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Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 μg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

Purpose: To compare the effects of recombinant human activated protein C (APC) and glucocorticoids alone and in combination in non-anesthetized resuscitated septic shock induced by cecal ligation and puncture (CLP) on (a) survival, (b) hemodynamics, and (c) vascular reactivity. The effects of treatments on major cellular pathways likely implicated were also studied., Methods: Four hours after CLP, rats were continuously infused with either saline (10 ml/kg/h), saline + APC, saline + dexamethasone (Dexa), or saline + APC + Dexa. Eighteen hours after CLP, arterial pressure, cardiac output, nitrite/nitrate ratio, and lactate concentrations were measured. Aortic rings and mesenteric arteries were isolated and mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase (NOS) or cyclooxygenase (COX) inhibitors. Protein expression was assessed by Western blotting. Aorta NO and superoxide anion content were measured by electron paramagnetic resonance., Results: All treatments improved hemodynamic parameters and vascular reactivity and decreased lactate and nitrite/nitrate levels. In treated aorta and mesenteric arteries, contractility and endothelial dysfunction were improved. This effect was associated with an increase in the phosphorylated form of protein kinase B as well as an increase in COX vasodilatory pathways and a decrease in iNOS expression suggesting that these pathways are implicated in the vascular effect of the treatments. CLP was associated with a marked increase in aortic NO and superoxide anion content (p < 0.05), which were decreased by APC and Dexa and totally abolished by APC + Dexa (p < 0.01). Survival length was significantly increased by the APC-Dexa combination., Conclusions: Both APC and Dexa improve arterial contractility and endothelial dysfunction resulting from septic shock in rats. Moreover, their combination increased the length of survival. These findings provide important insights into the mechanisms underlying APC- and/or Dexa-induced improvements of arterial dysfunction during septic shock.

An acquired resistance to activated protein C (APC) has been demonstrated in patients with antiphospholipid antibodies (aPL). Recent studies report interactions between beta2 glycoprotein I (beta2GPI) and prothrombin-binding antibodies and the protein C system. Some aPL in patients recognize one or more conformational epitopes shared by beta2GPI and catalytic domains of APC. Both beta2GPI and anti-prothrombin antibodies are associated with APC resistance. Several clinical studies have focused on an association between aPL and APC resistance, determined by classic activated partial thromboplastin time-based tests. It has been shown in most studies that APC resistance was associated with lupus anticoagulants. APC resistance is also associated with thrombosis, especially venous thromboembolism. Several recent studies have reported a novel integrated approach of coagulation using calibrated automated thrombography. This technique allows an approach of APC sensitivity without interference with lupus anticoagulants. Clinical associations between APC resistance and thromboembolic events have been demonstrated.

Background: Recombinant human activated Protein C (APC) is used as an adjunctive therapeutic treatment in septic shock. APC seemingly acts on coagulation-inflammation interaction but also by decreasing proinflammatory gene activity, thus inhibiting subsequent production of proinflammatory cytokines, NO and NO-induced mediators, reactive oxygen species production and leukocyte-endothelium interaction. The hemodynamic effects of APC on arterial pressure and cardiac function are now well established in animal models. However, the specific effects of APC on heart and vessels have never been studied., Objectives: To investigate the potential protective properties of therapeutic ranges of APC on a rat endotoxic shock model in terms of anti-inflammatory and cytoprotective pathways., Design: Laboratory investigation., Setting: University medical center research laboratory., Interventions: Rats were exposed to lipopolysaccharide (LPS) (10 mg/Kg intravenous). Endotoxic shock was treated with infusion of saline with or without APC (33 microg/kg/h) during 4 hrs. Hemodynamic parameters were continuously assessed and measurements of muscle oxygen partial pressures, NO and superoxide anion (O2(-)) by spin trapping, of NF-kappaB, metalloproteinase-9 (MMP-9) and inducible NO synthase (iNOS) by Western blotting, as well as leukocyte infiltration and MMP-9 activity were performed at both the heart and aorta level (tissue)., Main Results: APC partially prevented the reduction of blood pressure induced by LPS and improved both vascular hyporeactivity and myocardial performance. This was associated with a decreased up-regulation of NF-kappaB, iNOS and MMP-9. LPS-induced tissue increases in NO and O2(-) production were decreased by APC. Furthermore, APC decreased tissue leukocyte infiltration/activation as assessed by a decrease in myeloperoxidase and matrix metalloproteinase 9 activity., Conclusions: These data suggest that APC improves cardiovascular function: 1) by modulating the endotoxin induced-proinflammatory/prooxidant state, 2) by decreasing endothelial/leukocyte interaction and 3) by favoring stabilization of the extracellular matrix.

Endothelium dysfunction is one of the hallmarks of sepsis. Looney and Mattay, in the previous issue of Critical Care, highlight the role of activated protein C (APC) as a protective endothelial drug in septic situations. Nevertheless, the results of in vivo studies are less explicit and it remains uncertain whether these properties are relevant in human septic shock. Before considering recombinant APC (rAPC) as a therapeutic drug for the endothelium, we have to demonstrate its efficiency to protect or to reduce endothelium injury when infused a long time after the septic challenge. Nevertheless, if rAPC is efficient when infused in the early phase of septic challenge, we thus need to treat our patients earlier. At the least, genetically engineered variants have been designed with greater anti-apoptotic activity and reduced anticoagulant activity relative to wild-type APC. Further studies are needed to demonstrate the usefulness of these variants in septic shock therapy.

Determination of the quantity and activity of the Protein C molecule is of the utmost importance in highly purified concentrates prepared for replacement therapy. A multicenter study was undertaken to evaluate the comparability and accuracy of Protein C assays from commercial sources. Significant between-assay and interlaboratory differences were found for both functional and immunological assays. The interlaboratory variability is explained in part by the use of different control plasmas. The results also indicate the importance of the diluent used. This study emphasizes the need for standardized methods for determining the characteristics of Protein C concentrates.

Human protein C, isolated by conventional multistep methods, was used for immunization of mice. Monoclonal antibodies were prepared and screening of antibodies to human protein C was achieved using an immunoblotting technique. Five monoclonal anti-protein C antibodies were compared as affinity ligands. Different parameters were studied (adsorption capacity, specificity of adsorption, possibility of desorption under mild conditions) and two antibodies were selected. One antibody allows preparation of highly purified protein C in a single-step procedure from a fraction of plasma containing high levels of coagulation factors whereas the other can be used for preparation of protein C deficient plasma.

A variety of protein C assays are available as commercial kits. A collaborative study was undertaken to evaluate the performance of protein C assays. Various samples including calibrated plasmas and high purity concentrates destined to therapy were distributed among five laboratories. This comparison of protein C assays indicates that protein C levels measured by different functional or immunological assays and by five laboratories are very close for calibrated plasmas but not for high purity concentrates. The selection of the standard and the dilution buffer for protein C concentrates have important implications for the interpretation of the results. Dilution of purified protein C concentrates in protein C deficient plasma which restaure a total protein level similar to that of normal plasma improve the accuracy of functional protein C assays.

Our objective was to study acquired Activated Protein C (APC) resistance in patients with antiphospholipid antibodies (aPL) using a thrombin generation based assay. We compared patients with and without lupus (systemic lupus erythematosus, SLE). A parameter summarizing APC inhibition of thrombin generation with increasing APC concentrations (IC50-APC) was increased in all patient groups compared to controls: median values were 15.3 (interquartile range, IQR, 9.7 to 34.0) in patients with primary antiphospholipid syndrome (APS), 27.3 (IQR 23.5 to 43.5) in patients with SLE without APS, 64.1 (IQR 25.9 to 65.0) in patients with SLE/APS compared to 10.4 [IQR 8.5 to 15.8] in controls, respectively p = 0.003, p = 0.0001 and p = 0.0001.In conclusion, patients with SLE and primary APS displayed a hypercoagulable state characterized by APC resistance. [ABSTRACT FROM AUTHOR]

Le blocage du récepteur aux minéralocorticoides (MR) améliore la fonction artérielle et s’accompagne d’une réduction indépendante du risque cardiovasculaire. Notre objectif a été d’examiner si une hyperexpression endothéliale du MR était associée à une modification des variables hémostatiques et des marqueurs endothéliaux. Nous avons utilisé la souris double transgénique avec hyperexpression conditionnelle du MR dans l’endothélium (VE-Cadh-tTA/tetO-hMR). A l’âge de 3 mois, les souris mutantes (MR-EC) ont une concentration plasmatique en facteur von Willebrand (vWF) significativement plus élevée que les souris contrôles (CT) sans modification du CD146 soluble qui témoigne d’une dysfonction endothéliale globale sans anomalie des jonctions intercellulaires. Elle s’accompagne d’une augmentation des phospholipides anioniques dans le plasma qui reflète une réponse cellulaire apoptotique (426±59 versus 219±23nM équivalents phosphatidylsérine). La concentration en thrombine active mesurée par thrombinographie in vitro dans le plasma et l’activité de l’inhibiteur de la voie du facteur tissulaire des souris ne sont pas modifiées par rapport aux souris CT dans les conditions de base. En présence du système anticoagulant de la protéine C activée, la génération de thrombine est significativement plus faible chez les souris MR-EC (p=0,018). Les modifications portent sur la vitesse de génération explosive de thrombine (p=0,043) et la concentration maximale en thrombine (p=0,024). Ces résultats suggèrent une anomalie de la fonction endothéliale chez les souris MR-EC et un rôle du MR sur le système anticoagulant de la protéine C activée en rapport avec la réponse apoptotique. [Copyright &y& Elsevier]