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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., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: Potential conflict of interest: The project was in part funded by a Development grant from the National Health and Medical Research Council of Australia and by Q-Sera Pty Ltd, Australia., (©Croatian Society of Medical Biochemistry and Laboratory Medicine.)

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.

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.

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.

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., (Copyright © 2017. Published by Elsevier Ltd.)

: 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 ± 10. 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.

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 B 1 ; 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 B 1 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.

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., (© 2016 by The American Society of Hematology.)

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., Competing Interests: The authors declare no conflict of interest.

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.

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., (© 2015 International Society on Thrombosis and Haemostasis.)

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., (© 2014 John Wiley & Sons Ltd.)

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., (© 2015 S. Karger AG, Basel.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2013.)

Objective: Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not have increased circulating prothrombotic biomarkers, their plasma demonstrates increased tissue factor-dependent thrombin generation in vitro. The objectives of this study were to determine the pathological role of elevated prothrombin in venous and arterial thrombosis in vivo, and distinguish thrombogenic mechanisms in these vessels., Approach and Results: Prothrombin was infused into mice to raise circulating levels. Venous thrombosis was induced by electrolytic stimulus to the femoral vein or inferior vena cava ligation. Arterial thrombosis was induced by electrolytic stimulus or ferric chloride application to the carotid artery. Mice infused with prothrombin demonstrated increased tissue factor-triggered thrombin generation measured ex vivo, but did not have increased circulating prothrombotic biomarkers in the absence of vessel injury. After venous injury, elevated prothrombin increased thrombin generation and the fibrin accumulation rate and total amount of fibrin ≈ 3-fold, producing extended thrombi with increased mass. However, elevated prothrombin did not accelerate platelet accumulation, increase the fibrin accumulation rate, or shorten the vessel occlusion time after arterial injury., Conclusions: These findings reconcile previously discordant findings on thrombin generation in hyperprothrombinemic individuals measured ex vivo and in vitro, and show elevated prothrombin promotes venous, but not arterial, thrombosis in vivo.

Background and Objectives: Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown., Results: APCC (0.05 U mL(-1) ) increased FVIII activity ~ 4-fold within 1 min in one-stage clotting assays, followed by a return to initial levels within 10 min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~ 3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) ., Conclusion: Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors., (© 2013 International Society on Thrombosis and Haemostasis.)

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., Objectives: This study was designed to determine in an animal model whether a PCC (Beriplex P/N) can effectively reverse the effects of dabigatran. An additional objective was to evaluate markers of dabigatran-associated bleeding diathesis., Methods: Anesthetized rabbits were treated with 0.4 mg kg(-1) dabigatran followed by PCC doses of 20, 35 or 50 IU kg(-1) or placebo. After a standardized kidney incision, volume of blood loss and time to hemostasis were determined., Results: From an initial mean of 29 mL, blood loss progressively declined by 5.44 mL with a 95% confidence interval (CI) of 2.21-8.67 mL per 10 IU kg(-1) increment in PCC dose (P = 0.002). At a PCC dose of 50 IU kg(-1) blood loss was fully normalized. Increasing PCC doses shortened the median time to hemostasis from 20.0 to 5.7 min (P < 0.001). The rate of hemostasis was nearly trebled with each 10 IU kg(-1) increment in PCC dose (rate ratio, 2.89; CI, 1.64-5.09)., Conclusions: In this animal study, PCC showed potential as an agent for reversing the effects of dabigatran. Further investigation is warranted., (© 2012 International Society on Thrombosis and Haemostasis.)

Vitamin K was discovered fortuitously in 1929 as part of experiments on sterol metabolism and was immediately associated with blood coagulation. In the decade that followed, the principal K vitamers, phylloquinone and the menaquinones, were isolated and fully characterized. In the early 1940s, the first vitamin K antagonists were discovered and crystallized with one of its derivatives, warfarin, still being widely used in today's clinical setting. However, major progress in our understanding of the mechanisms of action of vitamin K came in the 1970s with the discovery of γ-carboxyglutamic acid (Gla), a new amino acid common to all vitamin K proteins. This discovery not only provided the basis to understanding earlier findings about prothrombin but later led to the discovery of vitamin K-dependent proteins (VKDPs) not involved in hemostasis. The 1970s also saw an important breakthrough with respect to our understanding of the vitamin K cycle and marked the discovery of the first bone VKDP, osteocalcin. Important studies relating to the role of vitamin K in sphingolipid synthesis were also underway at that time and would pave the way to further work 15 years later. The decades that followed saw the discovery of additional VKDPs showing wide tissue distribution and functional scope, the latest members having been identified in 2008. The 1990s and 2000s were also marked by important epidemiological and intervention studies that focused on the translational impact of recent vitamin K discoveries, notably with respect to bone and cardiovascular health. This short review presents an overview of the history of vitamin K and of its recent developments., (Copyright © 2012 S. Karger AG, Basel.)

Around 20 % of the patients with severe hemophilia develop inhibitory antibodies against the factor they lack. In these patients the administration of FVIII/FIX-concentrates are not hemostatically effective. Since FVIIa is not enzymatically active unless complexed with tissue factor (TF) exposed following an injury to the vessel wall, it was considered an attractive candidate for improved treatment of patients with inhibitors. Plasma-derived FVIIa was purified and shown to induce hemostasis in two hemophilia A patients with inhibitors. Later recombinant FVIIa (rFVIIa) was developed and pharmacological doses have an efficacy rate of around 90 % in serious bleedings and permit major orthopaedic surgery. These findings were a breakthrough in understanding the FVII-TF pathway in hemostasis. The initially formed FVIIa-TF complexes provide a limited amount of thrombin, activating FVIII, FV, FXI as well as platelets. On the activated platelet surface the full burst of thrombin necessary for generating a firm fibrin hemostatic plug occurs. In case of impaired thrombin generation, loose fibrin plugs easily dissolved are formed. Extra rFVIIa enhances thrombin generation and generates tight fibrin plugs.

Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, prothrombin purified from porcine plasma was modified through PEGylation at N-terminal residue using 40 kDa PEG-phenyl-isothiocyanate (PIT-PEG40K). The monoPEGylated prothrombin enhanced biostability and remarkably prolonged circulating half-life in mice as compared with that of the nonmodified prothrombin. Moreover, the immunogenicity of PEGylated prothrombin in mice is significantly decreased compared to nonmodified prothrombin. These studies demonstrated the feasibility of PEGylating prothrombin as a promising way for the development of new prothrombin drugs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Des-γ-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, has been shown to be associated with the biological malignant potential of HCC. The aim of this study was to evaluate the effect of DCP on HCC cell growth and metastasis, and to explore the underlying molecular mechanisms. DCP significantly stimulated HCC cell growth, as measured by cell counting kit-8 assay. Transwell chamber assay showed that DCP increased HCC cell migration through reconstituted extracellular matrix (Matrigel). Gelatin zymography assay and Western blot analysis demonstrated that DCP increased the secretion and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in the supernatant of cultured HCC cells and on tumour cell membranes. DCP was found to bind to the cell surface receptor Met, resulting in Met phosphorylation and subsequent activation of the epidermal growth factor receptor (EGFR). Western blot analysis demonstrated that DCP stimulated a sequential kinase phosphorylation cascade including ERK1/2, MEK1/2 and c-Raf, indicating activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK1/2 MAPK) signalling pathway. Furthermore, blocking ERK1/2 MAPK activation with ERK1/2 inhibitor PD98059 essentially abolished the DCP-induced MMP-2 and MMP-9 activity, confirming the signalling pathway of DCP stimulation. Taken together, these results suggested that DCP stimulates HCC growth and promotes HCC metastasis by increasing the activity of MMP-2 and MMP-9 through activation of the ERK1/2 MAPK signalling pathway., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy., Methods: In 50 anesthetized pigs, ~60% of the blood volume was withdrawn and replaced with hydroxyethyl starch. Pigs were randomized to receive either 200 mg kg(-1) fibrinogen (n = 10), fibrinogen and 35 IU kg(-1) prothrombin complex concentrate (PCC) (n = 10), fibrinogen and 4 mg kg(-1) recombinant human factor II (rhFII) concentrate (n = 10), fibrinogen and a three-factor combination (3F) of 4 mg kg(-1) rhFII, 0.006 mg kg(-1) recombinant human FVIIa and 0.32 mg kg(-1) recombinant human FX (n = 10), or saline (n = 10). Thereafter, a standardized liver laceration was performed to induce uncontrolled hemorrhage. Survival time and blood loss were determined, and standard coagulation tests and thrombelastometry were performed., Results: Fibrinogen combined with rhFII or PCC improved survival. Blood loss was significantly decreased in all groups as compared with the animals receiving saline. Clotting time was significantly shortened in the animals treated with fibrinogen and PCC, as well as in those treated with fibrinogen and 3F. One animal died after administration of fibrinogen and PCC., Conclusion: Following hemodilution, a combination of fibrinogen and PCC, rhFII or 3F enhances coagulation and final clot strength. Mortality was reduced statistically significantly only in the animals treated with fibrinogen and rhFII or PCC, whereas administration of the combination of fibrinogen and PCC caused a fatal thromboembolic complication. The combination of fibrinogen and rhFII might be effective in reversing dilutional coagulopathy and may reduce blood loss in cases of dilutional coagulopathy., (© 2011 International Society on Thrombosis and Haemostasis.)

c-Met, a type of receptor tyrosine kinase, may be significantly associated with the progression of hepatocellular carcinoma (HCC). In addition, des-γ-carboxyprothrombin (DCP) has been found to interact with c-Met and activate HCC cell growth. Therefore, the functional inhibition of c-Met expressed on HCC cells should arrest HCC cell growth. The present study found that the c-Met inhibitor SU11274 suppressed HCC cell growth by inhibiting the activation of c-Met. Furthermore, this inhibitor also neutralized the activation of HCC cell growth resulting from the addition of DCP. These results suggest that the functional inhibition of c-Met might be a target for the development of chemotherapeutic agents for HCC, and especially those that are positive for expression of DCP.

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p<0.0001), Hoplocephalus stephensii (Stephen's-banded snake; p<0.0001) and Notechis scutatus (tiger snake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

The action of the coumarin-type drugs and related compounds is reviewed to their VKOR antagonistic effects. In our study, twenty 3-pyridinyl, pyrimidinyl and pyrazolyl-4-hydroxycoumarin derivatives were synthesized. A comparative in vivo (CT, PT determination) and in vitro (measurement of PIVKA-II levels) anticoagulant study with respect to warfarin showed that the synthesized compounds have different anticoagulant activities, the most prospective compounds were the 3-pyrazolyl-4-hydroxycoumarin derivatives., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Meizothrombin efficiently activates mechanically triggered platelets and enhance collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma. Thus, in clotting system activation zone meizothrombin is able to enhance process of involving platelets in clotformation. Pre-thrombin 1 inhibits collagen-, ADP- and adrenalin-induced aggregation of platelet-rich blood plasma and so regulates not only plasm but platelet hemostasis by reverse negative relation.

In neurodegenerative diseases, such as Alzheimer's and Parkinson's, microglial cell activation is thought to contribute to CNS injury by producing neurotoxic compounds. Prothrombin and kringle-2 increase levels of NO and the mRNA expression of iNOS, IL-1beta, and TNF-alpha in microglial cells. In contrast, the human prothrombin kringle-2 derived peptide NSA9 inhibits NO release and the production of pro-inflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in LPS-activated EOC2 microglia. In this study, we investigated the anti-inflammatory effects of NSA9 in human prothrombin- and kringle-2-stimulated EOC2 microglia. Treatment with 20-100 muM of NSA9 attenuated both prothrombin- and kringle-2-induced microglial activation. NO production induced by MAPKs and NF-kappaB was similarly reduced by inhibitors of ERK (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), and NSA9. These results suggest that NSA9 acts independently as an inhibitor of microglial activation and that its effects in EOC2 microglia are not influenced by the presence of kringle-2.

Des-gamma-carboxy prothrombin (DCP) is an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells. Serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC. However, the role of DCP in the development of angiogenesis is not well understood. Herein, we report the effects of DCP on growth and migration of human vascular endothelial cells. DCP significantly stimulated the proliferation of HUVEC (ECV304) cells in a dose and time dependent manner, as measured by the MTT assay. A continuous rapid migration of ECV304 cells was observed in the presence of DCP measured by the scratch wound assay. The continuous rapid invasive activity, measured by transwell chamber assay also showed that DCP increased endothelial cells migration through the reconstituted extracellular matrix (Matrigel). Further, the tube formation of vascular endothelial cells on 3-D Matrigel showed an increased number of branch points of ECV304 cells induced by DCP in a dose dependent manner. The levels of vascular endothelial cell growth-related angiogenic factors and matrix metalloproteinase were also examined. DCP significantly stimulated the expression levels of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP)-2 (latent and active). Together, these data suggest that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities in angiogenesis of HCC.

Aims: Des-gamma-carboxyl prothrombin (DCP) is a serum protein produced by hepatocellular carcinoma (HCC) cells. The aim of this study was to evaluate the angiogenic activity of DCP in HCC cells., Main Methods: The proliferation of HCC cells was measured by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. The growth of HCC cells was also evaluated in vivo by using the xenografts in nude mice. The enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of angiogenic factors in supernatant of cell culture. The expression of angiogenic factors was examined by Western blot analysis and immunohistochemical staining., Key Findings: DCP displayed the stimulation of HCC cell growth in a dose (5-80 ng/ml) and time (24-96 h) dependent manner. The increase of cell growth was also observed in nude mice bearing well-established, palpable HepG2 and SMMC-7721 xenografts after 2 weeks administration of DCP. HCC cell growth was accompanied by the elevated levels of angiogenic factors. The levels of vascular endothelial growth factor (VEGF), transforming growth factor-alpha (TGF-alpha) and basic fibroblast growth factor (bFGF) in supernatant of SMMC-7721 cells were increased from 47, 126, and 60 pg/10(6) cells/24 h to 400, 208, and 298 pg/10(6) cells/24 h, respectively, after 72 h incubation with 80 ng/ml of DCP. The results of Western blot analysis and immunohistochemical staining of HCC xenografts also showed the significant increase of VEGF, TGF-alpha, and bFGF in HCC cells., Significance: These results provide the information that DCP is a type of growth factor in progression of HCC.

Background: Thrombin concentration modulates fibrin structure and fibrin structure modulates clot stability; however, the impact of localized, cell surface-driven in situ thrombin generation on fibrin structure and stability has not previously been evaluated., Methods and Results: Human fibroblasts were incubated with factors Xa, Va, prothrombin and fibrinogen, or plasma. Fibrin formation, structure, and lysis were examined using laser scanning confocal microscopy and transmission electron microscopy. In situ thrombin generation on the cell surface produced clots with a significantly denser fiber network in a 10-microm region proximal versus distal to (40 to 50 microm) the cell surface. This morphology was not altered by addition of integrin-blocking RGDS peptide and was not apparent in clots made by exogenous thrombin addition, suggesting that spatial morphology was dictated predominantly by localized thrombin generation on the fibroblast surface. The fibrin network lysed more rapidly distal versus proximal to the cell surface, suggesting that the structural heterogeneity of the clot affected its fibrinolytic stability., Conclusions: In situ thrombin generation on the cell surface modulates the three-dimensional structure and stability of the clot. Thrombus formation in vivo may reflect the ability of the local cell population to support thrombin generation and, therefore, the three-dimensional structure and stability of the fibrin network.

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.

Prothrombin (PT) is an RGD-containing bone-residing precursor to the serine protease thrombin (TH), which acts as an agonist for a variety of cellular responses in osteoblasts and osteoclasts. We show here that PT, TH, osteopontin (OPN) and fibronectin (FN) promoted adhesion of isolated neonatal rat long bone osteoclasts. However, the cells that adhered to PT and TH were smaller in size, rounded and contained 3-4 nuclei, in comparison to the cells adhering to OPN and FN, which were larger with extended cytoplasmic processes and 6-7 nuclei. Attachment of the larger osteoclasts to OPN and FN was inhibited by antibodies towards beta 3 and beta 1 integrin subunits, respectively. Whereas an RGD-containing peptide inhibited adhesion of the smaller osteoclasts to PT and TH, this was not seen with the beta 3 or beta 1 antibodies. In contrast, the beta 1 antibody augmented osteoclast adhesion to PT and TH in an RGD-dependent manner. Small osteoclasts were less efficient in resorbing mineralized bovine bone slices, as well as expressed lower mRNA levels of MMP-9 and the cathepsins K and L compared to large osteoclasts. The small osteoclast adhering to PT and TH may represent either an immature, less functional precursor to the large osteoclast or alternatively constitute a distinct osteoclast population with a specific role in bone.

Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Prothrombin is activated in vivo by the prothrombinase complex to form fragment 1.2 and thrombin. Fragment 1.2 has an amino-terminal gla domain and two kringle domains. The second kringle domain (kringle 2) binds to the exosite II on thrombin. Nascent thrombin generated on platelet surface remains non-covalently bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate coagulant activity of thrombin, we labeled thrombin at the active site with fluorescein-Phe-Pro-Arg chloromethylketone and monitored the fluorescence changes upon ligand binding. Anionic phospholipid-bound fragment 1.2 and fragment 2 bound to FPR-thrombin and induced changes in the active site with half maximal effects at 7.2 microM and 8.8 microM, respectively. We also tested the effect of anionic phospholipid-bound fragment 1.2 (0-10 microM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on amidolytic activity towards S2238, suggesting a competitive inhibition of the fibrinogen binding site. Furthermore, fragment 1.2 inhibited FPR-thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that the membrane-bound prothrombin fragment 1.2 may play a role in hemostasis by down regulating the procoagulant activity of newly formed thrombin.

Prothrombin is a plasma protein, which after tissue injury is converted to alpha-thrombin and is mainly involved in blood clot formation. It has also been shown to have a mitogenic effect on primary endothelial cells, vascular smooth muscle cells, fibroblasts and some tumor cells, but is an inhibitor of rat hepatocyte DNA synthesis on fibronectin matrix in cell culture. We now report that prothrombin is converted to alpha-thrombin by primary cultures of normal adult rat hepatocytes and alpha-thrombin is also a potent inhibitor of hepatocytes DNA synthesis. In contrast, rat hepatoma cells cultured under similar conditions were resistant to alpha-thrombin mediated DNA synthesis inhibition. The inhibitory effect of alpha-thrombin on DNA synthesis was antagonized by hirudin and antithrombin, two specific alpha-thrombin inhibitors or by the presence of collagen-I matrix. A thrombin receptor activating peptide (TRAP6) also inhibited EGF-mediated rat hepatocyte DNA synthesis, suggesting a role of the thrombin receptors in this process. Matrix fibronectin was degraded by alpha-thrombin. However, no appreciable cell detachment was observed. These results suggest a role of alpha-thrombin as a potent growth inhibitor of normal hepatocytes, possibly through control of fibronectin or other matrix protein(s).

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.

Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However, although bites can be deadly, snake venom is a natural biological resource that contains several components of potential therapeutic value. Venom has been used in the treatment of a variety of pathophysiological conditions in Ayurveda, homeopathy and folk medicine. With the advent of biotechnology, the efficacy of such treatments has been substantiated by purifying components of venom and delineating their therapeutic properties. This review will focus on certain snake venom components and their applications in health and disease.

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC(50) (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.

Objectives: To investigate the inhibitory role of gamma (gamma)-carboxyglutamic acid (Gla) in the Gla domain of urinary prothrombin in the formation of calcium oxalate crystals., Methods: Morpholine and formaldehyde at different concentrations were added to the solution of prothrombin to cause the conversion of Gla to gamma-methyleneglutamic acid (MGlu). The extent of the modification was controlled by the relative amount of modification reagents to prothrombin. The 100-fold molar excess of modification reagents to prothrombin produced the prothrombin molecule containing 2gamma-MGlu residues, and the 10,000-fold molar excess produced the molecule containing 8gamma-MGlu residues. The inhibitory activities of prothrombin and modified prothrombin to calcium oxalate crystallization were evaluated by the seeded crystallization technique., Results: The inhibitory index of Gla prothrombin to calcium oxalate crystallization was 14.2%, that of 2gamma-MGlu prothrombin was 12.8%, decreased by about 10%, and that of 8gamma-MGlu prothrombin was 5.0%, decreased by about 65%., Conclusions: The Gla in the Gla domain of urinary prothrombin may play an important role in inhibiting the formation of renal calcium oxalate calculus.

Background: The abnormal response to activated protein C could be the mechanism to explain the prothrombotic role of elevated coagulation factor levels., Objective: We evaluated the effect of factor VIII, II, or X (FVIII, FII, or FX) levels on activated protein C resistance technique and its association with the resistant phenotype., Materials and Methods: The correlation between APCR and FVIII was assessed in 36 samples, after Desmopressin infusion and the correlation between FII or FX and APCR in 15 patients with plasma levels between 100-125 U/dl. Also, the effect of the addition of purified human factors (FII, FX) to a normal plasma pool (final concentration: 100, 120, 140, 180, 220 U/dl) was estimated on the APCR technique., Results: APCR values correlated with FVIII increase (r(Spearman) = 0.839; p < 0.001); APCR was abnormal (<2.4) in 9/36 samples, showing higher FVIII values in the abnormal group (VIII(abnormalAPCR) = 176.7 +/- 14.2; VIII(normalAPCR) = 103.5 +/- 8.0). APCR did not correlate with endogenous FII (r(Spearman) = 0.423) or FX (r(Spearman) = -0.169). However, the addition of human FII or FX to the normal plasma pool caused a decrease in APCR (r(SpearmanFII) = -0.843; r(SpearmanFX) = -0.958) without reaching abnormal (<2.4) results. FVIII levels may be associated with a resistant phenotype at values >153.0 U/dl, according to the linear regression analysis. Exogenous FII or FX levels greater than 120 U/dl would affect the APCR, without obtaining abnormal results., Conclusions: The data do not allow the direct association of the FII or FX increase with a defect in the protein C system in the current conditions.

Des-gamma-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma (HCC). In the present study, we demonstrate that DCP has a mitogenic effect on HCC cell lines. Purified DCP stimulated DNA synthesis of Hep3B and SK-Hep-1 cells in a dose-dependent manner. DCP was found to bind with cell surface receptor Met causing Met autophosphorylation and also to activate STAT3 signaling pathway through Janus kinase 1. Luciferase gene reporter analysis showed that DCP induced STAT3-related transcription. Small interfering RNAs against both STAT3 and Met abrogated DCP-induced cell proliferation. DCP did not affect the mitogen-activated protein kinase pathway, Myc signaling pathway, or phosphoinositide 3-kinase/Akt pathway. Based on these results, we believe that DCP acts as an autologous mitogen for HCC cell lines. The Met-Janus kinase 1-STAT3 signaling pathway may be a major signaling pathway for DCP-induced cell proliferation.

Activation of prothrombin to thrombin is the key reaction in blood coagulation cascade. We have recently shown that Australian rough scaled snake, Tropidechis carinatus, possesses two parallel prothrombin activator systems. Trocarin D, a venom prothrombin activator produced in the venom gland, plays an offensive role as a toxin, whereas factor X is produced in the liver and plays a role in the hemostatic mechanism. These two proteins are structurally similar and have identical domain architecture. Because of the differences in their physiological roles, and tissue-specific expression, we determined the gene structure of these closely related proteins. Both the genes have eight exons similar to all mammalian factor X genes. All the exon-intron boundaries of these two genes are at the same position and the splice junctions are almost identical. Partial sequencing of the introns shows that they share a very high degree of sequence identity indicating that the gene duplication is a recent event. Further studies on the characterization of these two genes particularly the promoter regions are in progress.

A number of studies indicate that coagulation proteases play significant roles in cancer biology. Melanoma is a highly metastatic cancer, and there is evidence that thrombin contributes to this aggressive pattern. However, few studies correlate this type of cancer with formation of the prothrombinase complex, which is responsible for conversion of prothrombin into thrombin in the coagulation system. The aim of this study was to investigate the assembly and regulation of prothrombinase complex on the murine melanoma cell line, B16F10. B16F10 cells were unable to activate prothrombin except when previously incubated with factor Xa. This effect was dependent on factor Xa binding to cell membranes, since no activation was detected with Gla-domainless factor Xa. The thrombin formation by B16F10-bound factor Xa was enhanced approximately 10 fold in the presence of factor Va, indicating the assembly of prothrombinase complex. Differently from platelets, B16F10-assembled prothrombinase complex was inhibited by prothrombin fragment 1 but not by fragment 2. In addition, bothrojaracin, a specific ligand of proexosite I on prothrombin, caused a significant decrease in the zymogen activation. Our data demonstrate that B16F10 melanoma cells generate thrombin by promoting assembly of the prothrombinase complex. This ability might be correlated with the increased metastatic potential of this cell line. Moreover, B16F10-assembled prothrombinase complex seems to be modulated in a different way from that found for the physiological complex assembled on platelets.

A staging system for hepatocellular carcinoma was reported from Italy (CLIP). In this study, we evaluate the CLIP scoring system and establish a new scoring system for predicting the prognosis of patients with hepatocellular carcinoma. Patients (n=141) who were diagnosed and who underwent initial treatment at our single institution were recruited retrospectively into this study. We evaluated markers for prognosis, using a stratified Cox proportional hazard regression model and Kaplan-Meier survival analysis. CLIP score differentiated patients with different survival experiences by Kaplan-Meier estimated survival analysis. However, with respect the CLIP score, more than two thirds of patients were included in the early stage (CLIP 0-1), and the group with better prognosis than the survival rate of all patients was the only one with CLIP 0. Multivariate analysis revealed that des-gamma-carboxy prothrombin (DCP) >/=100 mAU/ml (relative risk, 2.06; P=0.0218) was statistically significant as a predictor of poor survival. A new prognostic scoring system included DCP classified patients to 6 well-balanced groups (score 0-5). The new prognostic scoring system 0 group (14.9% of the cohort) and the CLIP score 0 group (34.0% of the cohort) had a median survival of 66.9 and 61.6 months. The new prognostic scoring system performs better for prediction of survival than either the CLIP score or the Child-Pugh stage. In conclusion, the described scoring system provides more accurate prognostic information than the CLIP scoring system. It may help physicians decide more appropriate clinical and therapeutic management.