Dominique Arnoux

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

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 Russells 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.

Intraoperative evolution of coagulation parameters and t-PA/PAI balance in orthotopic liver transplantation.

The changes in coagulation and fibrinolysis were investigated in 10 patients undergoing orthotopic liver transplantation (OLT) which is known to be frequently associated with perturbations of haemostasis. The coagulation profile, already deteriorated before surgery in most patients, showed no appreciable further alteration. On the other hand, important modifications of fibrinolytic parameters occurred, essentially concerning tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI). t-PA activity constantly increased in the course of transplantation, reaching a maximum at the end of anhepaty. Large interindividual variations were noted in the level of t-PA activity (7.5 to 135 IU/ml). Free PAI activity followed a reverse kinetics, remaining low during the anhepatic stage, and dramatically increasing after allograft reperfusion. Despite the fibrinolytic potential related to high circulating t-PA levels, no biologic nor clinical evidence of systemic fibrinolysis was observed peroperatively. These findings suggest that PAI release could represent an early process making the use of antifibrinolytic drugs during OLT unnecessary.

Differential Inhibitory Effects of Isosorbide Dinitrate and its Mononitrate Metabolites on Platelet Aggregation and Thromboxane Formation

The effects of isosorbide dinitrate (ISDN) and its 2- and 5-mononitrate metabolites (2-ISMN and 5-ISMN) against platelet aggregation and thromboxane release were investigated by analysis of platelet aggregation curves. ISDN, 2-ISMN and 5-ISMN (isosorbide nitrates, ISN) inhibited both ADP- and epinephrine (EPI)-induced platelet aggregation. ISN affected specifically the extent of ADP-induced aggregation and the velocity of EPI-induced effects. 2-ISMN was more potent against platelet aggregation compared to ISDN and 5-ISMN. The isosorbide nitrates were poor inhibitors of both arachidonic acid-induced aggregation and platelet TxB2 release. The differential inhibition by the three isosorbide nitrates of endogenous TxB2 release during ADP-induced aggregation further indicates that 2-ISMN is a significantly more potent platelet inhibitor than either ISDN or 5-ISMN. These studies suggest a role of the metabolites in modulating the pharmacological effects of ISDN on platelet activity.

Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy

Tissue factor pathway inhibitor (TFPI) plays a crucial role in haemostasis by regulating TF-induced initiation of coagulation. Since it is expressed by endothelial and trophoblastic cells, TFPI is of particular importance at the placental level and might be involved in the occurrence of gestational vascular complications (GVC). In the present study, we investigated plasma free TFPI antigen in four groups of women: healthy non-pregnant women without history of pregnancy complications; women at the beginning (<12 weeks) and women during the third trimester of a normal pregnancy; women with late pregnancy complications (pre-eclampsia / HELLP syndrome, intra-uterine fetal death, fetal growth retardation) at the time of obstetrical event and/or at distance from pregnancy. In normal pregnancy, TFPI increased between first trimester and delivery (median 5.0 ng/ml vs. 7.1 ng/ml; p<0.0001) but remained lower than in non-pregnant controls (median 8.2 ng/ml; p<0.0001). In patients, when measured concomitantly to the obstetrical event, TFPI showed no difference with normal late pregnancy levels. In contrast, at distance from pregnancy, in the absence of any hormonal influence, TFPI was significantly lower than in non-pregnant controls (median 5.9 vs. 8.2ng/ml, p < 0.0001). After categorisation into quartiles, an inverse dose-effect relationship was demonstrated between TFPI categories recorded apart from pregnancy and GVC risk, with a crude odds ratio of 43.5 (95% confidence interval 8.2-230) for patients with TFPI values in the lowest quartile (< 5.7 ng/ml). In conclusion, low free TFPI at distance from pregnancy appears to be a strong indicator of GVC risk.