Jean-Marc Massé

Of mice and men: Comparison of the ultrastructure of megakaryocytes and platelets

OBJECTIVEnMice provide an excellent model for studying platelet and megakaryocyte (Mk) biology in vivo. Given the increasing use of transgenic and knockout mice, it is important that any similarities and differences between murine and human platelet/Mk biology be well defined. Therefore the objective of this study was to compare and contrast in detail any significant morphological differences between Mks, platelets, and mechanisms of thrombopoiesis in humans and mice.nnnMETHODSnThe distinctive structural and ultrastructural features of murine and human platelets and Mks are reviewed. Several platelet and Mk glycoproteins were also localized in murine cells by immunoelectron microscopy using polyclonal antibodies directed against human platelet proteins and compared to existing human data. Finally, the ultrastructure of maturing murine and human Mks in culture and bone marrow were examined in detail to facilitate a comparison of either in vivo or in vitro platelet production.nnnRESULTSnHuman and murine platelets exhibit significant but well-established morphological differences. Murine platelets are smaller and more numerous and display much greater granule heterogeneity than their human counterparts. Immunoelectron microscopy also demonstrated that murine platelet alpha-granules are highly compartmentalized. In fact, they are remarkably similar to human alpha-granules, with asymmetrical distribution of von Willebrand factor (vWF), and labeling of alpha(IIb)beta(3) and P-selectin (CD62P) in the granule limiting membrane. In vivo, murine but not human Mks are also consistently localized within the spleen. Subcellular events accompanying platelet formation and release by murine Mks are presented for the first time, and compared to human. Consistent differences were found in the pathway of redistribution of demarcation membranes preceding platelet formation, which may be important for the clarification of the mechanism of platelet release.nnnCONCLUSIONnHuman and murine platelets and Mks display several characteristic ultrastructural differences (size, number, histological distribution, platelet shedding) which have been emphasized and analyzed in this report. Nevertheless, since there are also many close similarities (organelle and glycoprotein subcellular distribution) mice offer an excellent in vivo model to study various aspects of human Mk and platelet biology.

Lentivirus degradation and DC-SIGN expression by human platelets and megakaryocytes.

Summary.u2002 Background and Aim:u2002As 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:u2002Genetically 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:u2002When 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 α‐granule markers showed that lentiviruses were degraded in the SCCS after contact with α‐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:u2002This 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.

Absence of incorporation of plasma von Willebrand factor into porcine platelet a‐granules

Summary. In order to study the relationship between plasma and platelet von Willebrand factor (vWF), we used an experimental model of crossed bone marrow transplantation (BMT) between SLA immunocompatible normal and homozygous von Willebrand (vWD) pigs. A normal pig received bone marrow from a vWD pig and a second pig with vWD was engrafted with marrow from a normal pig.

Polymorphonuclear Neutrophil and Megakaryocyte Mutual Involvement in Myelofibrosis Pathogenesis

The study presented here, performed on the bone marrow from patients with idiopathic myelofibrosis (MF) and on a murine model of MF, demonstrates a pathological interaction between PMN leukocytes and megakaryocyte (Mk), correlated with MF development. The data obtained revealed abnormal subcellular P-selectin distribution, which appeared to correlate with excessive and pathological emperipolesis of PMN leukocytes within Mk, leading to the destruction of Mk storage organelles and leakage of α -granular contents into the bone marrow microenvironment. The prominent role of growth factors, PDGF and TGF β, stored in the Mk α -granular compartment in the generation of MF has been previously largely documented. Both growth factors are essential for the Mk-dependent fibroblast proliferation. The destructive mutual cellular interaction of Mk and PMN leading to the pathological release of PDGF and TGF β within the bone marrow microenvironment may participate, through fibroblast activation, to the generation of MF. Therefore, this study provides insight into the possible pathophysiological mechanisms for the genesis of MF.

Analyses of cellular multimerin 1 receptors: in vitro evidence of binding mediated by αIibβ3 and αvβ3

Multimerin 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 αIibβ 3 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 aIibβ 3 and αvβ3.

The storage defects in grey platelet syndrome and αδ‐storage pool deficiency affect α‐granule factor V and multimerin storage without altering their proteolytic processing

Among proteins stored in α‐granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in α‐granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (αδ‐SPD) and grey platelet syndrome (GPS, α‐SPD), two conditions known to impair α‐granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in αδ‐SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin‐factor V complexes in plasma. Like normal platelets, GPS and αδ‐SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and αδ‐SPD platelets, indicating that these conditions preserve some aspects of normal α‐granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.

Studies of α-granule proteins in cultured human megakaryocytes

α-Granule protein storage is important for producing platelets with normal haemostatic function.The low to undetectable levels of several megakaryocyte-synthesized α-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. α-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34+ cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal α-granule protein processing and storage. ELISA indicated that cultured megakaryocytes con-Correspondence tained the a -granule proteins multimerin, von Willebrand factor, thrombospondin-1, β-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant pro-portion of the α-granule protein in megakaryocyte cultures was contained within the cells (averages: 41 – 71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that α-granule proteins were processed to mature forms and were predominantly located in the α-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying α-granule protein processing and storage.

Molecular study of the hematopoietic zinc finger gene in three unrelated families with gray platelet syndrome

Summary.u2002 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.

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

BACKGROUNDnHereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Lebers Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.nnnMETHODSnA specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.nnnRESULTSnTMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.nnnCONCLUSIONSnTMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.nnnGENERAL SIGNIFICANCEnLocal translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.

Neutrophil secretory defect in the gray platelet syndrome: A new case

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) α-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.