Procoagulant Phenotype Research Articles

L-carnitine inhibits a subset of platelet activation responses in chronic uraemia

Activated uraemic platelets expose the aminophospholipid phosphatidylserine (PS) at their outer surface, which generates a cell procoagulant phenotype and seems at least partly due to an increase in cell caspase-3 activity. L-carnitine (LC) may decrease surface-exposed PS in stored apheresis platelets and inhibit the activity of recombinant caspases, but its effects on platelet activation response with PS externalization have not been ascertained in chronic renal failure. In the present study, we investigated in vitro and in vivo the effects of LC on PS exposure in platelets from chronic uraemic patients. Platelet PS-exposure was assayed by flow cytometry using annexin V. Caspase activity in platelets was determined by the cleaving activity of the specific substrate DEVD-pNA and by a flow cytometric assay using rhodamine-fluorescence. The effects of LC in vivo were examined in a prospective cross-over trial including 10 patients on maintenance haemodialysis (HD) who were randomly allocated to two different treatment groups: LC (2 g i.v.) for 4 months followed by placebo (2 g i.v.) for another 4 months (group A), or placebo followed by LC (group B). PS-exposing platelets in blood samples obtained from HD patients were significantly higher than in healthy subjects (P<0.001) under both unstimulated and agonist-stimulated conditions. When uraemic platelets were pre-incubated with LC before agonist stimulation, platelet PS exposure proved to be significantly reduced (-13.7% for 0.5 mM LC and -25% for 5 mM LC). Pre-incubation of uraemic platelets with LC again significantly decreased the cells' caspase activity (P<0.05). In HD patients (Group A), LC supplementation was associated with a significant decrease (P<0.05) in platelet PS exposure followed by a progressive increase during treatment with placebo. In the other group of patients, while no change in platelet PS exposure was observed during the first 4 months of treatment with placebo, a significant reduction (P<0.05) in PS-positive platelets occurred after 2 and 4 months of LC therapy. Our data show that LC may reduce, possibly via inhibition of caspase activity, the exposure of PS in activated uraemic platelets. These findings may have implications for the thrombophilic tendency of uraemia.

Hypercoagulability resulting from opposite effects of lupus anticoagulants is associated strongly with thrombotic risk

Interference of antiphospholipid antibodies (aPL) with coagulation was investigated in 40 aPL-patients (24 with thrombosis) using thrombography. Impairment of the activated protein C anticoagulant pathway was partially offset by the genuine anticoagulant effect. The net result, a procoagulant phenotype, was associated with a 7-fold increased risk of thrombosis in aPL-patients.

Fibronectin-adherent monocytes express tissue factor and tissue factor pathway inhibitor whereas endotoxin-stimulated monocytes primarily express tissue factor: physiologic and pathologic implications.

Monocytes are critical cells in initiating physiologic and/or pathologic tissue factor (TF)-induced intravascular and extravascular coagulation. Monocytes constitutively express small amounts of TF and tissue factor pathway inhibitor (TFPI). Non-adherent lipopolysaccharide (LPS)-stimulated monocytes express significant amounts of TF; however, increased expression of TFPI by these cells is controversial. Further, whether fibronectin-adherent monocytes (mimicking conditions in the extravascular space) express sufficient TFPI to inhibit TF-procoagulant activity (PCA) is unknown. To compare TF and TFPI expression by fibronectin-adherent and LPS-stimulated non-adherent monocytes. Monocytes were isolated from normal peripheral blood, adhered to fibronectin or stimulated with lipopolysaccharide (LPS) under non-adherent conditions and examined for expression of TF and TFPI using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), ELISA and factor X (FX) activation. Under LPS-free conditions, the fibronectin-adherent monocyte TF mRNA, antigen and activity were markedly upregulated. Notably, cell and microparticle (MP)-associated TF and alternatively spliced TF (asTF) were all upregulated. TFPI mRNA and antigen were also upregulated in the fibronectin-adherent monocytes, which significantly inhibited TF-PCA. TFPI mRNAs for both alpha and beta forms were detected. The peak in TFPI activity occurred in tandem with the peak in TF-PCA. In contrast, LPS-stimulated monocytes, which expressed cell and MP-associated TF and asTF, demonstrated only minimal expression of TFPI as determined by mRNA, antigen or inhibition of TF activity. Both LPS-stimulated and fibronectin-adherent monocytes demonstrate a procoagulant phenotype by expressing TF but only fibronectin-adherent monocytes express significant amounts of TFPI to control thrombin generation and fibrin formation in the context of extravascular space.

α 1G T‐type Ca 2+ channel controls plasma membrane P‐selectin expression in pulmonary microvascular endothelial cells

The objective of this study was to determine the in vivo relevance of our previous in vitro observation, that CaV3.1 (α1G) T-type Ca2+ channel controls the thrombin-stimulated exocytosis of Weibel-Palade bodies in pulmonary microvascular endothelial cells. We utilized confocal fluorescence microscopy and isolated perfused lung to characterize the surface expression of P-selectin, a Weibel-Palade body membrane protein, at the apical plasma membrane of lung endothelial cells following thrombin stimulation in α1G knockout mice and their wild-type littermate mice. We found that in wild-type mice, thrombin stimulation induced membrane P-selectin expression in lung endothelial cells in both septal capillaries and extra-alveolar vessels. Pharmacological T-type Ca2+ channel blockade using mibefradil prevented thrombin stimulated-membrane P-selectin expression in septal but not extra-alveolar endothelia. In α1G knockout mice, thrombin stimulated-membrane P-selectin expression was attenuated in septal capillary endothelial cells, but not extra-alveolar vessel endothelial cells, compared to those of wild-type mice. Taken together, these results suggest that α1G T-type Ca2+ channel importantly contributes to agonist-induced lung microvascular endothelial cell procoagulant phenotype. Supported by HL-66299.

PO-83 Differential procoagulant phenotype of pancreatic and breast cancer cells related to different tissue factor activity

PO-83 Differential procoagulant phenotype of pancreatic and breast cancer cells related to different tissue factor activity

Progress in xenotransplantation following the introduction of gene-knockout technology

The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited progress in pig-to-primate organ xenotransplantation for many years. Survival of GT-KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti-nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild-type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T-cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ-source pigs is therefore required to overcome these problems. GT-KO pigs expressing a human complement-regulatory protein, e.g. decay-accelerating factor, and/or an 'anti-coagulant' gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti-coagulant to a procoagulant phenotype, and protect the islets from early loss.

Homocysteine targeting of plasma proteins in hemodialysis patients

Hyperhomocysteinemia, an independent, modifiable risk factor for cardiovascular disease, is found in most patients with end-stage renal disease. In this issue, Perna et al. examine the extent of protein-S-linked and protein-N-linked homocysteinylation in uremic patients on hemodialysis and the effect of folate treatment on protein homocysteinylation. Their findings show that protein-N-linked homocysteinylation, but not S-linked homocysteinylation, can be normalized by folate therapy.

The inhibitory effects of anticoagulation on in vivo gene transfer by adeno-associated viral or adenoviral vectors

Identifying factors that influence gene transfer efficacy is critical for a successful gene-based clinical study. Here we demonstrate that in vivo AAV-2-mediated gene transfer is efficiently inhibited by unfractionated heparin, but not by a heparin preparation containing mainly low-molecular-weight forms (LMWH). Surprisingly, inhibitors of thrombin or factor Xa (F.Xa) significantly reduced AAV-2 transduction in a dose-dependent manner. These effects were independent of the vector promoter, transgene, or strain of mice. Expression by alternate AAV serotypes 5 and 8 was not affected by anticoagulant drugs, which suggests an AAV-2-specific effect. Moreover, AAV-2-mediated gene expression was diminished in mice with deficiency in thrombin generation (factor IX deficiency) and enhanced in mice with procoagulant phenotype due to factor V Leiden. In addition, inhibitors of F.Xa diminished adenovirus-mediated gene expression. These results demonstrated that coagulation activity itself is critical to ensure optimal viral vector transduction. Since intravascular delivery of vectors often requires the use of anticoagulants, the use of LMWH appears to be safe. These observations are of relevance for approaches using AAV-2 or adenoviral vectors, especially in early phase studies designed to identify the minimum therapeutic doses.

Le CD154 plaquettaire : Une nouvelle interface dans l’hémostase et la réaction inflammatoire

Blood platelets play a crucial part in the blood clotting process by forming the platelet plug. Recent evidence indicates that they are likely to play a key role in the inflammatory reaction via CD154/CD40 interactions. CD40 was known to be widely expressed, for instance on cells of the vasculature including endothelial cells, smooth muscle cells and macrophages. It was also known that the triggering of CD40 on these cells led to the acquisition of an activated pro-inflammatory and pro-coagulant phenotype. It was subsequently shown that platelets express CD154 which is cryptic in unstimulated platelets but is expressed at the platelet surface upon platelet activation. When expressed at the platelet surface and exposed to CD40-expressing vascular cells, the platelet-associated CD154 triggers a variety of pro-inflammatory and pro-coagulant responses including induction of adhesion receptors, release of cytokines and chemokines, induction of tissue factor and of metalloproteinases. Platelet-associated CD154 is also involved in platelet/platelet interactions during platelet aggregation. Furthermore, in vivo models have emphasized the critical role of the platelet-associated CD154 in the progression of atherosclerotic disease and in the stabilization of arterial thrombi. Recent data show that CD40-bearing cells involved in fibrosis such as hepatic stellate cells and glomerular mesangial cells also respond to platelet-associated CD154, thus suggesting a new mechanism by which platelets may be instrumental in the inflammatory diseases of the liver or the kidney. Finally, platelet-associated CD154 has been shown to have immune competence both in vitro and in vivo, observations that open new fields of research on the potential implications of platelets in the immune response and auto-immune diseases.

Thrombose portale compliquant une infection à cytomégalovirus aiguë chez un sujet immunocompétent

The cytomegalovirus (CMV) infection is most often asymptomatic. The grave forms concern the immunocompromised patients. We report a new case pf acute CMV hepatitis complicated with portal thrombosis in an immunocompetent patient. A 29 year old man has presented a CMV hepatitis proved by the presence of pp65 protein and the viral DNA in serum. This infection was complicated by a portal thrombosis and the evolution was rapidly favourable under anticoagulant treatment. Eleven cases of major thrombosis complicating acute CMV infection in immunocompetent patients were previously reported in the English and French literature. The absence of local and general cause, the remission without anticoagulation, the elevated risk of thrombosis in both HIV and CMV seropositive patients, and in CMV seropositive renal transplant patients suggest a causal relation. Various pathogenic hypotheses were raised: presence of antiphospholipid antibodies, absent in our case, procoagulant phenotype induction of infected endothelial cells, proliferation induction of smooth cells. The acute CMV infection can be considered such as a possible cause of major thrombosis.

Cardiac involvement in the antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, associated with a hypercoagulable state and fetal loss and with other clinical manifestations including cardiac involvement. Cardiac manifestations of APS are valve abnormalities (valve thickening and vegetations), occlusive arterial disease (atherosclerosis and myocardial infarction), intracardiac emboli, ventricular dysfunction, and pulmonary hypertension. Antiphospholipid antibodies (aPLs) may have a role in the accelerated atherosclerotic arterial disease observed in APS, related to their ability to induce endothelial activation. aPLs have been incriminated in the pathogenesis of heart valve lesions in APS patients. Markers of endothelial cell activation are up-regulated with prominent deposition of aPL in heart valves, suggesting aPL deposition initiates an inflammatory process that recruits complement leading to the valve lesion. Autoantibody-mediated endothelial cell activation probably plays a role in sustaining a proadhesive, proinflammatory, and procoagulant phenotype. The heterogeneity of APS clinical manifestations is likely linked to the varied effects that aPL can induce on endothelial cells and to the different functions that endothelial cells display depending on the anatomic localization.

Even Flow

The appreciation that biomechanical forces induce signaling responses that regulate vascular tone and inflammatory responses has had a paradigm-shifting effect on our understanding of vascular biology. Endothelial cells are constantly exposed to rheologically imposed forces that vary due to vascular anatomic features and hemodynamic parameters, and of course shear fluctuates cyclically during the cardiac cycle. Simple laminar flow and high shear forces elicit a cellular response characterized by quiescence and alignment of endothelial cells in the direction of fluid flow, whereas disturbed flow and low shear forces induce an inflammatory procoagulant phenotype. These cellular effects are mediated through well-defined signaling pathways and gene expression responses. The cellular sensors or receptors for shear forces are incompletely characterized, but detection of stress seems to depend on the transmission of forces to points of cell–matrix and cell–cell attachment and requires specific conformational activation of integrins.1 From a pathophysiological perspective, disturbed laminar flow has been linked to inflammatory responses that initiate atheroma formation and elicit plaque instability. It is also generally accepted that flow is required for some aspects of vascular remodeling during vascular development and in adult angiogenic responses; however, specific roles for flow in vascular developmental programs have been incompletely characterized. See page 1817 Two recent reports, including one published in this issue of Arteriosclerosis, Thrombosis, and …

Procoagulant phenotype of endothelial cells after coculture with biomaterial‐treated blood cells

Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH(2)) terminal group at 5.4 or 54 x 10(4) beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils.

Red blood cells may contribute to hypercoagulability in uraemia via enhanced surface exposure of phosphatidylserine

The exposure of phosphatidylserine (PS) on the outer leaflet of the erythrocyte membrane may have several pathophysiological consequences, including the development of a procoagulant phenotype, a finding that seems relevant to the thrombotic risk seen in many disorders. Because PS externalization increases in erythrocytes from patients suffering from chronic uraemia, which is frequently associated with a prothrombotic state, the possible relationship between erythrocyte PS exposure, erythrocyte procoagulant activity and plasma levels of several haemostatic markers was studied in a group of haemodialysed patients. Uraemic erythrocytes displayed increased procoagulant activity, which proved to be correlated directly with erythrocyte PS exposure. Pre-incubation of uraemic erythrocytes with annexin V, a protein with high affinity and specificity for PS, strongly inhibited in vitro thrombin generation induced by erythrocytes as compared with untreated red cells. Thrombin generation and activation of fibrinolysis were found to occur in uraemic patients, as substantiated by increased plasma levels of markers for thrombin generation (prothrombin fragment F1.2 and thrombin-antithrombin complex) and fibrinolysis (D-dimer and plasmin-antiplasmin complex), respectively. Significant correlations between prothrombin fragment F1.2 and D-dimer suggested that hyperfibrinolysis was secondary to thrombin generation. Correlations were also found between erythrocyte PS levels and plasma levels of haemostatic markers, including prothrombin fragment F1.2 (P = 0.007), thrombin-antithrombin complex (P = 0.00009), plasmin-antiplasmin complex (P = 0.0009) and D-dimer (P = 0.005). Our study suggests that increased PS exposure may cause a pathological erythrocyte procoagulant phenotype, which may be a factor inducing a hypercoagulable state in uraemia.

Agonist Costimulation Increases Recruitment of Platelet Subpopulations to a Procoagulant Phenotype.

Previously we determined that platelets stimulated with PAR 1 agonists, even at levels eliciting maximal responses for support of factor Xa generation, showed only a subpopulation (~10%) that exposed binding sites for factor IXa. Now we show that the same subpopulation of platelets supported binding of both factor Xa and factor IXa. While stimulation of platelets with GPVI agonists collagen or convulxin also caused exposure of factor Xa binding sites on a small subpopulation (~10%) of platelets, simultaneous stimulation with PAR 1 and GPVI agonists resulted in synergistic recruitment of platelets to expose factor Xa binding sites (30–60%). In kinetic studies of platelet-supported thrombin generation, platelets preactivated with thrombin or collagen showed roughly equivalent initial rates of prothrombinase activity (5nM/min, 10nM/min, and 12nM/min for low, medium and high agonist concentrations) that increased with reaction time up to 20 minutes reflecting platelet stimulation by the thrombin generated. Platelets preactivated with both collagen and thrombin, even at high concentrations (collagen 50μg/ml and thrombin 2U/ml), supported additive rates of prothrombinase in contrast to the synergy achieved in exposure of factor Xa binding sites. These data indicate that although single strong agonists recruit only a small subpopulation of activated platelets to undergo procoagulant surface changes, co-stimulation acts synergistically to recruit a larger population. It was previously shown that treatment of platelets with 100μM of the protein kinase C inhibitor RO318220 permitted an increased (three-fold) PAR 1-stimulated platelet support of prothrombinase and an almost 20-fold increase in the size of the population exposing annexin V binding sites. We now show that agonist-stimulated platelets pretreated with 100μM RO318220, whether stimulated with PAR 1 or GPVI agonists separately or combined, recruited ~80% of platelets to bind factor Xa. These data suggest that exposure of a procoagulant phenotype is negatively regulated by a pathway containing a target of high concentrations of RO318220, and that both PAR 1 and GPVI pathways normally converge in relief of that regulation.

Endothelial microparticles: a potential contribution to the thrombotic complications of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) refers to persistent anti-phospholipid antibodies (aPL) associated with thrombotic and/or obstetrical complications. The endothelial cell is a target of aPL which can induce a procoagulant and proinflammatory endothelial phenotype, as reported both in vivo and in vitro. Microparticle production is a hallmark of cell activation. In the present study, the presence of endothelial microparticles (EMP) in the plasma of APS patients was investigated. To determine if there is a correlation with certain biological and clinical features, EMP levels were measured in thrombosis-free patients with systemic lupus erythematosus (SLE) patients, with and without aPL, in patients with non aPL-related thrombosis, as well as in healthy controls. Compared to healthy subjects, elevated plasma levels of EMP were found in patients with APS and in SLE patients with aPL, but not in SLE patients without aPL or in non aPL-related thrombosis. EMP levels were also associated with Lupus Anticoagulant (LA) detected by a positive Dilute Russell's Viper Venom time (DRVVT). In parallel, we analyzed the capacity of these plasma to induce vesiculation of cultured endothelial cells. We demonstrated an increase of EMP generated in response to plasma from patients with auto-immune diseases. Interestingly, only APS plasma induced the release of EMP with procoagulant activity. These ex vivo and in vitro observations indicate that generation of EMP in APS and SLE patients results from an autoimmune process involving aPL. Production of procoagulant microparticles in APS patients may represent a new pathogenic mechanism for the thrombotic complications of this disease.

An in vitro study on the mechanisms of coagulation activation in acute myelogenous leukemia (AML): role of tissue factor regulation by cytotoxic drugs and GM-CSF

AML patients may suffer from a disseminated coagulopathy, which can aggravate a pre-existing bleeding tendency due to thrombocytopenia and platelet dysfunction. The cellular and molecular mechanisms underlying this coagulopathy, however, are not completely understood. Indeed, the broad and increasing therapeutic use of cytotoxic drugs and growth factors is likely to contribute to the complexity of hemostatic abnormalities encountered in this hematologic malignancy. The nature of coagulation activation in AML was therefore investigated in vitro using the human leukemic cell line, HL60. Tissue factor (TF) was almost entirely located on the cell surface and bound factor VIIa, but only 15-25% of this TF was primarily functionally active. Treatment with increasing concentrations of daunorubicin or cytosine-beta-D-arabinofuranoside, two cytotoxic drugs commonly used in AML therapy, induced apoptosis and secondary necrosis of HL60 cells and resulted in marked decryption of TF PCA independent of de novo protein synthesis. This PCA-modulating effect was concomitant with and functionally dependent on the exposure of phosphatidylserine on the outer membrane leaflet. Similar observations were made in analogous ex vivo studies on patient-derived myeloblasts. Incubation of HL60 cells with GM-CSF, a cytokine expressed in the bone marrow microenvironment and used as an adjunct to AML treatment, evoked a cellular response, which included both enhanced TF production and release of VEGF-A and uPA into the culture medium. We conclude that both decryption of pre-formed TF PCA by chemotherapeutic drugs and de novo induction of TF by cytokines such as GM-CSF can regulate the pro-coagulant phenotype of HL60 cells in vitro.

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis.

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.

Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis

Histological findings in small-vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs) suggest that damaged endothelial cells undergo necrosis and detachment from the basement membrane. We postulated that isolation of these cells from peripheral blood might provide a novel marker of the disease and elucidate pathogenetic events. 18 patients with active ANCA-associated vasculitis, 20 patients in remission, 20 healthy controls, 12 patients with infection, and 12 patients with glomerular disease not associated with ANCA were studied. Endothelial cells were isolated from peripheral blood by use of Dynabeads coated with antibodies against CD146, and were stained for von Willebrand factor (vWF), CD31, and Ulex Europaeus lectin 1 (UEA-1). Tissue-factor immunocytochemistry and assays for markers of apoptosis and necrosis were also done. Few circulating endothelial cells were seen in healthy controls (0-20 cells/mL, median 5 cells/mL), patients with infection (0-16 cells/mL, median 8 cells/mL), and patients with non-ANCA glomerulonephritis (0-21 cells/mL, median 4 cells/mL). By contrast, large numbers of circulating endothelial cells were detected in patients with active vasculitis (20-5700 cells/mL, median 136 cells/mL, p<0.0001 when compared with healthy controls). Cell numbers fell substantially during 6 months of successful immunosuppressive treatment among those with active disease. Patients in remission had moderately raised cell numbers (0-60 cells/mL, median 16 cells/mL). 84% of cells obtained from patients with active disease stained positive for annexin/propidium iodide and 86% stained tissue factor positive, indicating a necrotic and procoagulant phenotype. Circulating endothelial cells are a novel marker of active ANCA-associated small-vessel vasculitis. The clinical use of this tool and the pathogenic mechanisms leading to these findings require further investigation.

Endothelium as a target for antiphospholipid antibodies

Several, and not necessarily alternative, pathogenic mechanisms have been suggested to sustain the thrombophilic diathesis of the anti-phospholipid syndrome. Among them, interference of anti-phospholipid antibodies with cell acting in the coagulation cascade likely plays a major role. Anti-phospholipid antibodies have been shown to react with endothelial cells mainly by reacting with beta 2 glycoprotein I expressed on the cell membrane surface. Beta 2 glycoprotein I can adhere to endothelial cell surface through the Annexin II receptor and through negatively charged structures (heparin-like molecules) that are bound by the phospholipid-binding site of the molecule. The autoantibody binding involves a yet unknown receptor that activates a signalling pathway able to translocate NFkappaB from the cytoplasm to the nucleus and to activate genes for adhesion molecule, pro-inflammatory cytokine and Tissue Factor up-regulation. The ultimate effect is the induction of a pro-inflammatory and a pro-coagulant endothelial phenotype that has been reproduced both in vitro and in vivo experimental models. Additional effects of anti-phospholipid-mediated endothelial cell activation are the interference with the protein C/S system, with the Annexin V binding, the up-regulation of endothelin I synthesis and the induction of apoptosis. Altogether these effects cooperate in switching endothelium from an anti-coagulant to a pro-coagulant surface.