Hydrocortisone plus fludrocortisone for community acquired pneumonia-related septic shock: a subgroup analysis of the APROCCHSS phase 3 randomised trial.

BACKGROUND: Glucocorticoids probably improve outcomes in patients hospitalised for community acquired pneumonia (CAP). In this a priori planned exploratory subgroup analysis of the phase 3 randomised controlled Activated Protein C and Corticosteroids for Human Septic Shock (APROCCHSS) trial, we aimed to investigate responses to hydrocortisone plus fludrocortisone between CAP and non-CAP related septic shock. METHODS: APROCCHSS was a randomised controlled trial that investigated the effects of hydrocortisone plus fludrocortisone, drotrecogin-alfa (activated), or both on mortality in septic shock in a two-by-two factorial design; after drotrecogin-alfa was withdrawn on October 2011, from the market, the trial continued on two parallel groups. It was conducted in 34 centres in France. In this subgroup study, patients with CAP were a preselected subgroup for an exploratory secondary analysis of the APROCCHSS trial of hydrocortisone plus fludrocortisone in septic shock. Adults with septic shock were randomised 1:1 to receive, in a double-blind manner, a 7-day treatment with daily administration of intravenous hydrocortisone 50 mg bolus every 6h and a tablet of 50 µg of fludrocortisone via the nasogastric tube, or their placebos. The primary outcome was 90-day all-cause mortality. Secondary outcomes included all-cause mortality at intensive care unit (ICU) and hospital discharge, 28-day and 180-day mortality, the number of days alive and free of vasopressors, mechanical ventilation, or organ failure, and ICU and hospital free-days to 90-days. Analysis was done in the intention-to-treat population. The trial was registered at ClinicalTrials.gov (NCT00625209). FINDINGS: Of 1241 patients included in the APROCCHSS trial, CAP could not be ruled in or out in 31 patients, 562 had a diagnosis of CAP (279 in the placebo group and 283 in the corticosteroid group), and 648 patients did not have CAP (329 in the placebo group and 319 in the corticosteroid group). In patients with CAP, there were 109 (39%) deaths of 283 patients at day 90 with hydrocortisone plus fludrocortisone and 143 (51%) of 279 patients receiving placebo (odds ratio [OR] 0·60, 95% CI 0·43-0·83). In patients without CAP, there were 148 (46%) deaths of 319 patients at day 90 in the hydrocortisone and fludrocortisone group and 157 (48%) of 329 patients in the placebo group (OR 0·95, 95% CI 0·70-1·29). There was significant heterogeneity in corticosteroid effects on 90-day mortality across subgroups with CAP and without CAP (p=0·046 for both multiplicative and additive interaction tests; moderate credibility). Of 1241 patients included in the APROCCHSS trial, 648 (52%) had ARDS (328 in the placebo group and 320 in the corticosteroid group). There were 155 (48%) deaths of 320 patients at day 90 in the corticosteroid group and 186 (57%) of 328 patients in the placebo group. The OR for death at day 90 was 0·72 (95% CI 0·53-0·98) in patients with ARDS and 0·85 (0·61-1·20) in patients without ARDS (p=0·45 for multiplicative interaction and p=0·42 for additive interaction). The OR for observing at least one serious adverse event (corticosteroid group vs placebo) within 180 days post randomisation was 0·64 (95% CI 0·46-0·89) in the CAP subgroup and 1·02 (0·75-1·39) in the non-CAP subgroup (p=0·044 for multiplicative interaction and p=0·042 for additive interaction). INTERPRETATION: In a pre-specified subgroup analysis of the APROCCHSS trial of patients with CAP and septic shock, hydrocortisone plus fludrocortisone reduced mortality as compared with placebo. Although a large proportion of patients with CAP also met criteria for ARDS, the subgroup analysis was underpowered to fully discriminate between ARDS and CAP modifying effects on mortality reduction with corticosteroids. There was no evidence of a significant treatment effect of corticosteroids in the non-CAP subgroup. FUNDING: Programme Hospitalier de Recherche Clinique of the French Ministry of Health, by Programme d'Investissements d'Avenir, France 2030, and IAHU-ANR-0004.

Tie2-mediated vascular remodeling by ferritin-based protein C nanoparticles confers antitumor and anti-metastatic activities.

BACKGROUND: Conventional therapeutic approaches for tumor angiogenesis, which are primarily focused on the inhibition of active angiogenesis to starve cancerous cells, target the vascular endothelial growth factor signaling pathway. This aggravates hypoxia within the tumor core and ultimately leads to increased tumor proliferation and metastasis. To overcome this limitation, we developed nanoparticles with antiseptic activity that target tumor vascular abnormalities. METHODS: Ferritin-based protein C nanoparticles (PCNs), known as TFG and TFMG, were generated and tested in Lewis lung carcinoma (LLC) allograft and MMTV-PyMT spontaneous breast cancer models. Immunohistochemical analysis was performed on tumor samples to evaluate the tumor vasculature. Western blot and permeability assays were used to explore the role and mechanism of the antitumor effects of PCNs in vivo. For knocking down proteins of interest, endothelial cells were transfected with siRNAs. Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett's multiple comparison test. RESULTS: PCNs significantly inhibited hypoxia and increased pericyte coverage, leading to the inhibition of tumor growth and metastasis, while increasing survival in LLC allograft and MMTV-PyMT spontaneous breast cancer models. The coadministration of cisplatin with PCNs induced a synergistic suppression of tumor growth by improving drug delivery as evidenced by increased blood prefusion and decreased vascular permeability. Moreover, PCNs altered the immune cell profiles within the tumor by increasing cytotoxic T cells and M1-like macrophages with antitumor activity. PCNs induced PAR-1/PAR-3 heterodimerization through EPCR occupation and PAR-1 activation, which resulted in Gα13-RhoA-mediated-Tie2 activation and stabilized vascular tight junctions via the Akt-FoxO3a signaling pathway. CONCLUSIONS: Cancer treatment targeting the tumor vasculature by inducing antitumor immune responses and enhancing the delivery of a chemotherapeutic agent with PCNs resulted in tumor regression and may provide an effective therapeutic strategy.

Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects - impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.

Activated Protein C for Ischemic Central Retinal Vein Occlusion: One-Year Results.

Intravitreally administered activated protein C in 10 eyes with ischemic central retinal vein occlusion significantly improved macular edema; in 5 eyes, the reperfusion of the retinal nonperfused areas exceeded 50% of baseline.

Activated Protein C in Cutaneous Wound Healing: From Bench to Bedside.

Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.

Evaluation of 3K3A-Activated Protein C to Treat Neonatal Hypoxic Ischemic Brain Injury in the Spiny Mouse.

Neonatal hypoxic ischemic encephalopathy (HIE) resulting from intrapartum asphyxia is a global problem that causes severe disabilities and up to 1 million deaths annually. A variant form of activated protein C, 3K3A-APC, has cytoprotective properties that attenuate brain injury in models of adult stroke. In this study, we compared the ability of 3K3A-APC and APC (wild-type (wt)) to attenuate neonatal brain injury, using the spiny mouse (Acomys cahirinus) model of intrapartum asphyxia. Pups were delivered at 38 days of gestation (term = 39 days), with an intrapartum hypoxic insult of 7.5 min (intrapartum asphyxia cohort), or immediate removal from the uterus (control cohort). After 1 h, pups received a subcutaneous injection of 3K3A-APC or wild-type APC (wtAPC) at 7 mg/kg, or vehicle (saline). At 24 h of age, pups were killed and brain tissue was collected for measurement of inflammation and cell death using RT-qPCR and histopathology. Intrapartum asphyxia increased weight loss, inflammation, and apoptosis/necrosis in the newborn brain. 3K3A-APC administration maintained body weight and ameliorated an asphyxia-induced increase of TGFß1 messenger RNA expression in the cerebral cortex, immune cell aggregation in the corpus callosum, and cell death in the deep gray matter and hippocampus. In the cortex, 3K3A-APC appeared to exacerbate the immune response to the hypoxic ischemic insult. While wtAPC reduced cell death in the corpus callosum and hippocampus following intrapartum asphyxia, it increased markers of neuro-inflammation and cell death in control pups. These findings suggest 3K3A-APC administration may be a useful therapy to reduce cell death and neonatal brain injury associated with HIE.

Final Results of the RHAPSODY Trial: A Multi-Center, Phase 2 Trial Using a Continual Reassessment Method to Determine the Safety and Tolerability of 3K3A-APC, A Recombinant Variant of Human Activated Protein C, in Combination with Tissue Plasminogen Activator, Mechanical Thrombectomy or both in Moderate to Severe Acute Ischemic Stroke.

OBJECTIVE: Agonism of protease-activated receptor (PAR) 1 by activated protein C (APC) provides neuro- and vasculoprotection in experimental neuroinjury models. The pleiotropic PAR1 agonist, 3K3A-APC, reduces neurological injury and promotes vascular integrity; 3K3A-APC proved safe in human volunteers. We performed a randomized, controlled, blinded trial to determine the maximally tolerated dose (MTD) of 3K3A-APC in ischemic stroke patients. METHODS: The NeuroNEXT trial, RHAPSODY, used a novel continual reassessment method to determine the MTD using tiers of 120, 240, 360, and 540 µg/kg of 3K3A-APC. After intravenous tissue plasminogen activator, intra-arterial mechanical thrombectomy, or both, patients were randomized to 1 of the 4 doses or placebo. Vasculoprotection was assessed as microbleed and intracranial hemorrhage (ICH) rates. RESULTS: Between January 2015 and July 2017, we treated 110 patients. Demographics resembled a typical stroke population. The MTD was the highest-dose 3K3A-APC tested, 540 µg/kg, with an estimated toxicity rate of 7%. There was no difference in prespecified ICH rates. In exploratory analyses, 3K3A-APC reduced ICH rates compared to placebo from 86.5% to 67.4% in the combined treatment arms (p = 0.046) and total hemorrhage volume from an average of 2.1 ± 5.8 ml in placebo to 0.8 ± 2.1 ml in the combined treatment arms (p = 0.066). INTERPRETATION: RHAPSODY is the first trial of a neuroprotectant for acute ischemic stroke in a trial design allowing thrombectomy, thrombolysis, or both. The MTD was 540 µg/kg for the PAR1 active cytoprotectant, 3K3A-APC. A trend toward lower hemorrhage rate in an exploratory analysis requires confirmation. CLINICAL TRIAL REGISTRATION: Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT02222714. ANN NEUROL 2019;85:125-136.

Activated protein C as disease-modifying therapy in antenatal preeclampsia: An open-label, single arm safety and efficacy trial.

OBJECTIVES: Preeclampsia is characterized by maternal systemic inflammation and coagulation activation, akin to the sepsis syndrome. Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) may modify disease progression to safely prolong pregnancies and improve perinatal outcomes. Both maternal and perinatal risks are highest remote from term. STUDY DESIGN: Open-label, single arm safety and efficacy trial of rhAPC in consenting pregnant women with severe early-onset preeclampsia. Disease severity-matched rhAPC-naïve controls were identified from an existing database. An additional six women were recruited as biomarker controls. MAIN OUTCOME MEASURES: Primary safety outcome: incidence of peripartum bleeding; primary efficacy outcome: duration of pregnancy after enrolment. RESULTS: Twelve (31.6%) of 38 eligible women consented; 3 did not receive the infusion due to staffing. Therefore, 9 women received rhAPC (24 µg/kg/hr for ≤96 h antenatally). No safety issues were identified. There was a marginal prolongation in eligibility-to-delivery intervals for women receiving rhAPC (Mantel-Cox p = 0.052; Gehan-Breslow-Wilcoxon p = 0.049). Compared with both the pre-infusion phase in the rhAPC-treated women themselves and with fullPIERS rhAPC-naïve women, rhAPC was associated with increased urine output during the infusion (6/9 vs 1/9 had urine output >100 mL/h during the infusion, Fisher's exact p = 0.003). CONCLUSIONS: These data support further investigation of APC in women with severe early-onset preeclampsia; recombinant and purified human APC is available. In addition, these data will inform the design and implementation of randomized controlled trials aiming to modify and/or moderate the proinflammatory and proacoagulant state of preeclampsia.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V.

Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti-FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once-weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab-mediated haemostasis remain to be explored. We investigated the role of APC-mediated reactions in these circumstances. APC dose-dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII-deficient plasmas, and in normal plasmas preincubated with an anti-FVIII antibody (FVIII-depleted). FVIIIa-independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC-induced down-regulation of emicizumab-dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII-depleted FV-deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab-related TG in FVIII-depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab-driven haemostasis was down regulated by APC-mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.

Homozygous protein C deficiency presenting as neonatal purpura fulminans: management with fresh frozen plasma, low molecular weight heparin and protein C concentrate.

Purpura fulminans in neonates is a rapidly progressive thrombotic disorder manifesting as hemorrhagic skin infarction and disseminated intravascular coagulation. Being inherited in an autosomal dominant manner, it is a medical emergency. Clinical presentations of patients may vary depending on the genetic mutations. Retinal and intracranial hemorrhages are the worst clinical scenarios with persistent morbidity. During acute phase, fresh frozen plasma, protein C concentrates and anticoagulant therapy should be administered rapidly. Here we report a patient with homozygous protein C deficiency.

Perioperative management of severe congenital protein C deficiency.

: Perioperative care of congenital protein C deficiency has not been well established. Here, we describe a patient with congenital protein C deficiency who underwent laparoscopic fundoplication and gastrostomy at 2 years of age. Preoperatively, we stopped warfarin, administered fresh frozen plasma, and activated protein C. These procedures were performed without bleeding or clotting events, and at 3 days after the procedures, we restarted warfarin. Several episodes of abdominal hemorrhage and purpura fulminans occurred 2-4 weeks postoperatively, and the events were managed conservatively. We conclude that an invasive procedure can be performed in patients with protein C deficiency with appropriate supportive therapy, and postoperative observation for a sufficient length of time is essential to minimize the risk of complications.

Early antithrombotic treatment with warfarin oral suspension in severe neonatal protein C deficiency.

Neonatal severe protein C deficiency is a serious disease. There is no uniform approach for long-term preventive treatment of thrombotic events. We report the case of neonatal severe protein C deficiency treated with warfarin oral suspension. An international normalized ratio (INR) from 2.5 to 3.5 was expected. The INR was measured by home monitoring using the Coaguchek XS® (Roche Diagnostics, Mannheim, Germany) monitor. During 2years of warfarin treatment, there were only two minor episodes of purpuric access and no bleeding was reported. This case suggests that the early introduction of warfarin oral suspension, home-care monitoring, and parental education programs may be a beneficial treatment option for children with protein C deficiency.

Refining anti-inflammatory therapy strategies for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a severe lung disease of preterm infants, which is characterized by fewer, enlarged alveoli and increased inflammation. BPD has grave consequences for affected infants, but no effective and safe therapy exists. We previously showed that prophylactic treatment with interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD induced by perinatal inflammation and hyperoxia. Here, we used the same BPD model to assess whether an alternative anti-inflammatory agent, protein C (PC), is as effective as IL-1Ra against BPD. We also tested whether delayed administration or a higher dose of IL-1Ra affects its ability to ameliorate BPD and investigated aspects of drug safety. Pups were reared in room air (21% O2 ) or hyperoxia (65% or 85% O2 ) and received daily injections with vehicle, 1200 IU/kg PC, 10 mg/kg IL-1Ra (early or late onset) or 100 mg/kg IL-1Ra. After 3 or 28 days, lung and brain histology were assessed and pulmonary cytokines were analysed using ELISA and cytokine arrays. We found that PC only moderately reduced the severe impact of BPD on lung structure (e.g. 18% increased alveolar number by PC versus 34% by IL-1Ra); however, PC significantly reduced IL-1ß, IL-1Ra, IL-6 and macrophage inflammatory protein (MIP)-2 by up to 89%. IL-1Ra at 10 mg/kg prevented BPD more effectively than 100 mg/kg IL-1Ra, but only if treatment commenced at day 1 of life. We conclude that prophylactic low-dose IL-1Ra and PC ameliorate BPD and have potential as the first remedy for one of the most devastating diseases preterm babies face.

Selecting Patients for Intra-Arterial Therapy in the Context of a Clinical Trial for Neuroprotection.

BACKGROUND AND PURPOSE: The advent of intra-arterial neurothrombectomy (IAT) for acute ischemic stroke opens a potentially transformative opportunity to improve neuroprotection studies. Combining a putative neuroprotectant with recanalization could produce more powerful trials but could introduce heterogeneity and adverse event possibilities. We sought to demonstrate feasibility of IAT in neuroprotectant trials by defining IAT selection criteria for an ongoing neuroprotectant clinical trial. METHODS: The study drug, 3K3A-APC, is a pleiotropic cytoprotectant and may reduce thrombolysis-associated hemorrhage. The NeuroNEXT trial NN104 (RHAPSODY) is designed to establish a maximally tolerated dose of 3K3A-APC. Each trial site provided their IAT selection criteria. An expert panel reviewed site criteria and published evidence. Finally, the trial leadership designed IAT selection criteria. RESULTS: Derived selection criteria reflected consistency among the sites and comparability to published IAT trials. A protocol amendment allowing IAT (and relaxed age, National Institutes of Health Stroke Scale, and time limits) in the RHAPSODY trial was implemented on June 15, 2015. Recruitment before and after the amendment improved from 8 enrolled patients (601 screened, 1.3%) to 51 patients (821 screened, 6.2%; odds ratio [95% confidence limit] of 4.9 [2.3-10.4]; P<0.001). Gross recruitment was 0.11 patients per site month versus 0.43 patients per site per month, respectively, before and after the amendment. CONCLUSIONS: It is feasible to include IAT in a neuroprotectant trial for acute ischemic stroke. Criteria are presented for including such patients in a manner that is consistent with published evidence for IAT while still preserving the ability to test the role of the putative neuroprotectant. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02222714.

A Compelling Case for the Use of Perioperative Zymogen Protein C for Increased Patient Safety.

It is imperative to maintain normal blood flow to provide adequate oxygen supply to specific organs and cells, as well as for the removal of metabolic byproducts. Therefore, any situation that results in blood clotting can injure or kill living tissues. In this paper, we describe a case where a protein C deficient subject who would, by all medical indicators, be at 100 % risk of experiencing thrombophlebitis, deep vein thrombosis, and or lung emboli, is able to escape all pathologies by using perioperative zymogen protein C (ZPC). This protein C deficient patient has a long history of blood clotting, particularly from surgical procedures. The patient is 81 years old and first experienced clotting due to hernia surgery in 1964, when he was hospitalized for 16 days post-surgery with life threatening complications. It was later determined in 1980, after many episodes, that the patient had hereditary protein C deficiency at the 38 % level. In his hernia surgery, perioperative ZPC was used along with accepted anticoagulation procedures with no blood clots or other related side effects occurring. This procedure can greatly benefit protein C deficient patients, and could potentially find use for non-PC deficient patients in surgeries and a variety of other medical treatments. This particular case helps to validate the importance of ZPC in effecting safer surgery in high-risk patients. It also supports the mechanism of ZPC acting as an anticoagulant without causing bleeding. Most importantly, each clinical case study represents a unique combination of surgeon, hematologist, medical staff, and patient functioning as a coordinated team. In this case, smaller amounts of very expensive ZPC achieved safe and efficacious results, which is hugely important for future clinical applications when considering the production cost of ZPC. More studies must be done to establish minimum dosing while achieving safe and efficacious outcomes.

Protein C zymogen in severe sepsis: a double-blinded, placebo-controlled, randomized study.

PURPOSE: To determine whether protein C zymogen (protein C concentrates or human protein C) improves clinically relevant outcomes in adult patients with severe sepsis and septic shock. METHODS: This is a randomized, double-blind, placebo-controlled, parallel-group trial that from September 2012 to June 2014 enrolled adult patients with severe sepsis or septic shock and high risk of death and of bleeding (e.g., APACHE II greater than 25, extracorporeal membrane oxygenation or disseminated intravascular coagulopathy). All patients completed their follow-up 90 days after randomization and data were analyzed according to the intention-to-treat principle. Follow-up was performed at 30 and 90 days after randomization. The primary endpoint was a composite outcome of prolonged intensive care unit (ICU) stay and/or 30-day mortality. Secondary endpoints included mortality. RESULTS: The study was stopped early in a situation of futility for the composite outcome of prolonged ICU stay and/or 30-day mortality that was 79 % (15 patients) in the protein C zymogen group and 67 % (12 patients) in the placebo group (p = 0.40) and for a concomitant safety issue: ICU mortality was 79 % (15 patients) in the protein C zymogen group vs 39 % (7 patients) in the placebo group (p = 0.020), and 30-day mortality was 68 vs 39 % (p = 0.072). CONCLUSION: Protein C zymogen did not improve clinically relevant outcomes in severe sepsis and septic shock adult patients. Given its high cost and the potential increase in mortality, the use of this drug in adult patients should be discouraged.

Severe Congenital Protein C Deficiency: Practical Aspects of Management.

Subcutaneous (SC) protein C (PC) was used in a child with purpura fulminans secondary to severe congenital PC deficiency. For maintenance, PC 80-120 IU/kg, given over 60-90 min SC Q48hr, has been successful as a home therapy for more than 3 years. The treatment was monitored by measuring trough PC chromogenic activity (target ≥15%) and D-dimer levels. No change in clinical course was appreciated after discontinuing enoxaparin (and leaving the patient on prophylactic PC replacement alone). A significant discrepancy between clotting-based and chromogenic-based PC activity is shown.

Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood.

INTRODUCTION: Acute traumatic coagulopathy has been associated with shock and tissue injury, and may be mediated via activation of the protein C pathway. Patients with acute traumatic coagulopathy have prolonged PT and PTT, and decreased activity of factors V and VIII; they are also hypocoagulable by thromboelastometry (ROTEM) and other viscoelastic assays. To test the etiology of this phenomenon, we hypothesized that such coagulopathy could be induced in vitro in healthy human blood with the addition of activated protein C (aPC). METHODS: Whole blood was collected from 20 healthy human subjects, and was "spiked" with increasing concentrations of purified human aPC (control, 75, 300, 2000 ng/mL). PT/PTT, factor activity assays, and ROTEM were performed on each sample. Mixed effect regression modeling was performed to assess the association of aPC concentration with PT/PTT, factor activity, and ROTEM parameters. RESULTS: In all subjects, increasing concentrations of aPC produced ROTEM tracings consistent with traumatic coagulopathy. ROTEM EXTEM parameters differed significantly by aPC concentration, with stepwise prolongation of clotting time (CT) and clot formation time (CFT), decreased alpha angle (α), impaired early clot formation (a10 and a20), and reduced maximum clot firmness (MCF). PT and PTT were significantly prolonged at higher aPC concentrations, with corresponding significant decreases in factor V and VIII activity. CONCLUSION: A phenotype of acute traumatic coagulopathy can be induced in healthy blood by the in vitro addition of aPC alone, as evidenced by viscoelastic measures and confirmed by conventional coagulation assays and factor activity. This may lend further mechanistic insight to the etiology of coagulation abnormalities in trauma, supporting the central role of the protein C pathway. Our findings also represent a model for future investigations in the diagnosis and treatment of acute traumatic coagulopathy.

Comparative Study on the Cytoprotective Effects of Activated Protein C Treatment in Nonsteatotic and Steatotic Livers under Ischemia-Reperfusion Injury.

UNLABELLED: Activated protein C (APC) has cytoprotective effects on liver ischemia-reperfusion injury (IRI). However, it is unclear whether APC is beneficial in steatotic liver IRI. We compared the cytoprotective effects of APC in nonsteatotic and steatotic liver IRI. METHODS: Mice fed either normal diets (ND mice) or high fat diets (HF mice), were treated with APC or saline (control) and were performed 60 min partial IRI. Moreover, primary steatotic hepatocytes were either untreated or treated with APC and then incubated with H2O2. RESULTS: APC significantly reduced serum transaminase levels and the inflammatory cells infiltration compared with control at 4 h in ND mice and at 24 h in HF mice. APC inhibited sinusoidal endothelial injury in ND mice, but not in HF mice. In contrast, APC activated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in HF mice, but not in ND mice. In the in vitro study, APC significantly increased AMPK phosphorylation, ATP concentration, and survival rates of hepatocytes compared with control. CONCLUSION: During IRI in normal liver, APC attenuated initial damage by inhibiting inflammatory cell infiltration and sinusoidal endothelial injury, but not in steatotic liver. However, in steatotic liver, APC might attenuate late damage via activation of AMPK.