Shedding Of Microparticles Research Articles

Pathophysiology of Trousseau’s syndrome

SummaryClinically relevant clotting abnormalities in cancer patients are referred to as Trousseau’s syndrome. While thrombotic complications such as venous thromboembolism are most frequent in every day’s practice, cancer patients may also experience severe bleeding symptoms due to complex systemic coagulopathies, including disseminated intravascular coagulation, haemolytic thrombotic microangiopathy, and hyperfibrinolysis. The pathophysiology of Trousseau’s syndrome involves all aspects of Virchow’s triad, but previous basic research has mainly focused on the cellular and molecular mechanisms underlying blood hypercoagulability in solid cancers and haematological malignancies. In this regard, over-expression of tissue factor (TF), the principal initiator of the extrinsic coagulation pathway, by primary tumour cells and increased shedding of TF-bearing plasma microparticles are critical to both thrombus formation and cancer progression. However, novel findings on intrinsic contact activation in vivo, such as the release of polyphosphates or DNA by activated platelets and neutrophils, respectively, have pointed to additional pathways in the complex pathophysiology of Trousseau’s syndrome.

Diet, microparticles and atherothrombosis.

Cardiovascular disease is the main cause of death worldwide, and is principally caused by atherosclerosis, with subsequent thrombus formation, eventually provoking an acute myocardial infarction or a stroke. The formation and progression of the atherosclerotic plaques responds to multiple factors including certain diets. Intensive research has elucidated the role of diet in cardiovascular disease and has led to public health policies focusing on educating the population on the role of nutrition in cardiovascular health. Compelling evidence shows that a healthy diet, rich in fruit and vegetables with moderate consumption of fish and a low consumption of animal by-products and processed foods, decreases low-grade inflammation and oxidation, leukocyte activation, platelet aggregation and microparticle shedding. Thus, following such a diet decreases the incidence of cardiovascular disease, lowers mortality and delays the progression of atherothrombosis. Identifying novel risk factors for cardiovascular disease and understanding how food impacts on the disease helps in the development of novel preventive measures.

Procoagulant Activity of Blood and Endothelial Cells via Phosphatidylserine Exposure and Microparticle Delivery in Patients with Diabetic Retinopathy

Background/Aims: The mechanisms for thrombosis in diabetic retinopathy (DR) are complex and need to be further elucidated. The purpose of this study was to test phosphatidylserine (PS) exposure on microparticles (MPs) and MP-origin cells from the circulation and to analyze cell-/MP-associated procoagulant activity (PCA) in DR patients. Methods: PS-positive MPs and cells from healthy controls (n = 20) and diabetic patients (n = 60) were analyzed by flow cytometry and confocal microscopy. Clotting time and purified coagulation complex assays were used to measure PCA. Results: PS exposure on platelets and monocytes was higher in proliferative DR (PDR) patients than in non-PDR patients or controls. The highest levels of MPs (derived from platelets [30%], erythrocytes [13%], leukocytes [28%], and endothelial cells [10%]) were found in patients with PDR. In addition, PS exposure on blood cells and shed MPs in DR patients led to significantly increased FXa and FIIa generation, fibrin formation, and markedly shortened coagulation time. Moreover, lactadherin reduced 70% of PCA by blocking PS, while an anti-tissue factor antibody had a smaller effect. Conclusion: Our results confirmed that PCA in DR patients may be partly ascribed to PS exposure and MP release from blood and endothelial cells. Lactadherin may act as an efficient anticoagulant factor in this process.

Vitamin D Reduces Oxidative Stress-Induced Procaspase-3/ROCK1 Activation and MP Release by Placental Trophoblasts.

Increased microparticle (MP) shedding by placental trophoblasts contributes to maternal vascular inflammatory response and endothelial dysfunction in preeclampsia. Vitamin D has beneficial effects in pregnancy; however, its effect on trophoblast MP release has not been investigated. To investigate if vitamin D could protect trophoblasts from oxidative stress-induced MP release. Placental trophoblasts were isolated from uncomplicated and preeclamptic placentas. Effects of vitamin D on MP release induced by oxidative stress inducer CoCl2 were studied. Annexin V+ MPs were assessed by flow cytometry. Expression of caveolin-1, endothelial nitric oxide synthase (eNOS), procaspase-3, cleaved caspase-3, and Rho-associated coiled-coil protein kinase 1 (ROCK1) in trophoblasts and trophoblast-derived MPs were determined by Western blot. Trophoblasts from preeclamptic pregnancies released significantly more MPs than cells from uncomplicated pregnancies (P < 0.01). CoCl2-induced increase in MP release was associated with upregulation of caveolin-1 and downregulation of eNOS expression in trophoblasts (P < 0.05), which could be attenuated by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 could also inhibit CoCl2-induced procaspase-3 cleavage and ROCK1 activation in trophoblasts. Consistently, CoCl2-induced upregulation of procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs were also reduced in cells treated with 1,25(OH)2D3. Placental trophoblasts from preeclamptic pregnancies released more MP than cells from uncomplicated pregnancies. Oxidative stress-induced increase in MP shedding is associated with upregulation of caveolin-1 and downregulation of eNOS expression in placental trophoblasts. Inhibition of caspase-3 cleavage and ROCK1 activation, together with upregulation of eNOS expression, could be the potential cellular/molecular mechanism(s) of vitamin D protective effects on placental trophoblasts.

Circulating microparticle subpopulations in systemic lupus erythematosus are affected by disease activity

BackgroundImmune cells under chronic inflammation shed microparticles (MPs) that could fuel the inflammatory responses and atherosclerosis typically presented in systemic lupus erythematosus (SLE). This study analyzes total and subset-specific MPs from SLE patients and their possible influence on clinical features, leukocyte activation and serum cytokines. MethodsTotal MPs and those derived from platelets, endothelial cells, monocytes, granulocytes and T-cells were quantified in plasma of 106 SLE patients and 33 healthy controls by flow cytometry. MP amounts were analyzed according to clinical manifestations, blood leukocyte populations and circulating cytokines (IFNα, TNFα, IL-10, BLyS, IL-17, IL-1β, CXCL10, CCL-2, CCL3, leptin). Finally, the in vitro effect of SLE-isolated MPs on the leukocyte activation status was analyzed. ResultsTotal circulating MPs in SLE patients were related to increased disease duration and the presence of cardiovascular disease. Furthermore, patients displayed increased counts of MPs from platelets, monocytes and T-lymphocytes, especially in SLE patients with disease activity or with TNFαhigh-profile. Accordingly, MPs were associated with increased expression of activation markers in blood T-cells and monocytes. Finally, analyses propose a role of glucocorticoids in MPs generation and leukocyte activation since both fresh and cultured T-cells under this treatment presented higher IL-10 and CD25 production. ConclusionsThe altered profile of subset-specific SLE-MPs was influenced by the disease activity and altered status of leukocyte native cells, also associated with a TNFαhigh-profile. Translational resultsSLE patients, especially those with disease activity, displayed increased counts of MPs derived from platelets, monocytes and T-lymphocytes, which were more frequently found in TNFαhigh-type patients. The origin of such SLE-MP subsets seems to be related to the over-activated status of T-cells and monocytes characteristic of these patients. Ex vivo and in vitro analyses propose a role of glucocorticoids in the generation of circulating MPs and leukocyte activation in SLE patients.

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated β-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. Senescence, as assessed by senescence-associated β-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated β-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II-induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS.

Circulating Microparticles As Predictive Biomarkers of Acute Graft Versus Host Disease

Background Graft-versus-host disease (GvHD) remains a leading cause of morbidity and mortality among allogeneic stem cell transplantation (SCT) patients. New research focusing on diagnostic methods and therapeutic interventions is required. Microparticles (MP) are structures ranging from 0.1 to 1 µm that are derived from the cell membranes of different cellular subtypes. The majority of circulating microparticles are derived from megakaryocytes (platelets) and erythrocytes. These structures have been described as biomarkers of cellular damage, activation, and intercellular signaling in in several autoimmune and vascular diseases. Whether the biologic events that characterize GvHD can alter the shedding of microparticles has not been described.Methods Patients who underwent allogeneic stem cell transplantation between august 2012 and September 2014 at the Hospital Sirio Libanes (Sao Paulo, Brazil) were included. Adult and pediatric patients were consecutively enrolled if neutrophil engraftment was achieved. Circulating MP were isolated and quantified by flow citometry. Annexin V-positive (total microparticles - TMP), CD61+ platelet-derived microparticles (PMP) and CD235+ erythrocyte-derived microparticles (EMP) were measured at neutrophil engraftment.Results MP were studied in the plasma of 48 patients. The levels of PMP were not associated with the risk of acute GvHD. However, the cumulative incidence (CI) of aGVHD was significantly increased among the patients with EMP counts above 360/µL at engraftment (59% vs. 26%, P=0.04). Higher EMP counts were associated with increased incidence of skin aGVHD (53% vs. 17%,P=0.02) and liver aGVHD (33% vs. 0%, P=0.002). There was no significant association between higher EMP count and incidence of gastrointestinal (GI) aGVHD (42% vs. 20%, P=0.10). The microparticles counts at engraftment were influenced by the intensity of the conditioning regimen. When compared with reduced intensity regimens, myeloablative regimens were associated with significantly higher EMP counts (152 vs. 349/µL, P=0.03). In patients exposed to reduced intensity conditioning regimens, a stronger association of a high EMP count and acute GvHD was observed (89% vs. 23%; p=0.002). A high EMP count was predictive of skin aGVHD (78% vs. 14%, P=0.002), liver aGVHD (67% vs. 0%, P<0.001), and GI aGVHD (72% vs. 20%, P=0.006).Conclusion Our data suggest that higher counts of erythrocyte-derived microparticles at engraftment may be associated with a greater risk of acute GVHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RhoB/ROCK mediates oxygen-glucose deprivation-stimulated syncytiotrophoblast microparticle shedding in preeclampsia.

Increased circulating syncytiotrophoblast microparticles (STBMs) are often associated with preeclampsia (PE) but the molecular mechanisms regulating STBM shedding remain elusive. Experimental evidence has shown that actin plays a key role in STBM shedding and that Rho/ROCK is important in regulating actin rearrangement. To investigate the role of RhoB/ROCK-regulated actin arrangement in STBM shedding in PE, chorionic villous explants were prepared from placenta of patients with normotensive or PE pregnancies and BeWo cells were fused to imitate syncytiotrophoblasts. The oxygen-glucose deprivation (OGD) conditions were applied to imitate the pathophysiology of PE in vitro. The results showed that RhoB and ROCK were activated in the preeclamptic placenta, accompanied by increased actin polymerization and decreased outgrowing microvilli. In villous tissue cultures or BeWo cells, OGD activated RhoB, ROCK1 and ROCK2 and promoted STBM shedding and actin stress fibers formation. In BeWo cells, RhoB overexpression activated ROCK1 and ROCK2, leading to F-actin redistribution and STBM shedding and the OGD-induced actin polymerization and STBM shedding could be reversed by RhoB or ROCK knockdown. These results reveal that RhoB and ROCK play a key role in PE by targeting STBM shedding through actin rearrangement and that RhoB/ROCK intervention may be a potential therapeutic strategy for PE.

Microparticle Shedding by Erythrocytes, Monocytes and Vascular Smooth Muscular Cells Is Reduced by Aspirin in Diabetic Patients

Introduction and objectivesDiabetes mellitus is associated with an enhanced risk for cardiovascular disease and its prevalence is increasing. Diabetes induces metabolic stress on blood and vascular cells, promoting platelet activation and vascular dysfunction. The level of vascular cell activation can be measured by the number and phenotype of microparticles found in the circulation. The aim of this study was to investigate the effect of a platelet-inhibitory dose of aspirin on the number and type of microparticles shed to the circulation. MethodsForty-three diabetic patients were enrolled in the study and received a daily dose of 100mg of aspirin for 10 days to cover the average platelet life-span in the circulation. Before and after the intervention period, circulating microparticles were characterized and quantified by flow cytometry. ResultsType 1 diabetic patients had about twice the number of tissue factor-positive circulating microparticles (derived both from platelets and monocytes) and endothelial-derived E-selectin positive microparticles than type 2 diabetic patients. Aspirin therapy significantly inhibited platelets since cyclooxygenase 1 derived thromboxane generation levels were reduced by 99%. Microparticles derived from erythrocytes, activated monocytes, and smooth muscle cells were significantly reduced after 10 days of aspirin administration. ConclusionsThese results indicate that: a) vascular and blood cells in type 1 diabetic patients are exposed to more sustained stress shown by their specific microparticle origin and levels; b) aspirin therapy inhibits vascular wall cell activation and microparticle shedding, and c) the effects of aspirin are similar in type 1 and 2 diabetes.

El ácido acetilsalicílico reduce la liberación de micropartículas eritrocitarias, monocitarias y de células del músculo liso vascular en pacientes diabéticos

Introduction and objectivesDiabetes mellitus is associated with an enhanced risk for cardiovascular disease and its prevalence is increasing. Diabetes induces metabolic stress on blood and vascular cells, promoting platelet activation and vascular dysfunction. The level of vascular cell activation can be measured by the number and phenotype of microparticles found in the circulation. The aim of this study was to investigate the effect of a platelet-inhibitory dose of aspirin on the number and type of microparticles shed to the circulation. MethodsForty-three diabetic patients were enrolled in the study and received a daily dose of 100mg of aspirin for 10 days to cover the average platelet life-span in the circulation. Before and after the intervention period, circulating microparticles were characterized and quantified by flow cytometry. ResultsType 1 diabetic patients had about twice the number of tissue factor-positive circulating microparticles (derived both from platelets and monocytes) and endothelial-derived E-selectin positive microparticles than type 2 diabetic patients. Aspirin therapy significantly inhibited platelets since cyclooxygenase 1 derived thromboxane generation levels were reduced by 99%. Microparticles derived from erythrocytes, activated monocytes, and smooth muscle cells were significantly reduced after 10 days of aspirin administration. ConclusionsThese results indicate that: a) vascular and blood cells in type 1 diabetic patients are exposed to more sustained stress shown by their specific microparticle origin and levels; b) aspirin therapy inhibits vascular wall cell activation and microparticle shedding, and c) the effects of aspirin are similar in type 1 and 2 diabetes.Full English text available from: www.revespcardiol.org/en

Plasma Levels of Endothelial Microparticles Bearing Monomeric C-reactive Protein are Increased in Peripheral Artery Disease.

C-reactive protein (CRP) as an indicator of cardiovascular disease (CVD) has shown limited sensitivity. We demonstrate that two isoforms of CRP (pentameric, pCRP and monomeric, mCRP) present in soluble form or on microparticles (MPs) have different biological effects and are not all measured by clinical CRP assays. The high-sensitivity CRP assay (hsCRP) did not measure pCRP or mCRP on MPs, whereas flow cytometry did. MPs derived from endothelial cells, particularly those bearing mCRP, were elevated in peripheral artery disease (PAD) patients compared to controls. The numbers of mCRP(+) endothelial MPs did not correlate with hsCRP measurements of soluble pCRP, indicating their independent modulation. In controls, statins lowered mCRP(+) endothelial MPs. In a model of vascular inflammation, mCRP induced endothelial shedding of MPs and was proinflammatory, while pCRP was anti-inflammatory. mCRP on endothelial MPs may be both an unmeasured indicator of, and an amplifier of, vascular disease, and its detection might improve risk sensitivity.

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells.Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution.Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50–1000nm).Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

PurposeIschemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.MethodsForty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.ResultsCompared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.ConclusionsStroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.

New Insights in the Potential Effect of Transfusing Red Blood Cells that Have Been Stored for Different Periods

Red Blood Cells (RBCs) units which can be stored for up to 42 days in the US undergo biochemical and morphological changes known as the storage lesion. The clinical significance of these changes is unclear. Results from &gt;55 observational studies have produced conflicting results: some are negative while others report associations between transfusion of longer-stored RBCs and mortality, infections, lung injury, deep vein thrombosis, multiple organ failure, and a decrease in tissue oxygenation. Recent advances are shedding some light on this controversy. While some elements of the RBC storage lesion such as pH and cationic changes and decreases in adenosine triphosphate and 2,3-diphosphoglycerate are well known, the recent application of "omics" technologies is revealing complex changes in metabolites, proteins, and lipids during storage. RBCs storage causes dysregulations in several metabolic (e.g., glycolytic) pathways which vary with unit processing, additive solution, storage period, and blood donor characteristics. Longer-stored RBCs demonstrate decreased antioxidant activity and impaired energy metabolism. Kinases and proteolytic enzymes become activated which affect Band 3 and structural proteins and result in remodeling of the RBCs' cytoskeleton; leading to increasing osmotic fragility and shedding of microparticles in the supernatant. The timing and extent of these changes need to be further elucidated; some appear to occur immediately (e.g., reduction in S-nitrosohemoglobin) while most appear after 2 weeks. These changes lead one to question the safety and efficacy profiles of longer-stored RBC transfusions. Animal models have recently evaluated potential consequences and possible mechanisms that could underlie adverse events in "susceptible" hosts. Two major hypotheses have been corroborated by animal studies. The first relates to the potential inhibition of Nitric Oxide (NO)-mediated vasodilatory effects as a result of NO scavenging by excess cell-free hemoglobin or because of a loss of RBC-mediated hypoxic vasodilation. The second is based on the fact that transfusion of a 42-day old RBC unit provides a large iron bolus to the mononuclear phagocyte system. Such a bolus can result in acute increases in non-transferrin bound iron (NTBI) which can cause oxidative damage and potentiate bacteria proliferation. Both the NO and Iron hypotheses appear at play in a study in septic canines that showed that transfusion of 42-day RBCs resulted in increases in cell free hemoglobin, NTBI, and plasma labile iron resulting in increased shock, lung injury, and mortality. However, two recent clinical trials in 377 premature infants and 2430 intensive care patients, respectively, did not demonstrate differences in outcomes following transfusion of &lt;7 days vs 2-42 days RBCs. Another trial randomized 1098 complex cardiac surgery patients to ≤10 days or ≥ 21 days RBCs. No significant clinical differences were observed. These trials are reassuring because shorter-stored RBCs do not appear to have a better safety profile than standard-issue RBCs. Additional clinical trials are underway to test similar hypotheses. However, it is unlikely that these studies will have the power to evaluate transfusions of ≥35 day-old RBCs (when the storage lesion is at its maximum) or the effect of older-stored blood in rarer populations such as highly transfused septic patients. Additional research to minimize the RBC storage lesion and develop biomarkers of RBC transfusion effectiveness is warranted. Investigations of the impact on blood availability of limiting RBC storage to 35 days should also be considered. Disclosures Triulzi: Fresenius kabi: Membership on an entity's Board of Directors or advisory committees.

Membrane Remodeling By Pathogenic Antibodies Underlies Monocyte Activation in Heparin-Induced Thrombocytopenia

Heparin induced thrombocytopenia (HIT) is an iatrogenic antibody-mediated disorder with a paradoxically high propensity for thrombosis. We have shown previously that human HIT IgGs and the HIT-like monoclonal antibody (MAb) KKO bind to platelet factor 4 (PF4) complexed with glycosaminoglycans (GAGs) on the surface of platelets and monocytes, initiating cell activation in vitro, thrombocytopenia in a transgenic mouse model, and thrombus formation in a laser microvascular injury model in vivo even in the absence of exogenous heparin. Monocytes bind PF4 and HIT Ab more readily than platelets because they express higher affinity GAGs, heparan sulfate and dermatan sulfate, in addition to chondroitin sulfate found on both cell types. To study changes in the structure of the monocytes that accompany HIT, we used scanning electron microscopy, confocal microscopy and flow cytometry to characterize the morphology and function of isolated human monocytes and mouse transgenic Fcg receptor IIA positive (FcγRIIA+) or wt (FcγRIIA-) monocytes in the absence or presence of platelets. We show by scanning electron microscopy that upon binding of pathogenic HIT Abs to PF4/GAG complexes on FcgRIIA expressing monocytes, they initiate profound remodeling of the cell membrane. Addition of 100 μg/ml recombinant human PF4 in the absence of HIT Abs initiates the activation process with the appearance of 177 ± 53 nm "knobs" on the surface of 70% of monocytes. Subsequent addition of the HIT-like monoclonal antibody KKO at 50 μg/ml dramatically alters the cellular surface with the appearance of large 701 ± 208 nm membrane "blebs" that were not seen on FcγRIIA-mouse monocytes. These large, membrane-associated structures likely engage FcγRIIA, clustering them in proximity to cell-bound immune complexes, which promotes cell activation that leads to thrombosis. These blebs increase in size over time and are then shed from the cells as monocyte-derived microparticles, which self-aggregate. As a result of shedding of these blebs, the monocytes lose much of their typical ruffled surface (only 67% of monocytes maintain ruffles in the presence of PF4 plus KKO, compared to 97% of control monocytes) and appear smoother, sometimes with pores indicating degranulation. In the presence of platelets, monocytes exposed to PF4 and KKO formed heterocellular aggregates in addition to these subcellular changes. In contrast to KKO, addition of the non-pathogenic MAb RTO not only did not induce blebbing, but largely inhibited PF4-induced changes in the monocyte surface. This suggests that RTO might prevent monocyte activation by interfering with PF4 tetramerization. Structural analysis of the shed microparticles by microscopy revealed that they had an average diameter of 356 ± 307 nm, with many larger particles and aggregates. Flow cytometry confirmed that the shed particles contain cell membrane lipids and receptors. Confocal microscopy showed uniform binding of labeled PF4 to the monocyte cell membrane followed by rapid clustering into large complexes after the addition of KKO, but not RTO. These studies affirm the centrality of cell surface PF4/GAG complexes in the pathogenesis of HIT and provide quantitative morphometric characteristics of the changes in the monocyte membrane structure. We propose that PF4 released from activated platelets binds to the surface of GAG-expressing monocytes in vivo, forming clusters of PF4/GAG complexes that likely promote antibody binding and cause monocyte activation through FcγRIIA along with large-scale remodeling of the cell membrane and shedding of procoagulant microparticles. DisclosuresNo relevant conflicts of interest to declare.

β cell membrane remodelling and procoagulant events occur in inflammation‐driven insulin impairment: a GLP‐1 receptor dependent and independent control

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and β-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f β-cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross-talk model. Methyl-β-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative β-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events.

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO−, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO− (25–300µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000µM stressor. Concentrations of ONOO− affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO− (threshold conc. 100µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO− (threshold conc. 100µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO− concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO− were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO− (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO−, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO− concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

Procoagulant Platelets

Abnormalities in coagulation are a leading cause of disease and death worldwide because of thrombotic events such as myocardial infarction, stroke, etc. It has been estimated that the cost of treatment of these disorders will rise to >820 billion by the year 2030.1 At the center of thrombus formation is the platelet, a cell that is seen as the cornerstone of hemostasis and thrombosis. Platelets mainly function to secure hemostasis by acting as the band-aids of the blood. They are the first responders to sites of vascular injury, bringing with them a membrane surface that provides the glue for clot formation, and a number of proteins essential for coagulation, as well. Platelet involvement ultimately leads to thrombin generation and clot stabilization through fibrin formation. Article see p 1414 The process of thrombus formation is highly orchestrated, but, although much is known about the steps needed to complete the task, holes in our understanding still exist. At sites of vessel injury where the arterial wall shear rate is high, platelets adhere to von Willebrand factor via the glycoprotein VI receptor and collagen. Subsequently, platelet activation occurs and platelets aggregate together via the fibrinogen (glycoprotein IIb/IIIa) receptor that creates platelet/fibrinogen bridges, leading to thrombus formation.2,3 A subgroup of platelets, however, respond to collagen exposure without glycoprotein IIb/IIIa activation, and instead undergo a direct transformation from resting surveillance cells to active procoagulant cells. This process is characterized by an initial shape change that results in membrane asymmetry and resultant exposure of surface anionic phospholipids including phosphatidylserine (PS). Next, the platelets disintegrate, leading to shedding of procoagulant microparticles that provide further hemostatic support. The mechanism underlying this PS exposure is dependent on cystolic Ca2+ elevation via phospholipase-Cγ and phosphatidylinositol 3-kinase signaling. The exposure of PS results in increased surface …

P75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia.

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75NTR expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75NTR activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte–endothelial crosstalk in microvascular diabetic complications.