Microvesicles (MV) are submicrometric membrane fragments (0.1 to 1 microm), released from the plasma membrane of activated or apoptotic cells. They are characterized by most of the antigenic profile of the cells they originate from, and by the presence of procoagulant phospholipids at their surface. MV are detectable in the peripheral blood of mammals and considered as efficient effectors in the haemostatic or thrombotic responses, able to remotely initiate or amplify beneficial or deleterious processes, depending on the circumstances. Variations in their level and phenotype make them relevant pathogenic markers of thrombotic disorders and vascular damage. To date, MV are recognized as mediators of communication allowing cells to influence a target present in the local microenvironment as well as to at distant sites. The mechanisms by which MV interact with target cells are still unclear, but a number of studies suggest involvement of MV-cell fusion or ligand-receptor interactions. More importantly, MV have been shown implicated in horizontal transfer of genetic material. This review focuses on the role of MV in the context of cancer, and their possible part in cancer associated thrombosis.

When subjected to stimulation, cells from the vascular compartment show a spontaneous collapse of the plasma membrane phospholipid asymmetry and phosphatidylserine is exposed at the external leaflet. Thus, phosphatidylserine externalization is essential for normal hemostasis and phagocytosis. The mechanism governing the migration of phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. Here, interaction of [Ca(2+)](i), cAMP and cGMP pathways and phosphatidylserine exposure was examined in human megakaryocytic cells. The membrane permeable cAMP and cGMP analogues, pCPT-cAMP and pCPT-cGMP, enhanced the Ca(2+) signal induced by ionophore and SOCE. Responses to pCPT-cAMP and pCPT-cGMP were independent of protein kinase A, protein kinase G (PKG) or ERK pathways. Inhibition of small G-proteins reduced or abolished the increase of [Ca(2+)](i) induced by pCPT-cAMP or pCPT-cGMP, respectively. pCPT-cGMP but not pCPT-cAMP enhanced the ability of cells to expose phosphatidylserine. This effect was not prevented by the inhibition of PKG or small G-proteins. These results show the differential role of cyclic nucleotides in the Ca(2+)-dependent membrane remodeling. Hence, pCPT-cGMP is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells through a mechanism implicating small G-proteins.

Microparticles are plasma membrane-derived vesicles shed from stimulated cells, in the broad sense of the term. Their presence is interpreted by proximal or remote cells in fundamental physiological processes including intercellular communication, hemostasis, and immunity. On the other hand, variations of their number or characteristics are frequently observed in pathophysiological situations.

Plasma membrane remodelling and cell stimulation For a longtime the plasma membrane has been considered as a simple barrier between the extracellular and intracellular milieu. Now, it is well accepted that it plays a pivotal role in many physiological processes allowing the communication of cells with their environment. On the one hand, the plasma membrane directly participates in intracellular signaling, on the other hand, changes in membrane structure contribute to the transcellular transfer of biological information. This review analyses the most recent features concerning the plasma membrane plasticity, with a special focus on the intracellular signaling pathways involved in the regulation of the loss of membrane phospholipid asymmetry during cell activation. The pathophysiologic consequences of microparticle/microvesicle shedding from membrane blebs are briefly exposed.

Once exposed at the external surface of activated platelets or apoptotic cells, phosphatidylserine, an anionic phospholipid mostly sequestered in the inner leaflet of the plasma membrane, plays essential roles in hemostasis and phagocytosis. The mechanism governing the migration of the phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. ERK pathway is among the elements modulating SOCE and phosphatidylserine externalization in megakaryocytic HEL cells. Here, we investigated the role of small GTPase Rho A, which may interact with the ERK pathway. Specific inhibitors of Rho A (exoenzyme C3 and toxin B) reduced both SOCE and phosphatidylserine-dependent procoagulant activity. Simultaneous inhibition of Rho A and extracellular signal-regulated kinase (ERK) pathways did not elicit further reduction with respect to each individual one. Rho A can regulate SOCE and phosphatidylserine exposure through the reorganization of actin cytoskeleton, but not through ROCK pathway. Hence, Rho A is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells.

. Because expressed at a significant level at the membrane of human T cells, we made the hypothesis that the cellular prion protein (PrP c ) could behave as a receptor, and be responsible for signal transduction. PrP c engagement by specific antibodies was observed to induce an increase in cytosolic calcium concentration and led to enhanced activity of Src protein tyrosine kinases. Antibodies to CD4 and CD59 did not influence calcium fluxes or signaling. The effect was maximal after the formation of a network involving avidin and biotinylated antibody to PrP c and was inhibited after raft disruption. PrP c localization was not restricted to rafts in resting cells but engagement was a prerequisite for signaling induction, with concomitant PrP c recruitment into rafts. These results suggest a role for PrP c in signaling pathways, and show that lateral redistribution of the protein into rafts is important for subsequent signal transduction.

Coagulation factor VIIa (FVIIa) is a key protease initiating the coagulation cascade in the presence of its receptor, tissue factor (TF). FVIIa elicits several cellular responses, probably involving other receptors(s) than TF. This study investigates the implication of recombinant FVIIa on the apoptosis of K562 erythroleukemia cells. These cells undergo apoptosis when induced to differentiate towards the erythroid lineage by hemin. They do not express TF, but can be transfected to do so. FVIIa treatment significantly reduced the degree of hemin-induced apoptosis in K562 cells, but not in TF+ derived transfectants. Induction of apoptosis by hemin also elicited decrease in intracellular Ca2+ concentration ([Ca2+]i), but FVIIa restored this [Ca2+]i close to that of non-treated cells. These results suggest that FVIIa acts via a TF-independent pathway to counteract apoptosis by a mechanism involving its Gla domain and linked to the maintenance of Ca2+ homeostasis in K562 cells.

IntroductionApoptosis, the term introduced 27 years ago to characterize a particular form of cell death distinct from necrosis,1 is now considered a genetically-controlled and energy-dependent process of fundamental significance in the development and maintenance of homeostasis in multicellular organisms.2-4 For instance, in the nematode Caenorhabditis elegans, a model widely used for the study of programmed cell death, 131 of the 1,090 somatic cells generated during hermaphrodite development undergo this form of death.5 Embryologists have suspected cell death of being instrumental in the “sculpture” of parts of the body well before the initial definition of apoptosis. In fact, cell proliferation can no longer be dissociated from apoptosis and it is obvious that variety of disorders involve either an excess of cell death for those referred to as disorders of cell loss, or a defect of apoptosis for those resulting in cell accumulation. Substantial information has been gained from studies of the hierarchical control of lymphocyte survival.6 Apoptosis is accompanied by characteristic changes in cell morphology, among which shrinkage, membrane blebbing, and nucleus condensation are the most frequently evoked (Fig. 1). Budding and disintegration by fragmentation in multiple bodies is the ultimate stage of this death process.7 Alterations are induced by external signals as different as physical (radiation, mechanical stress), chemical (oxidants, xenobiotics) or biological (granzymes, receptor-mediated signals, ceramide), and also by survival factor deprivation. Interestingly, some of these signals can result from subnecrotic damage. In the so-called induction phase, each agent exerts its proapoptotic action through a “private” pathway, leading to the common pathways composed of the effector and degradation phases. The effector phase consists of a mitochondrial checkpoint involving the Bcl-2/Bax anti/proapoptotic balance, immediately after which cytochrome c is released from the injured mitochondrion and binds to adaptor proteins to activate the caspase cascade. The degradation phase is achieved by reactive oxygen species (ROS) generated at the mitochondrial level, cytoplasmic changes (depletion of glutathione and variations of cytosolic calcium), and by caspases.Caspases, also referred to as interleukin-1-converting enzyme (ICE)-like proteases, are a family of cysteine proteinases showing specificity for Asp residue and having various cytoplasmic or nuclear substrates, such as cytoskeletal proteins or proteins involved in DNA repair or control of endonucleases. The latter mechanism explains why DNA ladders, multiples of the 180 bp nucleosomal unit, constitute one of the hallmarks of apoptotic cells.8 Plasma membrane remodeling, resulting in the occurrence of phosphatidylserine (PS) in the exoplasmic leaflet and the shedding of membrane microparticles, are other hallmarks worth considering.9-12 The caspase cascade can, alternatively, be directly activated by granzyme B, which penetrates into the cytoplasm through perforin channels, or after Fas (CD95) or tumor necrosis factor (TNF) receptor 1 (TNFR1) ligation. The generation of caspase-3 (CPP32) appears to be a pivotal step, since this enzyme mediates both the activation of CAD (caspase-activated deoxyribonuclease) and PS externalization.8,13 A number of determinants, including PS, are expressed in apoptotic cells and derived fragments for their noninflammatory engulfment by phagocytes, whereas tissue necrosis is accompanied by proinflammatory events.9,11,14,15 Despite extensive investigations, major gaps still exist in trying to connect and define the relative contribution of the different components of this basic process, but recently, apoptotic features have been described in unicellular, primitive eukaryotes, such as yeast,16,17 which could be used as model organisms to expand our knowledge. Owing to the presence of the effector machinery for programmed cell death in virtually all nucleated cell types, it is obvious that mechanisms have evolved in parallel for a tight regulation of apoptosis, as detailed in most of the references quoted in this section.In such an active context, the impact of apoptosis has not escaped the attention of cardiovascular biologists. Recent reviews emphasize the role of programmed cell death in cardiac development, heart failure and ischemic heart disease,18-21 and in vascular disease. Of these, a majority deal with atherosclerosis and concern endothelial or smooth muscle cells and leukocytes.22-25 To avoid redundancy, then, the purpose of the present state-of-the-art review is to focus on aspects related to plasma membrane modifications contributing to the acquisition of hemorrhagic or thrombogenic phenotypes or to the development of (auto)immune response, in vitro and in vivo, in the vascular compartment.