormal spreading and stress fibers, meaning that actin-myosin network is unstable. We next used a shRNA strategy to knock down MYH9 expression during normal MK differentiation and compared shRNA-treated MKs (with 50% residual MYH9 expression) with those derived from patient CD34+ cells. Treatment with shRNA decreased in vitro PPF, as previously observed for cells from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as MKs from patients. Normal platelet production is dependent on the formation of branched long proplatelets. Surprisingly the defect in PPF formation seen in patients was rescued by blebbistatin, an inhibitor of class II myosin. In its presence the proplatelets increased by 2 to 5 fold, suggesting that the remaining myosin IIA might be hyperactivated and inhibiting PPF. Ultrastructural studies by electron microscopy performed on cells with blebbistatin treatment corrected abnormalities seen in the patients. Parallel to blebbistatin, ROCK (Y27632) or MLCK inhibitors (P18 or ML7) led to a threefold increase in proplatelet formation. We therefore compared by western blot the status of pMLC2 in patients and control MKs. In 3 out of 6 patients we found an increased pMLC2. In addition, we also found an increased pMLC2 in MKs treated with shRNA with 50% residual myosin expression. Altogether our results show that reducing the protein expression by half was sufficient to recreate features characteristic of the disease in megakaryocytes, while the increased PPF, relative to control, in response to rho/ROCK inhibitors and to myosin inhibitors, suggest that the defective PPF in patients may be related to an activated rho/ROCK pathway and/or increase of the contractile force. Disclosures: No relevant conflicts of interest to declare.

The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

BCR-ABL negative myeloproliferative neoplasms (MPNs; polycythemia vera, essential thrombocythemia, primary myelofibrosis) are malignant diseases arising from a multipotent hematopoietic progenitor, frequently altered by JAK2 V617F or other JAK/STAT activating mutations. The thrombopoietin receptor (TpoR, MPL) is one of the major dimeric cytokine receptors that use JAK2 in the myeloid lineage, and was found to be down-modulated in certain MPN patients. We searched for negative regulators of MPL expression. Here we report that miR-28 targets the 3' untranslated (3'UTR) region of MPL, inhibiting its translation, as well as other proteins potentially involved in megakaryocyte differentiation, such as E2F6. Expression of miR-28 in CD34-derived megakaryocytes inhibited terminal differentiation. miR-28 was found to be overexpressed in platelets of a fraction of MPN patients, while it was expressed at constant low levels in platelets from healthy subjects. Constitutive activation of STAT5 leading to autonomous growth of hematopoietic cell lines was associated with increased miR-28 expression. We discuss how down-modulating MPL and other targets of miR-28, and of related miR-708 and miR-151, could contribute to MPN pathogenicity.

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.

Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and alpha granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell-derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9.

Abstract Abstract 4596 Normal platelet production is dependent on the formation of branched long cytoplasmic extensions, called proplatelets (PPT). Mutations of the Myh9 gene (encoding for the nonmuscle myosin heavy chain IIA) result in autosomal dominant disorders, where patients develop various degrees of macrothrombocytopenia, with sometimes glomerular impairment, hearing loss and cataracts. There has been questioning as to whether the mechanism for the macrothrombocytopenia is haploinsufficiency, or a dominant negative effect of the mutated gene. We performed an in vitro study to investigate PPF from patient megakaryocytes (MK). By this approach, a decrease in PPF from patient CD34 derived MKs was observed in comparison to normal cultured MK. Surprisingly this defect of PPF observed in patients was rescued by blebbistatin, an inhibitor of class II myosin. Immunofluorescence studies performed showed that besides clusterization of GPIb in patient's platelets, no major repartition abnormalities were seen in cultured MKs derived from patient's CD34 for myosin, actin, tubulin, vWF, and Rac (except in one patient where actin and Rac formed aggregates to some extend, in a small number of MKs). In order to better understand the role of myosin during normal and abnormal PPF, we used a shRNA strategy to disrupt the Myh9 expression during normal MK differentiation and compared shRNA-treated MKs with MKs derived from patient CD34. Megakaryocytes treated with a shRNA that knocks down the protein of about 50%, did not alter MK ploidization, but decreased in vitro PPF, as previously observed for cells issued from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as patient MKs. Addition of Blebbistatin to shRNA treated MKs led to an increase of PPF, suggesting that the remaining myosin II might be hyperactivated and inhibit PPF. Altogether this study strongly suggests that the thrombocytopenia of the Myh9 syndrome is essentially related to haploinsufficiency in myosin II. Disclosures: No relevant conflicts of interest to declare.

Thrombocytopenia is a frequent complication of many viral infections (hepatitis C virus, adenoviruses, lentiviruses, etc,), showing that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms arc, involved depending on the nature of the infecting viruses: immunological platelet destruction, inappropriate platelet activation and consumption, or impaired megakaryopoiesis. Viruses bind platelets through various links, specific receptors and identified ligands. A review of the specific interactions of platelets with other types of viruses and of the different receptors involved in these interactions is presented. We also report on a study, performed in our laboratory, of the reciprocal effects between platelets, megakaryocytes (MKs) and human immunodeficiency virus (HIV)-1, trying to understand the consequences Of Such interactions. HIV-1 (and replication-deficient HIV-1, insuring optimal biosecurity) were incubated in vitro with human platelets and MKs. HIV-1 internalization was found within two distinct endocytic. compartments and occurred preferentially in activated platelets: firstly, endocytic vesicles devoid of platelet secretion contained intact and potentially infectious viruses; secondly, viruses with altered structure were enclosed in the platelet surface-connected canalicular system (SCCS) in contact with platelet secretary products. Similar dual compartments were identified in MKs. Images suggesting that virus internalization by platelets also occurs in vivo were occasionally found of platelets from patients with acquired immune deficiency syndrome (AIDS) and thrombocytopenia. Finally, the pathogen receptor DC-SIGN was identified on platelets and MKs and facilitates HIV-1 internalization within platelets. In conclusion, virus particles can be specifically internalized in platelets and MKs where they can be sheltered, or get into contact with secretory products that lead to their destruction, This occurs in activated platelets (able to interact with macrophages), potentially leading to platelet clearance from the circulation and to the transfer of their viral load into target organs, Moreover, and similarly to platelets, MKs are also potentially colonized by intact infectious viruses, and the relationship between this observation and the occurrence of thrombocytopenia needs to be further examined. Finally, the understanding of the close platelet interaction with viruses emphasizes the importance of pursuing research on pathogen inactivation in platelet concentrates. (C) 2008 S. Karger GmbH, Freiburg i.Br.

La fonction des plaquettes s'exerce principalement au cours de l'hemostase primaire. De nouvelles fonctions ont ensuite ete mises en evidence au cour; des processus inflammatoires. Dans ce travail de these nous nous sommes interesses a cet aspect moins connu des plaquettes en particulier leur interaction avec les particules infectieuses ainsi que leur capacite de migration. Nous avons pu montre que les plaquettes sont capables d'endocyter les bacteries par un phenomene actif et d'interagir specifiquement avec elles. Puis nous avons etudie l'interaction des plaquettes avec des virus VIH et avons elabore une construction virale deletee des sequences codantes assurant une biosecurite optimale. Les resultats montrent que les VIH sont internalisees dans deux compartiments plaquettaires differents ou ils sont soit heberges et preserves dans leur integrite, soit degrades par les proteines secretaires plaquettaires. Dans les megacaryocytes deux compartiments d'internalisation ont ete identifies, l'un ou les lentivirus restent intacts, l'autre ou les lentivirus sont degrades. De plus nous avons mis en evidence l'expression du recepteur des pathogenes DC-SIGN dans les megacaryocytes et les plaquettes et demontres que ce recepteur medie Pendocytose des lentivirus dans les plaquettes. Par ailleurs, l'etude du chimiotactisme plaquettaire a montre que celles-ci migrent en reponse a la chimiokine SDF-1 via son recepteur CXCR4. Des anomalies de migration des megacaryocytes en reponse a SDF-1 ayant ete decrites dans le syndrome de Wiskott-Aldrich, nous avons etudie un modele murin deficient en proteine WASp et montre que la thrombopenie est associee a une liberation ectopique des plaque

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 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.

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