Blood Coagulation Assays Research Articles

Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

Tantalum-containing mesoporous bioactive glass powder for hemostasis.

This study evaluates the hemostatic properties of tantalum-containing mesoporous bioactive glasses (Ta-MBGs) through a suite of in-vitro methods: hemolysis percentage, zeta potential, blood coagulation assays (Activated Partial Thromboplastin Time - APTT and Prothrombin Time - PT) and cytotoxicity tests. Five compositions of Ta-MBG, with x mol% Ta2O5 added to the glass series (80-x)SiO2-15CaO-5P2O5-xTa2O5 where x=0 (0Ta), x=0.5 (0.5Ta), x=1 (1Ta), x=5 (5Ta), and x=10 (10Ta) mol%, were synthesised. The hemostatic potential of all the Ta-MBGs was confirmed by their negative zeta potential (-23 to -31 mV), which enhances the intrinsic pathway of blood coagulation. The hemolysis percentages of all Ta-MBGs except 10Ta showed statistically significant reductions compared to the same experiments carried out both in the absence of a sample ('no treatment' group) and in the presence of 10Ta. These observations validate the consideration of Ta-MBGs as hemostatic agents as they do not cause significant lysis of red blood cells. Cytotoxicity analysis revealed that Ta-MBGs had no effect on bovine fibroblast viability. Furthermore, a reduction in both APTT (a test to evaluate the intrinsic pathway of coagulation) and PT (a test to evaluate the extrinsic pathway) signified enhancement of hemostasis: 5Ta caused a significant reduction in APTT compared to 'no treatment', 1Ta and 10Ta and a significant reduction in PT compared to 0Ta. Therefore, we conclude that 5mol% of Ta optimised the hemostatic properties of these mesoporous bioactive glasses.

Референсные интервалы параметров ротационной тромбоэластометрии у беременных и родильниц: одноцентровое рандомизированное исследование

Введение: В последнее время стали востребованы интегральные тесты оценки системы гемостаза у женщин во время беременности, а также во время акушерских операций, связанных с кровотечением. В нашей стране отсутствуют работы по определению популяционных норм ротационной тромбоэластометрии (РОТЕМ) у беременных, рожениц и родильниц. Цель исследования: определить пределы референсных интервалов показателей РОТЕМ во время физиологически протекающей беременности и в раннем послеродовом периоде и провести корреляционный анализ со стандартными показателями коагулограммы. Материалы и методы: Обследовано 229 женщин Красноярского края, которые были поделены на 5 групп: небеременные (контроль), беременные II и III триместров, женщины раннего послеродового периода после операции кесарева сечения и естественных родов. Все женщины были сопоставимы по возрасту, индексу массы тела, сопутствующей патологии. Референсные интервалы рассчитывали либо через среднее и стандартное отклонение (для нормального распределения), либо через медиану и 2,5 и 97,5% перцентили (для ненормального распределения). Результаты: В процессе исследования подтвердилось, что во время беременности и в раннем послеродовом периоде происходят изменения в системе гемостаза в сторону гиперкоагуляции. Показатели ROTЕM у женщин в III триместре беременности и раннем послеродовом периоде существенно отличаются от показателей небеременных в популяции. У женщин после родов вне зависимости от способа родоразрешения различий между параметрами ROTЕM не установлено. Корреляция между параметрами ROTЕM и традиционными лабораторными тестами коагуляции выявила взаимосвязь между амплитудой плотности сгустка в тесте FIBTEM и уровнем фибриногена по Клаусу. Заключение: Представлены референсные интервалы параметров РОТЕМ у женщин Красноярского края. Найдена корреляция между параметрами РОТЭМ и уровнем фибриногена. Background: Recently, integral tests for hemostasis assessment in pregnant women as well as during obstetric surgery associated with bleeding have become popular. In our country, there are no studies of population norms for rotational thromboelastometry (ROTEM) in pregnant women, parturients and puerperants. Objectives: to identify reference intervals limits for ROTEM parameters during physiological pregnancy and in early postpartum period and to carry out correlation analysis with standard coagulogram parameters. Patients/Methods: We examined 229 women in Krasnoyarsk region; they were divided into 5 groups: nonpregnant (control), pregnant women in II and III trimesters, women in early postpartum period after cesarean section and after vaginal delivery. All women were comparable in age, body mass index, and concomitant pathology. Reference intervals were calculated either as the mean and standard deviation (for normal distribution), or as the median and 2.5 and 97.5% percentiles (for abnormal distribution). Results: The study confirmed hemostasis hypercoagulation changes during pregnancy and in early postpartum period. ROTEM parameters in women in III trimester of pregnancy and in early postpartum period differ significantly from those in non-pregnant women. Differences between ROTEM parameters in puerperants, regardless of the delivery method, not found. The correlation between ROTEM parameters and traditional laboratory coagulation tests revealed a relationship between clot density amplitude in FIBTEM test and Klaus fi brinogen level. Conclusions: Reference intervals of ROTEM parameters in women of Krasnoyarsk region are presented. A correlation was found between ROTEM parameters and fibrinogen level

Fabrication of Chitosan-Reinforced Multifunctional Graphene Nanocomposite as Antibacterial Scaffolds for Hemorrhage Control and Wound-Healing Application.

Accidents on battlefields and roads often lead to hemorrhage and uncontrolled bleeding. Hence, immediate hemorrhage control remains of great importance to reduce mortality and socioeconomic loss. Herein, nanobiocomposite scaffolds (film and sponge) have been fabricated for the first time through the incorporation of a graphene-silver-polycationic peptide (GAP) nanocomposite into chitosan (Cs). Ten different scaffolds viz. Cs, Cs-GAP25, Cs-GAP50, Cs-GAP75, and Cs-GAP100 were prepared in the form of films and sponges. Cs-GAP100 nanobiocomposite sponge exhibited excellent porosity, fluid absorption, and blood clotting capacity, whereas Cs-GAP100 nanobiocomposite film showed excellent mechanical strength and poor degradation property. The presence of graphene in GAP provided a unique mechanical property and prevented the natural degradation, whereas silver nanoparticles and polycationic peptide provided an efficient antimicrobial property to the scaffolds. The high surface area of graphene and the hydrophilic nature of the polycationic peptide also imparted high fluid and blood absorption capacity to Cs-GAP nanobiocomposite scaffolds. The in vitro whole blood clotting assay demonstrated that clotting efficacy improved with the concentration of GAP nanocomposite and Cs-GAP100 nanobiocomposite sponge significantly (p value <0.003) reduced the clotting time to 60 s, as compared to the pristine chitosan dressings. On the other side, the Cs-GAP100 nanobiocomposite film showed an excellent wound-healing property. The Cs-GAP100 nanobiocomposite demonstrated profound antibacterial activity against Escherichia coli and Staphylococcus aureus. The intracellular reactive oxygen species (ROS) assay explained the interfacial interaction of Cs-GAP100 nanobiocomposite and bacterial cells, resulting in cell damage and finally cell death. The obtained information thus provided a novel safe-by-design concept for fabrication of Cs-GAP100 nanobiocomposite scaffolds and demonstrated potential development of antibacterial hemostatic and wound dressing in traumacare management.

Rotational thrombelastometry (ROTEM) improves hemostasis assessment compared to conventional coagulation test in ACLF and Non-ACLF patients

BackgroundPatients with liver cirrhosis typically exhibit abnormal coagulation parameters in conventional coagulation tests (CCTs). Rotational thromboelastometry (ROTEM) is a holistic blood coagulation assay. This method provides an insight into the global hemostatic capabilities and has been suggested to provide a better overview of the coagulation system in liver cirrhosis.MethodsThe goal of this study was to examine hemostasis in patients with stable liver cirrhosis (Non-ACLF) and in acute-on-chronic liver failure (ACLF) by CCT and ROTEM including agreement of both tests and the prospective assessment of test performance based on clinical outcomes in ACLF patients. Therefore, ACLF patients were additionally subgrouped by bleeding events. Fifty-five Non-ACLF patients and twenty-two patients with ACLF were analysed in this prospective cohort study.ResultsCoagulation parameters analysed by CCT were outside the normal range in Non-ACLF and ACLF patients, but were significantly more aberrant in ACLF patients. Non-ACLF patients analysed by ROTEM revealed parameters largely within the normal limits, while significantly more ROTEM parameters in ACLF patients were affected. Maximum clot firmness (MCF) was significantly divergent between both patient groups and correlated well with levels of fibrinogen and platelet count. Using Cohen’s Kappa coefficient κ, the strength of agreement between CCT and ROTEM analyses was determined to be fair for Non-ACLF patients and moderate for ACLF patients. Bleeding events occurred significantly more often in ACLF group with significantly reduced A10 and MCF.ConclusionsFor assessing hemostasis in Non-ACLF and ACLF patients the underlying dataset shows advantages of ROTEM over CCT. A10 and MCF represent suitable prognostic parameters in predicting bleeding events in ACLF group.

Porous 3D Printed Scaffolds For Guided Bone Regeneration In a Rat Calvarial Defect Model

The application of 3D printed scaffolds for bone tissue regeneration has been explored in previous studies. In this study, we combined 3D printing with porogen leaching to develop scaffolds with dual-scale porosity and investigated their capability in guided bone regeneration in a rat critical size calvarial defect model. The scaffolds were additively manufactured from medical grade polycaprolactone (mPCL) doped with porogen microparticles having an average size of 22 μm, which were subsequently leached to create microscale porosity. Morphological analysis revealed an interconnected macroscale porosity of about 60% with an average pore size of 700 µm and intra-strut microscale pores with a porosity of nearly 40% and average pore size of 20–70 µm. The microscale porosity resulted in a 3-fold increase in the scaffolds’ surface area, a 2-fold enrichment in negatively charged surface groups, which did lead to significantly increased protein adsorption and faster hydrolysis-driven degradation in vitro. An in vitro blood clotting assay demonstrated an increased TGF-β1 release from the clots formed on the dual-scale porous scaffolds. In a rat calvarial defect, bone formation was found in both the macro- and microscale pores and was at a similar level when compared to calcium phosphate coated mPCL scaffolds.

Serum amyloid A4 is a procoagulant apolipoprotein that it is elevated in venous thrombosis patients

BackgroundSerum amyloid A4 (SAA4) is an apolipoprotein that is in the SAA family and it is constitutively translated. Previously, acute‐phase SAA1 and SAA2 levels were associated with venous thromboembolism (VTE). ObjectiveWe investigated the association of plasma SAA4 with VTE and the role of SAA4 in coagulation. Patients and MethodsThe association of SAA4 with VTE in a case‐control study of adult VTE subjects (N = 113 each group) and the effects of recombinant SAA4 on plasma blood coagulation assays and prothrombin activation initiated by factor Xa were evaluated. ResultsPlasma SAA4 levels in VTE subjects were higher vs. controls (48.1 vs. 38.4 µg/mL; P < .001). Elevated plasma SAA4 level (above the 90th percentile of controls) was associated with increased VTE occurrence (odds ratio, 3.8; 95% confidence interval, 1.8‐8.0). This association remained significant after the adjustment for acute‐phase SAA level, suggesting that SAA4 associated with VTE is independent of acute‐phase SAA. Two isoforms of SAA4, that is, glycosylated and nonglycosylated SAA4 isoforms, were each higher in VTE patients. When recombinant SAA4 was added to plasma, it shortened factor Xa‐1‐stage clotting times, showing that it enhances clotting in plasma. In reaction mixtures containing purified factors Xa and Va and prothrombin, recombinant SAA4 increased prothrombin activation, showing that it enhances prothrombinase activity. ConclusionElevated plasma constitutive SAA4 levels were linked to VTE in adults, and SAA4 can enhance thrombin generation in plasma. Our data highlight a previously unknown procoagulant activity of SAA4 that appears to be related to risk of venous thrombotic events.

Age- and gender-related reference ranges for thromboelastography from a healthy Indian population.

Thromboelastography (TEG) is a whole blood clotting assay largely used in major surgeries and trauma to monitor patients' in vivo hemostatic status. Standardization of kaolin-activated citrated whole blood thromboelastography is not done in the Indian population. This study primarily aims to derive reference ranges of kaolin-activated TEG for healthy volunteers in the Indian population. Secondarily, it aims to study the age- and gender-related hemostatic changes in the study population. A total of 120 healthy volunteers were enrolled (55 adult males, 32 adult females, and 33 children). The volunteers were interviewed for any bleeding history or drug intake which affects coagulation. Kaolin-activated TEG was performed on citrated whole blood, and parameters including R-time, K-time, angle, MA, LY30, and CI were analyzed. Derived reference range for total volunteers irrespective of age and sex were as follows: R-time: 3.8-10.6, K-time: 1.2-3.1, angle: 44.9-72.0, MA: 41.2-64.5, LY30: 0-9.9, and CI: -3.7 to 3.4. Statistically significant difference was observed in different age and sex groups for R-time, K-time, and angle. About 40% of the volunteers had at least one abnormal parameter according to the manufacturer's reference range which decreased to 12.5% when the derived reference ranges were considered. Gender- and age-related variances were observed in reference ranges of our population and which was also differed from the other ethnic population. Many of our healthy volunteers were categorized as coagulopathic when manufacturer's reference range was considered. So, it is important to derive the reference range of the target population before using the TEG into clinical practice.

Comprehensive assessment of Nile tilapia skin collagen sponges as hemostatic dressings.

Nile tilapia skin collagen sponge was fabricated by lyophilization and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide (EDC/NHS). The physicochemical properties were examined. The EDC/NHS cross-linked collagen sponge presented an enhanced water absorption capacity. In addition, biocompatibility and hemostatic efficiency were evaluated by acute systemic toxicity assay, dermal irritation test, intradermal reaction test, sensitization test, cytotoxicity, blood clotting assay in vitro, and liver and femoral artery hemorrhage models in vivo. Results showed that the produced collagen sponges before and after EDC/NHS cross-linking had excellent biocompatibility. Furthermore, EDC/NHS cross-linking promoted fibroblast cells viability and proliferation reflected by the MTT reduction assay. Meanwhile, EDC/NHS cross-linked collagen sponge exhibited the best blood clotting ability and hemostatic efficiency in rat femoral artery hemorrhage model in comparison with non-crosslinked and commercial collagen sponges. Our results demonstrated that the fabricated collagen sponges could be used as perfect hemostatic dressings.

A synthetic heparin mimetic that allosterically inhibits factor XIa and reduces thrombosis in vivo without enhanced risk of bleeding

BackgroundHuman factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long‐standing hypothesis has been that allosterism originating from heparin‐binding site(s) on coagulation enzymes is a promising approach to yield safer agents. ObjectivesTo develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk. MethodsWe employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work. ResultsSulfated chiro‐inositol (SCI), a non‐saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin‐binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway‐based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum‐dose‐tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl3‐induced arterial and thromboplastin‐induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 μg/animal, which matched enoxaparin at 2500 μg/animal. ConclusionsOverall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

Relationship of thromboelastography and conventional clotting test values with severe bleeding in critically ill patients with coagulopathy: A prospective study.

This study aimed to ascertain the associations of thromboelastography (TEG® ) and standard laboratory test (SLTs) values with the presence of bleeding in critically ill patients with known coagulopathy. Three groups of coagulopathic patients with (a) hepatic failure, (b) postoperative period after prolonged cardiac surgery, and (c) complex abdominal surgery with sepsis were prospectively included in this study. On intensive care unit (ICU) admission, patients were stratified into two groups according to whether they had major bleeding (MB) (evident overt bleeding, important bleeding apparent on imaging studies, and/or need for moderate-massive blood transfusion and hemodynamic instability). Blood samples were drawn for the SLTs (international normalized ratio [INR], activated partial thromboplastin time [aPTT], platelet count, and fibrinogen level [Clauss]) and TEG whole blood coagulation assays. Receiver operating characteristic (ROC) curves were generated to determine the efficiency of TEG and SLTs for detecting bleeding. The correlations between SLTs and TEG parameters with similar coagulation profiles were evaluated by Spearman rank-order analysis. Eighty-three patients were included, and bleeding was confirmed in 45 (54%). The fibrinogen level demonstrated the best accuracy for detecting bleeding with an area under the curve and 95% confidence intervals [AUC (95% CI)] of 0.74 (0.63-0.85) with the best cutoff value of≤2g/L. Regarding TEG-MA, the AUC (CI) obtained with the optimal cutoff value of≤51mm was 0.68 (0.56-0.80). Both conventional clotting tests and TEG values were poorly associated with bleeding in this critically ill cohort of patients with coagulopathy.

Abstract 416: Approach of mRNA Delivery by Using Nanoparticles to Recover Endocardial Notch Signaling

Notch signaling plays a significant role in cardiac development and morphogenesis, especially for cardiac chamber development. Failure of mechanotransduction of endocardial Notch signaling in ventricular chamber causes lack of trabeculation. Advent of nanoparticle (NP) technology offers a therapeutic approach of targeted delivery. Although delivering mRNA is an effective method, it is challenging due to stability and cell defense mechanisms. To overcome the limitations and effectively deliver mRNA to endocardium, we used Poly(lactic-co-glycolic acid) (PLGA) synthesized NPs, which have been used in various drug delivery systems due to its biocompatibility, efficient encapsulation of hydrophilic and hydrophobic materials, specific targeting effects, and lower payload concentration. We hypothesized our PLGA NPs are able to deliver NICD mRNA to target endocardium to overexpress Notch signaling in a Notch inhibited zebrafish animal model. Using double emulsion, we synthesized PLGA NPs at 55–65 kDa and performed in vitro test on HUVECs. 69.8 ± 4.3% of Rhodamine B was successfully loaded into the NPs, and after incubation with HUVECs the signal lasted for 28 days after burst release. Hemolysis and whole blood clotting assays proved that NPs did not interfere with the blood clotting process or lyse blood cells. We characterized mRNA incorporated PLGA nanoparticles and incubated with HUVECs after antibody conjugation. To test in vivo study, we injected gata1a morpholino oligomer (MO) to Tg(fli1:gfp) transgenic line to inhibit hematopoiesis, which reduces wall shear stress (WSS) of ventricular chamber leading lack of trabeculae. At 2 days post fertilization (dpf), NICD mRNA incorporated nanoparticles were injected into the circulatory system of gata1a MO injected zebrafish to rescue trabeculation. At 5 dpf, zebrafish hearts were harvested and analyzed via qRT-PCR to compare Notch related gene expression among control, gata1a MO injected, and rescue group. The NICD mRNA injected group successfully rescued the expression level of Notch components despite a lack of WSS and mechanosensitive Notch signaling via gata1a MO injection. This finding will provide an initial therapeutic approach of NICD mRNA for trabecular related congenital heart disease.

Modified ROTEM for the detection of rivaroxaban and apixaban anticoagulant activity in whole blood: A diagnostic test study.

Rapid detection of the anticoagulant effect of oral factor Xa (FXa) inhibitors may be essential in several emergency clinical situations. Specific assays quantifying the drugs are performed in plasma and require a turnaround time that is too long to be useful in emergency situations. Rotational thromboelastometry (ROTEM) is a whole blood coagulation assay of blood viscoelasticity and could be of interest for FXa inhibitor detection in emergency. However, conventional ROTEM reagents only detect high amounts of inhibitors. The aim of this study was first to assess the effect of whole blood components on the viscoelastic measurement of the effects of FXa inhibitors, and second to evaluate whether a modified ROTEM, triggered with a low amount of tissue factor and a saturating amount of phospholipid vesicles, can reliably detect low levels of FXa inhibitor activity in whole blood. Diagnostic test study. A university research laboratory. From November 2014 to April 2016. Sixty-six patients: 30 treated with rivaroxaban, 17 with apixaban and 19 without treatment. ROTEM was triggered with 2.5 pmol l of tissue factor and 10 μmol l of phospholipid vesicles. Modified ROTEM parameters were measured in different experimental conditions: platelet-poor plasma (PPP), platelet-rich plasma, PPP supplemented with fibrinogen and reconstituted whole blood with various haematocrit levels adjusted between 30 and 60%. Modified ROTEM was further validated using whole blood from patients who were either treated or not treated with FXa inhibitors. Modified ROTEM allowed detection of as little as 25 ng ml FXa inhibitors in PPP, with at least a 1.4-fold increase of the clotting time (P ≤ 0.02). Neither changes of fibrinogen concentration nor variations of platelet count or haematocrit precluded FXa inhibitor detection. A lengthened modified ROTEM clotting time of more than 197 s allowed detection of FXa inhibitor concentrations above 30 ng ml in whole blood with 90% sensitivity and 85% specificity. Modified ROTEM may be applicable in emergency situations for the detection of FXa inhibitors in whole blood.

Isolation and purification of anticoagulant enzymes from plant latex

The plant latex is a complex mixture of organic, inorganic and hydrolytic enzymes such as proteases which acts as anticoagulant and finds its uses in surgeries, heart diseases and blood clot treatment. In the present study, latex was collected from Euphorbia milii, Jatropha malacophylla, Thevetia peruviana, Euphorbia pulcherimma and Artocarpus altilis to evaluate the best source of anticoagulant enzyme though four other plants were originally screened as well but failed to show the enzyme. Among the five, A. altilis and J. malacophylla showed the highest anticoagulant activity by milk clotting, blood clotting and APTT assay. The samples were subjected to three-step purification, i.e., salt precipitation, dialysis, ion exchange and gel filtration. The fold purification of A. altilis was found to be 5.37 and 2.31 for J. malacophylla respectively after the gel filtration. The percentage of yield of A. altilis was found to be 63.23% and 26.4% for J. malacophylla. Molecular weight of J. malacophylla sample and A. altilis was found to be ~80kDa and ~105kDa respectively, determined by SDS PAGE. Both enzymes showed optimum activity at pH 7. A. altilis showed optimum activity at 35°C, incubation time of 40 minutes, substrate concentration of 60mM, with MgCl2 as activator the activity increased at 600?L and with EDTA as inhibitor the activity increased at 400?L. J. malacophylla showed optimum activity at 45°C, incubation time of 10 minutes, substrate concentration of 80mM and stable at 35 °C which is the human body temperature. A. altilis showed optimum conditions for human administration making it therapeutically viable.

Coagulation Factor XIIIa Inhibitor Tridegin: On the Role of Disulfide Bonds for Folding, Stability, and Function.

Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the 15 possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogues using a targeted protecting group strategy to investigate the impact of the C19-C25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogues was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild-type tridegin and the analogues as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C19-C25 bond on conformational stability and binding mode. The strategy of selectively reducing disulfide bonds to facilitate large-scale synthesis, while retaining the functionality of disulfide-bonded peptides, has been demonstrated with our present study.

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate.

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.

Use of blood clotting assays to assess potential anticoagulant rodenticide exposure and effects in free-ranging birds of prey

Non-target wildlife, particularly birds of prey, are widely exposed to and acutely poisoned by anticoagulant rodenticides (ARs). An unresolved issue surrounding such exposure, however, is the potential for sublethal effects. In particular, the consequences of AR exposure and resulting coagulopathy on health and survival of unintentionally exposed animals, which often encounter a multitude of anthropogenic stressors, are understudied. In a wildlife rehabilitation setting, AR intoxication may be masked by more obvious injuries related to collision with vehicles or electrocution, thereby obfuscating proximate from ultimate cause of mortality. An assessment of coagulation function of admitted wildlife may provide a means of identifying animals exhibiting sublethal coagulopathy, and ultimately ensuring provision of appropriate and swift treatment. In conjunction with routine diagnostics for injury and disease, we performed two blood clotting assays (prothrombin time, Russell's viper venom time) affected by vitamin K-dependent coagulopathy of samples from six species of live raptors admitted to a rehabilitation facility. We also measured clotting time in pre-fledgling barn owl chicks (Tyto furcata) from 10 nest sites in Lower Mainland Canada. Prolonged clotting time or failure to form a clot altogether was observed in 23.0% of 61 sampled raptors admitted to the rehabilitation facility. This is a biologically significant proportion of individuals given the fortuitous and likely biased nature by which raptors are found and admitted to rehabilitation facilities. In contrast, there was little evidence of coagulopathy in 19 pre-fledgling barn owl chicks. The utility of avian coagulation tests for diagnosing AR exposure is promising, yet there remains a need to establish species specific reference values and standardize assay methodologies among testing facilities.

Rational truncation of aptamer for cross-species application to detect krait envenomation

In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent).

Neutrophil KLF2 Regulates Arterial and Venous Thrombosis

Experimental, clinical and pathological studies support an important link between inflammation and thrombosis. Although accumulating evidence suggests that cells of the innate immune system contribute to the thrombotic process, the identity of nodal molecular determinants operative in immune cells remains poorly understood. Our previous work in mice bearing global deletion of the transcription factor KLF2 identifiedthis factor as a critical mediator of thrombosis). Here, using cell-specific KLF2 deleted murine models (endothelial, platelet, and myeloid- deleted KLF2) we identify myeloid KLF2 as the major determinant of thrombosis. Complete blood counts and coagulation assays in myeloid KLF2 deleted mice (MY-K2-KO) were similar to those in control LysM cre mice. Using two models of vascular thrombosis (carotid artery thrombosis assay, Rose Bengal model and complete inferior vena cava ligation model of venous thrombosis) we observed a robust prothrombotic phenotype in the MY-K2-KO mice as compared to control Cre mice. As LysM Cre deletes in both neutrophils and monocyte/macrophages, we next sought to determine whether one subset was dominantly responsible for the observed phenotype. Adoptive transfer of KLF2-KO neutrophils in control mice conferred both the arterial and venous pro-thrombotic phenotype. Conversely, neutrophil depletion with Ly6G antibody (>90% depletion achieved) reversed the prothrombotic phenotype in MY-K2-KO mice. By contrast, depletion of monocytes/macrophages using clodronate liposome injection did not affect arterial or venous thrombosis. Next, we evaluated the effect of KLF2 deficiency on critical neutrophil functions that may contribute to the pro-thrombotic phenotype. As compared to controls, MY-K2-KO neutrophils demonstrated more pronounced recruitment to the endothelium. In microflow chamber assays, MY-K2-KO neutrophils showed decreased rolling and increased P-selectin induced adhesion. The importance of this was affirmed by the observation that adoptive transfer of MY-K2-KO neutrophils incubated with PSGL-1 antibody was unable to generate a prothrombotic phenotype in control mice. In addition, MY-K2-KO neutrophils demonstrated enhanced neutrophil extracellular traps generation associated with increased myeloperoxidase and neutrophil elastase expression. And finally, MY-K2-KO neutrophils (but not monocytes) demonstrated increased tissue factor expression and activity. Collectively, these studiesidentify neutrophil KLF2 as a critical determinant of both arterial and venous thrombosis and suggest that modulation of neutrophil KLF2 may be a fruitful approach to the management of thrombosis. DisclosuresSchmaier:Alnylam: Research Funding; Biomotiv: Consultancy; Cleveland Clinic Foundation: Research Funding; Temple University: Patents & Royalties; Enzyme Research Laboratories: Honoraria; Shire: Consultancy, Honoraria, Research Funding.