Mario Colucci

Cultured human endothelial cells generate tissue factor in response to endotoxin.

Bacterial infection is associated with disseminated intravascular coagulation and fibrin deposition in the microcirculation; the mechanism of these effects in humans is still unclear. We have studied the generation of procoagulant activity (PCA) by cultured human endothelial cells (EC) in response to endotoxin. Cells from umbilical cord veins were grown in Eagles minimum essential medium with 20% fetal calf serum till confluence. Absence of fibroblasts and macrophages was carefully checked. Endotoxin (Salmonella enteritidis lipopolysaccharide (LPS) W or Escherichia coli 0111:B4 LPS W, 0.01-1.0 micrograms/ml) was added to culture dishes for 4-6 h. PCA of EC was measured by a one-stage clotting assay and/or a two-stage amidolytic assay with the chromogenic substrate S-2222. In the absence of endotoxin, EC generated little, if any PCA (2-5 units/10(5) cells). In contrast, the addition of endotoxin resulted in generation of strong PCA that reached a maximum within 4-6 h (185-241 units/10(5) cells) and was dose-dependent between 1 and 0.01 microgram endotoxin/ml of culture medium. The generation of PCA required RNA and protein synthesis but did not require the presence of serum. No activity was found in the culture medium. The activity was of tissue thromboplastin type, as indicated by biological and immunological criteria. These endotoxin effects were observed in the absence of endothelial damage, as shown by phase-contrast microscopy and lack of 51Cr release. These data could contribute to elucidate the pathogenesis of vascular complications associated with endotoxemia in man.

Sepsis, thrombosis and organ dysfunction.

Sepsis is often associated with haemostatic changes ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by widespread microvascular thrombosis and subsequent consumption of platelets and coagulation proteins, eventually causing bleeding manifestations. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. The latter, along with the micro-organism and its derivatives are now believed to drive the major changes responsible for massive thrombin formation and fibrin deposition, namely 1) the aberrant expression of the TF by different cells (especially monocytes-macrophages), 2) the impairment of physiological anticoagulant pathways, orchestrated mainly by dysfunctional endothelial cells (ECs) and 3) the suppression of fibrinolysis due to overproduction of plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). The ensuing microvascular thrombosis and ischemia are thought to contribute to tissue injury and multiple organ dysfunction syndrome (MODS). Recent evidence indicates that extracellular nuclear materials released from activated and especially apoptotic or necrotic cells, e.g. High Mobility Group Box-1 (HMGB-1) and histones, are endowed with cell toxicity, proinflammatory and clot-promoting properties and thus, during sepsis, they may represent late mediators that propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenesis of DIC and MODS may have implications for the development of new therapeutic agents potentially useful for the management of severe sepsis.

Effectiveness of Hepatitis B Vaccination in Babies Born to Hepatitis B Surface Antigen-Positive Mothers in Italy

This study examined 522 children born to hepatitis B surface antigen (HBsAg)-positive mothers from 1985 through 1994 and evaluated the protection provided by anti-hepatitis B virus (HBV) immunization at birth. Babies were given hepatitis B immunoglobulin and hepatitis B vaccine at birth. At 5-14 years after immunization, 17 children (3.3%) were anti-HB core antigen positive, and 3 also were HBsAg positive. One carrier child had a double mutation, with substitution of proline-->serine at codons 120 (P120S) and 127 (P127S) within the a determinant of HBsAg. Of the 522 children, 400 (79.2%) of 505 still had protective anti-HBsAg titers > or =10 mIU/mL. Thus, HBV vaccination of children born to HBsAg-positive mothers is effective and confers long-term immunity. There is no evidence that the emergence of HBV escape mutants secondary to the immune pressure against wild-type HBV is of concern.

Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein c reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation.

Activated protein C (APC) is a potent physiologic anticoagulant with profibrinolytic properties, and has been shown to prevent thrombosis in different experimental models. We investigated the effect of human APC on thrombin-induced thromboembolism in mice, a model of acute intravascular fibrin deposition leading to death within minutes. APC given intravenously (i.v.) as a bolus 2 min before thrombin challenge (1,250 U/kg) reduced mortality in a dose-dependent manner despite the lack of thrombin inhibitor activity. Significant inhibition of thrombin-induced death was observed at the dose of 0.05 mg/kg, and maximal protection was obtained with 2 mg/kg (> 85% reduction in mortality rate). Histology of lung tissue revealed that APC treatment (2 mg/kg) reduced significantly vascular occlusion rate (from 89.2 to 46.6%, P < 0.01). The protective effect of APC was due to the inhibition of endogenous thrombin formation as indicated by the fact that (a) the injection of human thrombin caused a marked decrease in the coagulation factors of the intrinsic and common pathways (but not of Factor VII), suggesting the activation of blood clotting via the contact system; (b) APC pretreatment reduced markedly prothrombin consumption; (c) the lethal effect of thrombin was almost abolished when the animals were made deficient in vitamin K-dependent factors by warfarin treatment, and could be restored only by doubling the dose of thrombin, indicating that the generation of endogenous thrombin contributes significantly to death; and (d) APC failed to protect warfarin-treated animals, in which mortality is entirely due to injected thrombin, even after protein S supplementation. Other results suggest that APC protects from thrombin-induced thromboembolism by rendering the formed fibrin more susceptible to plasmin degradation rather than by reducing fibrin formation: in thrombin-treated mice, fibrinogen consumption was not inhibited by APC; and inhibition of endogenous fibrinolysis by epsilon-aminocaproic or tranexamic acid resulted in a significant reduction of the protective effect of APC. Since APC did not enhance plasma fibrinolytic activity, as assessed by the measurement of plasminogen activator (PA) or PA inhibitor (PAI) activities, PAI-1 antigen, or 125I-fibrin degrading activity, we speculate that the inhibition of additional (endogenous) thrombin formation by APC interrupts thrombin-dependent mechanisms that make fibrin clots more resistant to lysis, so that the intravascular deposited fibrin can be removed more rapidly by the endogenous fibrinolytic system.

Dabigatran enhances clot susceptibility to fibrinolysis by mechanisms dependent on and independent of thrombin‐activatable fibrinolysis inhibitor

Summary.  Background: Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin‐activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial. Objective: To evaluate the profibrinolytic effect of dabigatran, a new, direct thrombin inhibitor, using different in vitro models. Methods and Results: The resistance of tissue factor‐induced plasma clots to fibrinolysis by exogenous tissue‐type plasminogen activator (t‐PA) (turbidimetric method) was reduced by dabigatran in a concentration‐dependent manner, with ≥ 50% shortening of lysis time at clinically relevant concentrations (1–2 μm). A similar effect was observed in the presence of low (0.1 and 1 nm) but not high (10 nm) concentrations of thrombomodulin. Acceleration of clot lysis by dabigatran was associated with a reduction in TAFI activation and thrombin generation, and was largely, although not completely, negated by an inhibitor of activated TAFI, potato tuber carboxypeptidase inhibitor. The assessment of the viscoelastic properties of clots showed that those generated in the presence of dabigatran were more permeable, were less rigid, and consisted of thicker fibers. The impact of these physical changes on fibrinolysis was investigated using a model under flow conditions, which demonstrated that dabigatran made the clots markedly more susceptible to flowing t‐PA, by a mechanism that was largely TAFI‐independent. Conclusions: Dabigatran, at clinically relevant concentrations, enhances the susceptibility of plasma clots to t‐PA‐induced lysis by reducing TAFI activation and by altering the clot structure. These mechanisms might contribute to the antithrombotic activity of the drug.

Helicobacter pylori Neutrophil-Activating Protein Stimulates Tissue Factor and Plasminogen Activator Inhibitor-2 Production by Human Blood Mononuclear Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that activates phagocytic NADPH-oxidase. The effect of HP-NAP on the production of tissue factor (TF), plasminogen activator inhibitor-2 (PAI-2), and urokinase-type plasminogen activator (u-PA) by human blood mononuclear cells (MNC) was evaluated by using functional and immunological assays and mRNA analysis. HP-NAP induced time- and dose-dependent increases in TF and PAI-2, with a maximal effect at 300 nmol/L (>15-fold increase in antigens). No changes in u-PA were observed. When whole bacteria were used, an H. pylori mutant lacking HP-NAP was significantly less active than the wild-type strain. MNC from a patient with chronic granulomatous disease behaved as do normal cells, which indicates that HP-NAP effects can occur independently of NADPH-oxidase. HP-NAP, by inducing the coordinate expression of cell procoagulant and antifibrinolytic activities, might favor fibrin deposition and contribute to the inflammatory reaction of gastric mucosa elicited by H. pylori.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression1

Summary.  Background: NO‐releasing statins are new chemical entities, combining HMG‐CoA reductase inhibition and slow NO release, that possess stronger anti‐inflammatory and antiproliferative activities than the native statins. Objective: We evaluated the antithrombotic effects of nitropravastatin (NCX‐6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. Methods and results: In vitro, NCX‐6550 inhibited (1) U46619‐ and collagen‐induced platelet aggregation in buffer and plasma; (2) collagen‐induced P‐selectin expression in whole blood and (3) platelet adhesion to collagen‐coated coverslips under high shear stress. These effects were displayed at concentrations of NCX‐6550 ranging from 25 to 100 μm, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 μm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS‐ and PMA‐induced TF expression by blood mononuclear cells was also inhibited by NCX‐6550 (IC50 13 μm), but not by pravastatin, as assessed by functional and immunological assays and by real‐time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX‐6550 (24–48 mg kg−1) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. Conclusions: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Warfarin inhibits both procoagulant activity and metastatic capacity of Lewis Lung Carcinoma cells: Role of vitamin K deficiency

Chronic vitamin K deficiency, either dietary or pharmacologically induced with warfarin, depressed significantly the growth of lung secondaries in a spontaneously metastasizing murine tumor, the Lewis Lung Carcinoma. This effect was associated with a marked depression of the procoagulant activity of cancer cells, which could contribute to fibrin deposition around the tumor. Cellular anticoagulation may thus be an important mechanism in the antimetastatic effect of warfarin.

Factors affecting the compliance of the antenatal hepatitis B screening programme in Italy

The effectiveness in the prevention of perinatally transmitted HBV infection was assessed in 11858 pregnant women consecutively recruited in public and private hospitals in six Italian regions during a 2 months period in 2001. Of them 10881 (91.8%) attended HBsAg antenatal screening. The overall HBsAg prevalence was 1.7% (CI 95%: 1.4-1.9); it was 1.4% (CI 95%: 1.2-1.7) in pregnant women born in Italy but 5.9% (CI 95%: 4.1-8.1) in those born in Asia, Africa, central and south America, and eastern Europe. Results of multiple logistic regression analysis indicate that birth in foreign countries (OR 2.0; CI 95%: 1.3-3.0), family size with more than 4 members in the household (OR 3.5; CI 95%:2.7-4.6), and birth in a private hospital (OR 1.9; CI 95%: 1.3-2.8) were all independent predictors of lack of adherence to HBsAg screening. Out of the 182 new-borns of HBsAg positive mothers 172 (95.0%) were given active plus passive immunisation; this figure was 100% in new-borns of foreign mothers. These findings evidence a good effectiveness in the prevention of perinatally transmitted HBV in Italy. More efforts should be addressed to improve the effectiveness of the programme among foreign pregnant women who have high rate of HBsAg and more likely escape HBsAg screening than Italian pregnant women.

Thrombin activatable fibrinolysis inhibitor: At the nexus of fibrinolysis and inflammation

TAFI (thrombin activatable fibrinolysis inhibitor) is the precursor of a basic carboxypeptidase (TAFIa) with strong antifibrinolytic and anti-inflammatory activity. Compelling evidence indicates that thrombin, either alone or in complex with thrombomodulin, is the main physiological activator of TAFI. For this reason derangements of thrombin formation, whatever the cause, may influence the fibrinolytic process too. Experimental models of thrombosis suggest that TAFI may participate in thrombus development and persistence under certain circumstances. In several models of pharmacological thrombolysis, the administration of TAFI inhibitors along with the fibrinolytic agent leads to a marked improvement of thrombus lysis, underscoring the potential of TAFI inhibitors as adjuvants for thrombolytic therapy. The role of TAFI in inflammatory diseases is more complex as it may serve as a defense mechanism, exacerbate the disease, or have no influence, depending on the nature of the model and the role played by the mediators controlled by TAFIa. Finally, the numerous clinical studies in patients with thrombotic disease support the idea that increased levels of TAFI and/or the enhancement of TAFI activation may represent a new risk factor for venous and arterial thrombosis.