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Context.--Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity.Objective.--To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a 'real-world' population of patients undergoing rivaroxaban treatment and hypercoagulation testing.Design.--During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/ taking rivaroxaban, wild-type/taking rivaroxaban, FVLHET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32).Results.--The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients (P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 [+ or -] 0.12, versus 1.64 [+ or -] 0.3 in FVL-HET patients not taking rivaroxaban (P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL (P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone.Conclusions.--Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.(Arch Pathol Lab Med. 2018;142:70-74; doi: 10.5858/arpa.2016-0616-OA), Rivaroxaban is a direct, antithrombin-independent factor Xa inhibitor, which inhibits not only free factor Xa, but also clot-bound factor Xa and the prothrombinase complex. It is used for treatment and [...]

In skinned myocardium, cyclic AMP--dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin--binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the [Ca.sup.2+] responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the [Ca.sup.2+] sensitivity of force ([pCa.sub.50]) and the activation dependence of the rate of force redevelopment ([k.sub.tr]) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C--null background ([cMyBP-C.sup.-/-]), (c) nonphosphorylatable cTnI with [serines.sup.23/24/43/45] and [threonine.sup.144] mutated to alanines ([cTnI.sub.Ala5]), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background ([cTnI.sub.Ala5]/[cMyBP-C.sup.-/-]). Here, PKA treatment decreased [pCa.sub.50] in WT, [cTnI.sub.Ala5], and [cMyBP-C.sup.-/-] myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In WT and [cTnI.sub.Ala5] myocardium, PKA treatment also increased [k.sub.tr] at submaximal levels of activation; however, PKA treatment did not have an effect on [k.sub.tr] in [cMyBP-C.sup.-/-] or [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In addition, reconstitution of [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium with recombinant cMyBP-C restored the effects of PKA treatment on [pCa.sub.50] and [k.sub.tr] reported in [cTnI.sub.Ala5] myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in [pCa.sub.50] mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment. 10.1085/jgp.201010448

The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothefial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high ([HV.sub.T]; 20 ml/kg) or low ([LV.sub.T]; 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HVT induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. [HV.sub.T] also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from [HV.sub.T]-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-[[beta].sub.6]) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection. endothelial protein C receptor; mechanical stress; vascular barrier function; ventilator-induced lung injury

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-[alpha], interferon (IFN)-[gamma], and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-[alpha] and IFN-[gamma] or stimulation with LPS demonstrated a time-and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and + 128 bp was found to mediate IFN-[gamma], TNF-[alpha], and LPS responsiveness as well as the synergistic effect of IFN-[gamma]/TNF-[alpha] on hBf promoter activity. Site-directed mutagenesis of a IFN-[gamma]-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-[gamma] responsiveness. Mutagenesis of a nuclear factor (NF)-[kappa]B cis element at -466 to -456 bp abrogated TNF-[alpha] and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-[gamma] TNF-[alpha], and LPS is through GAS and NF-[kappa]B cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway. septic shock; monocytes/macrophages; complement factor B; cytokines

Byline: Fabio Baratto (1), Flavio Michielan (1), Muzio Meroni (1), Antonella Dal Palu (1), Annalisa Boscolo (1), Carlo Ori (1) Keywords: Sepsis; Therapy; Shock; Septic; Protein C Abstract: Objective To describe the efficacy and safety of protein C (PC) concentrate to restore physiological values in adult septic patients having clinical contraindications to activated PC. Design Case series (pilot study). Setting Three adult ICUs of a University Hospital. Patients and participants Twenty adult patients affected by severe sepsis or septic shock with plasma values of PC < 50%. Interventions Patients were treated with PC concentrate (Ceprotin .sup.(r)----Baxter) with a starting bolus followed by a continuous infusion for 72 h [3 IU/(kg h)]. Measurements and results PC activity, WBC, platelets, d-Dimer, fibrinogen, PT, aPTT, AT III, lactate, Sepsis-related Organ Failure Assessment (SOFA), Disseminated Intravascular Coagulation (DIC) score, adverse events, and mortality were measured. Baseline plasma PC activity was 34.5 +- 9.1%. PC concentrate normalized the PC activity in all patients within 48 h, and then remained stable for the following days. At baseline, several patients showed abnormal PT, aPTT, platelets values, and lactate levels. During the study period, there was a significant increase of platelets, fibrinogen, PT, AT III, and a significant decrease of d-Dimer, aPTT, DIC score, and lactate. No adverse reactions (hemorrhage or thrombosis) were observed. Mortality at 28 days was 35%. Conclusions Our pilot study shows that the administration of PC concentrate to patients having contraindications to the treatment with activated PC was safe and possibly useful to control the coagulopathy triggered and sustained by sepsis. A randomized, double blind study in patients with severe sepsis and contraindications to activated PC administration would be advisable to state the safety and the possible role of this product in the treatment of severe sepsis. Author Affiliation: (1) Department of Pharmacology and Anesthesiology, University of Padua, Padua, Italy Article History: Registration Date: 21/04/2008 Received Date: 19/03/2007 Accepted Date: 17/04/2008 Online Date: 06/05/2008