Retrospective Comparison of Andexanet Alfa and 4-Factor Prothrombin Complex for Reversal of Factor Xa-Inhibitor Related Bleeding.

The aim of this retrospective study was to compare andexanet alfa and 4-factor prothrombin complex (4F-PCC) for reversal of factor Xa (FXa)-inhibitor bleeding. Patients that received andexanet alfa for reversal were included. An equivalent number of patients administered 4F-PCC for FXa-inhibitor bleeding were randomly selected as historical controls. The primary outcome was effective hemostasis achievement within 12 h, defined using ANNEXA-4 criteria. Thromboembolic events and mortality within 30 days were also evaluated. A total of 32 patients were included. Baseline characteristics were not statistically different between andexanet alfa (n = 16) and 4F-PCC (n = 16). Intracranial bleeding was the primary reversal indication in 43.8% versus 62.5% of patients, respectively. Effective hemostasis was reached in 75.0% of andexanet alfa patients compared to 62.5% of 4F-PCC patients (P = .70). Thromboembolic events occurred in 4 (25.0%) patients and 3 (18.8%) patients, respectively (P = .99). Mortality incidence was 12.5% and 31.3%, respectively (P = .39). Andexanet alfa and 4F-PCC attained hemostasis in a majority of patients. A high, but a similar rate of thromboembolic events was seen with both treatments. Prospective studies are needed to elucidate comparative risks and benefits of the 2 agents.

Andexanet Alfa or Prothrombin Complex Concentrate for Factor Xa Inhibitor Reversal in Acute Major Bleeding: A Systematic Review and Meta-Analysis.

OBJECTIVES: To combine evidence on andexanet alfa and prothrombin complex concentrates for factor Xa inhibitor-associated bleeding to guide clinicians on reversal strategies. DATA SOURCES: Embase, Pubmed, Web of Science, and the Cochrane Library. STUDY SELECTION: Observational studies and randomized clinical trials studying hemostatic effectiveness of andexanet alfa or prothrombin complex concentrate for acute reversal of factor Xa inhibitor-associated hemorrhage. DATA EXTRACTION: Two independent reviewers extracted the data from the studies. Visualization and comparison of hemostatic effectiveness using Sarode et al or International Society of Thrombosis and Hemostasis Scientific and Standardization Committee criteria at 12 and 24 hours, (venous) thrombotic event rates, and inhospital mortality were performed by constructing Forest plots. Exploratory analysis using a logistic mixed model analysis was performed to identify factors associated with effectiveness and venous thromboembolic event. DATA SYNTHESIS: A total of 21 studies were included (andexanet: 438 patients; prothrombin complex concentrate: 1,278 patients). The (weighted) mean effectiveness for andexanet alfa was 82% at 12 hours and 71% at 24 hours. The (weighted) mean effectiveness for prothrombin complex concentrate was 88% at 12 hours and 76% at 24 hours. The mean 30-day symptomatic venous thromboembolic event rates were 5.0% for andexanet alfa and 1.9% for prothrombin complex concentrate. The mean 30-day total thrombotic event rates for andexanet alfa and prothrombin complex concentrate were 10.7% and 3.1%, respectively. Mean inhospital mortality was 23.3% for andexanet versus 15.8% for prothrombin complex concentrate. Exploratory analysis controlling for potential confounders did not demonstrate significant differences between both reversal agents. CONCLUSIONS: Currently, available evidence does not unequivocally support the clinical effectiveness of andexanet alfa or prothrombin complex concentrate to reverse factor Xa inhibitor-associated acute major bleeding, nor does it permit conventional meta-analysis of potential superiority. Neither reversal agent was significantly associated with increased effectiveness or a higher rate of venous thromboembolic event. These results underscore the importance of randomized controlled trials comparing the two reversal agents and may provide guidance in designing institutional guidelines.

The sweet side of venom: Glycosylated prothrombin activating metalloproteases from Dispholidus typus (boomslang) and Thelotornis mossambicanus (twig snake).

Dispholidus typus and Thelotornis mossambicanus are closely related rear-fanged colubrid snakes that both possess strongly procoagulant venoms. However, despite similarities in overall venom biochemistry and resulting clinical manifestations, the underlying venom composition differs significantly between the two species. As a result, the only available antivenom-which is a monovalent antivenom for D. typus-has minimal cross reactivity with T. mossambicanus and is not a clinically viable option. It was hypothesised that this lack of cross reactivity is due to the additional large metalloprotease protein within T. mossambicanus venom, which may also be responsible for faster coagulation times. In this study, we found that T. mossambicanus venom is a more powerful activator of prothrombin than that of D. typus and that the SVMP transcripts from T. mossambicanus form a clade with those from D. typus. The sequences from D. typus and T. mossambicanus were highly similar in length, with the calculated molecular weights of the T. mossambicanus transcripts being significantly less than the molecular weights of some isoforms on the 1D SDS-PAGE gels. Analyses utilising degylcosylating enzymes revealed that T. mossambicanus SVMPs are glycosylated during post-translational modification, but that this does not lead to the different molecular weight bands observed in 1D SDS-PAGE gels. However, differences in glycosylation patterns may still explain some of the difference between the enzymatic activities and neutralization by antivenom that have been observed in these venoms. The results of this study provide new information regarding the treatment options for patients envenomated by T. mossambicanus as well as the evolution of these dangerous snakes.

Rapid serum tube technology overcomes problems associated with use of anticoagulants.

INTRODUCTION: Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake proth-rombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients. MATERIALS AND METHODS: Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes. RESULTS: Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined. CONCLUSIONS: Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.

Prothrombin, alone or in complex concentrates or plasma, reduces bleeding in a mouse model of blood exchange-induced coagulopathy.

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

The use of new procoagulants in blunt and penetrating trauma.

PURPOSE OF REVIEW: Uncontrolled bleeding in trauma secondary to a combination of surgical bleeding and trauma-induced complex coagulopathy is a leading cause of death. Prothrombin complex concentrates (PCCs), recombinant activated factor seven (rFVIIa) and recombinant human prothrombin act as procoagulants by increasing thrombin generation and fibrinogen concentrate aids stable clot formation. This review summarizes the current evidence for procoagulant use in the management of bleeding in trauma, and data and evidence gaps for routine clinical use. RECENT FINDINGS: Retrospective and prospective studies of PCCs (±fibrinogen concentrate) have demonstrated a decreased time to correction of trauma coagulopathy and decreased red cell transfusion with no obvious effect on mortality or thromboembolic outcomes. PCCs in a porcine model of dilutional coagulopathy demonstrated a sustained increase in thrombin generation, unlike recombinant human prothrombin which showed a transient increase and has been studied only in animals. In other retrospective studies, there is a suggestion that lower doses of PCCs may be effective in the setting of acquired coagulopathy. SUMMARY: There is increasing evidence that early correction of coagulopathy has survival benefits, and the use of procoagulants as first-line therapy has the potential benefit of rapid access and timely treatment. This requires confirmation in prospective studies.

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples.

Background Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals. Methods Plasma clotting assays were performed using a Hyland-Clotek instrument. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay. Results We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood. Conclusions These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.

In vitro exploration of latent prothrombin mutants conveying antithrombin resistance.

INTRODUCTION: Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). MATERIALS AND METHODS: We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. RESULTS: The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). CONCLUSIONS: Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.

Use of four-factor prothrombin complex concentrate in the reversal of warfarin-induced and nonvitamin K antagonist-related coagulopathy.

: To evaluate the efficacy of international normalized ratio (INR) reversal using four-factor prothrombin complex concentrate (4F-PCC) in nonmedication-induced coagulopathy. We performed a single-site, retrospective cohort study of patients receiving off-label use of 4F-PCC. Cohorts included liver dysfunction if they had acute liver decompensation or cirrhosis without other causative factors of liver failure such as sepsis, coagulopathy of acute sepsis (CAS) if they had documentation of sepsis and no underlying liver disorder, known factor deficiencies, or medication-induced coagulopathy, or warfarin if they were taking warfarin. Patients with unknown medication or direct oral anticoagulant usage were excluded. 4F-PCC was administered 32 times in 26 patients for nonvitamin-K antagonist related coagulopathy (11 CAS and 21 liver dysfunction) and 47 administrations were in warfarin patients. Liver dysfunction patients had a mean model for end-stage liver disease score of 28 ±â€Š10. CAS and warfarin patients had significant INR reductions (ΔINR 1.9, P < 0.01; ΔINR 3.9, P < 0.01, respectively). Liver dysfunction patients mean change in INR trended toward significance (ΔINR 0.7, P = 0.09). Patients who received 4F-PCC based upon previously established dosing guidelines for moderate elevations in INR (20-30 IU/kg) doing demonstrated similar reductions in INR between CAS and warfarin patients (ΔINR 1.3, P = 0.03, ΔINR 1.0 P < 0.01, respectively). 4F-PCC significantly reduces the INR in CAS patients and trended toward significant reductions in liver dysfunction patients. Adequately powered, prospective trials are needed to demonstrate 4F-PCC efficacy in reversal of these coagulopathies.

Reduced anticoagulation variability in patients on warfarin monitored with Fiix-prothrombin time associates with reduced thromboembolism: The Fiix-trial.

Fiix-prothrombin time (Fiix-PT) differs from traditional PT in being affected by reduced factor (F) II or FX only. In the randomized controlled Fiix-trial, patients on warfarin monitored with Fiix-PT (Fiix-warfarin patients) had fewer thromboembolisms (TE), similar major bleeding (MB) and more stable anticoagulation than patients monitored with PT (PT-warfarin patients). In the current Fiix-trial report we analyzed how reduced anticoagulation variability during Fiix-PT monitoring was reflected in patients with TE or bleeding. Data from 1143 randomized patients was used. We analyzed the groups for anticoagulation intensity (time within target range; TTR), international normalized ratio (INR) variability (variance growth rate B1; VGR) and dose adjustment frequency. We assessed how these parameters associated with clinically relevant vascular events (CRVE), ie TE or MB or clinically relevant non-MB. TTR was highest in Fiix-warfarin patients without CRVE (median 82%;IQR 72-91) and lowest in PT-warfarin patients with TE (62%;56-81). VGR was lowest in Fiix-warfarin patients without CRVE (median VGR B1 0.17; 95% CI 0.08-0.38) and with TE (0.20;0.07-0.26) and highest in PT-warfarin patients with TE (0.50;0.27-0.90) or MB (0.59;0.07-1.36). The mean annual dose adjustment frequency was lowest in Fiix-warfarin patients with TE (mean 5.4;95% CI 3.9-7.3) and without CRVE (mean 6.0; 5.8-6.2) and highest in PT-warfarin patients with TE (14.2;12.2-16.3). Frequent dose changes predicted MB in both study arms. Compared to patients monitored with PT, high anticoagulation stability in Fiix-warfarin patients coincided with their low TE rate. Those with bleeding had high variability irrespective of monitoring method. Thus, although further improvements are needed to reduce bleeding, stabilization of anticoagulation by Fiix-PT monitoring associates with reduced TE.

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy.

Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.

Des-gamma-carboxy prothrombin antagonizes the effects of Sorafenib on human hepatocellular carcinoma through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways.

Despite significant progress, advanced hepatocellular carcinoma (HCC) remains an incurable disease, and the overall efficacy of targeted therapy by Sorafenib remains moderate. We hypothesized that DCP (des-gamma-carboxy prothrombin), a prothrombin precursor produced in HCC, might be one of the reasons linked to the low efficacy of Sorafenib. We evaluated the efficacy of Sorafenib in HLE and SK-Hep cells, both of which are known DCP-negative HCC cell lines. In the absence of DCP, Sorafenib effectively inhibited the growth of HCC and induced cancer cell apoptosis. In the presence of DCP, HCC was resistant to Sorafenib-induced inhibition and apoptosis, as determined by in vitro assays and in mice xenografted with HLE cells. Molecular analysis of HLE xenografted-nude mice showed that DCP activates the transduction of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR cascades. DCP might stimulate the formation of compensatory feedback loops in the intricately connected signaling pathways when kinases are targeted by Sorafenib. Our results indicate that DCP antagonizes the inhibitory effects of Sorafenib on HCC through activation of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR signaling pathways. Taken together, our findings define a DCP-mediated mechanism of inhibition of Sorafenib in HCC, which is critical for targeting therapy in advanced HCC.

Induction of microglial toll-like receptor 4 by prothrombin kringle-2: a potential pathogenic mechanism in Parkinson's disease.

Microglia-mediated neuroinflammation may play an important role in the initiation and progression of dopaminergic (DA) neurodegeneration in Parkinson's disease (PD), and toll-like receptor 4 (TLR4) is essential for the activation of microglia in the adult brain. However, it is still unclear whether patients with PD exhibit an increase in TLR4 expression in the brain, and whether there is a correlation between the levels of prothrombin kringle-2 (pKr-2) and microglial TLR4. In the present study, we first observed that the levels of pKr-2 and microglial TLR4 were increased in the substantia nigra (SN) of patients with PD. In rat and mouse brains, intranigral injection of pKr-2, which is not directly toxic to neurons, led to the disruption of nigrostriatal DA projections. Moreover, microglial TLR4 was upregulated in the rat SN and in cultures of the BV-2 microglial cell line after pKr-2 treatment. In TLR4-deficient mice, pKr-2-induced microglial activation was suppressed compared with wild-type mice, resulting in attenuated neurotoxicity. Therefore, our results suggest that pKr-2 may be a pathogenic factor in PD, and that the inhibition of pKr-2-induced microglial TLR4 may be protective against degeneration of the nigrostriatal DA system in vivo.

Effects of recombinant human prothrombin on thrombin generation in plasma from patients with hemophilia A and B.

BACKGROUND: The present study was carried out to investigate the impact of FII levels, and their increase, on the hemostatic potential in plasma from hemophilia A and B patients with and without inhibitors. METHOD: Recombinant human factor (F) II (rhFII) was added ex vivo to plasma from 68 patients with hemophilia A and B, with or without inhibitors. The hemostatic potential as measured by thrombin generation (calibrated automated thrombogram [CAT]) was focused on the endogenous thrombin potential (ETP) as it has been shown to correlate with the clinical phenotype of bleeding in hemophilia patients and has also been used to guide bypassing therapy in hemophilia patients with inhibitors before elective surgery. The factor eight inhibitor bypassing agent (FEIBA(®) ) was used as a reference to the clinical situation. RESULTS: The study shows that rhFII concentration-dependently increased ETP by a similar magnitude in hemophilia A and B, both with and without inhibitors. Compared with FEIBA, rhFII showed a shallower concentration-response curve. In both types of hemophilia 100 mg L(-1) of rhFII roughly doubled the ETP. A corresponding response was obtained by 0.5 U mL(-1) of FEIBA. CONCLUSION: These data support the theory that FII is one of the major components responsible for the efficacy of FEIBA. The data also indicate that rhFII may be useful, alone or in combination with other coagulation factors, in some of the conditions for which FEIBA is used today, although more data are needed to substantiate this.

Des-gamma-carboxy prothrombin (DCP) antagonizes the effects of gefitinib on human hepatocellular carcinoma cells.

BACKGROUND/AIMS: Des-gamma-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, is known as a marker for HCC. Recent studies indicated that high levels of DCP are associated with the malignant potential of HCC. In this study, we aimed to investigate the association of DCP with gefitinib treatment failure in HCC and whether DCP counteracts gefitinib-induced growth inhibition and apoptosis of HCC. METHODS: The experiments were performed in HCC cell lines HepG2 and PLC/PRF/5. The effects of gefitinib on HCC in the presence or absence of DCP were evaluated by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Apoptotic cells were identified by Annexin V-FITC/PI staining. Western blotting was performed to analyze the expressions of molecules related to the apoptotic caspase-dependent pathway and epidermal growth factor receptor (EGFR) pathway. RESULTS: Gefitinib inhibited HCC cell proliferation and induced apoptosis in HCC cells. The effects of gefitinib on HCC cells were antagonized by DCP. In the presence of DCP, HCC cells were resistant to the gefitinib-induced inhibition of proliferation and stimulation of apoptosis. DCP prevented the activation of the apoptotic caspase-dependent pathway induced by gefitinib. These antagonistic effects of DCP also arose from its ability to up-regulate EGFR, c-Met and hepatocyte growth factor (HGF) in HCC cells. CONCLUSION: DCP antagonized gefitinib-induced HCC cell growth inhibition by counteracting apoptosis and up-regulating the EGFR pathway. High levels of DCP might thus lead to low response rates or possibly no response to gefitinib in patients with HCC.

The effect of recombinant and plasma-derived prothrombin on prothrombin time in human plasma.

INTRODUCTION: When investigating coagulation assays to measure the effect of infused prothrombin (FII) in in vivo coagulopathy models, we found that addition of FII, plasma-derived human FII (pd-hFII) or recombinant human FII (r-hFII), to normal plasma resulted in a concentration-dependent increase in prothrombin time (PT) initiated with Innovin(®) . METHODS: The effect on PT by addition to plasma of either pd-hFII or r-hFII, using different commercial PT reagents, was studied both by turbidimetry and viscometry. RESULT: Addition of FII to plasma resulted in increased PT when initiated with Innovin(®) : PT increased with 20% by doubling the concentration. The prolongation of PT became more pronounced with 2-6000 times diluted Innovin(®) . However, by adjustment of the final free Ca(2+) concentration in the assay with diluted Innovin(®) from 8.3 to 1.3 mmol/L, no FII-dependent increase in PT was found. In contrast, no prolongation of PT was found with other commercial PT reagents. A KM = 3 nmol/L was obtained with pd-hFII, respectively, r-hFII with FII-depleted plasma using Thromborel(®) to initiate PT. CONCLUSION: At normal plasma concentration of FII, addition of FII should not have an effect on PT. The prolonged PT with Innovin(®) , but not with other PT reagents, at supranormal FII concentration is an artefact.

Prothrombin complex concentrates as reversal agents for new oral anticoagulants: lessons from preclinical studies with Beriplex.

Although new oral anticoagulants (NOACs) represent an advance in anticoagulant therapy over vitamin K antagonists (VKAs), they nevertheless have a low, but significant risk for bleeding complications. Reversal agents for VKAs, such as prothrombin complex concentrates (PCCs), are currently being evaluated in preclinical studies for NOAC reversal. This article reviews the preclinical data for the most extensively studied PCC for NOAC reversal, Beriplex, a 4-factor PCC. The results from the Beriplex studies are also compared with those obtained with other reversal agents, including different nonactivated PCCs, activated PCCs, and recombinant activated factor VII.

Interleukin-13/Interleukin-4-induced oxidative stress contributes to death of prothrombinkringle-2 (pKr-2)-activated microglia.

The present study examined whether Interleukin-13 (IL-13) or IL-4, an anti-inflammatory cytokine, could induce cell death of activated microglia by prothrombin kringle-2 (pKr-2) which is a domain of prothrombin distinct from thrombin. Microglia cell death was detected at eight days after co-treatment of pKr-2 with IL-13/IL-4 in vitro. This cell death was assessed by live assay, dead assay, TUNEL and MTT assay. In parallel, reactive oxygen species (ROS) production was evident as assessed by superoxide assay, WST-1 and analyzing DCF in combination of pKr-2 and IL-13 or IL-4 treated microglia. The IL-13/IL-4-enhanced ROS production and cell death in pKr-2 activated microglia was partially inhibited by an NADPH oxidase inhibitor, apocynin and/or by several antioxidants. Moreover, Western blot analysis showed a significant increase in cyclooxygenase-2 (COX-2) expression in combination of pKr-2 and IL-13 or IL-4 treated microglia, which was partially inhibited by apocynin and an antioxidant, trolox. Additional studies demonstrated that microglia cell death was reversed by treatment with COX-2 inhibitor, NS398. Our data strongly suggest that oxidative stress and COX-2 activation through NADPH oxidase may contribute to IL-13/IL-4 induced cell death of pKr-2 activated microglia.