Drotrecogin Alfa Research Articles

Single-Cell RNA-seq reveals transcriptomic modulation of Alzheimer's disease by activated protein C.

Single-Cell RNA sequencing reveals changes in cell population in Alzheimer's disease (AD) model 5xFAD (5x Familial AD mutation) versus wild type (WT) mice. The returned sequencing data was processed through the 10x Genomics CellRanger platform to perform alignment and form corresponding matrix to perform bioinformatic analysis. Alterations in glial cells occurred in 5xFAD versus WT, especially increases in microglia proliferation were profound in 5xFAD. Differential expression testing of glial cells in 5xFAD versus WT revealed gene regulation. Globally, the critical genes implicated in AD progression are upregulated such as Apoe, Ctsb, Trem2, and Tyrobp. Using this differential expression data, GO term enrichment was completed to observe possible biological processes impacted by AD progression. Utilizing anti-inflammatory and cyto-protective recombinant Activated Protein C (APC), we uncover inflammatory processes to be downregulated by APC treatment in addition to recuperation of nervous system processes. Moreover, animal studies demonstrated that administration of recombinant APC significantly attenuated Aβ burden and improved cognitive function of 5xFAD mice. The downregulation of highly expressed AD biomarkers in 5xFAD could provide insight into the mechanisms by which APC administration benefits AD.

Abstract 13370: Single-Cell RNA-seq Reveals Transcriptomic Modulation of Alzheimer’s Disease by Activated Protein C

Introduction: Alzheimer’s Disease (AD) causes neuroinflammation characterized by degradation of neurological functions resulting in memory loss and confusion. Activated Protein C (APC), a plasma zymogen, has shown promising cytoprotection, anti-inflammation, and anti-apoptotic properties. ScRNA-seq allows for examining the underlying mechanisms by which APC therapy on AD at the cellular level. Hypothesis: APC treatment can ameliorate AD symptoms of 5xFAD transgenic mice. The scRNA Seq analysis could provide detailed responses of various cell populations to Alzheimer's disease pathogenesis and molecular aspects of APC therapy. Methods: Whole brain tissue were collected at 7 months, APC-treated 5xFAD mice received daily recombinant APC or saline injections. The 10x Genomics Next Gem kit was used to dissociate the tissue for sequencing. Bioinformatic analysis was performed using Seurat in R and an integrated dataset was used to compare multiple single-cell samples. Results: Bioinformatic analysis of 5xFAD versus wild type (WT) C57BL/6J mouse brain revealed much of the sample consists of microglial cells. Interestingly, 5xFAD versus WT brains have notable decreases in astrocyte and neuron populations. Administration of recombinant APC rebounds these populations to comparable levels as WT while dramatically reduces the microglial population in 5xFAD versus WT brain. The largest proportion of oligodendrocytes are also recorded after APC treatment. Differential expression testing conveys transcriptomic regulation of known AD critical genes such as Apoe , Ctsb , Trem2 , and Tyrobp by APC. Consequently, GO term enrichment uncovers inflammatory biological processes such as microglial activation to be downregulated by APC-treated 5xFAD. Additionally, recuperation of nervous system developmental processes was found in APC-treated 5xFAD mice. Conclusions: APC treatment drastically impacts the cellular pathophysiology of AD. Differential expression testing of APC treated 5xFAD versus WT mice revealed transcriptomic downregulation of AD implicated genes throughout multiple cell-types. Administration of APC significantly impacts AD implicated genes associated with AD pathogenesis through enrichment analysis.

Analysis of the urinary metabolic profiles in irradiated rats treated with Activated Protein C (APC), a potential mitigator of radiation toxicity

Purpose The goal of the current study was to identify longitudinal changes in urinary metabolites following IR exposure and to determine potential alleviation of radiation toxicities by administration of recombinant APC formulations. Materials and methods Female adult WAG/RijCmcr rats were irradiated with 13.0 Gy leg-out partial body X-rays; longitudinally collected urine samples were subject to LC-MS based metabolomic profiling. Sub-cohorts of rats were treated with three variants of recombinant APC namely, rat wildtype (WT) APC, rat 3K3A mutant form of APC, and human WT APC as two bolus injections at 24 and 48 hours post IR. Results Radiation induced robust changes in the urinary profiles leading to oxidative stress, severe dyslipidemia, and altered biosynthesis of PUFAs, glycerophospholipids, sphingolipids, and steroids. Alterations were observed in multiple metabolic pathways related to energy metabolism, nucleotide biosynthesis and metabolism that were indicative of disrupted mitochondrial function and DNA damage. On the other hand, sub-cohorts of rats that were treated with rat wildtype-APC showed alleviation of radiation toxicities, in part, at the 90-day time point, while rat 3K3A-APC showed partial alleviation of radiation induced metabolic alterations 14 days after irradiation. Conclusions Taken together, these results show that augmenting the Protein C pathway and activity via administration of recombinant APC may be an effective approach for mitigation of radiation induced normal tissue toxicity.

Activated protein C inhibits mesothelial-to-mesenchymal transition in experimental peritoneal fibrosis

BackgroundIn addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) in endothelial cells. ObjectivesTo investigate the protective effect of APC in a chlorhexidine gluconate (CG)–induced peritoneal fibrosis model. MethodsPeritoneal fibrosis was induced in wild-type as well as EPCR- and PAR1-deficient mice via daily injection of CG (0.2 mL of 0.1% CG in 15% ethanol and 85% saline) for 21 days with or without concomitant injection of recombinant human APC derivatives (50 μg/kg of bodyweight). The expression of proinflammatory cytokines and profibrotic markers as well as collagen deposition were analyzed using established methods. ResultsCG significantly upregulated the expression of transforming growth factor-β1 in peritoneal tissues, which culminated in the deposition of excessive extracellular matrix proteins, thickening of the peritoneal membrane, and mesothelial-to-mesenchymal transition in damaged tissues. APC potently inhibited CG-induced peritoneal fibrosis and downregulated the expression of proinflammatory cytokines, collagen deposition, Smad3 phosphorylation, and markers of mesothelial-to-mesenchymal transition (α-smooth muscle actin, vimentin, and N-cadherin). APC also inhibited transforming growth factor-β1–mediated upregulation of α-smooth muscle actin, Smad3, and fibronectin in human primary mesothelial cells. Employing signaling-selective and anticoagulant-selective variants of APC and mutant mice deficient for either EPCR or PAR1, we demonstrated that the EPCR-dependent signaling function of APC through PAR1 activation was primarily responsible for its antifibrotic activity in the CG-induced peritoneal fibrosis model. ConclusionAPC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.

Abstract 10402: Activated Protein C Rescues Sepsis-Associated Cardiac Dysfunction and Renal Injury in Aging

Introduction: Sepsis is a progressive life-threatening condition in which mortality has been consistently linked to increasing organ dysfunction. We have found that activated protein C (APC) has cytoprotective and anti-inflammatory properties in cardiovascular disease. Whether APC is critical for maintaining systemic homeostasis under sepsis in aging remains unknown. Hypothesis: Administration of recombinant APC ameliorates sepsis-associated renal injury and cardiac dysfunction in aging via EPCR (endothelial cell protein C receptor). Methods: Young (3-6 months) and aged (18-21 months) C57BL/6J wild type (WT), and EPCR point mutation (EPCR R84A/R84A ) knock in C57BL/6J mice were subjected to cecal ligation and puncture (CLP) surgery. APC (0.2 μg/g) was injected (i.p.) 1 hour after CLP surgery and every 24 hours to determine the therapeutic effects of APC on CLP-induced sepsis. Cardiac function was assessed daily via echocardiogram. Results: The Kaplan-Meier survival curve showed that the survival rate of young mice is 90% and of aged mice is 0% on day 7 of post-CLP. Moreover, the spleens were significantly enlarged in both young and aged CLP WT mice. Intriguingly, administration of recombinant APC can keep all young animals' survival and increased survival rate of aged animals to 65% on day 7 of post-CLP. The spleen size of young/aged CLP groups were significantly reduced by APC treatment. However, the beneficial effects of APC on the survival rate and spleen enlargement caused by CLP were abolished in EPCR R84A/R84A mice versus EPCR WT/WT littermates. The renal tubules necrosis score and cardiac dysfunctions caused by CLP were worse in aged versus young mice, but APC treatment significantly rescued CLP-caused renal pathology and cardiac dysfunctions of young/aged mice and the rescuing capacity of APC for both renal pathology and cardiac dysfunctions in aged versus young mice was stronger. In addition, APC treatment reduced circulating inflammatory monocytes and neutrophils during CLP-induced sepsis in young/aged WT but not EPCR R84A/R84A mice Conclusions: Sepsis associated mortality is increased in aging. APC treatment can ameliorate age-related sepsis renal injury and cardiac dysfunctions through receptor EPCR to reduce sepsis related mortality.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat Frequency of mESC-Derived Cardiomyocyte Monolayers.

ABSTRACTBackground:Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells.Methods:Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies.Results:Twelve hours LPS treatment (5 μg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 μg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples.Conclusion:LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.

1179. Septic shock in Coccidioides immitis Infection

Background Coccidiodes can cause severe or disseminated disease in a minority of patients, but coccidioidomycosis resulting in septic shock is rare. We describe a case of fulminant C. immitis infection whose diagnosis was delayed by a markedly elevated serum procalcitonin. We review the published literature of coccidioidomycosis complicated by septic shock.Case ReportA 74-year-old man presented with cough, fever, and three weeks of progressive decline. He was febrile at 39.1°C, tachycardic, and tachypneic with crackles in the left lung; an initial procalcitonin (PCT) was 1.73 ng/mL. Broad-spectrum antibacterials were administered. His condition worsened on the 2nd day, requiring vasopressors, endotracheal intubation, and hemodialysis. PCT rose to > 400 ng/mL. Respiratory cultures grew fungi consistent with Coccidioides. Liposomal amphotericin B (AmB) was initiated. His shock progressed, and he died on hospital day 6.MethodsWe performed a MEDLINE and Google Scholar search using the terms “coccidioidomycosis”, “Coccidioides”, “sepsis”, and “septic shock”. Indexed articles and conference abstracts were included if the patient had confirmed coccidioidomycosis and vasopressor-dependent shock with no alternate cause. Data on age, sex, ethnicity, comorbidities, antifungal therapy, and outcome were tabulated.ResultsWe identified 18 reports describing 31 patients with vasopressor-dependent septic shock due to Coccidioides since 1993 (table 1). Of these cases, 23/31 (74.2%) died. Of those with reported race/ethnicity, 20/23 were Latino, 4/23 of African descent, 5/23 Asian-Pacific Islander, and 3/23 white. 27/31 (87.1%) were male. All but two were adults (median age 51.5 years). 21/31 (67.7%) had reported comorbid conditions. 20/31 patients (64.5%) and all of the survivors received AmB-based therapy.Table 1 - Published cases of coccidioidomycosis complicated by septic shock. All ages are in years. Abbreviations: HIV/AIDS = human immunodeficiency virus/acquired immunodeficiency syndrome; CAD = coronary artery disease; COPD = chronic obstructive pulmonary disease; ESRD = End stage renal disease; SLE = Systemic Lupus Erythematosus; APC = recombinant human activated protein C (drotrecogin alfa). ConclusionCoccidioidomycosis is an elusive diagnosis in critically-ill patients due to its rarity, the lack of rapid diagnostics, and its propensity to mimic other infections. Mortality is high, potentially due to delays in diagnosis. The marked elevation in PCT has not been previously noted in coccidioidomycosis and may further confound diagnosis. Improved diagnostics and the rapid institution of AmB may reduce mortality in patients with coccidioidal sepsis.Disclosures All Authors: No reported disclosures

Prone positioning in non-intubated patients with COVID-19: raising the bar

Prone positioning in non-intubated patients with COVID-19: raising the bar

Health economic evaluations of sepsis interventions in critically ill adult patients: a systematic review

BackgroundSepsis is a global health priority. Interventions to reduce the burden of sepsis need to be both effective and cost-effective. We performed a systematic review of the literature on health economic evaluations of sepsis treatments in critically ill adult patients and summarised the evidence for cost-effectiveness.MethodsWe systematically searched MEDLINE, Embase, and the Cochrane Library using thesaurus (e.g. MeSH) and free-text terms related to sepsis and economic evaluations. We included all articles that reported, in any language, an economic evaluation of an intervention for the management of sepsis in critically ill adult patients. Data extracted included study details, intervention details, economic evaluation methodology, and outcomes. Included studies were appraised for reporting quality using the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist.ResultsWe identified 50 records representing 46 economic evaluations for a variety of interventions including antibiotics (n = 5), fluid therapy (n = 2), early goal-directed therapy and other resuscitation protocols (n = 8), immunoglobulins (n = 2), and interventions no longer in clinical use such as monoclonal antibodies (n = 7) and drotrecogin alfa (n = 13). Twelve (26%) evaluations were of excellent reporting quality. Incremental cost-effectiveness ratios (ICERs) ranged from dominant (lower costs and higher effectiveness) for early goal-directed therapy, albumin, and a multifaceted sepsis education program to dominated (higher costs and lower effectiveness) for polymerase chain reaction assays (LightCycler SeptiFast testing MGRADE®, SepsiTest™, and IRIDICA BAC BSI assay). ICERs varied widely across evaluations, particularly in subgroup analyses.ConclusionsThere is wide variation in the cost-effectiveness of sepsis interventions. There remain important gaps in the literature, with no economic evaluations identified for several interventions routinely used in sepsis. Given the high economic and social burden of sepsis, high-quality economic evaluations are needed to increase our understanding of the cost-effectiveness of these interventions in routine clinical practice and to inform decision makers.Trial registrationPROSPERO CRD42018095980

The effect of activated protein C in the experimental disseminated intravascular coagulation model formed by lipopolysaccharide infusion.

Purpose:In this experimental study, activated protein C (APC), which has anticoagulant, antithrombotic, profibrinolytic, anti-inflammatory and antiapoptotic properties, was used to prevent coagulopathy in a disseminated intravascular coagulation (DIC) model formatted with lipopolysaccharide (LPS) infusion.Methods:Twenty-five Wistar albino rats weighting 280 – 320 g each were used. They were randomly divided into three groups: sham, control and study groups. To sham group (n = 5), only normal saline was infused. To control (n = 10) and study groups (n = 10), 30 mg/kg LPS was infused for 4 h from femoral vein. After LPS infusion, 100 µg/kg recombinant APC was given during 4 h in study group. Eight hours later, blood samples were taken from abdominal aorta and the animals sacrificed. From these samples, platelet, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were studied.Results:Platelet counts and fibrinogen levels were significantly lower in control and study groups than sham group (p < 0.05). The PT, aPTT and D-dimer levels were significantly higher in control and study groups than in sham group (p < 0.05). When comparing control and study groups, platelet counts were not statistically different (p = 0.36). However, the difference of the fibrinogen levels was significant between these groups (p = 0.0001). While PT and aPTT were longer in the study group compared to the control group (p < 0.05), D-dimer levels were lower in the study group than in control (p = 0.0001).Conclusion:Use of APC can prevent hypercoagulation and consumption coagulopathy in the DIC as a result of correcting hematological parameters other than prolongation of coagulation time.

Orphan designation: drotrecogin alfa (activated), Treatment of acute respiratory distress syndrome (ARDS)

Orphan designation: drotrecogin alfa (activated), Treatment of acute respiratory distress syndrome (ARDS)

Orphan designation: drotrecogin alfa (activated), Treatment of acute respiratory distress syndrome (ARDS)

Orphan designation: drotrecogin alfa (activated), Treatment of acute respiratory distress syndrome (ARDS)

Neuroprotection and vasculoprotection using genetically targeted protease-ligands

Thrombin and activated protein C (APC) are known coagulation factors that exhibit profound effects in brain by acting on the protease activated receptor (PAR). The wild type (WT) proteases appear to impact cell survival powerfully, and therapeutic forms of APC are under development. Engineered recombinant thrombin or APC were designed to separate their procoagulant or anticoagulant effects from their cytoprotective properties. We measured vascular disruption and neuronal degeneration after a standard rodent filament stroke model. For comparison to a robust anticoagulant, we used a GpIIb/IIIa inhibitor, GR144053. During 2 h MCAo both WT murine APC and its mutant, 5A-APC, significantly decreased neuronal death 30 min after reperfusion. During 4 h MCAo, only 5A-APC significantly protected neurons but both WT-APC and 5A-APC exacerbated vascular disruption during 4 h MCAo. Human APC mutants appeared to reduce 24 h neuronal injury significantly when given after 2 h delay after MCAo. In contrast, 24 h vascular damage was worsened by high doses of WT and mutant APCs, although only statistically significantly for high dose 3K3A-APC. Mutated thrombin worsened vascular damage significantly without affecting neuron damage. GR144053 failed to ameliorate vascular disruption or neuronal injury despite significant anticoagulation. Differential effects on neurons and the vasculature were demonstrated using wild-type and mutated proteases. The mutants murine 3K3A-APC and 5A-APC protected neurons in this rodent model but in high doses worsened vascular leakage. Cytoactive effects of plasma proteases may be separated from their coagulation effects. Further studies should explore impact of dose and timing on cytoactive and vasculoactive properties of these drugs.

Is tri-iodothyronine a better choice than activated protein C in sepsis treatment?

Sepsis can be defined as a life-threatening organ dysfunction due to a dysregulated host response to infection. In sepsis, the coagulation cascade is activated and the balance shifts to the procoagulant side. Recently, the use of protein C is proposed for the treatment of sepsis. Another therapeutic agent that has been intensively studied is tri-iodothyronine. This study aimed to compare the effects of activated protein C and tri-iodothyronine, which are administered at a single dose to sepsis-induced rats at the late phase. Leukocyte, platelet, hemoglobin and antithrombin-III concentrations and histopathological changes in the small intestine, liver and lung were evaluated at 24 hours. Single-dose intraperitoneal recombinant human APC (activated protein C) has a partial curative effect on hematological parameters in the late phase, while it is possible to state that it has significant therapeutic effects on hepatic and intestinal tissues, but more remarkably on the lung tissue. Tri-iodothyronine is also considered to be used for the treatment and has a strong potential to be a therapeutic agent. We observed that the T3 hormone has significantly limited and reduced the sepsis-related damage to hepatic and intestinal tissues, but especially the lung tissue. Tri-iodothyronine can be a good alternative to APC, which is partially allowed due to high cost and complication of bleeding in the treatment of sepsis.

Pharmacological Differentiation of Thrombomodulin Alfa and Activated Protein C on Coagulation and Fibrinolysis In Vitro.

Although thrombomodulin alfa (TM alfa), recombinant human soluble thrombomodulin, exerts antithrombogenic effects through activated protein C (APC), clinical trials suggested that TM alfa has a lower bleeding risk than does recombinant human APC. To address the mechanism explaining this difference, effects of TM alfa and APC on thrombogenic, coagulation, and fibrinolytic processes were compared in vitro. TM alfa and APC inhibited generation of thrombogenic markers, thrombin, and prothrombin fragment F1+2 and prolonged coagulation parameters, activated clotting time (ACT), and activated partial thromboplastin time (APTT). Concentrations of TM alfa effective for thrombin and F1+2 generation inhibition were comparable to those of APC. However, effects of TM alfa on ACT and APTT were clearly weaker than those of APC. TM alfa significantly prolonged clot lysis time (CLT) and decreased LY30, a parameter of degree of fibrinolysis in thromboelastography, whereas APC significantly shortened CLT and increased LY30. These results suggested that while the antithrombogenic effects of TM alfa were similar to those of APC, its anticoagulant effects were lower. In addition, effects of TM alfa were antifibrinolytic, while those of APC were profibrinolytic.

Hydrocortisone plus Fludrocortisone for Adults with Septic Shock

BackgroundSeptic shock is characterized by dysregulation of the host response to infection, with circulatory, cellular, and metabolic abnormalities. We hypothesized that therapy with hydrocortisone plus fludrocortisone or with drotrecogin alfa (activated), which can modulate the host response, would improve the clinical outcomes of patients with septic shock.MethodsIn this multicenter, double-blind, randomized trial with a 2-by-2 factorial design, we evaluated the effect of hydrocortisone-plus-fludrocortisone therapy, drotrecogin alfa (activated), the combination of the three drugs, or their respective placebos. The primary outcome was 90-day all-cause mortality. Secondary outcomes included mortality at intensive care unit (ICU) discharge and hospital discharge and at day 28 and day 180 and the number of days alive and free of vasopressors, mechanical ventilation, or organ failure. After drotrecogin alfa (activated) was withdrawn from the market, the trial continued with a two-group parallel design. The analysis compared patients who received hydrocortisone plus fludrocortisone with those who did not (placebo group).ResultsAmong the 1241 patients included in the trial, the 90-day mortality was 43.0% (264 of 614 patients) in the hydrocortisone-plus-fludrocortisone group and 49.1% (308 of 627 patients) in the placebo group (P=0.03). The relative risk of death in the hydrocortisone-plus-fludrocortisone group was 0.88 (95% confidence interval, 0.78 to 0.99). Mortality was significantly lower in the hydrocortisone-plus-fludrocortisone group than in the placebo group at ICU discharge (35.4% vs. 41.0%, P=0.04), hospital discharge (39.0% vs. 45.3%, P=0.02), and day 180 (46.6% vs. 52.5%, P=0.04) but not at day 28 (33.7% and 38.9%, respectively; P=0.06). The number of vasopressor-free days to day 28 was significantly higher in the hydrocortisone-plus-fludrocortisone group than in the placebo group (17 vs. 15 days, P<0.001), as was the number of organ-failure–free days (14 vs. 12 days, P=0.003). The number of ventilator-free days was similar in the two groups (11 days in the hydrocortisone-plus-fludrocortisone group and 10 in the placebo group, P=0.07). The rate of serious adverse events did not differ significantly between the two groups, but hyperglycemia was more common in hydrocortisone-plus-fludrocortisone group.ConclusionsIn this trial involving patients with septic shock, 90-day all-cause mortality was lower among those who received hydrocortisone plus fludrocortisone than among those who received placebo. (Funded by Programme Hospitalier de Recherche Clinique 2007 of the French Ministry of Social Affairs and Health; APROCCHSS ClinicalTrials.gov number, NCT00625209.)

Pharmacogenomic biomarkers do not predict response to drotrecogin alfa in patients with severe sepsis

PurposeTo explore potential design for pharmacogenomics trials in sepsis, we investigate the interaction between pharmacogenomic biomarkers and response to drotrecogin alfa (activated) (DrotAA). This trial was designed to validate whether previously identified improved response polymorphisms (IRPs A and B) were associated with an improved response to DrotAA in severe sepsis.MethodsPatients with severe sepsis at high risk of death, who received DrotAA or not, with DNA available were included and matched to controls adjusting for age, APACHE II or SAPS II, organ dysfunction, ventilation, medical/surgical status, infection site, and propensity score (probability that a patient would have received DrotAA given their baseline characteristics). Independent genotyping and two-phase data transfer mitigated bias. The primary analysis compared the effect of DrotAA in IRP+ and IRP− groups on in-hospital 28-day mortality. Secondary endpoints included time to death in hospital; intensive care unit (ICU)-, hospital-, and ventilator-free days; and overall DrotAA treatment effect on mortality.ResultsSix hundred and ninety-two patients treated with DrotAA were successfully matched to 1935 patients not treated with DrotAA. Genotyping was successful for 639 (DrotAA) and 1684 (nonDrotAA) matched patients. The primary hypothesis of a genotype-by-treatment interaction (assessed by conditional logistic regression analysis) was not significant (P = 0.30 IRP A; P = 0.78 IRP B), and there was no significant genotype by treatment interaction for any secondary endpoint.ConclusionsNeither IRP A nor IRP B predicted differential response to DrotAA on in-hospital 28-day mortality.ClinicalTrials.gov registration NCT01486524

The Immunoregulatory Activities of Activated Protein C in Inflammatory Disease.

Tissue injury prompts the initiation of host defense responses to limit blood loss, restrict pathogen entry, and promote repair. Biochemical and cellular pathways that lead to blood coagulation serve a fundamental role in generating a physical barrier at the wound site, but have also evolved to promote immune response to injury. Similarly, anticoagulant pathways that attenuate clot formation also regulate innate and adaptive immune responses. Of particular importance is activated protein C (APC) which serves as a principal regulator of thrombin generation, shapes the innate immune response to infection, and has been shown to contribute to the adaptive immune response in several preclinical models of autoimmune disease. APC controls blood coagulation by proteolytic degradation of procoagulant activated cofactors essential for fibrin clot development, but also cleaves multiple additional substrates and interacts with cell surface receptors to exert additional physiologically important roles. In this review, we focus on the molecular mechanisms utilized by APC to limit inflammation and, in particular, current understanding of the basis for APC anticoagulant and signaling activities. In particular, we provide an overview of established and emerging signaling pathways initiated by APC on endothelial cells, monocytes, neutrophils, dendritic cells, and T cells to control and regulate immune cell physiology. Finally, we consider the impact of APC activity in the context of both acute and chronic inflammatory disease, and the continuing efforts to harness the immunoregulatory properties of recombinant APC for therapeutic use.

Differential Effects of Activated Protein C on Synovial Fibroblasts in Response to Hypoxia and Normoxia

Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints and their lining or synovium. Previous studies showed that the synovium in RA patients is more hypoxic than normal synovium. Activated protein C (APC) has anticoagulant and anti-inflammatory effects and is highly expressed in the joints of RA patients. We examined the effect of APC on RA and normal synovial fibroblasts under hypoxic conditions. Human synovial fibroblasts were isolated from the synovial tissues of RA patients and normal controls and cells were exposed to recombinant APC under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. Cell proliferation was measured using MTT assays. Cell lysates and conditioned media were collected and assayed for matrix metalloproteinase (MMP)-2, MMP-9 and p38 using zymography and western blots. Proliferation of both normal and RA synovial fibroblasts dose-dependently increased after APC treatment in normoxic conditions. Under hypoxia, APC enhanced RA cell proliferation but had no effect on normal fibroblasts. MMP-2 production and activation were significantly augmented by APC in both cell types under normoxia and hypoxia conditions. However, activated MMP-2 was more reduced in cells under hypoxia than normoxia. APC substantially reduced the phosphorylation of p38 in normal and RA synovial fibroblasts under hypoxia. No difference in p38 phosphorylation was observed under normoxia. The receptor for APC, endothelial protein C receptor (EPCR), was elevated in normal fibroblasts under hypoxic conditions whereas in RA cells, EPCR was highly expressed under both normoxic and hypoxic conditions. We found that hypoxia enhanced the effect of APC on RA synovial fibroblasts through activation of MMP2 and inhibition of p38 phosphorylation. Our results suggested that APC may suppress joint destruction and progression of inflammation in a hypoxic RA environment.