Prothrombin Time Assay Research Articles

LDH and NLR, as inflammatory markers, the independent risk factors for COVID-19 complicated with respiratory failure in elderly patients.

Lactate dehydrogenase (LDH) is an enzyme that is responsible for the production of lactic acid, which is a necessary byproduct when the body does not have enough oxygen. LDH levels in the blood can be used as a marker to predict mortality in patients with ARDS, severe COVID-19, and cancer. To analyze the clinical characteristics of COVID-19 in the elderly and the correlation between LDH and respiratory failure in COVID-19 patients, to improve the identification and management of this type of pneumonia by clinicians. This was a single-center retrospective study. We performed routine laboratory tests in 105 COVID-19 patients admitted to the affiliated hospital of Qingdao University (Qingdao, China) from October 1, 2022 to February 1, 2023. The diagnosis of respiratory failure was established based on the results of blood gas analysis upon admission. The median age was 79 years. Among all univariable parameters, LDH, neutrophil to lymphocyte ratio (NLR) and Prothrombin Time (PT) were significantly independent risk factors of RF in elderly COVID-19 patients. LDH (AUC=0.829) also had a maximum specificity (96.5%), with the cutoff value of 280.5. The levels of LDH, NLR, and PT may serve as potential indicators for elderly COVID-19 patients combined with respiratory failure. LDH, NLR and PT assays can be beneficial for patients who need closer respiratory monitoring and more aggressive supportive care to prevent a negative prognosis.

Edoxaban enfolded beta-1,4-poly-d-glucosamine nanoparticles for targeting eponym Stuart–Prower factor for treatment of venous thrombosis

The present research looked for ways to develop shielded nanoparticles (NPs)-drug transporters made of chitosan (CS) to enhance the bioavailability of edoxaban tosylate monohydrate (ETM) for oral administration by examining the correlation among design aspects and data from experiments using response surface methodology (RSM). ETM-loaded CS nanoparticles (ETM-CS-NPs) were developed using the ionic gelation of CS with tripolyphosphate (TPP). Utilising Zeta-sizer and scanning electron microscopy, the ETM-CS-NPs were evaluated for particle size (PS), zeta potential (ZP), surface morphology, polydispersity index (PDI), entrapment efficiency (EE) and drug loading (DL). Drug and polymer interactions in NPs were assessed using Fourier transform infra-red spectroscopy. The response surface approach and Design-Expert software optimised the ETM-CS-NPs. Using RSM, the effects of independent variables such as the amount of CS, the amount of TPP, and the amount of glacial acetic acid on PS, PDI and ZP were analysed. The optimal combination of PS (354.8 nm), PDI (0.509), ZP (43.7 + mV), % EE (70.3 ± 1.3) and % DL (9.1 ± 0.4) has been identified for the optimised ETM-CS-NPs. ETM-CS-NPs’ anticoagulant activity was evaluated using activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT) assays. In conclusion, a practical and consistent method has been established, and its application has been proven in vitro, indicating its utility for future studies of the biological distribution of ETM-CS-NPs in vivo for specific antithrombotic treatments.

Minimal interference of concizumab with standard clinical coagulation laboratory assays - An in vitro study.

Non-factor replacement therapies are emerging as prophylactic treatment options in haemophilia A or B (HA/HB) with and without inhibitors. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody preventing factor (F)Xa inhibition and enhancing thrombin generation. Based on experience with other non-factor therapies and extended half-life products, there is a focus on potential interference with common clinical coagulation assays used to monitor patients treated with concizumab. To evaluate the impact of concizumab on standard clinical coagulation assays. Plasma samples (normal, HA/HB with/without inhibitors) in the presence/absence of added concizumab (250-16,000ng/mL) were analysed in clinical assays including activated partial thromboplastin time (aPTT), prothrombin time (PT), FVIII and FIX one-stage clot and chromogenic substrate assay, assays for detecting FVIII or FIX inhibitors and other assays for coagulation factors. Concizumab did not impact PT assays, but resulted in a small shortening of aPTT (up to 5 s in haemophilia plasma and 0.4 s in normal plasma). Concizumab had no, or only a minor impact on FVIII and FIX activity assays or Bethesda inhibitor assays. FXI and FXII activity in normal plasma, as measured by single factor aPTT-based assay, was significantly increased in the presence of concizumab (+11% each). This was also the case for FVII and FX measured by PT-based assays using plasma with 25% of FVII or FX (+64% and +22%, respectively). The presence of concizumab did not, or only slightly, influence the outcome of standard clinical coagulation assays relevant for HA and HB.

AAV mediated genome engineering with a bypass coagulation factor alleviates the bleeding phenotype in a murine model of hemophilia B

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20–42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.

Fitusiran reduces bleeding in factor X–deficient mice

AbstractFactor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 μL to 590 ± 335 μL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Abstract 3128: HRA00184-C004, preclinical characterization of a novel tissue factor-targeted antibody-drug conjugate

Abstract The physiological function of tissue factor (TF) is to initiate coagulation cascade by forming complex with plasma Factor VII after vascular injury. For Tisotumab vedotin (Tivdak), a TF targeting antibody-drug conjugate (ADC) approved for the treatment of cervical cancer, bleeding events were prespecified as adverse effects (AEs) of interest, besides ocular toxicity and peripheral neuropathy (known MMAE payload related AE). In order to mitigate the bleeding risk, a next generation of TF-targeting ADC is developed by Hengrui Pharmaceuticals. HRA00184-C004 is a novel ADC comprising a differentiated TF-targeted antibody (HRP07132) conjugated to a proprietary topoisomerase I inhibitor (Dxh, an exatecan derivative) via a protease-cleavable linker. In contrast to most TF-targeting antibodies, the naked HRP07132 does not interfere the interaction between TF and Factor VII or Factor X, has no impact on coagulation function in prothrombin time and thrombin generation assay, therefore reduced bleeding incidence is expected for HRA00184-C004. Meanwhile, HRA00184-C004 exhibited not only potent growth inhibition against multiple TF-expressing cancer cell lines but also bystander-killing effect in vitro. HRA00184-C004 also displayed robust and sustained anti-tumor activities in the PDAC HPAFII (TF high), TNBC MDA-MB-231 (TF moderate) and NSCLC H358 (TF low) TF-expressing xenograft models. Furthermore, in cynomolgus monkey, HRA00184-C004 is well-tolerated over a 6-week period with 12 mg/kg dosing every 3 weeks with acceptable PK profile. Notably, neither coagulation disorder nor skin rash was observed, which are superior to the historical NHP data reported by Tivdak. In conclusion, with the encouraging efficacy in preclinical study and promising safety profiles in NHPs, HRA00184-C004 has demonstrated the best-in-class potential and is scheduled to advance into clinical development. Citation Format: Hanxiao Ying, Lingfeng You, Ning He, Shaoyu Xu, Pu Pu, Sophie Lin, Jun Feng, Zhe Zhang, Xin Ye, Min Hu, Feng He. HRA00184-C004, preclinical characterization of a novel tissue factor-targeted antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3128.

New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants.

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure-activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases.

A comparative in vitro study of the anticoagulant effect of branded versus generic rivaroxaban

BackgroundSeveral generic formulations of rivaroxaban were recently marketed to be used interchangeably with their branded equivalent. However, there have been no previously published studies that directly compared the in vitro anticoagulant effect of branded vs. generic rivaroxaban. The aim of this in vitro study was to compare the effects of three raw rivaroxaban materials, obtained from the branded (Xarelto®) and two generic (Rivarolto® and Rivaroxaban Sandoz®) rivaroxaban formulations on an array of coagulation assays. MethodsA pool of normal plasma was spiked with several concentrations of the three rivaroxaban (range 50–750 ng/ml). The concentrations were assessed with a rivaroxaban calibrated anti-Xa assay and confirmed by ultra-high-performance liquid chromatography-mass spectrometry coupled with tandem mass spectrometry (UHPLC-MS/MS). The following assays were performed: Prothrombin time (PT), activated Partial Thromboplastin time (aPTT), Diluted Russell's Viper Venom Test (dRVVT), Thrombin time (TT), Clauss Fibrinogen, Factor VII, VIII and IX assays, and thromboelastography. ResultsThe results obtained by the three rivaroxaban at similar concentrations were comparable. Increasing concentrations of the three rivaroxaban showed a strong positive correlation with the PT, aPTT and dRVVT assays (r > 0.95, p < 0.01 for all), and a strong negative correlation with the Factors assays (r < −0.95, p < 0.01 for all). TT and Clauss Fibrinogen were not affected by rivaroxaban. No significant difference was identified in the mean assays' results obtained by the three rivaroxaban. ConclusionThis study showed that the branded and generic rivaroxaban exert an identical in vitro anticoagulant effect across a wide range of concentrations.

Characterization and Performance Evaluation of Magnesium Chloride-Enriched Polyurethane Nanofiber Patches for Wound Dressings.

Wound patches are essential for wound healing, yet developing patches with enhanced mechanical and biological properties remains challenging. This study aimed to enhance the mechanical and biological properties of polyurethane (PU) by incorporating magnesium chloride (MgCl2) into the patch. The composite patch was fabricated using the electrospinning technique, producing nanofibers from a mixture of PU and MgCl2 solutions. The electrospun PU/MgCl2 was then evaluated for various physico-chemical characteristics and biological properties to determine its suitability for wound healing applications. Tensile strength testing showed that the mechanical properties of the composite patch (10.98 ± 0.18) were significantly improved compared to pristine PU (6.66 ± 0.44). Field scanning electron microscopy (FESEM) revealed that the electrospun nanofiber patch had a smooth, randomly oriented non-woven structure (PU - 830 ± 145 nm and PU/MgCl2 - 508 ± 151 nm). Fourier infrared spectroscopy (FTIR) confirmed magnesium chloride's presence in the polyurethane matrix via strong hydrogen bond formation. Blood compatibility studies using coagulation assays, including activated partial thromboplastin time (APTT), prothrombin time (PT), and hemolysis assays, demonstrated improved blood compatibility of the composite patch (APTT - 174 ± 0.5 s, PT - 91 ± 0.8s, and Hemolytic percentage - 1.78%) compared to pristine PU (APTT - 152 ± 1.2s, PT - 73 ± 1.7s, and Hemolytic percentage - 2.55%). Antimicrobial testing showed an enhanced zone of inhibition (Staphylococcus aureus - 21.5 ± 0.5 mm and Escherichia coli - 27.5 ± 2.5 mm) compared to the control, while cell viability assays confirmed the non-cytotoxic nature of the developed patches on fibroblast cells. The study concludes that adding MgCl2 to PU significantly improves the mechanical, biological, and biocompatibility properties of the patch. This composite patch shows potential for future wound healing applications, with further studies needed to validate its efficacy in-vivo.

Cystoseira myrica: from beach-cast seaweed to fucoidan with antioxidant and anticoagulant capacity

This study highlights the potential of the brown algae Cystoseira myrica, collected from raw beach seaweed wastes, as a reliable source of bioactive fucoidan. Fucoidans are natural bioactive sulfated polysaccharides that are gaining popularity worldwide due to their diverse biochemical composition, attractive physical properties, and functional pharmacological activities. The aim of this work is to valorize the beach-Accumulated brown algae C. myrica by fucoidan extraction and to investigate its anticoagulant and antioxidant activity. Fucoidan was extracted using several steps of papain digestion followed by cetyltrimethylammonium bromide (CTAB) precipitation and calcium chloride treatment to avoid the coextraction of other polysaccharides. Structural features of the extracted fucoidan were investigated by Fourier transform infrared spectroscopy (FT-IR), solid-state nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy. Agarose gel electrophoresis was used to confirm the purity of the isolated fucoidan from C. myrica. Anticoagulant properties were studied in vitro by activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays. Furthermore, the antioxidant activity was investigated by 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and Fe chelating ability. Our results indicate that the approach used was effective in extracting fucoidan with a yield of 3.07%, a high amount of sulfate (27.79%), and fucose was found to be the major monosaccharide component. The extracted fucoidan showed an interesting anticoagulant activity. It prolonged aPTT significantly at a concentration of 10 µg/ml and prolonged PT at high doses. This demonstrated that fucoidan from C. myrica may affect intrinsic pathways while having little impact on the extrinsic mechanism of coagulation. However, the extracted fucoidan significantly exhibited an interesting antioxidant capacity as shown by the higher ABTS radical scavenging activity and Ferrous ion-chelating effect. The current findings suggest that fucoidan isolated from C. myrica has unique structural, antioxidant and anticoagulant properties and offers innovative therapeutic possibilities.

CT-001, a novel fast-clearing factor VIIa, enhanced the hemostatic activity in postpartum samples

AbstractThe hemostatic system is upregulated to protect pregnant mothers from hemorrhage during childbirth. Studies of the details just before and after delivery, however, are lacking. Recombinant factor VIIa (rFVIIa) has recently been granted approval by the European Medicines Agency for the treatment of postpartum hemorrhage (PPH). A next-generation molecule, CT-001, is being developed as a potentially safer and more efficacious rFVIIa-based therapy. We sought to evaluate the peripartum hemostatic status of pregnant women and assess the ex vivo hemostatic activity of rFVIIa and CT-001 in peripartum blood samples. Pregnant women from 2 study sites were enrolled in this prospective observational study. Baseline blood samples were collected up to 3 days before delivery. Postdelivery samples were collected 45 (±15) minutes after delivery. Between the 2 time points, soluble fibrin monomer and D-dimer increased whereas tissue factor, FVIII, FV, and fibrinogen decreased. Interestingly, the postdelivery lag time and time to peak in the thrombin generation assay were shortened, and the peak thrombin generation capacity was maintained despite the reduced levels of coagulation proteins after delivery. Furthermore, both rFVIIa and CT-001 were effective in enhancing clotting activity of postdelivery samples in activated partial thromboplastin time, prothrombin time, thrombin generation, and viscoelastic hemostatic assays, with CT-001 demonstrating greater activity. In conclusion, despite apparent ongoing consumption of coagulation factors at the time of delivery, thrombin output was maintained. Both rFVIIa and CT-001 enhanced the upregulated hemostatic activity in postdelivery samples, and consistent with previous studies comparing CT-001 and rFVIIa in vitro and in in vivo, CT-001 demonstrated greater activity than rFVIIa.

Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2'-Deoxyguanosine and 2'-Substituted RNA Analogues.

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2'-deoxyguanosine (GBr) in syn positions, while in the anti-positions, locked nucleic acid guanosine (GLNA) in the analogue TBABL, 2'-O-methylguanosine (GOMe) in TBABM, and 2'-F-riboguanosine (GF) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (Tm approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer.

Critical Appraisal on the Role of Warfarin in the Current Era.

Warfarin has been the most extensively used oral anticoagulant (OAC) in medical settings for over 60 years. Its uses, potential adverse effects, and methods for reversing its effects have been firmly established, rendering it a routine medication in medical settings where most professionals feel at ease employing it. Compared to other vitamin K antagonists (VKAs), such as acenocoumarol, warfarin offers benefits like diminished prothrombin time (PT) assays leading to enhanced oral anticoagulation. Observations over the past few years have seen the inclusion of novel/direct OACs (NOACs/DOACs) in the anticoagulant armamentarium. Although DOACs have several advantages, warfarin still has an important role in subsets of patients where DOACs are contraindicated, not well-tolerated, or cannot afford DOACs due to higher costs. Moreover, there are patient profiles where warfarin is still considered a superior choice compared to DOACs, such as age group of >75 years, kidney failure with creatinine clearance (CrCl) below 30 mL/minute, and prosthetic mechanical valve replacement. Precise management of the international normalized ratio (INR) is crucial for the effectiveness of warfarin treatment. INR monitoring is the major concern in the Indian context due to the lack of laboratories for standardized measurement. Adopting strategies such as point-of-care INR monitoring devices and anticoagulation clinics can help to improve clinical outcomes with warfarin therapy. The present review provides a critical overview of the role of warfarin therapy in the current OAC arsenal and strategies for improving therapeutic control and patient adherence. How to cite this article: Nair T. Critical Appraisal on the Role of Warfarin in the Current Era. J Assoc Physicians India 2023;71(10):31-36.

Establishment of reference values for prothrombin time, activated partial thromboplastin time and fibrinogen in adults in Burkina Faso.

The ordering of clinical haemostasis tests is increasing in Burkina Faso due to the newly emergence of cardiovascular and metabolic diseases. However, appropriate local reference values (RV) are lacking. Our study aimed to establish RV for prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen assays. In 2020, we carried out a cross-sectional study at the transfusion centre of Ouagadougou and included 280 healthy blood donors (140 males and 140 females) as reference subjects (RS) according to CLSI guidelines (C28 A3). From each RS a 5 mL blood sample had been withdrawn in citrated tubes. We performed PT, aPTT and fibrinogen assays using the Sysmex™ CA660 coagulometer and Siemens™ reagents. RV were calculated using the "central 95 percentile" method. Reference values of PT, aPTT and Fibrinogen were respectively [73.84%-117.50%], [20,01-29.45] seconds and [2.04-3.83] g/L for females and [58.81%-112,31%] seconds, [20,9-29,98] seconds and [1.58-3.35] g/L for males. We report for the first time locally appropriate haemostasis RV for the Burkina Faso adult's population. They will be of clinical use to our health care professionals.

Hemostatic system in Takotsubo patients at long-term follow-up: A hidden activation?

BackgroundTakotsubo cardiomyopathy (TTS) has long been considered a benign condition, despite recurrent events and long-term adverse outcomes are often reported. Endothelial damage, blood hyperviscosity, and platelet activation described in acute phase persist in long-term follow-up; however, TTS pathophysiology is still not fully understood. Here, we explored the hemostatic system at a median of 3.1 years after TTS to uncover additional long-lasting changes in these patients. MethodsWe assessed hemostatic parameters in women with TTS (n = 23) or coronary artery disease (CAD; n = 31) and in control women (n = 26) age-matched, by thromboelastographic analysis, prothrombin time (PT) and partial thromboplastin time (aPTT) coagulation assays and microparticle exposing Tissue Factor (MP-TF). Functional fibrinogen and fibrin polymerization were analyzed by Clauss method and spectrophotometry, respectively. Platelet reactivity was evaluated by light transmission aggregometry, whereas plasminogen activator inhibitor-1 (PAI-1) and brain-derived neurotrophic factor (BDNF) were measured by ELISA kit. ResultsCompared with control subjects, TTS patients exhibit an accelerated clot formation, higher percentage of fibrin polymerization and higher PAI-1 levels. Compared with CAD, TTS patients showed sustained residual platelet activation but decreased functional fibrinogen, fibrin polymerization and MP-TF levels, prolonged aPTT and a marked BDNF increase. ConclusionsThe long-term activation of hemostatic system observed in TTS patients compared to control subjects suggests a persistent humoral abnormality that may be related to the propensity for TTS recurrence. The higher residual platelet activity observed in TTS than in CAD patients invites investigation on TTS-tailored antiplatelet therapy potentially needed to prevent TTS adverse outcomes.

Comparative analysis of platelet depleted plasma prepared on the Roche 8100 automation line and manually centrifuged platelet poor plasma for routine coagulation assays

ObjectivesTo evaluate whether the routine coagulation tests can be performed using platelet depleted plasma (PDP, residual platelet count <40000/μL) to achieve maximum efficiency of the automated workflow and compare results of these tests performed with platelet poor plasma (PPP residual platelet count <10,000/μL) prepared manually ‘offline’. Design and MethodsThe PDP was obtained first following ‘on line’ centrifugation at 4150 RPM (3000g) for 7 min. The routine coagulation tests, Prothrombin Time (PT), Activated Partial Thromboplastin Clotting Time (aPTT), D-dimer (DD), Antithrombin III (AT3) and Fibrinogen (FBG) were performed. The PPP was obtained from an aliquot of PDP samples with additional ‘manual off line’ centrifugation at 7700 RPM (3314g) for 3 min (total 10 min, online + offline) and the same tests were performed. The statistical analysis was carried out using EP Evaluator v11 to compare results from both methods. ResultsThe results from both PPP and PDP samples demonstrated strong correlation. For example, PT (R = 0.9989; N = 55, and of Bias −0.12 (−0.67%), aPTT(R = 0.9957; N = 60, Bias 0.26 (0.58%)), AT3(R = 0.9800; N = 49, Bias −2.0 (−2.2%)), FBG (R = 0.9956; N = 57, Bias −1.9 (−0.5%)) and DD (R = 0.9981; N = 38, Bias 0.005 (0.373%)) with insignificant bias. ConclusionsThe utilization of the Roche cobas® 8100 automated ‘online’ centrifugation helps achieve optimal workflow efficiency without impacting analytical performance of the PT, aPTT, DD, AT3 and FBG assays. The use of PDP can be superior method to PPP for routine coagulation tests.

Sandwich-like electro-conductive polyurethane-based gelatin/soybean oil nanofibrous scaffolds with a targeted release of simvastatin for cardiac tissue engineering

Cardiac tissue engineering (CTE) is a promising way for the restoration of injured cardiac tissue in the healthcare system. The development of biodegradable scaffolds with appropriate chemical, electrical, mechanical, and biological properties is an unmet need for the success of CTE. Electrospinning is a versatile technique that has shown potential applications in CTE. Herein, four different types of multifunctional scaffolds, including synthetic-based poly (glycerol sebacate)-polyurethane (PGU), PGU-Soy scaffold, and a series of trilayer scaffolds containing two outer layers of PGU-Soy and a middle (inner) layer of gelatin (G) as a natural and biodegradable macromolecule without simvastatin (S) and with simvastatin (GS), an anti-inflammatory agent, were fabricated in the sandwich-like structure using electrospinning technique. This approach offers a combination of the advantages of both synthetic and natural polymers to enhance the bioactivity and the cell-to-cell and cell-to-matrix intercommunication. An in vitro drug release analysis was performed after the incorporation of soybean oil (Soy) and G. Soy is used as a semiconducting material was introduced to improve the electrical conductivity of nanofibrous scaffolds. The physicochemical properties, contact angle, and biodegradability of the electrospun scaffolds were also assessed. Moreover, the blood compatibility of nanofibrous scaffolds was studied through activated partial thromboplastin time (APTT), prothrombin time (PT), and hemolytic assay. The results showed that all scaffolds exhibited defect-free morphologies with mean fiber diameters in the range of 361 ± 109 to 417 ± 167 nm. A delay in blood clotting was observed, demonstrating the anticoagulant nature of nanofibrous scaffolds. Furthermore, rat cardiomyoblast cell lines (H9C2) were cultured on scaffolds for 7 days, and the morphology and cell arrangement were monitored. Data indicated an appropriate cytocompatibility. Of note, in the PGU-Soy/GS nanofibrous scaffold, a high survival rate was indicated compared to other groups. Our findings exhibited that the simvastatin-loaded polymeric system had positive effects on cardiomyoblasts attachment and growth and could be utilized as a drug release carrier in the field of CTE.Graphical

Phyllanthus emblica (Amla) methanolic extract regulates multiple checkpoints in 15-lipoxygenase mediated inflammopathies: Computational simulation and in vitro evidence

Amla (Phyllanthus emblica) has long been used in traditional folk medicine to prevent and cure a variety of inflammatory diseases. In this study, the antioxidant activity (DPPH scavenging and reducing power), anti-inflammatory activity (RBC Membrane Stabilization and 15-LOX inhibition), and anticoagulation activity (Serin protease inhibition and Prothrombin Time assays) of the methanolic extract of amla were conducted. Amla exhibited a substantial amount of phenolic content (TPC: 663.53 mg GAE/g) and flavonoid content (TFC: 418.89 mg GAE/g). A strong DPPH scavenging effect was observed with an IC50 of 311.31 µg/ml as compared to standard ascorbic acid with an IC50 of 130.53 µg/ml. In reducing power assay, the EC50 value of the extract was found to be 196.20 µg/ml compared to standard ascorbic acid (EC50 = 33.83 µg/ml). The IC50 value of the RBC membrane stabilization and 15-LOX assays was observed as 101.08 µg/ml (IC50 of 58.62 µg/ml for standard aspirin) and 195.98 µg/ml (IC50 of 19.62 µg/ml for standard quercetin), respectively. The extract also strongly inhibited serine protease (trypsin) activity with an IC50 of 505.81 µg/ml (IC50 of 295.44 µg/ml for standard quercetin). The blood coagulation time (PTT) was found to be 11.91 min for amla extract and 24.11 min for standard Warfarin. Thus, the findings of an in vitro study revealed that the methanolic extract of amla contains significant antioxidant, anti-inflammatory, and anticoagulation activity. Furthermore, in silico docking and simulation of reported phytochemicals of amla with human 15-LOXA and 15-LOXB were carried out to validate the anti-inflammatory activity of amla. In this analysis, epicatechin and catechin showed greater molecular interaction and were considerably stable throughout the 100 ns simulation with 15-lipoxygenase A (15-LOXA) and 15-lipoxygenase B (15-LOXB) respectively.

In vitro and in silico investigation of garlic’s (Allium sativum) bioactivity against 15-lipoxygenase mediated inflammopathies

Introduction: Garlic (Allium sativum) is widely used as a flavor-enhancing dietary ingredient and exhibits a wide spectrum of pharmacological effects. This study aimed to investigate the therapeutic effects of aqueous garlic extract to explore the bioactivity against 15-lipoxygenase (15-LOX) mediated inflammopathies. Methods: In this study, the antioxidant (DPPH free radical scavenging assay and reducing power assay), anti-inflammatory (hypotonicity-induced hemolysis assay and 15-LOX inhibition assay) and anticoagulation (serine protease inhibition assay and prothrombin time assay) effects of the aqueous garlic extract were investigated. Furthermore, in silico molecular docking and dynamic simulation analysis of reported small compounds of garlic against 15-LOX1 and 15-LOX2 were performed to figure out the most efficient phytochemical ligands and validate the anti-inflammatory potential. Results: The DPPH scavenging effect and the reducing power of the extract were found with the IC50 of 213.87 ± 1.49 μgmL-1 and EC50 of 124.78 ± 3.39 μgmL-1, respectively. In the hypotonicity-induced hemolysis and 15-LOX inhibition assay, the IC50 values were observed as 147.59 ± 2.98 μgmL-1 and 250.05 ± 8.48 μgmL-1, respectively. The extract inhibited serine protease activity with an IC50 of 301.33 ± 1.31 μgmL-1 and prevented blood coagulation for 10.05 ± 0.35 minutes in prothrombin time assay. The in silico study identified Rhamnetin as a potential 15-LOX1 and 15-LOX2 inhibitor, and it exhibited a stable interaction with the targets throughout the 100 ns dynamic simulation. Conclusion: The findings of this study provide molecular insights into garlic’s medicinal properties as well as its bioactive compounds, which can be potential therapeutic interventions for 15-LOX mediated inflammations.

Estimating the measurement uncertainties of the international sensitivity index of 12 thromboplastins through Monte Carlo simulation

BackgroundMeasurement uncertainty (MU) estimation has become an important process in clinical laboratories; however, calculating the MUs of the international sensitivity index (ISI) of thromboplastins is difficult because of the complex mathematical calculations required in calibration. Therefore, this study quantifies the MUs of ISIs through the Monte Carlo simulation (MCS), which involves random sampling of numerical values to solve a complex mathematical calculation. MethodsEighty blood plasmas and commercially available certified plasmas (ISI Calibrate) were used to assign the ISIs of each thromboplastin. Prothrombin times were measured using reference thromboplastin and 12 commercially available thromboplastins (Coagpia PT-N, PT Rec, ReadiPlasTin, RecombiPlasTin 2G, PT-Fibrinogen, PT-Fibrinogen HS PLUS, Prothrombin Time Assay, Thromboplastin D, Thromborel S, STA-Neoplastine CI Plus, STA-Neoplastine R 15, and STA-NeoPTimal) with two automated coagulation instruments: ACL TOP 750 CTS (ACL TOP; Instrumentation Laboratory, Bedford, MA, USA) and STA Compact (Diagnostica Stago, Asnières-sur-Seine, France). Then, the MUs of each ISI were simulated through MCS. ResultsThe MUs of ISIs ranged from 9.7 % to 12.1 % and 11.6 % to 12.0 % when blood plasma and ISI Calibrate were used, respectively. For some thromboplastins, the ISI claimed by manufacturers significantly differed from the estimated results. ConclusionsMCS is adequate to estimate the MUs of ISI. These results would be clinically useful for estimating the MUs of the international normalized ratio in clinical laboratories. However, the claimed ISI significantly differed from the estimated ISI of some thromboplastins. Therefore, manufacturers should provide more accurate information about the ISI value of thromboplastins.