Multimerin 1.

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.

Unusual evolution of Waldenström's macroglobulinemia into osteolytic myeloma.

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.

Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment.

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.

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) 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.

Analyses of cellular multimerin 1 receptors: in vitro evidence of binding mediated by alphaIIbbeta3 and alphavbeta3.

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 alphaIIbbeta3 and alphavbeta3. Endothelial cell binding to MMRN1 was predominantly mediated by alphavbeta3 and did not require the MMRN1 RGD site or cellular activation. Like many other alphavbeta3 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 alphaIIbbeta3 and alphavbeta3.

Endocytosis and storage of plasma factor V by human megakaryocytes.

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.

Platelet shape change and subsequent glycoprotein redistribution in human stenosed arteries.

Shear stress encountered in stenosed human arteries is able to induce a certain range of platelet activation. In order to determine the extent of platelet shape change induced by high shear rate conditions, we used electron microscopy (EM) and immuno-EM to study platelet ultrastructure from blood flowing in vivo through stenosed arteries. Then it was compared with platelets from healthy controls exposed in vitro to a shear rate of 4000 s(-1). Six patients with stenosed arteries (iliac, femoral and renal) were investigated at the time of transcutaneous angiography. Blood was harvested from the same catheter in the stenosed artery and in the abdominal aortic artery (control sample), each patient being its own control. The percentage of platelets with shape changes (loss of discoid form, pseudopod emission, organelle centralisation) significantly increased in samples from stenosed arteries. Shape change was concomitant with the membrane glycoprotein IIb-IIIa distribution at the pseudopod extremities. These activated platelets had not completed secretion and were maintained in a reversible activation state. Similar results were obtained on platelets from healthy donors submitted in vitro to a high shear rate. In conclusion, this study shows that the high shear rate encountered in human stenosed arteries is able to induce shape change and reversible activation of platelets in vivo.

Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis.

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 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. 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 microM 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.

Studies of alpha-granule proteins in cultured human megakaryocytes.

alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-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 alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-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 alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage.

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.

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 alpha-granular contents into the bone marrow microenvironment. The prominent role of growth factors, PDGF and TGFbeta, stored in the Mk alpha-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 TGFbeta 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.

Tissue-type plasminogen activator (t-PA) is stored in Weibel-Palade bodies in human endothelial cells both in vitro and in vivo.

Vascular endothelial cells are thought to be the main source of plasma tissue-type plasminogen activator (t-PA) and von Willebrand factor (VWF). Previous studies have suggested that both t-PA and VWF are acutely released in response to the same stimuli, both in cultured endothelial cells and in vivo. However, the subcellular storage compartment in endothelial cells has not been definitively established. We tested the hypothesis that t-PA is localized in Weibel-Palade (WP) bodies, the specialized endothelial storage granules for VWF. In cultured human umbilical vein endothelial cells (HUVECs), t-PA was expressed in a minority of cells and found in WP bodies by immunofluorescence. After up-regulation of t-PA synthesis either by vascular endothelial growth factor (VEGF) and retinoic acid or by sodium butyrate, there was a large increase in t-PA-positive cells. t-PA was exclusively located to WP bodies, an observation confirmed by immunoelectron microscopy. Incubation with histamine, forskolin, and epinephrine induced the rapid, coordinate release of both t-PA and VWF, consistent with a single storage compartment. In native human skeletal muscle, t-PA was expressed in endothelial cells from arterioles and venules, along with VWF. The 2 proteins were found to be colocalized in WP bodies by immunoelectron microscopy. These data indicate that t-PA and VWF are colocalized in WP bodies, both in HUVECs and in vivo. Release of both t-PA and VWF from the same storage pool likely accounts for the coordinate increase in the plasma level of the 2 proteins in response to numerous stimuli, such as physical activity, beta-adrenergic agents, and 1-deamino-8d-arginine vasopressin (DDAVP) among others.

Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation.

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.

Pathogenic effects of anti-glycoprotein Ib antibodies on megakaryocytes and platelets.

Antibodies directed against the glycoprotein (GP) Ib have been identified as the potential cause of various platelet disorders: Immune thrombocytopenic purpura (ITP) may be caused by such autoantibodies; Anti-thrombotic drugs targeting GPIb also induce thrombocytopenia. In order to elucidate the potential mechanism(s) of the anti-GPIb effects, we have examined by electron microscopy (EM) the effect of several antibodies directed against GPIb and GPIIb-IIIa on human culture megakaryocytes (MK). Virtually all antibodies to GPIb enhanced the interaction of newly formed platelets with MK when compared to other antibodies. These effects were retrieved when antibodies were tested on platelets. We conclude that antibodies to GPIb can potentially inhibit platelet release by MK, and can also induce homotypic platelet adhesion. These results may have implications in the pathophysiology of thrombocytopenia and platelet recovery in ITP, and shed light on the pathological effect of anti-GPIb antibodies used as antithrombotic drugs.