Whole-Exome Sequencing in a Family with an Unexplained Tendency for Venous Thromboembolism: Multicomponent Prediction of Low-Frequency Variant Deleteriousness and of Individual Protein Interaction.

Whole-exome sequencing (WES) in families with an unexplained tendency for venous thromboembolism (VTE) may favor detection of low-frequency variants in genes with known contribution to hemostasis or associated with VTE-related phenotypes. WES analysis in six family members, three of whom affected by documented VTE, filtered for MAF < 0.04 in 192 candidate genes, revealed 22 heterozygous (16 missense and six synonymous) variants in patients. Functional prediction by multi-component bioinformatics tools, implemented by a database/literature search, including ClinVar annotation and QTL analysis, prioritized 12 missense variants, three of which (CRP Leu61Pro, F2 Asn514Lys and NQO1 Arg139Trp) were present in all patients, and the frequent functional variants FGB Arg478Lys and IL1A Ala114Ser. Combinations of prioritized variants in each patient were used to infer functional protein interactions. Different interaction patterns, supported by high-quality evidence, included eight proteins intertwined in the "acute phase" (CRP, F2, SERPINA1 and IL1A) and/or in the "fibrinogen complex" (CRP, F2, PLAT, THBS1, VWF and FGB) significantly enriched terms. In a wide group of candidate genes, this approach highlighted six low-frequency variants (CRP Leu61Pro, F2 Asn514Lys, SERPINA1 Arg63Cys, THBS1 Asp901Glu, VWF Arg1399His and PLAT Arg164Trp), five of which were top ranked for predicted deleteriousness, which in different combinations may contribute to disease susceptibility in members of this family.

Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

Platelet activation state in early stages of COVID-19.

BACKGROUND: Platelet activation at the early stage of COVID-19 is poorly described. The need for antiplatelet therapy in patients with COVID-19 remains controversial. We characterized the platelet activation profile in hospitalized patients at the early stage of COVID-19 using the modified prothrombinase Platelet Activation State (PAS) Assay. METHODS: Sixteen patients admitted to the emergency department of the IRCCS San Raffaele Hospital (Milan, Italy) between February 8 and April 2021 were enrolled. All patients presented with respiratory symptoms and tested positive for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Platelet activation was measured via the PAS Assay within 24 hours from patients' hospital admission. Data were compared with those measured in N.=24 healthy subjects (controls). RESULTS: Platelet activation was significantly higher in COVID-19 patients with respect to controls (PAS=0.63 [0.58-0.98%] vs. 0.46 [0.40-0.65%], respectively; P=0.03). Of note, highest PAS values were measured in the two patients with the worst clinical outcome, i.e., death because of respiratory failure (PAS=2.09% and 1.20%, respectively). No differences in standard coagulation parameters were noted between these two patients and those who were later discharged home. CONCLUSIONS: This study provides evidence of significant platelet activation state at the early stage of COVID-19 and suggests that the patient-specific platelet activation profile is a reliable clinical marker to stratify COVID-19 patients at high risk of poor clinical outcome who might potentially benefit from antiplatelet therapy.

Bleeding in patients with continuous-flow left ventricular assist devices: acquired von Willebrand disease or antithrombotics?

OBJECTIVES: To evaluate the competing pro-haemorrhagic contribution of acquired von Willebrand (vW) disease and antithrombotic therapy in patients implanted with continuous-flow left ventricular assist devices (LVADs). METHODS: We compared the extent of vW factor (vWf) degradation [vWf antigen (vWf:Ag)] and a decrease of functional activity of large vWf multimers [vWf collagen binding (vWf:CB)] in LVAD patients who did and did not suffer from bleeding. Data were measured pre-implant, at short-term (t1: <3 months) and long-term (t2: >12 months) follow-up. The occurrence of primary bleeding events, as well as bleeding recurrence, was correlated with patient-specific vWf profile and antithrombotic regimen. Indeed, patients were discharged on warfarin (international normalized ratio: 2-2.5) and aspirin, with the latter withhold after a first bleeding episode. RESULTS: Fifty-three patients were enrolled. The median follow-up was 324 (226-468) days. We recorded 25 primary bleeding events (47% of patients). All primary events occurred in patients on warfarin and aspirin. Both vWf:Ag and vWf:CB decreased significantly post-implant (P = 0.0003 and P < 0.0001), and patients showing pathological vWf:CB/vWf:Ag ratio (<0.7) increased progressively over the time of support (pre-implant = 26%, t1 = 58%, t2 = 74%; P < 0.0001). Of note, activity of large vWf multimers of bleeders was significantly lower at t2 with respect to non-bleeders (vWf:CB: 61 (36-115) vs 100 (68-121), P = 0.04; vWf:CB/vWf:Ag ratio: 0.36 (0.26-0.61) vs 0.58 (0.33-0.96), P = 0.04). Despite these marked differences in the vWf profile, following aspirin discontinuation only 3 patients had bleeding recurrence. CONCLUSIONS: Aspirin contributes significantly to haemorrhagic events in the background of acquired vW disease; its discontinuation significantly reduces bleeding recurrence. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT03255928; ClinicalTrials.gov Identifier: NCT03255928.

The development of various forms of lung injury with increasing tidal volume in normal rats.

Sixty-three, open-chest normal rats were subjected to mechanical ventilation (MV) with tidal volumes (VT) ranging from 7.5-39.5ml kg-1 and PEEP 2.3 cmH2O. Arterial blood gasses and pressure, and lung mechanics were measured during baseline ventilation (VT = 7.5ml kg-1) before and after test ventilation, when cytokine, von Willebrand factor (vWF), and albumin concentration in serum and broncho-alveolar lavage fluid (BALF), wet-to-dry weight ratio (W/D), and histologic injury scores were assessed. Elevation of W/D and serum vWF and cytokine concentration occurred with VT > 25ml kg-1. With VT > 30ml kg-1 cytokine and albumin concentration increased also in BALF, arterial oxygen tension decreased, lung mechanics and histology deteriorated, while W/D and vWF and cytokine concentration increased further. Hence, the initial manifestation of injurious MV consists of damage of extra-alveolar vessels leading to interstitial edema, as shown by elevated vWF and cytokine levels in serum but not in BALF. Failure of the endothelial-epithelial barrier occurs at higher stress-strain levels, with alveolar edema, small airway injury, and mechanical alterations.

Influence of Different Antithrombotic Regimens on Platelet-Mediated Thrombin Generation in Patients with Left Ventricular Assist Devices.

We characterized the biologic background of prothrombotic platelet function in the setting of durable left ventricular assist devices (LVADs) evaluating the role of different antithrombotic regimens. Platelet-mediated thrombin generation was quantified using the Platelet Activity State (PAS) Assay and the Thrombin Generation Test (TGT) in 78 patients implanted with the HeartMate II (n = 10, 13%), the HeartMate 3 (HM3) (n = 30, 38%), or the HVAD (n = 38, 49%) and managed with oral anticoagulation plus aspirin (n = 46, 59%) or anticoagulation alone (n = 32, 41%). Coagulation parameters (platelet count, International Normalized Ratio (INR), activated Partial Thromboplastin Time, Fibrinogen and D-Dimer levels) and hemolysis (lactate dehydrogenase levels [LDH]) were also recorded to comprehensively characterize the hemostatic profile in the two groups. In patients without aspirin, the PAS assay revealed low-intensity increase in platelet prothrombinase activity (1.11-fold, p = 0.03). Similarly the TGT revealed moderate higher platelet reactivity when compared with patients receiving aspirin, consistent with reduction in lag time (0.87-fold, p < 0.001), increase in peak of thrombin generation (1.5-fold, p = 0.002) and thrombin generation rate (2-fold, p = 0.02), but comparable endogenous thrombin potential (p = 0.50). Coagulation parameters and LDH were comparable in the two groups (p > 0.05). Moreover, no differences were noted in platelet prothrombinase activity of patients implanted with the HM3 or HVAD. Our results suggest that, in the setting of durable LVADs, aspirin minimally modulates the biochemical pathway of platelet-mediated thrombin generation. Accordingly, re-evaluation of current antithrombotic management criteria in patients stratified according to bleeding/thromboembolic risk might be safe and beneficial to prevent adverse events.

Missense changes in the catalytic domain of coagulation factor X account for minimal function preventing a perinatal lethal condition.

INTRODUCTION: Inherited deficiencies in the coagulation pathway provide diversified models to investigate the molecular bases of perinatal lethality associated with null-like variants. Differently from X-linked haemophilias, homozygous/doubly heterozygous null variants in the rare autosomally inherited deficiency of factor X (FX) might be incompatible with perinatal survival. AIM: To provide experimental evidence about the null/close-to-null FX function. METHODS: The residual secreted (ELISA) and functional (thrombin generation assays) protein levels associated with the novel nonsense (c.1382G>A; p.Trp461Ter) and missense (c.752T>C; p.Leu251Pro) variants, found in the proposita with life-threatening symptoms at birth, were characterized through recombinant (r)FX expression. RESULTS: The rFX-461Ter showed very low secretion and undetectable function. Expression and function of the predicted readthrough-deriving missense variants (rFX-461Tyr, rFX-461Gln) were also severely impaired. These unfavourable features, due to nucleotide and protein sequence constraints, precluded functional readthrough over the 461 stop codon. Differently, the poorly secreted rFX-251Pro variant displayed residual function that was characterized by anti-TFPI aptamer-based amplification or selective inhibition of activated FX function by fondaparinux in plasma and found to be reduced by approximately three orders of magnitude. Similarly to the rFX-251Pro, a group of catalytic domain missense variants cause poorly secreted molecules with modest function in FX-deficient patients with life-threatening symptoms. CONCLUSIONS: Our data, contributing to the knowledge of the very severe FX deficiency forms, support life-saving requirement of trace FX function, clearly exemplified by the dysfunctional but not completely inactive rFX-251Pro variant that, albeit with severely reduced function, is compatible with a residual activity ensuring minimal haemostasis and permitting perinatal survival.

Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome.

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

Which Antiplatelet Therapy in Patients With Left Ventricular Assist Device and Aspirin Allergy?

In patients with left ventricular assist device support and aspirin allergy, the choice of effective antiplatelet strategy remains a challenge. We compared the antithrombotic effect of clopidogrel vs ticagrelor in an LVAD patient with aspirin allergy by using a modified protocol of the thrombin generation test, accounting selectively for the platelet contribution on thrombin generation. Our results demonstrate enhanced antithrombotic efficacy offered by ticagrelor. Consistent with experimental results, the patient has passed more than 300 days without thromboembolic complications. This study provides additional mechanistic rationale supporting clinical evidence and opens the perspective to identify individual poor responsiveness to drugs by specifically evaluating drug-mediated platelet function.

Platelet activation is a preoperative risk factor for the development of thromboembolic complications in patients with continuous-flow left ventricular assist device.

AIMS: To correlate the dynamics of platelet activation with the development of thromboembolic events in patients with continuous-flow left ventricular assist device (cf-LVAD). METHODS AND RESULTS: The platelet activity state (PAS) assay was utilized to evaluate platelet activation in 68 cf-LVAD patients implanted with the HeartMate II (n = 15, 22%), HeartMate 3 (n = 15, 22%), or HeartWare HVAD (n = 38, 56%). PAS was measured preoperatively, early post-implant, and at long-term follow-up (1, 3, 6, 12, 18, and 24 months post-implant). PAS was also measured at the occurrence of adverse events in patients who developed thrombotic complications. Data on patient demographics, medical history, antithrombotic therapy, and coagulation parameters were also analysed. Over a median follow-up of 602 (234-942) days, PAS values did not increase over time in the overall population (P = 0.15). However, PAS measured at event was 15-fold higher in the six patients (9%) who suffered pump thrombosis (n = 2) or ischaemic stroke (n = 4) vs. the rest of the population [6.67% (5.59%-11.98%) vs. 0.45% (0.33%-0.75%); P = 0.012], despite comparable coagulation profile. Pre-implant PAS values were 4.5-fold higher in these patients [1.90% (1.24%-3.17%) vs. 0.42% (0.32%-0.72%); P = 0.006]. Neither preoperative variables nor the type of the pump or the antiplatelet strategy were associated with a higher risk of complications. CONCLUSIONS: Thrombotic events are associated with altered PAS values. Moreover, baseline elevated PAS values in patients who developed thrombotic events suggest patient-specific tendency to post-implant thromboembolic complications. Prospectively, systematic monitoring of PAS might guide the development of refined patient-tailored antithrombotic strategies and the technological improvement of LVAD design.

Low-grade endotoxemia and clotting activation in the early phase of pneumonia.

BACKGROUND AND OBJECTIVE: Community-acquired pneumonia (CAP) is associated with an increased risk of arterial and venous thrombosis but the underlying pathophysiological mechanisms are still unclear. We investigated if, in patients with CAP, a pro-thrombotic state does exist and its relationship with serum levels of endotoxins. METHODS: A total of 104 consecutive patients with CAP were prospectively recruited and followed up until discharge. At admission and at discharge, serum endotoxins, systemic markers of clotting activation and zonulin, a marker of gut permeability, were analysed. Hospitalized patients matched for gender, age and comorbidities but without infections were used as control. RESULTS: At admission, CAP patients showed higher plasma levels of F1+2 , a marker of thrombin generation (P = 0.023), and lower levels of protein C (PC; P < 0.001) and activated PC (aPC) (P < 0.001) compared with controls. At discharge, plasma levels of both PC and aPC significantly increased while F1+2 significantly decreased (P < 0.001). Baseline serum endotoxins and zonulin were higher in CAP patients than controls (P < 0.001) and significantly decreased at discharge; a significant correlation between serum endotoxins and zonulin was detected (R = 0.575; P < 0.001) CONCLUSION: This study provides the first evidence that CAP patients disclose an ongoing pro-thrombotic state and suggests a role for endotoxemia in determining enhanced thrombin generation.

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.

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.

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.

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.

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-α, interleukin-1ß, 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-α, interleukin-1ß, 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.

Human protein C zymogen concentrate in patients with severe sepsis and multiple organ failure after adult cardiac surgery.

PURPOSE: To describe outcome and changes in clotting and inflammatory parameters in an uncontrolled case series of consecutive patients with severe sepsis who received protein C concentrate soon after cardiac surgery. METHODS: From January 2007 to January 2008 nine consecutive adult patients with severe sepsis or septic shock and two or more organ failure after cardiac surgery received protein C concentrate, 50 IU/kg as a bolus followed by continuous infusion of 3 IU/kg per hour for 72 h. RESULTS: The increase in protein C levels was accompanied by an early drop in interleukins and near-normalization of prothrombin time, activated partial thromboplastin time, antithrombin and thrombin-antithrombin complex levels (p < or = 0.03). No patient experienced drug-related side effects. Thirty-day mortality was 11% (1 patient) compared to the expected mortality of 68%. CONCLUSIONS: In this pilot, uncontrolled study of nine patients with sepsis-induced double organ failure following cardiac surgery, treatment with protein C concentrate was associated with significant improvement in clinical, inflammatory and clotting parameters, no bleeding and low 30-day mortality.

Cytokine release, small airway injury, and parenchymal damage during mechanical ventilation in normal open-chest rats.

Lung morpho-functional alterations and inflammatory response to various types of mechanical ventilation (MV) have been assessed in normal, anesthetized, open-chest rats. Measurements were taken during protective MV [tidal volume (Vt) = 8 ml/kg; positive end-expiratory pressure (PEEP) = 2.6 cmH(2)O] before and after a 2- to 2.5-h period of ventilation on PEEP (control group), zero EEP without (ZEEP group) or with administration of dioctylsodiumsulfosuccinate (ZEEP-DOSS group), on negative EEP (NEEP group), or with large Vt (26 ml/kg) on PEEP (Hi-Vt group). No change in lung mechanics occurred in the Control group. Relative to the initial period of MV on PEEP, airway resistance increased by 33 +/- 4, 49 +/- 9, 573 +/- 84, and 13 +/- 4%, and quasi-static elastance by 19 +/- 3, 35 +/- 7, 248 +/- 12, and 20 +/- 3% in the ZEEP, NEEP, ZEEP-DOSS, and Hi-Vt groups. Relative to Control, all groups ventilated from low lung volumes exhibited histologic signs of bronchiolar injury, more marked in the NEEP and ZEEP-DOSS groups. Parenchymal and vascular injury occurred in the ZEEP-DOSS and Hi-Vt groups. Pro-inflammatory cytokine concentration in the bronchoalveolar lavage fluid (BALF) was similar in the Control and ZEEP group, but increased in all other groups, and higher in the ZEEP-DOSS and Hi-Vt groups. Interrupter resistance was correlated with indexes of bronchiolar damage, and cytokine levels with vascular-alveolar damage, as indexed by lung wet-to-dry ratio. Hence, protective MV from resting lung volume causes mechanical alterations and small airway injury, but no cytokine release, which seems mainly related to stress-related damage of endothelial-alveolar cells. Enhanced small airway epithelial damage with induced surfactant dysfunction or MV on NEEP can, however, contribute to cytokine production.