Patrizia Della Valle

Activation of coagulation and fibrinolysis during coronary surgery: on-pump versus off-pump techniques.

Background The authors studied the changes in selected hemostatic variables in patients undergoing coronary surgery with on-pump coronary artery bypass grafting (CABG) or off-pump coronary artery bypass surgery (OPCAB) techniques. Methods Platelet counts and plasma concentrations of antithrombin, fibrinogen, D dimer, &agr;2 antiplasmin, and plasminogen were measured preoperatively, 5 min after administration of heparin, 10 min after arrival in the intensive care unit, and 24 h after surgery in patients scheduled to undergo OPCAB (n = 15) or CABG (n = 15). To correct for dilution, hemostatic variables and platelet counts were adjusted for the changes in immunoglobulin G plasma concentrations and hematocrit, respectively. Results Adjusting for dilution, antithrombin and fibrinogen concentrations decreased to a similar extent in patients undergoing OPCAB or CABG (pooled means and 95% confidence limits of the mean: 95.5% of baseline, 93–98%, P = 0.002, and 91.7% of baseline, 88–95%, P = 0.0001), respectively, whereas &agr;2-antiplasmin concentrations were unchanged. Only CABG was associated with a reduction in platelet counts (76% of baseline, 66–85%, P = 0.0001), plasminogen concentrations (96% of baseline, 91–99%, P = 0.011), and increased D-dimer formation (476%, 309–741%, P = 0.004). Twenty-four hours after surgery, platelet counts were still lower in patients undergoing CABG (P = 0.049), but all the investigated variables adjusted for dilution were similar in the two groups. Conclusions Coronary surgery causes a net consumption of antithrombin and fibrinogen. A transient decrease in platelet counts, with plasminogen activation and increased D-dimer formation, however, is only observed with CABG. Twenty-four hours after surgery, the hemostatic profiles of patients in both groups are similar.

Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.

Liver-directed lentiviral gene therapy in a dog model of hemophilia B

Gene therapy with lentiviral vectors targeting transgene expression to hepatocytes provides stable reconstitution of clotting activity in dogs with hemophilia B and does not show genotoxicity in tumor-prone mice. Advancing gene therapy for hemophilia Hemophilia is an inherited bleeding disorder caused by a deficiency in a blood clotting factor. The current treatment requires lifelong intravenous administration of the missing clotting factor every few days, a costly and demanding regimen for patients with hemophilia. Gene therapy has the potential to provide a single-shot treatment option by replacing a functional gene in liver cells that naturally produce the factor. Cantore et al. now report a study of the efficacy and safety of liver-directed in vivo gene therapy in large and small animal models using lentiviral vectors. This gene therapy strategy with lentiviral vectors may complement the use of other gene therapy vectors for treating hemophilia. We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.

Liver gene therapy by lentiviral vectors reverses anti-factor IX pre-existing immunity in haemophilic mice.

A major complication of factor replacement therapy for haemophilia is the development of anti‐factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre‐existing anti‐FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50–100% of normal. The treatment was effective in 75% of treated mice. FIX‐specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B‐cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX‐specific suppressive capacity were induced in gene therapy treated mice and controlled FIX‐specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high‐dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors‐positive haemophilia B mice. Liver gene therapy can thus reverse pre‐existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors‐positive haemophilic patients.

Maintaining end-expiratory transpulmonary pressure prevents worsening of ventilator-induced lung injury caused by chest wall constriction in surfactant-depleted rats.

Objective:To see whether in acute lung injury 1) compression of the lungs caused by thoracoabdominal constriction degrades lung function and worsens ventilator-induced lung injury; and 2) maintaining end-expiratory transpulmonary pressure by increasing positive end-expiratory pressure reduces the deleterious effects of chest wall constriction. Design:Experimental study in rats. Setting:Physiology laboratory. Interventions:Acute lung injury was induced in three groups of nine rats by saline lavage. Nine animals immediately killed served as a control group. Group L had lavage only, group LC had the chest wall constricted with an elastic binder, and group LCP had the same chest constriction but with positive end-expiratory pressure raised to maintain end-expiratory transpulmonary pressure. After lavage, all groups were ventilated with the same pattern for 1½ hrs. Measurements and Main Results:Transpulmonary pressure, measured with an esophageal balloon catheter, lung volume changes, arterial blood gasses, and pH were assessed during mechanical ventilation. Lung wet-to-dry ratio, albumin, tumor necrosis factor-&agr;, interleukin-1&bgr;, interleukin-6, interleukin-10, and macrophage inflammatory protein-2 in serum and bronchoalveolar lavage fluid and serum E-selectin and von Willebrand Factor were measured at the end of mechanical ventilation. Lavage caused hypoxemia and acidemia, increased lung resistance and elastance, and decreased end-expiratory lung volume. With prolonged mechanical ventilation, lung mechanics, hypoxemia, and wet-to-dry ratio were significantly worse in group LC. Proinflammatory cytokines except E-selectin were elevated in serum and bronchoalveolar lavage fluid in all groups with significantly greater levels of tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6 in group LC, which also exhibited significantly worse bronchiolar injury and greater heterogeneity of airspace expansion at a fixed transpulmonary pressure than other groups. Conclusions:Chest wall constriction in acute lung injury reduces lung volume, worsens hypoxemia, and increases pulmonary edema, mechanical abnormalities, proinflammatory mediator release, and histologic signs of ventilator-induced lung injury. Maintaining end-expiratory transpulmonary pressure at preconstriction levels by adding positive end-expiratory pressure prevents these deleterious effects.

Variable interference of activated protein C resistance in the measurement of protein S activity by commercial assays

Congenital and acquired deficiencies of vitamin K-dependent protein S (PS) are an established risk factor for thrombotic disease (1,2). Due to the multiple PS domains involved in the interaction with other plasma proteins and the expression of activated protein C (APC) cofactor activity, the existence of qualitative defects of PS is anticipated (3). Thus, measurement of PS anticoagulant activity should be preferred to PS antigens determination in the identification of both congenital and acquired deficiencies. At present, two commercial assays are available for the measurement of the APC cofactor activity of PS. with one of these assays qualitative protein S deficiencies had been erroneously identified (4), as originally indicated by a multicenter collaborative study comparing different functional PS assays (5). In the latter study, plasma samples obtained from patients reportedly affected by qualitative PS deficiencies showed virtual coincidence of free PS antigen and anticoagulant activity levels only when this was evaluated after immunoadsorption of PS (6). This observation raised the possibility of artifacts related to PS testing in whole plasma (5). Following the description of APC resistance a new marker of congenital thrombophilia characterized by defective clotting time prolongation upon additon of APC to plasma (7) -, the seemingly PS deficient patients have been correctly diagnosed as APC resistant cases, with the conclusion that APC resistance may interfere in PS activity testing by the available commercial methods (4). To explore the extent of such interference, we have conducted a prospective comparison of two commercial functional PS assays and our home-made assay (6) in a series of patients with APC resistance referred to our Institution for the evaluation of a thrombophilic state.

The protein C pathway and sepsis.

After the discovery of the key components of the protein C (PC) pathway a beneficial effect on survival of the infusion of activated protein C (APC) in animal models of sepsis was demonstrated, leading to the development of recombinant human activated protein C (rh-APC) as a therapeutic agent. It soon became clear that rather than the anticoagulant and profibrinolytic activities of APC, its anti-inflammatory and cytoprotective properties played a major role in the treatment of patients with severe sepsis. Such properties affect the response to inflammation of endothelial cells and leukocytes and are exerted through binding of APC to at least five receptors with intracellular signaling. The main APC protective mechanism involves binding of the Gla-domain to the endothelial protein C receptor (EPCR) and cleavage of protease activated receptor 1 (PAR-1), eliciting suppression of proinflammatory cytokines synthesis and of intracellular proapoptotic pathways and activation of endothelial barrier properties. However, thrombin cleaves PAR-1 with much higher catalytic efficiency, followed by pro-inflammatory, pro-apoptotic and barrier disruptive intracellular signaling, and it is unclear how APC can exert a protective activity through the cleavage of PAR-1 when thrombin is also present in the same environment. Interestingly, in endothelial cell cultures, PAR-1 cleavage by thrombin results in anti-inflammatory and barrier protective signaling provided occupation of EPCR by the PC gla-domain, raising the possibility that the beneficial effects of rh-APC might be recapitulated in vivo by administration of h-PC zymogen to patients with severe sepsis. Recent reports of h-PC infusion in animal models of sepsis support this hypothesis.

Disappearance of anti-PF4/heparin antibodies under prolonged fondaparinux administration in a patient with DVT associated with LMWH-induced thrombocytopenia

Disappearance of anti-PF4/heparin antibodies under prolonged fondaparinux administration in a patient with DVT associated with LMWH-induced thrombocytopenia -

The fall in exhaled nitric oxide with ventilation at low lung volumes in rabbits: an index of small airway injury.

The mechanisms involved in the fall of exhaled nitric oxide (NOe) concentration occurring in normal, anesthetized open chest rabbits with prolonged mechanical ventilation (MV) at low lung volume have been investigated. NOe, pH of exhaled vapor condensate, serum prostaglandin E(2), and F(2alpha), tumor necrosis factor (TNF-alpha), PaO(2), PaCO(2), pHa, and lung mechanics were assessed before, during, and after 3-4h of MV at zero end-expiratory pressure (ZEEP), with fixed tidal volume (9 ml kg(-1)) and frequency, as well as before and after 3-4h of MV on PEEP only. Lung histology and wet-to-dry ratio (W/D), and prostaglandin and TNF-alpha in bronchoalveolar lavage fluid (BALF) were also assessed. While MV on PEEP had no effect on the parameters above, MV on ZEEP caused a marked fall (45%) of NOe, with a persistent increase of airway resistance (45%) and lung elastance (12%). Changes in NOe were independent of prostaglandin and TNF-alpha levels, systemic hypoxia, hypercapnia and acidosis, bronchiolar and alveolar interstitial edema, and pH of exhaled vapor condensate. In contrast, there was a significant relationship between the decrease in NOe and bronchiolar epithelial injury score. This indicates that the fall in NOe, which occurs in the absence of an inflammatory response, is due to the epithelial damage caused by the abnormal stresses related to cyclic opening and closing of small airways with MV on ZEEP, and suggests its use as a sign of peripheral airway injury.

Effects of Various Modes of Mechanical Ventilation in Normal Rats

Background:Recent studies in healthy mice and rats have reported that positive pressure ventilation delivered with physiological tidal volumes at normal end-expiratory volume worsens lung mechanics and induces cytokine release, thus suggesting that detrimental effects are due to positive pressure ventilation per se. The aim of this study in healthy animals is to assess whether these adverse outcomes depend on the mode of mechanical ventilation. Methods:Rats were subjected to 4 h of spontaneous, positive pressure, and whole-body or thorax-only negative pressure ventilation (N = 8 per group). In all instances the ventilatory pattern was that of spontaneous breathing. Lung mechanics, cytokines concentration in serum and broncho–alveolar lavage fluid, lung wet-to-dry ratio, and histology were assessed. Values from eight animals euthanized shortly after anesthesia served as control. Results:No evidence of mechanical ventilation–dependent lung injury was found in terms of lung mechanics, histology, or wet-to-dry ratio. Relative to control, cytokine levels and recruitment of polymorphonuclear leucocytes increased slightly, and to the same extent with spontaneous, positive pressure, and whole-body negative pressure ventilation. Thorax-only negative pressure ventilation caused marked chest and lung distortion, reversible increase of lung elastance, and higher polymorphonuclear leucocyte count and cytokine levels. Conclusion:Both positive and negative pressure ventilation performed with tidal volumes and timing of spontaneous, quiet breathing neither elicit an inflammatory response nor cause morpho-functional alterations in normal animals, thus supporting the notion of the presence of a critical volume threshold above which acute lung injury ensues. Distortion of lung parenchyma can induce an inflammatory response, even in the absence of volotrauma.