Elisabeth M. Cramer

Induction of specific storage organelles by von Willebrand factor propolypeptide.

Endothelial cells store the multimeric adhesive glycoprotein von Willebrand factor (vWf), which promotes the formation of a platelet plug at the site of vessel injury. To investigate the packaging of vWf into the granules called Weibel-Palade bodies, we expressed pro-vWf cDNA and cDNA lacking the prosequence in a variety of cell lines. Storage granules formed only in cells that contain a regulated pathway of secretion. Furthermore, packaging required the prosequence. Pro-vWf, lacking the C-terminal region involved in interchain disulfide bonding, formed granules. We conclude that the signal for storage is universal in that an adhesive glycoprotein can be stored by a hormone-secreting cell; the storage of vWf is independent of its covalent multimeric structure; the unusual rod shape of Weibel-Palade bodies is due to vWf; and the vWf propolypeptide is necessary for the formation of vWf storage granules.

Paris-Trousseau syndrome : clinical, hematological, molecular data of ten new cases.

Paris-Trousseau syndrome (PTS) is an inherited disorder characterized by mild hemorragic tendency associated with 11q chromosome deletion. Here we report ten new patients (5 boys, 5 girls) with complete clinical history, biological data, ultra-structural and molecular investigations. Thrombocytopenia is chronic in all the patients except two boys in whom it disappeared during the two first years of life. On Romanovsky stained peripheral blood smears, abnormal platelets with giant granules were detected in all the children and confirmed by electron microscopy (EM). On bone marrow smears, dysmegakaryopoiesis with many micromegakaryocytes was constantly observed. Abnormal alpha-granules were virtually absent from bone marrow and cultured megakaryocytes, while EM detected numerous images of granule fusion within blood platelets. Molecular analyses evidenced that the fli-1 gene is deleted in all the patients except one confirming the crucial role of the transcription factor FLI-1 in megakaryopoiesis. In summary, this study documents ten new cases of PTS with characteristic alpha-granule abnormalities, and shows the putative pathogenic role of fli-1 gene in the pathophysiology of this syndrome.

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

OBJECTIVE Mice 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. METHODS The 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. RESULTS Human 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. CONCLUSION Human 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.

Platelet interaction with bacteria

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

Severe congenital dyserythropoietic anaemia type I: prenatal management, transfusion support and alpha‐interferon therapy

We report a case of congenital dyserythropoietic anaemia, type I, with severe pre‐ and postnatal manifestations. Exchange transfusions were required for fetal anaemia (3·5 g/dl) at 28 and 30 weeks of gestation. Transfusions were administered at birth (Caesarean section at week 35) and at regular intervals thereafter. At 14 months, α‐interferon therapy was initiated (106 units three times a week). This resulted in stabilization of the haemoglobin at or above 11 g/dl and a reduction in the percentage of erythroblasts with ultrastructurally abnormal heterochromatin. After 9 months, the dose of α‐interferon was decreased to 106 units twice a week. No relapse of anaemia was noted during an additional 4 months of follow‐up.

THE ORIGIN AND PHYSIOLOGICAL RELEVANCE OF α‐GRANULE ADHESIVE PROTEINS

Platelet a-granules are the principal intracellular reservoirs of proteins destined for release during primary haemostasis at the site of vessel wall injury. Despite the identification of many adhesive proteins within the a-granules, the biological significance and origin of these moieties have been subject to much speculation, particularly since high concentrations are also present in the plasma and within extracellular matrices.

7 Megakaryocytes and platelets in α-granule disorders

This chapter summarizes research data contributing to current understanding of disorders affecting α-granules of megakaryocytes and platelets. Diagnostic features of the gray platelet syndrome are well defined. Combined evidence suggests a defect, specific to the megakaryocyte cell lineage, causing a cytoskeletal abnormality and defective targeting of endogenously synthesized proteins to the α-granule. The abnormalities linked by signal transduction pathways. von Willebrand disease and afibrinogenaemia are disorders which highlight the functional importance of platelet storage pools of von Willebrand factor and fibrinogen, essential ligands in the process of adhesion and aggregation. The abnormality in the factor V Quebec disorder leads to a degradation of most proteins contained within the α-granule. The familial platelet disorder Paris-Trousseau thrombocytopenia is the only α-granule disorder associated with a cytogenetic abnormality, and it presents a useful model for exploring the genetic influence on regulation of thrombopoiesis. Study of these syndromes has elucidated aspects of the physiology of normal megakaryocyte maturation and platelet formation, including storage organelle biosynthesis.

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

Summary.  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 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: 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.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets

We recently identified a multi‐SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti‐Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti‐SERCA 2b, anti‐SERCA 3, anti‐SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α‐granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes.  Thus, the multi‐SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub‐compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays

Summary— Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three‐dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.