Prothrombin Time Assay Research Articles

Correlation of Thromboelastography with Apparent Rivaroxaban Concentration: Has Point-of-Care Testing Improved?

Concern remains over reliable point-of-care testing to guide reversal of rivaroxaban, a commonly used factor Xa inhibitor, in high-acuity settings. Thromboelastography (TEG), a point-of-care viscoelastic assay, may have the ability to detect the anticoagulant effect of rivaroxaban. The authors ascertained the association of apparent rivaroxaban concentration with thromboelastography reaction time, i.e., time elapsed from blood sample placement in analyzer until beginning of clot formation, as measured using TEG and TEG6S instruments (Haemonetics Corporation, USA), hypothesizing that reaction time would correlate to degree of functional factor Xa impairment. The authors prospectively performed a diagnostic accuracy study comparing coagulation assays to apparent (i.e., indirectly assessed) rivaroxaban concentration in trauma patients with and without preinjury rivaroxaban presenting to a single center between April 2016 and July 2018. Blood samples at admission and after reversal or 24 h postadmission underwent TEG, TEG6S, thrombin generation assay, anti-factor Xa chromogenic assay, prothrombin time (PT), and ecarin chromogenic assay testing. The authors determined correlation of kaolin TEG, TEG6S, and prothrombin time to apparent rivaroxaban concentration. Receiver operating characteristic curve compared capacity to distinguish therapeutic rivaroxaban concentration (i.e., greater than or equal to 50 ng/ml) from nontherapeutic concentrations. Eighty rivaroxaban patients were compared to 20 controls. Significant strong correlations existed between rivaroxaban concentration and TEG reaction time (ρ = 0.67; P < 0.001), TEG6S reaction time (ρ = 0.68; P < 0.001), and prothrombin time (ρ = 0.73; P < 0.001), however reaction time remained within the defined normal range for the assay. Rivaroxaban concentration demonstrated strong but not significant association with coagulation assays postreversal (n = 9; TEG reaction time ρ = 0.62; P = 0.101; TEG6S reaction time ρ = 0.57; P = 0.112) and small nonsignificant association for controls (TEG reaction time: ρ = -0.04; P = 0.845; TEG6S reaction time: ρ = -0.09; P = 0.667; PT-neoplastine: ρ = 0.19; P = 0.301). Rivaroxaban concentration (area under the curve, 0.91) and TEG6S reaction time (area under the curve, 0.84) best predicted therapeutic rivaroxaban concentration and exhibited similar receiver operating characteristic curves (P = 0.180). Although TEG6S demonstrates significant strong correlation with rivaroxaban concentration, values within normal range limit clinical utility rendering rivaroxaban concentration the gold standard in measuring anticoagulant effect.

Influence of hemolysis, icterus and lipemia on coagulation tests as performed on Cobas t511 new analyzer.

: In the coagulation laboratory, spurious hemolysis, icterus and lipemia (HIL) in test samples represent by far the leading diagnostic prenalytical challenges. The aim of this study was to assess the performance of the preanalytical module on the new hemostasis analyser Cobas Roche t511. We assessed the influence of HIL on prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib), antithrombin and D-dimer on plasma pools aliquots with different interference degrees. Moreover, we evaluated spontaneous hemolysis by comparing results on 50 paired samples (hemolysed versus nonhemolysed). Spurious hemolysis interference studies highlight the absence of a clinical significant impact on PT, APTT and antithrombin test results at all hemoglobin concentration investigated. For Fib and D-dimer assays a clinically significant difference was observed in the most hemolysed aliquot for Fib and in the two most hemolysed aliquots for D-dimer. Spontaneous hemolysis interference studies showed no clinical significant differences for PT and antithrombin assays, instead for APTT, Fib and D-dimer we found significant statistical and clinical differences between hemolysed and non hemolysed specimens. Bilirubin interference studies and lipemic samples interference studies enable us to confirm that the differences in the results obtained between the different aliquots and reference pool is not clinically significant for all assays. HIL check preanalytical module of Cobas Roche t511 analyzer displaied excellent performance for routine use in clinical laboratories. Regardless of analytical considerations, the type of interference encountered with spurious HIL is substantially different and requires different approaches.

Antioxidant, Anti-inflammatory, and Anticoagulation Properties of Aegiceras corniculatum and Acanthus ilicifolius

Free radical production from different biological and environmental sources is due to an imbalance of natural antioxidants, which further leads to inflammation. Antioxidant metabolites are often characterized by anti-inflammatory and anticoagulation activity. Mangrove plants synthesize different classes of metabolites, including antioxidants, to minimize the devastating effect of oxidation resulting from the elevated salinity, UV, and other unique geochemical components. Accordingly, this study aimed at investigating the antioxidant, anti-inflammatory, and anticoagulation properties, as well as polyphenol content of the two selected mangrove plant species: Aegiceras corniculatum and Acanthus ilicifolius. We used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, reducing power, ferric reducing antioxidant power (FRAP) assay, β-carotene-linoleic acid bleaching assay (BCB), total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) to determine antioxidant activity of the ethanol extract of A. corniculatum bark and leaves and A. ilicifolius leaves. Furthermore, human red blood cell (HRBC) membrane stabilization assay, lipoxygenase (LOX) inhibition assay, and prothrombin time (PT) test were performed for determining anti-inflammatory activity of the samples. A. corniculatum bark is a potent antioxidant (IC50 20.49 ± 2.14 µg/mL in DPPH assay) with anti-inflammatory (IC50 23.58 ± 1.75 µg/mL in LOX inhibition assay) and anticoagulation activity (18.19 ± 0.13 min in prothrombin time assay) compared to other extracts. All extracts were found with significant (P&lt;0.001) antioxidant, anti-inflammatory, and anticoagulation properties. Further studies on liquid chromatography-mass spectrometry (LC-MS) analysis, anti-inflammatory, and anticoagulation are recommended.

Hydroxyapatite/silk fibroin composite biomimetic scaffold for dental pulp repair

Dental pulp repair is a difficult clinical problem. In the present study, the authors aimed to mimic the extracellular matrix of dental pulp tissue structurally and compositionally. Nanofibrous silk fibroin (SF) scaffolds containing hydroxyapatite (HAp) nanoparticles were fabricated by using the freeze-drying approach. Rod-shaped HAp was successfully embedded in the composite scaffold, the diameter of which was about 100–200 nm as shown by transmission electron microscopy analysis. The three-dimensional microstructure of the composite scaffold prepared in various ratios of HAp to SF was observed by scanning electron microscopy and the pore size of the optimal scaffold was about 30–120 μm. Meanwhile, the hemocompatibility of the composite scaffolds was evaluated based on their impact on the clotting function by way of activated partial thromboplastin time, prothrombin time and thromboelastographic assays. The scaffolds possessed a low hemolysis rate of red blood cells. Furthermore, cell culture tests using dental pulp stem cells found that the scaffolds had good biocompatibility. There biomimetic HAp/SF composite scaffolds may serve as a promising biomaterial for dental pulp repair.

Identification of a Spontaneous Noncoding Mutation on Chromosome 18 Which Suppresses Thrombosis in Factor V Leiden Homozygous, TFPI Deficient Mice

Factor V Leiden (FVL) is an incompletely penetrant thrombosis susceptibility variant common in humans. This incomplete penetrance suggests there are modifiers of FVL (F5L) that alter the susceptibility to a thrombotic event. To identify modifiers, we performed a sensitized ethylnitrosourea (ENU) mutagenesis screen in mice to induce mutations suppressing the F5L/LTfpi+/- perinatal lethal thrombotic phenotype. Although we identified thrombosuppressor mutations in two of our 22 independent MF5L mouse lines thus far, the others are still unknown. Our mouse line MF5L16 has produced a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/LTfpi+/- mice. We performed the Prothrombin Time (PT) assay on plasma from a subset of these mice and observed a significantly longer PT in MF5L16F5L/LTfpi+/- mice compared to F5+/LTfpi+/- and Tfpi+/- control mice (q&amp;lt;0.01). The aim of our study was to identify the MF5L16 thrombosuppressor. First, we analyzed four mice by whole genome sequencing and identified a total of 44 candidate ENU mutations. Sanger re-sequencing analysis determined that 22 were false-positive calls. Seven arose in our F5L/L breeding colony and were introduced into MF5L16 by these mice. Three arose on the Tfpi- mouse background. The remaining 12 candidates could not be analyzed as they were in repetitive/low complexity regions. Due to the lack of ENU-induced mutations segregating in MF5L16, we next analyzed the seven mutations entering the pedigree from the F5L/L breeders in all 136 MF5L16 F5L/LTfpi+/- mice. We identified a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18A) and F5L/LTfpi+/- mouse survival (p=0.0031). Two additional mutations within intronic regions on Chromosomes 9 and 13 were also significantly associated with survival when co-inherited with the Chromosome 18 mutation (p=0.000113). Importantly, no coding variants were linked to these variants. To re-create the suppression of the lethal F5L/LTfpi+/- phenotype, we bred a F5L/L Chr18A/A mouse onto the Tfpi- background to produce F5+/L Chr18+/ATfpi+/- mice. We then bred these F5+/L Chr18+/ATfpi+/- triple heterozygous mice to F5L/L Chr18A/ATfpi+/+ mice to observe the effects of Chr18A on F5L/LTfpi+/- mouse survival. The first litter of six mice from this cross yielded one F5L/LTfpi+/- mouse that was also heterozygous for Chr18 (F5L/LTfpi+/- Chr18+/A: expected ratio ~1:8). This suggests that the Chr18A variant suppresses F5L/LTfpi+/- lethal thrombosis and that noncoding genomic variants can act as potent thrombosuppressors. Our genetic analyses suggest that thrombosuppressor variants arose in our F5L mouse colony due to positive selective pressure for the best F5L breeders. In addition, the genomic interactions between the putative thrombosuppressor variants described here illuminate the complexities of thrombosis genomics and could provide insights into human thrombosis. Disclosures No relevant conflicts of interest to declare.

In Vitro and In Vivo Characterization of Marstacimab, an Anti-TFPI Antibody

Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies of the intrinsic coagulation plasma proteins Factor VIII (FVIII) or Factor IX (FIX), respectively. An approach to achieving hemostatic pharmacology in hemophilia is to augment the extrinsic cascade is to neutralize Tissue Factor Pathway Inhibitor (TFPI). TFPI is a multi-Kunitz (K) domain inhibitor which binds to and inhibits Factor Xa via the K2 domain and Factor VIIa/Tissue Factor activity (K1 domain). Marstacimab is a fully human monoclonal antibody that binds to and neutralizes TPFI activity and is under development for treatment of hemophilia. To support lead identification during late discovery, five anti-tissue factor pathway inhibitor (TFPI) antibodies (EC50&lt;5nM) were evaluated in an in vivo rabbit pharmacokinetics/pharmacodynamics (PK/PD) study and in vitro using rabbit and human hemostatic assays. Marstacimab displayed potent in vitro activities, and potent pharmacology in shortening the dilute prothrombin time (dPT) assay (1.3 nM) and in the thrombin generation assay (TGA) (2.8 nM). Following an intravenous administration in rabbits, marstacimab and the other anti-TFPI antibodies exhibited target-mediated drug disposition (TMDD) with a half-life less than 2 days, when compared to a control IgG. The pharmacokinetics of anti-TFPI antibody can be described using a Michaelis-Menten mechanistic model in which in vivo Km and Vmax were estimated for the observed nonlinear clearance. The estimated pharmacokinetic parameters for marstacimab was 45 and 38 mL/kg for the central and peripheral volume of distribution, 23 nM for Km, 131 nM/kg/hr for Vmax. In addition, the correlation between in vitro Kd, in vivo Km and in vitro/in vivo potency were assessed. Compared to other anti-TFPI antibodies, marstacimab exhibited reduced clearance and a good in vitro-in vivo relationship. Second, the binding epitopes were further characterized using competitive surface plasmon resonance (SPR) and marstacimab has distinct binding epitopes from the other antibodies. Lastly, the potential human pharmacokinetics and efficacy were evaluated using allometry scaling and in vitro potency, the results predict a minimal target occupancy of 76% following a weekly subcutaneous administration of marstacimab. These studies indicate that the differential influence of the TFPI-binding epitopes on the pharmacokinetics, but not on the pharmacodynamics. The selection strategy could be applicable to the other antibody discovery and development. Disclosures Parng: Pfizer: Employment. Jin:Pfizer: Employment. Holsti:Pfizer: Employment. Benard:pfizer: Employment. Rakhe:Pfizer Inc.: Employment. Patel-Hett:Pfizer: Employment. Joyce:Pfizer: Employment. Webster:pfizer: Employment. Pittman:Pfizer Inc.: Employment.

Adeno-Associated Virus Mediated Delivery of a TFPI Antibody Restores Hemostasis in Mouse Model of Hemophilia

Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment of an active bleed. Some patients develop inhibitory antibodies making them refractory to replacement therapies. Although hemophilic patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. Augmenting the extrinsic tissue factor pathway is an attractive alternate approach to maintain hemostasis in hemophilic patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating protease functions of Factor Xa and Factor VIIa/Tissue Factor complex and as such, TFPI inhibition has been explored as an innovative option to restore thrombin generation. Previously, we demonstrated that antibody mediated TFPI inhibition restores hemostasis in mouse models of hemophilia (Jasuja et al, 2016). Here we investigated the impact of an adeno-associated virus (AAV) vector expressed anti-TFPI antibody in correcting bleeding disorders in hemophilic mice as a novel approach to restore hemostasis. An AAV vector expressing heavy and light chains of a neutralizing anti-TFPI monoclonal antibody was designed and constructed. In vitro evaluations confirmed that vector expressed antibody was functional using a dilute Prothrombin Time (dPT) assay. Next, male hemophilic A mice were systemically administered (intravenous) 1x1011 AAV vector genomes (vg) encoding anti-TFPI or control and had hemostasis evaluated 16 days post-treatment. Following recalcification of whole blood, thromboelastography (TEG) was used to measure clot formation times (R value), speed of clot formation (K value), as well as maximum amplitude. In hemophilia A mice treated with AAV-anti-TFPI, clot formation time (R value) was normalized to wild type levels, with a 10-fold reduction compared to hemophilia A mice administered a control Green fluorescent protein (GFP) encoding AAV vector. Mice administered the control AAV-GFP vector failed to correct additional parameters, including speed of clot formation (K value), angle and maximum amplitude, all of which remained similar to untreated hemophilia A mice. In contrast, for AAV-anti-TFPI dosed Hem A mice, all of these parameters were in the normal range similar to wild type mice. Our data demonstrate AAV mediated delivery of anti-TFPI antibody corrects coagulation abnormalities observed in a mouse model of Hemophilia A. Disclosures Rakhe: Pfizer Inc.: Employment. Jasuja:Pfizer Inc.: Employment. Shelke:Pfizer Inc.: Employment. Sawant:Pfizer Inc.: Employment. Somanathan:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment.

Procoagulant tumor microvesicles attach to endothelial cells on biochips under microfluidic flow.

Tumor patients are at a high risk of venous thromboembolism (VTE), and the mechanism by which this occurs may involve tumor-derived microvesicles (MVs). Previously, it has been shown that tumor MVs become attached to endothelial cells in static conditions. To investigate whether this process occurs under physiologically relevant flow rates, tumor MVs were perfused across a microfluidic device coated with growing human umbilical vein endothelial cells (HUVECs). Cell lines were screened for their ability to form tumor spheroids, and two cell lines, ES-2 and U87, were selected; spheroids formed were transferred to a microfluidic chip, and a second endothelial cell biochip was coated with HUVECs and the two chips were linked. Media flowed through the spheroid chip to the endothelial chip, and procoagulant activity (PCA) of the tumor media was determined by a one-stage prothrombin time assay. Tumor MVs were also quantified by flow cytometry before and after interaction with HUVECs. Confocal images showed that HUVECs acquired fluorescence from MV attachment. Labeled MVs were proportionally lost from MV rich media with time when flowed over HUVECs and were not observed on a control chip. The loss of MV was accompanied by a proportional reduction in PCA. Flow cytometry, confocal microscopy, and live flow imagery captured under pulsatile flow confirmed an association between tumor MVs and HUVECs. Tumor MVs attached to endothelial cells under physiological flow rates, which may be relevant to the VTE pathways in cancer patients.

In vitro haemostatic effect of amphibian crude skin secretions in rabbit blood plasma

In this research we have studied whether the amphibian crude skin secretions can modify some parameters of haemostatic system. We prepared the samples of crude skin secretions of Bombina bombina, Bombina variegata, Bufo bufo, and Bufotes viridis and investigated the plasma clotting function in vitro by performing activated partial thromboplastin time (aPTT), prothrombin time and thrombin time (TT) assays. The data indicate that the components of B. bombina and B. variegata skin secretions significantly prolonged aPTT clotting time, while the components of B. viridis skin secretions prolonged the time of plug formation in TT. The proteolytic activity and the ability of the skin secretions components to activate plasma proenzymes were assayed in vitro using synthetic chromogenic substrates. The components of B. variegata skin secretions cleaved all studied chromogenic substrates, whereas the B. viridis secretions intensively cleaved thrombin specific substrate and protein C specific substrate. The components of B. bombina and B. bufo skin secretions activated prothrombin and protein C in plasma. The components of B. variegata, B. bufo and B. viridis skin secretions in a dosedependent manner induced platelet aggregation.

Determination of the cut-off prothrombin time to estimate plasma rivaroxaban overdose status.

Laboratory monitoring of rivaroxaban (RIV) is required under certain conditions. Mass spectrometry and anti-factor Xa assays are the recommended methods, which may not be readily available. Prothrombin time (PT) is the most widely used and simple coagulation assay. To set the cutoff PT and international normalized ratio (INR) to estimate RIV overdose status. RIV-spiked pooled normal plasma was used. PT test was performed using a CA-7000 coagulometer and Thromborel S reagent. The precise measurement of RIV concentration at the cut-off PT was evaluated according to the Clinical and Laboratory Standard Institute (CLSI) EP12-A2 guideline. The RIV concentration at 275ng/mL was analyzed using 40 replicates. Receiver operating characteristic (ROC) analysis was performed to determine the cutoff value for the determination of RIV potential overdose status. An imprecision estimation of PT was conducted with 220.00ng/mL, 247.50ng/mL, 261.25ng/mL, 288.75ng/mL, 302.50ng/mL and 330.00ng/mL concentrations of RIV in 60 replicates. According to the ROC analysis, the cutoff clotting times and INR values to determine the overdose status of RIV were 13.45s and 1.39. With these values, there was a 92.6% probability that plasma samples with RIV concentration ≤ 247.50ng/mL yielded consistently negative (on-therapy dose) results, and those with ≥ 302.50ng/mL yield consistent positive (potential overdose) results using our PT assay. PT with a reliable cutoff clotting time and INR can be used to determine the potential overdose status of RIV to facilitate the diagnosis and treatment by controlling the dose.

Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Polyphenolics from Gymnocarpos decandrus Forssk roots and their biological activities

Phytochemical investigation of Gymnocarpos decandrus roots lead to the isolation, characterization and evaluation of four compounds: the bis-coumarin daphnoretin, two biflavonoids: wikstrol A and wikstol B in addition to β-sitosterol glucoside. Their structures were established via spectroscopic data. The crude root extract showed a significant antimicrobial activity against Bacillus subtilis. In addition, Coagulation activity of the same extract and daphnoretin were investigated via measuring their effect on prothrombin time (PT) and activated partial thromboplastin time (aPTT) assay in citrated plasma collected from healthy regular blood donors and they were found to prolong the PT and aPTT assays (p < 0.05). The three polyphenolics were described for the first time from the Caryophllaceae family. Furthermore, this is the first phytochemical and biological study to be carried on G. decandrus roots.

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites.

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.

Croatian Society of Medical Biochemistry and Laboratory Medicine: National recommendations for blood collection, processing, performance and reporting of results for coagulation screening assays prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer.

A modern diagnostic laboratory offers wide spectrum of coagulation assays utilized in the diagnosis and management of patients with haemostatic disorders, preoperative screening and anticoagulation therapy monitoring. The recent survey conducted among Croatian medical biochemistry and transfusion laboratories showed the existence of different practice policies in particular phases of laboratory process during coagulation testing and highlighted areas that need improvement. Lack of assay standardization together with non-harmonized test results between different measurement methods, can potentially lead to incorrect decisions in patient’s treatment. Consequently, patient safety could be compromised. Therefore, recommended procedures related to preanalytical, analytical and postanalytical phases of prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer testing are provided in this review, aiming to help laboratories to generate accurate and reliable test results.

Acute toxicity and antiproliferative and procoagulant activities of fractions derived from Thymus satureioides of the Moroccan High Atlas

This study aimed to determine the effects of crude extract (CE) and fractions derived from the Moroccan endemic plant Thymus satureioides (T. satureioides) on inflammation, coagulation and viability of a cell cancer line. The crude extract and fractions from the leaves of T. satureioides were analysed using HPLC and examined for their antioxidant capacity using mineral and biological substrates. The anti-inflammatory activity of the extracts was evaluated using croton oil- and carrageenan-induced oedema. The effect on blood coagulation in normal rat plasma was also determined using prothrombin time (PT) and activated partial thromboplastin time (APTT) assays. Furthermore, the oral acute toxicity and antiproliferative effects on the MCF-7 cell line of the CE were evaluated. The major identified polyphenol in the CE and polar fractions was rosmarinic acid. No mortalities due to acute toxicity were observed at 5 g/kg. All plant preparations protected the cell membranes of erythrocytes against oxidative damage and showed high scavenging activity, as measured using DPPH and FRAP. Moreover, the hydrophobic fractions demonstrated significant inhibitory effects against croton oil-induced oedema. The methanol fraction (60 mg/kg) significantly reduced oedema induced by carrageenan during the first phase. During the APTT and PT assays, the CE and fractions caused plasma coagulation even at low concentrations. An antiproliferative assay (MTT) showed a significant decrease in the number of viable cells in culture medium supplemented with CE at different concentrations. These results suggest that extracts from the leaves of T. satureioides constitute a valuable source of anti-inflammatory and anti-cancer metabolites.

Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum.

The currently available oral anti-Xa agents are claimed to produce their anticoagulant and antithrombotic effects solely by the inhibition of factor Xa. This study profiled various anti-Xa drugs in routinely used laboratory assays to demonstrate that their effects are not solely related to the anti-Xa activities. Apixaban, betrixaban, edoxaban, and rivaroxaban were obtained commercially. Native and citrated whole blood was used for the activated clotting time (ACT) and thromboelastography (TEG). Citrated plasma was used for monitoring the prothrombin time (PT), activated partial thromboplastin time (aPTT), Heptest, and prothrombinase-induced clotting time (PiCT) tests. An amidolytic method was used for the determination of anti-Xa effects. Thrombin-induced fibrinokinetics was monitored optically. Thrombin generation studies were carried out using the calibrated automated thrombogram. All of the anti-Xa agents produced concentration- and assay-dependent effects. In the ACT at 2.5 μg/mL and TEG at 1.0 μg/mL, edoxaban exhibited the strongest anticoagulation effect. In the PiCT, PT, and aPTT assay at 1 μg/mL, edoxaban showed stronger effects than other agents. The half maximal inhibitory concentration of these agents for the inhibition of factor Xa ranged from 340 to >1000 ng/mL. In the thrombin generation inhibition assay, apixaban showed the strongest activity. In the fibrinokinetics, different anti-Xa agents produced varying degrees of inhibition. These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents.

Pharmacokinetics and Pharmacodynamics of an Oral Formulation of Apixaban in Horses After Oral and Intravenous Administration.

Horses with inflammatory and infectious disorders are often treated with injectable heparin anticoagulants to prevent thrombotic complications. In humans, a new class of direct oral acting anticoagulants (DOAC) appear as effective as heparin, while eliminating the need for daily injections. Our study in horses evaluated apixaban, a newly approved DOAC for human thromboprophylaxis targeting activated factor X (Xa). Our goals were to: (1) Determine pharmacokinetics and pharmacodynamics of apixaban after oral (PO) and intravenous (IV) administration in horses; (2) Detect any inhibitory effects of apixaban on ex vivo Equid herpesvirus type 1 (EHV-1)-induced platelet activation, and (3) Compare an anti-Xa bioactivity assay with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) for measuring apixaban concentrations. In a blinded placebo-controlled cross-over study, five horses received a single dose (0.2 mg/kg) of apixaban or placebo PO or IV. Blood was collected before and at 3 (IV) or 15 (PO) min, 30 and 45 min, and 1, 2, 3, 4, 6, 8, and 24 h after dosing for measuring apixaban UPLC-MS concentrations and anti-Xa activity. Pharmacodynamic response was measured in a dilute prothrombin time (dPT) assay. Flow cytometric EHV-1-induced platelet P-selectin expression and clinical pathologic safety testing were performed at baseline, 2 and 24 h and baseline and 24 h, respectively. We found no detectable apixaban in plasma PO administration. After IV administration, plasma apixaban levels followed a two-compartment model, with concentrations peaking at 3 min and decreasing to undetectable levels by 8 h. The elimination half-life was 1.3 ± 0.2 h, with high protein binding (92–99%). The dPT showed no relationship to apixaban UPLC-MS concentration and apixaban did not inhibit EHV-1-induced platelet activation after IV dosing. Apixaban anti-Xa activity showed excellent correlation to UPLC-MS (r2 = 0.9997). Our results demonstrate that apixaban has no apparent clinical utility as an anticoagulant for horses due to poor oral availability.

Anticoagulant activity of two novel polysaccharides from flowers of Apocynum venetum L.

Two novel polysaccharides were obtained from flowers of Apocynum venetum L., and named as Vp2a-II and Vp3. Their average molecular weights were 7 kDa and 9 kDa, respectively. Methods of monosaccharide analysis, acid hydrolysis and methylation analysis, Fourier transform-infrared spectroscopy (FT-IR), gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR) spectroscopy were used to identify the structure of Vp2a-II and Vp3. Vp2a-II was composed of →6)-β-d-Glcp-(1 → 6)-α-d-Galp-(1→ residues. Vp3 was composed of α-d-GlcpA-(3 → α-d-GalpA residues. The anticoagulant activity was evaluated by activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) assays in vitro. Results indicated that Vp3 exhibited the anticoagulant activity.

Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum

Introduction: There are four oral anti-Xa drugs currently available for clinical use in various indications. These drugs are claimed to mediate their therapeutic effects by solely targeting factor Xa. While these agents are structurally similar, their biochemical properties and their effects on blood coagulation differ. Such differences may impact their safety and efficacy profile. The purpose of this study was to demonstrate the differences among factor Xa inhibitors in terms of their in vitro anticoagulant activity and other biochemical effects.Materials and Methods: Commercially obtained powdered forms of Apixaban, Betrixaban, Edoxaban and Rivaroxaban were profiled in this study. Stock solutions of each drug were prepared at 1mg/ml. To investigate the effect on the whole blood clotting profile, thromboelastographic studies were carried out over a concentration range of 0.5 - 2.5 ug/ml and whole blood activated clotting time (ACT) was measured at 1.0 and 2.5 ug/ml. The anticoagulant profile in citrated human pool plasma was measured at concentrations of 0.062-1.0 ug/ml using such tests as prothrombin time (PT) and activated partial thromboplastin time (aPTT). The anti-Xa effects of each agent were measured using a kinetic amydolytic method. The inhibitory potency was calculated in terms of IC-50. Thrombin generation inhibition studies on each drug were carried out in human pool plasma in a concentration range of 0.0-1.0 ug/ml using calibrated aotomated thrombogram (CAT) assay (Diagnostica Stago, Paris, France). Fibrinokinetics studies were carried out using an optical kinetic method, where thrombin was used to trigger clot formation. All results were compiled in terms of mean + 1 SD of 3-5 replicates.Results: All of the anti-Xa agents produced concentration and assay-dependent effects in these studies. The summary of each agent's effects at selected fixed concentrations and the IC-50 of the anti-Xa activity is given in the Table. In the whole blood ACT at 2.5ug/ml, Edoxaban showed the strongest anti-coagulant effects followed by Rivaroxaban > Betrixaban, whereas Apixaban showed minimal effects. In the TEG analysis at 1ug/ml, Edoxaban exhibited stronger anti-coagulant effects as measured by various TEG parameters, including R-time, K-time, alpha, and MA. Edoxaban and Rivaroxaban showed comparable effects followed by Betrixaban, whereas Apixaban exhibited weaker effects. In the PT assay at 1ug/ml, Edoxaban showed stronger effects, whereas Apixaban, Betrixaban and Rivaroxaban were comparable. aPTT at 1ug/ml revealed that Edoxaban was the strongest anti-Xa inhibitor followed by Betrixaban, whereas Apixaban and Rivaroxaban were comparable. In the anti-Xa assay Edoxaban was stronger (IC-50 = 340ng/ml, 0.62uM) than Apixaban (IC-50 =400ng, 0.87uM), Rivaroxaban (IC-50 = 840ng, 1.9uM) and Betrixaban (IC = >1000ng, >2.22 uM). In the thrombin generation assays at 1ug/ml, Apixaban showed the strongest inhibitory activity (IC-50 = 50ng/ml, 108nm) followed by Edoxaban (IC-50 = 58ng/ml, 108nm), Betrixaban (IC-50 = 60ngml, 133nm) while Rivaroxaban showed relatively weaker activity (IC-50 = 100ng/ml, 299nm). In the fibrinokinetics study at 1ug/ml, the anti-Xa agents produced varying degrees of inhibition with Rivaroxaban (67%), Edoxaban (42%), Apixaban (32%) and Betrixaban (12%).Summary and Conclusion: These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents. Assay dependent variations are exhibited by each of these drugs, revealing distinct individual profiles. Edoxaban was the only anti-Xa agent which consistently exhibited relatively stronger inhibitory profile which was proportional to its anti-Xa activity. These studies indicate that the oral anti-Xa drugs may modulate the hemostatic system through additional mechanisms independent of the inhibition of factor Xa. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Engineering electrospun multicomponent polyurethane scaffolding platform comprising grapeseed oil and honey/propolis for bone tissue regeneration.

Essential oils play an important role in reducing the pain and inflammation caused by bone fracture.In this study, a scaffold was electrospun based on polyurethane (PU), grape seed oil, honey and propolis for bone tissue-engineering applications. The fiber diameter of the electrospun PU/grape seed oil scaffold and PU/grape seed oil/honey/propolis scaffold were observed to be reduced compared to the pristine PU control. FTIR analysis revealed the existence of grape seed oil, honey and propolis in PU identified by CH band peak shift and also hydrogen bond formation. The contact angle of PU/grape seed oil scaffold was found to increase owing to hydrophobic nature and the contact angle for the PU/grape seed/honey oil/propolis scaffold were decreased because of hydrophilic nature. Further, the prepared PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold showed enhanced thermal stability and reduction in surface roughness than the control as revealed in thermogravimetric analysis (TGA) and atomic force microscopy (AFM) analysis. Further, the developed nanocomposite scaffold displayed delayed blood clotting time than the pristine PU in the activated prothrombin time (APTT) and partial thromboplastin time (PT) assay. The hemolytic assay and cytocompatibility studies revealed that the electrospun PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold possess non-toxic behaviour to red blood cells (RBC) and human fibroblast cells (HDF) cells indicating better blood compatibility and cell viability rates. Hence, the newly developed electrospun nanofibrous composite scaffold with desirable characteristics might be used as an alternative candidate for bone tissue engineering applications.