Florence Toti

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

Increased levels of procoagulant tissue factor-bearing microparticles within the occluded coronary artery of patients with ST-segment elevation myocardial infarction: role of endothelial damage and leukocyte activation.

OBJECTIVEnDuring myocardial infarction, platelet activation and endothelial apoptosis are responsible for the release of procoagulant membrane-derived microparticles (MPs) in the bloodstream. Few data are available on the potential role played by MPs in coronary atherothrombosis. In the present study, we investigated the levels and cellular origins of MPs within the occluded coronary artery of patients with ST-segment elevation myocardial infarction (STEMI) treated by primary angioplasty (PCI).nnnMETHODSnA total of 12 patients with STEMI treated by primary PCI within 24h of symptom onset were included in this study. MPs procoagulant activity and cellular origin were characterized within the occluded coronary artery before PCI (C(0)), after restoration of the epicardial blood flow (C(1)), and in blood collected from the femoral artery (F).nnnRESULTSnLevels of leukocyte-derived CD11a(+) MPs, endothelial-derived CD105(+) MPs, and tissue factor (TF)-bearing MPs were significantly higher within the occluded coronary artery than in peripheral blood samples. Restoration of the epicardial blood flow led to a significant reduction of procoagulant CD11a(+) and CD105(+) MPs by 30% and 42%, respectively (p<0.05).nnnCONCLUSIONSnElevation of procoagulant MPs within the occluded coronary artery of patients with STEMI suggests their pathophysiological role in coronary atherothrombosis.

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

Summary.u2002 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 (αIIbβ3) 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 (4.2u2003±u20030.6 vs. CP 15.5u2003±u20032.1u2003nm PhtdSer; Pu2003=u20030.001) together with sGPV (36u2003± 3 vs. CP 58u2003±u20038u2003ngu2003mL−1; Pu2003=u20030.02). Leukocyte‐derived MP decreased at day 6 (0.12u2003±u20030.04 vs. CP 0.56u2003±u20030.12u2003nm PhtdSer; Pu2003=u20030.01) 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.

Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics.

The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium‐ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine‐labelled proteins in platelets treated with thrombin or a thrombinu2003+u2003collagen mixture and in particular the phosphorylation of a 40u2003kD band were severely reduced. Furthermore, inhibition of thiol‐containing enzymes, including tyrosine‐phosphatases, by N‐ethyl maleimide did not lead to aminophospholipid exposure in the patients platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.

Scott syndrome: an inherited defect of the procoagulant activity of platelets.

Anionic phospholipids, chiefly phosphatidylserine, are essential for the assembly of the characteristic enzyme complexes of the blood coagulation cascade at the surface of stimulated platelets and derived microparticles. In the resting cell, these phospholipids are sequestered in the inner leaflet of the plasma membrane. Scott syndrome is an extremely rare bleeding disorder that confirms the essential role of these anionic procoagulant phospholipids. In Scott patients, phosphatidylserine externalization and microparticle shedding are dramatically impaired. This functional deficiency is clearly evidenced by the measurement of residual prothrombin in serum. The recent detection of a familial Scott syndrome testifies to the genetic origin of the defect. Symptomatic Scott patients present provoked hemorrhages and are probably homozygous for the disorder whereas asymptomatic children are probably heterozygous. The Scott phenotype can be detected in platelets, red cells and lymphocytes by functional prothrombinase assay and flow cytometry. Intermediate degrees of phosphatidylserine exposure and vesiculation are observed in cells from the asymptomatic heterozygous offspring when compared to those from their homozygous defective parent and healthy subjects. The functional and molecular characterization of mutated element(s) in Scott syndrome should be of valuable help for the understanding of phospholipid transmembrane migration, also termed flip-flop, its possible links with membrane vesiculation, and the eventual implications in thrombotic or apoptotic processes.

Circulating procoagulant microparticles in acute pulmonary embolism: A case–control study

We investigated whether circulating procoagulant microparticles (CPMPs) contributed to hypercoagulability in 45 patients with acute pulmonary embolism (APE) and in 45 controls with and 45 controls without cardiovascular risk factors. Concentrations of CPMPs and platelet-derived microparticles (PMPs) were statistically significantly higher in patients with APE than in controls without cardiovascular risk factors. PMPs appeared to be the main source of procoagulant microparticle release in APE, but this correlation disappeared when APE patients were compared to controls with cardiovascular risk factors. CPMPs may have a role in venous thrombosis as mediators of cardiovascular risk factors.

Factors influencing the level of circulating procoagulant microparticles in acute pulmonary embolism

BACKGROUNDnFlow cytometry has shown levels of platelet-derived microparticles (PMPs) and endothelial-derived microparticles (EMPs) to be elevated in deep-vein thrombosis. Cardiovascular risk factors can also contribute to hypercoagulability due to circulating procoagulant microparticles (CPMPs).nnnAIMSnTo investigate in a case-control study the respective contribution of pulmonary embolism and cardiovascular risk factors to the level of hypercoagulability due to CPMPs.nnnMETHODSnCPMP, PMP and EMP levels were measured in 45 consecutive patients (age 67.9 +/- 11.6 years; 66.7% men) admitted to an intensive care unit for acute pulmonary embolism (APE), 45 healthy control subjects with no history of venous thromboembolism or vascular risk factors (Controls(noCVRFs)), and 45 patients with cardiovascular risk factors (Controls(CVRFs)). APE was diagnosed by spiral computed tomography or scintigraphy. CPMP levels were assessed using a prothrombinase assay on platelet-depleted plasma (results expressed as nmol/L equivalent).nnnRESULTSnCPMP levels were higher in APE patients than in Controls(noCVRFs) (medians 4.7 vs 3.2 nmol/L, interquartile ranges [IQRs] 2.9-11.1 vs 2.3-4.6 nmol/L; p=0.02). Similar results were reported for PMPs (medians 2.2 vs 1.9 nmol/L, IQRs 1.7-5.8 vs 1.4-2.4 nmol/L; p=0.02), whereas EMP levels were not significantly different. However, CPMP procoagulant activity was not significantly different in APE patients and Controls(CVRFs).nnnCONCLUSIONSnCPMPs and PMPs were significantly elevated in APE patients vs Controls(noCVRFs), but this correlation was not significant when APE patients were compared with Controls(CVRFs). Our observations highlight the importance of adjusting for the presence of cardiovascular risk factors in conditions in which microparticle levels are raised.

Electrophoretic studies on molecular defects of von Willebrand factor and platelet glycoprotein IIb-IIIa with antibodies produced in egg yolk from laying hens.

Immunoglobulins isolated from egg yolk (IgYs) are a convenient source of polyclonal antibodies. Their purification is simple and the yields important (50 mg immunoglobulins/egg). Nevertheless their biochemical characteristics are different from those of rabbit antibodies generally used for the study of molecular defects of plasmatic and platelet proteins. Provided standardization is achieved, IgYs can be used for immunoelectrophoresis, immunoprecipitation or immunoblotting assays. We describe here the electrophoretic and immunoblotting conditions employed to explore human plasmatic and platelet von Willebrand factor (vWF) and human platelet GP IIb-IIIa using IgYs. These two proteins are involved in primary hemostasis and their absence or abnormality is responsible for hereditary bleeding disorders. The methods were applied to the characterization of patients with vWF or GP IIb-IIIa defects and compared to classical mammalian IgG immunoelectrophoretic techniques. Results were further confirmed by flow cytometric analysis.

Rescue of a pancreatic islet graft after steroid therapy

Allogeneic islet transplantation can restore insulin secretion in patients suffering from type 1 diabetes. However, the majority of islet transplant recipients experience a gradual decline in graft function (1). Several studies have implicated immune rejection in the loss of grafted islets (2, 3). Recently, our group reported a decline in islet graft function in association with human leukocyte antigen (HLA) sensitization in an islet transplant recipient and the recovery of islet function after treatment with an anti-CD20 monoclonal antibody (4). In this report, we document the decline of islet function in an islet transplant recipient and the recovery of the graft after steroid bolus therapy. The patient was a 43-year-old woman with a 37-year history of diabetes who underwent islet transplantation because of metabolic instability as part of a clinical trial (GRAGIL-TRIMECO). Tests for HLA antibodies using the Luminex technique were consistently negative for HLA classes I and II. The patient received a graft containing 7400 islet equivalents per kilogram body weight from a single donor by percutaneous intraportal infusion. A pretransplant complement-dependent Tand B-cell cross-match was negative. Immunosuppression was achieved by treating the patient with tacrolimus (Prograf; Fujisawa, Osaka, Japan; 2 mg/day), mycophenolate mofetil(CellCept;Roche,Basel,Switzerland; 2 g/day), antithymocyte globulin (Thymoglobulin; Genzyme SAS, Saint-Germain-enLaye, France; total dose of 6 mg/kg), and etanercept (Enbrel; Pfizer, Groton, CT; 50 mg intravenously at day 0, then 25 mg subcutaneously on days 3, 5, and 10) induction. Immediately after islet transplantation, fasting C-peptide levels increased from 0 to 1.2 ng/mL (normal range: 0.8–3 ng/mL). The patient was treated by subcutaneous insulin infusionwithexternalpump,andtheinsulin requirementsdecreasedslowlyfrom26to17 IU/day. One month after transplantation, a postprandial glucose peak greater than 11 mmol/L was observed, with C-peptide levels decreasing to 0.3 ng/mL and an increase in insulin requirements to 30 IU/day. Treatment with steroid boluses (methylprednisolone; Pfizer; 10 mg/kg for 3 days and then 6, 4 and 2 mg/kg for 1 day) was initiated 4 days after the decrease in Cpeptide. Continuous intravenous insulin therapy (Umuline Rapide; Eli Lilly, Indianapolis, IN; 3– 4.5 IU/hr) was administered to prevent hyperglycemia during the steroid therapy. Four days after steroid therapy, C-peptide levels were restored to 1.3 ng/mL, with insulin requirements under external pump slowly decreasing from 35 to 15 IU/day (Fig. 1). In the absence of an identified cause of islet loss, a diagnosis of acute cellular islet rejection was proposed. An assay for anti-HLA antibodies using the Luminex technique remained consistently negative for HLA classes I and II. In addition, cross-matches performed with sera collected at the time of the graft dysfunction episode remained negative. No islet autoantibodies against glutamate acid decarboxylase 65 (GAD65) or IA-2 were detected. Tacrolimus trough levels were 11.5 g/L. There was no clinical or biological sign of infection. Polymerase chain reaction using primers specific for cytomegalovirus and Epstein-Barr virus yielded no detectable product. At 3 months after the first islet injection, the patient received a second islet graft of 6545 islet equivalents per kilogram body weight with basilixumab (Simulect, Novartis, Basel, Switzerland; 20 mg intravenously at days 0 and 4) and etanercept (Enbrel; Pfizer) in induction. One month later, the patient was insulin-free and has remained so for 8 months. 1,8 8

Interference of activated factor VII in apoptotis of erytholeukemic K562 cells

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.