Pathophysiologic significance of procoagulant microvesicles in cancer disease and progression.

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.

[Markers of thrombotic disease: procoagulant microparticles]. / Marqueurs des pathologies thrombotiques. Les microparticules procoagulantes.

Thrombosis remains one of the leading causes of mortality and morbidity in developed countries. Relevant markers of the primary thrombotic risk however remain of limited accessibility, and clinicians are left with markers of essentially etiological nature. Fortunately, new entities, testifying to cellular activation or damage within the vascular compartment, have been recently described and are in the validation process. Microparticles (MP) are plasma membrane fragments released by stimulated or apoptotic cells. In the vascular compartment, they constitute a disseminated storage pool of bioactive effectors involved in inflammation, thrombosis, vascular tone, angiogenesis. Their biological characteristics are predetermined by the cytosolic and membraneous components hijacked from the activated cells. Their procoagulant properties are based on, (i) the accessibility of phosphatidylserine, a procoagulant aminophospholipid exposed after stimulation and necessary for the assembly of the blood clotting enzyme complexes, and (ii) the possible presence of tissue factor, the major initiator of the coagulation cascade. The incidence of MP in haemostatic processes has been demonstrated in physiology and pathology. They are now considered true pathogenic markers of the thrombotic risk.

Origin and biological significance of shed-membrane microparticles.

Microparticles (MPs) are small vesicles released from the membrane surface during eukaryotic cell activation or apoptosis. They originate from various cell types, displaying the typical surface cell proteins and cytoplasmic components of their cell origin. Their procoagulant properties are linked to phosphatidylserine exposed at their surface. Numerous reports have shown that MPs are able to mediate long-range signaling, acting on different targets from those of their own cellular origin. MPs-mediated binding to other cells occurs by integration into the membrane, by adhesion to the cell surface or by ligand-receptor interaction. Elevated levels of circulating MPs have been detected in cardiovascular and immune-mediated diseases. Despite extensive studies of the procoagulant and pro-inflammatory properties of MPs, little is known about their effect on vascular function. MPs accumulate in atherosclerotic plaques and injured vascular wall. Circulating MPs from patients with myocardial infarction induce endothelial dysfunction by impairing the endothelial nitric oxide (NO) pathway, without causing changes in endothelial NO-synthase (eNOS) expression. However, MPs from T-cells may induce endothelial dysfunction, altering gene expression of eNOS and caveolin-1. Moreover, MPs may promote the expression of pro-inflammatory proteins implicated in vascular contractility alterations. This review describes the origin of MPs and their biological role in physiological conditions and in various pathological states, with special reference to the possible linkage between their pro-inflammatory and procoagulant properties and vascular dysfunction.

[Microparticles during sepsis and trauma. A link between inflammation and thrombotic processes]. / Microparticules circulantes au cours des traumatismes graves et des sepsis: un élément du couplage inflammation-thrombose.

Sepsis and trauma lead to a sustained activation of monocytes and endothelium. In the vascular compartment, stimulated cells release microparticles. Circulating MP provide an additional procoagulant phospholipid surface enabling the assembly of the clotting enzymes complexes and thrombin generation. Their procoagulant properties rely on the exposition of phosphatidylserine, made accessible after cell stimulation and on the possible presence of tissue factor, the main cellular initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor activity. At sites of endothelium injury, enhanced release or recruitment of procoagulant MP through P-selectin-PSGL-1 pathway could concentrate TF activity above a threshold allowing blood coagulation to be triggered. Converging evidences from experimental or clinical data highlight a role for MP harboring tissue factor in the initiation of disseminated intravascular coagulopathy. In these settings, the pharmacological modulation of MP levels or biological functions through activated protein C or factor VIIa allows challenging issues.

Cyclic GMP modulates store-operated calcium entry inducing phosphatidylserine translocation at the surface of megakaryocytic cells.

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.

Elevated levels of procoagulant microparticles in a patient with myocardial infarction, antiphospholipid antibodies and multifocal cardiac thrombosis.

Circulating procoagulant microparticles (MP) are pathogenic markers of enhanced coagulability associated to a variety of disorders and released from stimulated vascular cells. When derived from endothelial cells, MP were found characteristic of thrombotic propensity in primary antiphospholipid syndrome (APS). The prothrombotic status of a patient with antiphospholipid antibodies (APL), a past history of mesenteric vein thrombosis and presenting myocardial infarction and extensive intracardiac thrombosis was examined by measurement of circulating procoagulant MP. MP of platelet and endothelial origins were highly elevated with respect to values detectable in patients with myocardial infarction and no history of APS (6- and 3-fold elevation, respectively) or in healthy volunteers (13- and 25-fold elevation, respectively). In this particular patient, with moderate APL titer, a drastic release of procoagulant MP could have contributed to thrombus growth and the development of extensive intracardiac thrombosis.

[The significance of circulating microparticles in physiology, inflammatory and thrombotic diseases]. / Les microparticules circulantes: rôles physiologiques et implications dans les maladies inflammatoires et thrombotiques.

BACKGROUND: In multicellular organisms, apoptosis and subsequent microparticle shedding play a key role in homeostasis. Having long been considered as << cellular dust >>, microparticles released in biological fluids upon cell activation or apoptosis appear as multifunctional bioeffectors involved in the modulation of key functions including immunity, inflammation, hemostasis and thrombosis, angiogenesis. MP constitute reliable markers of vascular damage, accessible to biological detection whilst the cells they originate from remain sequestered in tissues or are promptly submitted to phagocytosis. RECENT FINDINGS: MP modulate biological functions of target cells through the transfer of cytoplasmic content, lipids and membrane receptors. The pharmacological modulation of circulating levels of microparticles could be of particular interest in thrombotic or inflammatory diseases, cancer or hemophilia. CONCLUSION: MP can now be viewed not only as a hallmark of cell damage but also as a true biological tool.

[Microparticles and cardiovascular disease]. / Microparticules et pathologies cardiovasculaires.

Microparticles are membrane fragments liberated by activated or apoptopic cells. Thought for a long time to be cellular debris with no specific biologic function, in the vascular compartment they are a circulating reservoir of cellular effectors involved in thrombosis, inflammation, vascular remodelling and angiogenesis. High concentrations of circulating procoagulating microparticles found in many cardiovascular diseases indicate the importance of platelet, endothelial and monocytic activation and could contribute to the persistence of atherothrombotic disease. Pharmacological modulation of circulating microparticle concentrations could become a major therapeutic target in the future.

Modulation of signal transduction through the cellular prion protein is linked to its incorporation in lipid rafts.

Because expressed at a significant level at the membrane of human T cells, we made the hypothesis that the cellular prion protein (PrPc) could behave as a receptor, and be responsible for signal transduction. PrPc 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 PrPc and was inhibited after raft disruption. PrPc localization was not restricted to rafts in resting cells but engagement was a prerequisite for signaling induction, with concomitant PrPc recruitment into rafts. These results suggest a role for PrPc in signaling pathways, and show that lateral redistribution of the protein into rafts is important for subsequent signal transduction.

Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb-IIIa antagonists.

Circulating procoagulant microparticles (MP) were measured as markers of vascular damage and prothrombotic risk in patients undergoing ST-segment myocardial infarction (STEMI) treated by primary percutaneous transluminal coronary angioplasty (PTCA) and additional GPIIb-IIIa antagonists. Cells possibly more responsive to GPIIb-IIIa (alpha(IIb)beta(3)) antagonists were evidenced through MP phenotypes by comparison with healthy volunteers (HV) and STEMI patients treated by PTCA without GPIIb-IIIa antagonist (CP). In 50 STEMI patients, blood samples were collected at day 1 and day 6. Circulating procoagulant MP were captured on annexin V and quantified by prothrombinase assay as nanomolar phosphatidylserine equivalents (nm PhtdSer). Platelet activation by thrombin was confirmed through independent measurement of soluble GPV (sGPV). With respect to HV, procoagulant MP levels were high in patients with STEMI or unstable angina, platelet-derived MP and elevated sGPV testifying to significant platelet activation. A substantial release of endothelial-derived MP was evidenced simultaneously. In abciximab-treated patients, procoagulant MP, mainly of platelet origin, decreased precociously at day 1 (4.2 +/- 0.6 vs. CP 15.5 +/- 2.1 nm PhtdSer; P = 0.001) together with sGPV (36 +/- 3 vs. CP 58 +/- 8 ng mL(-1); P = 0.02). Leukocyte-derived MP decreased at day 6 (0.12 +/- 0.04 vs. CP 0.56 +/- 0.12 nm PhtdSer; P = 0.01) suggesting a possible effect on underlying inflammatory status. In patients presenting cardiovascular events at 6-month follow-up, procoagulant MP levels at day 1 could be indicative of a worsened outcome. MP could constitute a relevant parameter for the follow-up of STEMI patients treated by GPIIb-IIIa antagonists.

Rho A participates in the regulation of phosphatidylserine-dependent procoagulant activity at the surface of megakaryocytic cells.

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.

[Procoagulant membranous microparticles and atherothrombotic complications in diabetics]. / Microparticules membranaires procoagulantes et complications athérothrombotiques chez le patient diabétique.

Endothelial apoptosis and platelet activation play a key role in atherothrombotic event. These two mechanisms resulting membrane thickening leading to procoagulant microparticle (MP) liberation into the blood stream. In the vascular compartment, MP contribute to increased thrombin formation, to platelet activation, and prolong inflammation of the arterial wall by inducing the synthesis of cytokines and adhesion of glycoproteins by the endothelial cells. In diabetic patients, increased endothelial apoptosis associated with intense platelet and monocytic activation could contribute to accelerated atherothrombosis. Endothelial, platelet and monocytic derived MP, found in high concentrations in these patients, induce a prothrombotic, proadhesive and proinflammatory tendency in the vascular comportment which could directly impact on the vascular prognosis. In diabetes, increased platelet or monocytic MP is a marker for microvascular disease. Likewise, in acute coronary syndromes of diabetic patients, high concentrations of procoagulant MP could be associated with a poor cardiovascular prognosis. In these diabetic patients, many treatments (antioxidant, antiplatelet, lipid lowering, antihypertensive) significantly reduce the levels of MP and parameters associated with inflammation. MP are one of the key factors linking inflammation, oxidative stress, apoptosis and thrombosis in accelerated atherothrombotic disease of the diabetic. In future, the measurement of MP should help evaluate the efficacy of antioxidant and antiplatelet therapy, especially in diabetic patients.

Protective effects of vitamin C on endothelium damage and platelet activation during myocardial infarction in patients with sustained generation of circulating microparticles.

During myocardial infarction (MI), high levels of circulating procoagulant microparticles (MP) shed from endothelial cells and platelets diffuse prothrombotic and proinflammatory potentials crucial for the coronary prognosis. In addition to conventional treatments, we evaluated whether vitamin C treatment could modify circulating levels of procoagulant MP. Upon admission, 61 patients with MI were prospectively randomized for immediate additional vitamin C treatment. Circulating MP were quantified by functional prothrombinase assay before and after 5 days of vitamin C administration (1 g day-1). The cellular origin of MP was also assessed. In vitamin C-treated patients, the reduction in platelet-derived MP was 10% higher (P = 0.01). In patients with diabetes mellitus, dyslipidemia or more than two cardiovascular risk factors, vitamin C decreased endothelial and platelet-derived MP levels by approximately 70% and 13%, respectively. This early effect on circulating platelet and endothelial-derived MP, testifies to the importance of oxidative stress during MI. Vitamin C could prove beneficial for the outcome of patients at higher thrombotic risk.

Cellular microparticles: what are they bad or good for?

Microparticles are fragments released from the plasma membrane of most stimulated or apoptotic cells. After having long been considered inert cell debris, of possible value for the diagnosis of cell activation or death, there is increasing documented evidence that they can interact with neighboring or remote cells, in which case they acquire a pathophysiologic potential. On the one hand, deleterious microparticles stemming from activated cells can elicit an adverse response from other cells, themselves undergoing membrane vesiculation, leading to pathogenic amplification. On the other hand, since they are thought to reflect a balance between cell stimulation, proliferation, and death, it is conceivable that they are discerned as sensors for the maintenance of homeostasis in multicellular organisms. Because vesiculation is an integral part of the plasma-membrane remodeling process, with the transverse migration of procoagulant phosphatidylserine from the cytoplasmic to the exoplasmic leaflet as the central event, the majority of released microparticles are thought to fulfill a hemostatic function under physiologic conditions. This is particularly true when they originate from platelets, with possible deviation towards thrombosis when produced in excess. Owing to these procoagulant properties, the hemostasis laboratory offers the most appropriate tools for the assessment of the in vivo significance of microparticles.

Deciphering the plasma membrane hallmarks of apoptotic cells: phosphatidylserine transverse redistribution and calcium entry.

BACKGROUND: During apoptosis, Ca2+-dependent events participate in the regulation of intracellular and morphological changes including phosphatidylserine exposure in the exoplasmic leaflet of the cell plasma membrane. The occurrence of phosphatidylserine at the surface of specialized cells, such as platelets, is also essential for the assembly of the enzyme complexes of the blood coagulation cascade, as demonstrated by hemorrhages in Scott syndrome, an extremely rare genetic deficiency of phosphatidylserine externalization, without other apparent pathophysiologic consequences. We have recently reported a reduced capacitative Ca2+ entry in Scott cells which may be part of the Scott phenotype. RESULTS: Taking advantage of these mutant lymphoblastoid B cells, we have studied the relationship between this mode of Ca2+ entry and phosphatidylserine redistribution during apoptosis. Ca2+ ionophore induced apoptosis in Scott but not in control cells. However, inhibition of store-operated Ca2+ channels led to caspase-independent DNA fragmentation and decrease of mitochondrial membrane potential in both control and Scott cells. Inhibition of cytochrome P450 also reduced capacitative Ca2+ entry and induced apoptosis at comparable extents in control and Scott cells. During the apoptotic process, both control and more markedly Scott cells externalized phosphatidylserine, but in the latter, this membrane feature was however dissociated from several other intracellular changes. CONCLUSIONS: The present results suggest that different mechanisms account for phosphatidylserine transmembrane migration in cells undergoing stimulation and programmed death. These observations testify to the plasticity of the plasma membrane remodeling process, allowing normal apoptosis even when less fundamental functions are defective.

Vitamin C inhibits endothelial cell apoptosis in congestive heart failure.

BACKGROUND: Proinflammatory cytokines like tumor necrosis factor-alpha and oxidative stress induce apoptotic cell death in endothelial cells (ECs). Systemic inflammation and increased oxidative stress in congestive heart failure (CHF) coincide with enhanced EC apoptosis and the development of endothelial dysfunction. Therefore, we investigated the effects of antioxidative vitamin C therapy on EC apoptosis in CHF patients. METHODS AND RESULTS: Vitamin C dose dependently suppressed the induction of EC apoptosis by tumor necrosis factor-alpha and angiotensin II in vitro as assessed by DNA fragmentation, DAPI nuclear staining, and MTT viability assay. The antiapoptotic effect of vitamin C was associated with reduced cytochrome C release from mitochondria and the inhibition of caspase-9 activity. To assess EC protection by vitamin C in CHF patients, we prospectively randomized CHF patients in a double-blind trial to vitamin C treatment versus placebo. Vitamin C administration to CHF patients markedly reduced plasma levels of circulating apoptotic microparticles to 32+/-8% of baseline levels, whereas placebo had no effect (87+/-14%, P<0.005). In addition, vitamin C administration suppressed the proapoptotic activity on EC of the serum of CHF patients (P<0.001). CONCLUSIONS: Administration of vitamin C to CHF patients suppresses EC apoptosis in vivo, which might contribute to the established functional benefit of vitamin C supplementation on endothelial function.