Objectives Acquired coagulopathy may be associated with bleeding risk. Approaches to restore haemostasis include administration of coagulation factor concentrates, but there are concerns regarding potential prothrombotic risk. The present study assessed the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) versus activated PCC (aPCC) and recombinant factor VIIa (rFVIIa), using three preclinical animal models. Methods The first model was a modified Wessler model of venous stasis-induced thrombosis in rabbit, focusing on dilutional coagulopathy; the second model employed the same system but focused on direct oral anticoagulant reversal (i.e. edoxaban). The third model assessed the prothrombotic impact of 4F-PCC, aPCC and rFVIIa in a rat model of ferric chloride-induced arterial thrombosis. Results In the first model, thrombi were observed at aPCC doses ≥10 IU/kg (therapeutic dose 100 IU/kg) and rFVIIa doses ≥50 μg/kg (therapeutic dose 90 μg/kg), but not 4F-PCC 50 IU/kg (therapeutic dose 50 IU/kg). The impact of 4F-PCC (up to 300 IU/kg) on thrombus formation was evident from 10 minutes post-administration, but not at 24 hours post-administration; this did not change with addition of tranexamic acid and/or fibrinogen concentrate. 4F-PCC-induced thrombus formation was lower after haemodilution versus non-haemodilution. In the second model, no prothrombotic effect was confirmed at 4F-PCC 50 IU/kg. The third model showed lower incidence of thrombus formation for 4F-PCC 50 IU/kg versus aPCC (50 U/kg) and rFVIIa (90 μg/kg). Conclusions These results suggest that 4F-PCC has a low thrombotic potential versus aPCC or rFVIIa, supporting the clinical use of 4F-PCC for the treatment of coagulopathy-mediated bleeding.

Background Hemophilia B is caused by coagulation factor IX (FIX) deficiency. Recombinant fusion protein linking coagulation FIX with recombinant albumin (rIX-FP; Idelvion(R)) is used for replacement therapy with an extended half-life. A previous quantitative whole-body autoradiography (QWBA) study investigating the biodistribution of rIX-FP indicated equal biodistribution, but more prolonged tissue retention compared with a marketed recombinant FIX product. Objectives To complete and confirm the QWBA study data by directly measuring rIX-FP protein and activity levels in tissues following intravenous (i.v.) administration to normal rats and FIX-deficient (hemophilia B) mice. Methods After i.v. administration of rIX-FP at a dose of 2000 IU/kg, animals were euthanized at specific time points up to 72 hours postdosing. Subsequently, plasma and various tissues, which were selected based on the previous QWBA results, were harvested and analyzed for FIX antigen levels using an ELISA (both species) or an immunohistochemistry method (mice only), as well as for FIX activity levels (mice only) using a chromogenic assay. Results In rats, rIX-FP distributed extravascularly into all tissues analyzed (ie, liver, kidney, skin and knee) with peak antigen levels reached between 1 and 7 hours postdosing. In hemophilia B mice, rIX-FP tissue distribution was comparable to rats. FIX antigen levels correlated well with FIX activity readouts. Conclusions Our results confirm QWBA data showing that rIX-FP distributes into relevant target tissues. Importantly, it was demonstrated that rIX-FP available in tissues retains its functional activity and can thus facilitate its therapeutic activity at sites of potential injury.

Herzog, Eva; Kaspereit, Franz; Krege, Wilfried; Doerr, Baerbel; Mischnik, Marcel; Pragst, Ingo; Dickneite, Gerhard

Learning Objectives: Burn injuries cause physiologic changes in multiple organ systems in the body. Following major burns, mortality is usually attributable to pulmonary complications, which can occur in up to 41% of patients admitted to the hospital after burn injury. Patients with preexisting comorbidities such as chronic lung diseases may be more susceptible. We therefore sought to examine the impact of preexisting respiratory disease on burn injury outcomes. Methods:A retrospective analysis of patients admitted to a regional burn center from 2002–2012. Independent variables analyzed included basic demographics, burn mechanism, presence of inhalation injury, total body surface area (TBSA) of burn, pre-existing comorbidities, smoker status, length of hospital stay, and days of mechanical ventilation. Bivariate analysis was performed and Cox regression modeling using significant variables was utilized to estimate hazard of progression to mechanical ventilation and mortality. Results: There were a total of 7640 patients over the study period. Overall survival rate was 96%. 8% (n=672) had a preexisting respiratory disease. Chronic lung disease patients had a higher crude mortality rate (7%) compared to those without lung disease (4%, p <0.01). The adjusted Cox regression model to estimate the hazard of progression to mechanical ventilation in patients with respiratory disease was 21% higher compared to those without respiratory disease (HR =1.21, 95% CI = 1.01–1.44). The hazard of progression to mortality is 56% higher (HR = 1.56, 95% CI = 1.10–2.19) for patients with pre-existing respiratory disease compared to those without respiratory disease after controlling for patient demographics and injury characteristics. Conclusions: Preexisting respiratory disease significantly increases the hazard of progression to mechanical ventilation and mortality in patients following burn injury.Given the increasing number of Americans with acute or chronic respiratory diseases, there will likely be a greater number of individuals at risk for worse outcomes following burn injury.

INTRODUCTION:Rivaroxaban is an oral, selective direct factor Xa inhibitor approved for several indications in patients at risk of thrombotic events. One limitation of its clinical use is the lack of data pertaining to its reversal in situations where urgent response is critical (e.g. acute bleeding events or emergency surgery).MATERIALS AND METHODS:This study assessed the effectiveness of a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®)/Kcentra(®)) for the reversal of rivaroxaban-associated bleeding in an in vivo rabbit model, and evaluated the correlations between in vitro coagulation parameters and haemostasis in vivo.RESULTS:Administration of single intravenous doses of rivaroxaban (150-450 μg/kg) resulted in increased and prolonged bleeding following standardised kidney incision. Pre-incision treatment with 4F-PCC (25-100 IU/kg) resulted in a dose-dependent reversal of rivaroxaban (150 and 300 μg/kg)-associated increases in time to haemostasis and blood loss; no reversal was seen at the highest rivaroxaban dose (450 μg/kg). Of the in vitro biomarkers tested, thrombin generation and whole-blood clotting time correlated well with in vivo measures of 4F-PCC-mediated effects. Thrombin generation was highly reagent-dependent, with the assay initiated using the phospholipid-only reagent being the most predictive of effective haemostasis in vivo.CONCLUSIONS:In summary, in a rabbit model of acute bleeding, treatment with 4F-PCC reduced bleeding to control levels following rivaroxaban 150 μg/kg and 300 μg/kg administration.

BACKGROUND:Apixaban is a direct factor Xa inhibitor approved for the treatment and prevention of thromboembolic disease. There is a lack of data regarding its reversal in cases of acute bleeding or prior to emergency surgery that needs addressing.OBJECTIVES:This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®) /Kcentra(®) , CSL Behring) can effectively reverse apixaban-associated bleeding in an in vivo rabbit model and evaluated the correlations between in vivo hemostasis and in vitro coagulation parameters.METHODS:For dose-finding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800-1600 μg kg(-1) ) and, following a standardized kidney incision, volume of blood loss and time to hemostasis were measured. In a subsequent study phase, anesthetized rabbits were treated with apixaban 1200 μg kg(-1) followed by 4F-PCC (6.25-100 IU kg(-1) ), and the effects on the same bleeding parameters were assessed. In parallel, coagulation parameters were monitored.RESULTS:Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration. Preincision treatment with 4F-PCC resulted in a statistically significant reversal in bleeding time (all doses) and volume (doses ≥ 12.5 IU kg(-1) ). Of the coagulation parameters measured, thrombin generation initiated using the RD reagent (phospholipids only) was the most sensitive to in vivo measures of 4F-PCC's hemostatic efficacy, although some correlations were also observed for prothrombin time and whole blood clotting time.CONCLUSIONS:In this rabbit model of acute hemorrhage, 4F-PCC showed potential for reversing the bleeding effects of apixaban. Clinical data in apixaban-treated patients are needed to confirm these results.

This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can effectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].

Background:Edoxaban is an oral, selective direct factor Xa inhibitor approved in Japan for venous thromboembolism prevention after orthopedic surgery. Data are lacking regarding reversal strategies for edoxaban; this study assessed whether four-factor prothrombin complex concentrate (Beriplex®/Kcentra®; CSL Behring GmbH, Marburg, Germany) can effectively reverse its effects on hemostasis using a previously described rabbit model. Methods:The study comprised assessments of thrombin generation in vitro, pharmacokinetic parameters, and edoxaban reversal in vivo. In a blinded in vivo stage, a standardized kidney incision was performed in animals (n = 11 per group) randomized to receive vehicle + saline, edoxaban (1,200 &mgr;g/kg) + saline, or edoxaban (1,200 &mgr;g/kg) + four-factor prothrombin complex concentrate (50 IU/kg). Animals were monitored for treatment impact on hemostasis and coagulation parameters. Data are median (range). Statistical tests were adjusted for multiple testing. Results:Edoxaban administration increased blood loss (30 [2 to 44] ml) and time to hemostasis (23 [8.5 to 30.0] min) compared with the control group (3 [1 to 8] ml and 3 [2.0 to 5.0] min, respectively). Biomarkers of coagulation (prothrombin time, activated partial thromboplastin time, whole blood clotting time) and thrombin generation parameters (e.g., peak thrombin, endogenous thrombin potential, lag time) were also affected by edoxaban. Administration of four-factor prothrombin complex concentrate significantly reduced time to hemostasis (to 8 [6.5 to 14.0] min, observed P < 0.0001) and total blood loss (to 9 [4 to 22] ml, observed P = 0.0050) compared with the edoxaban + saline group. Of the biomarkers tested, prothrombin time, whole blood clotting time, and endogenous thrombin potential correlated best with clinical parameters. Conclusion:In a rabbit model of hemostasis, four-factor prothrombin complex concentrate administration significantly decreased edoxaban-associated hemorrhage.

The oral direct and selective factor Xa inhibitor edoxaban (Daiichi Sankyo) is currently available in Japan for the prophylaxis of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery and is undergoing investigation in phase III trials for the prevention of stroke in patients with atrial fibrillation and the treatment and secondary prevention of VTE. The primary complication of any available anticoagulant therapy is the risk of bleeding. Rapid reversal of anticoagulation may be necessary in patients requiring emergency treatment due to uncontrolled bleeding. Prothrombin complex concentrates (PCC) are frequently used to reverse the effect of vitamin K antagonists such as warfarin and have also been suggested to be potentially effective in reversing the effects of the new oral anticoagulants. The present study was therefore designed to determine whether the four-factor PCC Beriplex® can effectively reverse bleeding and normalize coagulation following edoxaban administration in a rabbit kidney injury model.

Summary.  Background:  One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation.

Introduction: Fluid resuscitation after trauma often results in dilutional coagulopathy that may hinder control of bleeding and, once initial hemostasis has been secured, heighten risk of perioperative bleeding when further surgery is required. Since multiple coagulation factor deficiencies typically accompany fluid resuscitation, prothrombin complex concentrate (PCC) containing factors II, VII, IX and X may potentially offer greater hemostatic efficacy than coagulation factor monotherapy.Materials and methods: Anesthetized normothermic rabbits were hemodiluted 50-60% by phased blood withdrawal and infusion of hydroxyethyl starch and erythrocytes. The animals were randomly assigned to receive saline placebo, 25 IU.kg(-1) PCC (Beriplex P/N) or 180 mu g.kg(-1) activated recombinant factor VII (rFVIIa; NovoSeven). Immediately thereafter, bleeding was precipitated by a standardized kidney incision.Results: PCC accelerated hemostasis compared both with saline and rFVIIa (p=0.002 for both comparisons). The median times to hemostasis in the PCC, saline and rFVIIa groups were 12, 19 and 28 min, respectively. PCC reduced blood loss by a median of 43 mL with a 95% confidence interval (CI) of 8.0-67.5 mL vs. saline and 82 mL (CI, 35.0-110.0 mL) vs. rFVIIa. PCC augmented peak thrombin generation by a median of 104.1 nM (CI, 78.3-142.3 nM) compared with saline and 105.8 nM (CI, 70.7-139.5 nM) relative to rFVIIa. At the respective 180 mu g.kg(-1) and 25 IU.kg(-1) doses tested, rFVIIa displayed thrombogenicity in the Wessler stasis model, while PCC did not.Conclusions: In an animal model of dilutional coagulopathy and kidney trauma, PCC accelerated hemostasis and diminished blood loss compared with rFVIIa monotherapy. (C) 2009 Elsevier Ltd. All rights reserved.

BACKGROUND Fluid resuscitation after traumatic injury may necessitate coagulation factor replacement to prevent bleeding complications of dilutional coagulopathy. Recombinant activated factor VII (rFVIIa) is being widely investigated as a hemostatic agent in trauma. Multicomponent therapy with prothrombin complex concentrate (PCC) containing coagulation factors II, VII, IX, and X might offer potential advantages. METHODS Anesthetized mildly hypothermic normotensive pigs were hemodiluted by substituting 65% to 70% of total blood volume in phases with hydroxyethyl starch and red cells. Thereafter, animals received 12.5 mL . kg isotonic saline placebo, 35 IU . kg PCC, or 180 microg x kg rFVIIa. Immediately afterward, a standardized spleen injury was inflicted, and prothrombin time (PT) and hemostasis were assessed. Thrombin generation was also determined. RESULTS Hemodilution depleted levels of factors II, VII, IX, and X markedly, prolonged PT and decreased thrombin formation. PCC and rFVIIa both fully normalized the hemodilution-induced lengthening of PT. In PCC recipients, peak thrombin generation was greater by a median of 60.7 nM (confidence interval 56.4-64.9 nM) compared with the rFVIIa group (p = 0.008). After spleen trauma, time to hemostasis was shortened to a median of 35 minutes in animals treated with PCC versus 94 minutes with rFVIIa (p = 0.016). CONCLUSIONS In a pilot study involving an in vivo large-animal model of spleen trauma, PCC accelerated hemostasis and augmented thrombin generation compared with rFVIIa. Further investigations are warranted on PCC as a hemostatic agent in trauma.

Introduction There is considerable uncertainty about the reproducibility of the various instruments used to measure dyspnea, their ability to reflect changes in symptoms, whether they accurately reflect the patient's experience and if its evolution is similar between acute heart failure syndrome patients and nonacute heart failure syndrome patients.URGENT was a prospective multicenter trial designed to address these issues.Methods Patients were interviewed within 1 hour of first physician evaluation, in the emergency department or acute care setting, with dyspnea assessed by the patient using both a five-point Likert scale and a 10-point visual analog scale (VAS) in the sitting (60º) and then supine (20º) position if dyspnea had not been considered severe or very severe by the sitting versus decubitus dyspnea measurement.Results Very good agreements were found between the five-point Likert and VAS at baseline (0.891, P <0.0001) and between changes (from baseline to hour 6) in the five-point Likert and in VAS (0.800, P <0.0001) in acute heart failure (AHF) patients.Lower agreements were found when changes from baseline to H6 measured by Likert or VAS were compared with the seven-point comparative Likert (0.512 and 0.500 respectively) in AHF patients.The worse the dyspnea at admission, the greater the amplitude of improvement in the first 6 hours; this relationship is stronger when dyspnea is measured with VAS (Spearman's rho coefficient = 0.672) than with the five-point Likert (0.272) (both P <0.0001) in AHF patients.By the five-point Likert, only nine patients (3% (1% to 5%)) reported an improvement in their dyspnea, 177 (51% (46% to 57%)) had no change, and 159 (46% (41% to 52%)) reported worse dyspnea supine compared with sitting up in AHF patients.The PDA test with VAS was markedly different between AHF and non-AHF patients.Conclusions Both clinical tools five-point Likert and VAS showed very good agreement at baseline and between changes from baseline to tests performed 6 hours later in AHF patients.The PDA test with VAS was markedly different between AHF and non-AHF patients.Dyspnea is improved within 6 hours in more than threequarters of the patients regardless of the tool used to measure the change in dyspnea.The greater the dyspnea at admission, the greater the amplitude of improvement in the first 6 hours. P2