TGA Assay Research Articles

Evaluation of covalent and supra-molecular interactions in phosphated galacturonic-glucuronic acid for network structure for use in wound dressings

Keeping in view the wound healing properties of sterculia gum (SG) and ceftriaxone in consideration, in the present work, antibiotic drug encapsulated SG and poly(vinylphosphonic acid) [poly(VPA)] based copolymeric hydrogel wound dressings were designed to enhance the wound healing. Copolymers were characterized by FESEM & EDS, AFM, FTIR, 13C NMR, XRD, TGA and biomedical assay of blood compatibility, mucoadhesion and antioxidant properties. AFM analysis revealed 76.5 nm average roughness and XRD analysis showed sharp peaks at 2θ = 21°. FTIR spectrum of copolymer indicated characteristic peaks at 1116 cm−1 due to stretching vibration of P = O groups of poly(VPA) and twin peaks at 985 and 920 cm−1 due to asymmetric and symmetric vibration of P-O respectively and depicted grafting of phosphated polymers onto gum. The hemolysis test (hemolysis index = 3.40 ± 0.18%) revealed biocompatible nature and the adhesion test demonstrated 84 ± 5.00 mN detachment force which was required to detach the copolymer from the mucosal membrane. Hydrogel dressings absorbed 6.0 ± 5.00 (g/g gel) simulated wound fluid to main the moist wound environment. They were found permeable to O2 and impermeable to microbes and also exhibited antioxidant character. The release profile of ceftriaxone elucidated sustained release of encapsulated ceftriaxone drug with non-Fickian diffusion mechanism and was best fitted in first order kinetic model. Covalent and supra-molecular interactions formed in phosphated galacturonic-glucuronic acid of sterculia gum (SG) were found useful for sustained drug delivery and mucoadhesive properties. These biophysical interactions demonstrated the suitability of antibiotic-encapsulated network structures for wound dressing applications.

Lupeol-loaded chitosan-Ag+ nanoparticle/sericin hydrogel accelerates wound healing and effectively inhibits bacterial infection

Lupeol, a pentacyclic triterpene, has demonstrated significant wound healing properties; however, its low water solubility has limited its clinical applicability. To overcome this limitation, we utilized Ag+-modified chitosan (CS-Ag) nanoparticles to deliver lupeol, resulting in the formation of CS-Ag-L-NPs. These nanoparticles were then encapsulated within a temperature-sensitive, self-assembled sericin hydrogel. Various analytical methods, including SEM, FTIR, XRD, HPLC, TGA assay, hemolysis and antibacterial activity tests, were employed to characterize the nanoparticles. Additionally, an infectious wound model was used to evaluate the therapeutic and antibacterial efficacy of the CS-Ag-L-NPs modified sericin hydrogel. Our results showed that the encapsulation efficiency of lupeol in CS-Ag-L-NPs reached 62.1 %, with good antibacterial activity against both gram-positive and gram-negative bacteria and a low hemolysis ratio (<5 %). The CS-Ag-L-NPs sericin gel exhibited multiple beneficial effects, including inhibiting bacterial proliferation in wound beds, promoting wound healing via accelerated re-epithelialization, reducing inflammation, and enhancing collagen fiber deposition. We conclude that the CS-Ag-L-NPs loaded sericin hydrogel has tremendous potential for development as a multifunctional therapeutic platform capable of accelerating wound healing and effectively suppressing bacterial infections in clinical settings.

Preparation and characterization of a new sustainable bio-based elastomer nanocomposites containing poly(glycerol sebacate citrate)/chitosan/n-hydroxyapatite for promising tissue engineering applications

Poly (glycerol sebacate citrate) (PGSC) has potential applications in tissue engineering due to its biodegradability and suitable elasticity. However, its applications are restricted owing to its acidity and high degradation rate. In this study, a new bio-nanocomposite based on PGSC has been synthesized by incorporating chitosan (CS) and various concentrations of hydroxyapatite nanoparticles (n-HA). It is assumed that the basicity of a CS and hydroxyl groups of n-HA will reduce the acidity of PGSC and control the rate of degradation. Also, the biocompatibility of n-HA and inherent hydrophilicity of CS can improve cell adhesion and proliferation of PGSC-based scaffolds. FTIR, XRD, FESEM, and EDX tests confirmed the synthesis of these nanocomposites and the interaction between each of the components. The results of the DMTA test also indicated the elastic behavior of the samples embedded with n-HA. The hydrophilicity assay demonstrated that the water contact angle of the scaffolds decreased as the concentration of n-HA augmented, and it reached the value of 44 ± 0.9° for nanocomposite containing 5 wt.% n-HA. The degradation rate of all PGSC nanocomposites was reduced due to the anionic groups of n-HA and CS. TGA assay indicated that the incorporation of n-HA led to the enhancement of scaffolds’ thermal stability. Furthermore, the synergistic effect of CS and n-HA on the enhancement of protein adsorption and cell proliferation was confirmed through protein adhesion and MTT assay, respectively. Consequently, the addition of n-HA and CS perform the new bio-nanocomposites scaffolds based on PGSC with sufficient hydrophilicity, flexibility, and thermal stability in tissue engineering applications.

Enhancing structural strength and improving cell survival through Polycaprolactone/(gelatin/hydroxyapatite) Core‐Shell nanofibers for tissue engineering

AbstractBone tissue engineering is a practical approach to repairing broken or damaged bones that combines scaffold, cells, and growth factors for treatment. In this study, polycaprolactone (PCL)/gelatin (Ge)/hydroxyapatite (HA) core‐shell nanocomposites have been produced by the coaxial electrospinning method. Coaxial electrospinning is an efficient method for scaffold preparation to provide an interconnected porous fibrous scaffold. The prepared nanocomposite simultaneously benefited from the good mechanical behavior of the core PCL polymer. The desired biological properties also originated from the outer layer of the Ge/HA nanocomposite. Nanofibrous scaffolds' properties were characterized using SEM, TEM, FTIR, TGA, DSC, tensile test, contact angle, and MTT assay. The morphology of the as‐electrospun nanofibers was investigated using SEM and TEM, which revealed a defect‐less fibrous morphology. TEM images showed the core‐shell structure of the prepared scaffold nanofibers. The contact angle test showed that the presence of HA nanoparticles has improved the wettability of fibrous composites. In addition, HA nanoparticles could effectively strengthen the polymer scaffolds. The highest UTS value of 4.1 MPa was obtained in the PCL/(Ge+10%HA) sample. The cytotoxicity results revealed that the prepared scaffolds were utterly biocompatible. Moreover, significant cell proliferation of osteosarcoma cells was observed at high HA contents. The interconnected pores allowed cells to migrate into the scaffolds and grow inside. Based on the obtained results, PCL/(Ge/ HA) core‐shell nanofibers could be a promising candidate for bone scaffolds.

Characterization of a Missense Mutation in the Catalytic Domain and a Splicing Mutation of Coagulation Factor X Compound Heterozygous in a Chinese Pedigree.

Background: Congenital coagulation factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million caused by mutations in the FX-coding gene(F10), leading to abnormal coagulation activity and a tendency for severe hemorrhage. Therefore, identifying mutations in FX is important for diagnosing congenital FX deficiency. Results: Genetic analysis of the proband identified two single-base substitutions: c.794T > C: p.Ile265Thr and c.865 + 5G > A: IVS7 + 5G > A. His FX activity and antigen levels were < 1% and 49.7%, respectively; aPTT and PT were prolonged to 65.3 and 80.5 s, respectively. Bioinformatics analysis predicted the two novel variants to be pathogenic. In-vitro expression study of the missense mutation c.794T > C: p.Ile265Thr showed normal synthesis and secretion. Activation of FXs by RVV, FVII/TF, and FVIII/FIX all showed no obvious difference between the variant and the reference. However, clotting activity by PT and aPTT assays and activity of thrombin generation in a TGA assay all indicated reduced activity of the mutant FX-Ile265Thr compared to FX-WT. Minigene assay showed a normal splicing mode c.865 + 5G > A: IVS7 + 5G > A, which is inconsistent with clinical phenotype. Conclusions: The heterozygous variants c.794T > C: p.Ile265Thr or c.865 + 5G > A: IVS7 + 5G > A indicate mild FX deficiency, but the compound heterozygous mutation of the two causes severe congenital FX deficiency. Genetic analysis of these two mutations may help characterize the bleeding tendency and confirm congenital FX deficiency. In-vitro expression and functional study showed that the low activity of the mutant FX-Ile265Thr is caused by decrease in its enzyme activity rather than self-activation. The minigene assay help us explore possible mechanisms of the splicing mutation. However, more in-depth mechanism research is needed in the future.

Direct Oral Anticoagulants Reduce Hypercoagulopathy and Preserve Podocyte Function in an Experimental Model of Glomerular Disease

Proteinuric glomerular diseases are a leading cause of chronic kidney disease (CKD). Both pre-CKD glomerular disease and established CKD are major risk factors for thrombosis. Glomerular capillary podocyte injury is a key determinant of CKD progression and results in massive proteinuria accompanied by an acquired hypercoagulopathy that drives thrombotic risk. Unfortunately, the routine use of anticoagulant prophylaxis during glomerular proteinuria (GP) remains controversial due to both a lack of agreement regarding indications and no randomized controlled trial data demonstrating both safety and efficacy. We have recently used rat glomerular disease models to reveal that: (1) Proteinuria is directly correlated with hypercoagulopathy and in vivo thrombosis and (2) Thrombin, the key effector enzyme of the coagulation system, directly injures podocytes during proteinuria. What is not yet known is the ability of direct oral anticoagulant (DOAC) therapy to improve these important CKD and thrombosis outcomes. Thus, the aim of the present study is to determine if DOACs simultaneously reduce podocytopathy and enable effective thromboprophylaxis during GP. We hypothesized that DOACs would simultaneously preserve podocyte function and reduce hypercoagulopathy, in a podocyte-specific rodent model of glomerular disease. We utilized the podocin promotor-human diphtheria toxin receptor (pDTR) transgenic rat model to induce highly specific podocyte injury following a single I.P. injection of 50 ng/kg diphtheria toxin (DT). DT-induced proteinuria was subsequently treated daily by oral gavage with 1) Dabigatran (20 mg/kg; Dabi), 2) Rivaroxaban (3 mg/kg; Riva), or 3) Sham (saline) and compared to healthy controls (n=3-6/group). Morning spot urine and citrated plasma samples were collected from each group at day 10 post-DT. Endogenous Thrombin Potential (ETP) was measured by Technothrombin TGA assay, without and with DOAC-Stop reagent. Glomeruli were isolated from the kidney, dissociated into single-cell suspensions and analyzed by flow cytometry following immunofluorescent antibody and TUNEL staining. Both Dabi and Riva significantly reduced proteinuria (Fig A) and podocytopathy (TUNEL positive podocyte fraction; Fig B), while concomitantly correcting elevated ETP levels (Fig C open symbols). Addition of DOAC-Stop (Fig C closed symbols) revealed an insignificant (P=0.18) trend toward partial ETP reduction, consistent with DOAC-mediated reduction of the underlying GP-mediated hypercoagulopathy (via indirect, antiproteinuric effects). In conclusion, dabigatran and rivaroxaban reduce proteinuria and enhance podocyte health in concert with alleviation of the acquired hypercoagulopathy in a podocyte-specific rodent model of glomerular disease. Overall these data suggest DOAC treatment as a novel approach to simultaneously reduce both podocytopathy and thrombotic co-morbidities during glomerular disease. Additional experiments using this model to determine DOAC efficacy on in vivo thrombosis are in progress. Results from these preclinical studies should inform subsequent randomized controlled DOAC trials that may transform care for patients with glomerular disease by mitigating their risk of both CKD progression and thrombosis. Disclosures No relevant conflicts of interest to declare.

Polyurethane nanocomposite impregnated with chitosan-modified graphene oxide as a potential antibacterial wound dressing.

In this study, a polyurethane nanocomposite film (PU/CS-GO) was synthesized by incorporating chitosan-modified GO nanosheets (CS-GO) into PU matrices. The successful synthesis of unmodified GO and CS-GO nanosheets was confirmed by FTIR, XRD, Raman, SEM, EDS, and TGA analyses. In comparison with polyurethane nanocomposite containing unmodified graphene oxide (PU/GO), the effect of the modification of GO with chitosan on different properties such as morphological, thermal, thermo-mechanical, wettability, antibacterial, and biocompatibility properties was evaluated by various analyses including XRD, FE-SEM, TGA, DMTA, contact angle, antibacterial studies, and MTT assay. FE-SEM images exhibited better dispersion of CS-GO nanosheets within polyurethane matrices compared to GO nanosheets. According to DMTA results, PU/CS-GO showed a higher glass transition temperature (Tg) and storage modulus values than PU/GO sample. Based on antibacterial and MTT assays, in comparison with PU/GO, the PU/CS-GO nanocomposite demonstrated better biocompatibility and also antibacterial activity against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. Based on results, the PU/CS-GO nanocomposite can be suggested as a promising material for antibacterial wound dressing.

Synthesis of PLA/SBA-15 Composite Scaffolds for Bone Tissue Engineering

Composite materials are used in bone tissue engineering because they mimic the structure of the extracellular matrix of bone. In this work, polylactic acid (PLA) fiber scaffolds prepared by air-jet spinning technique, were doped with different concentrations of SBA-15 ceramic (0.05, 0.1, and 0.15 wt%). The SBA-15 ceramic powder was made by the Sol-Gel process. Physicochemical characterization of PLA, SBA-15, and composite fiber scaffold was done by XRD, SEM, BET, FTIR, TGA, mechanical test, and biocompatibility assay, which were performed in a cell culture model with osteoblast cells. Our results showed a random nanofibers composite scaffold with an improvement in the physicochemical properties. The PLA fiber diameter increases as increases the content of SBA-15, and the mechanical properties were dose-dependent. SBA-15 shows the well-ordered mesoporous hexagonal structure with a pore size of 5.8 ± 0.2 nm and a specific surface area with a value of 1042 ± 89 m2/g. PLAfibers and composites have thermal stability up to 300°C, and thermal decomposition in the range 316-367°C. In vitro biocompatibility results showed that PLA/SBA-15 composite scaffold had no cytotoxicity effect in terms of cell adhesion and viability of osteoblast cells. Furthermore, the doped SBA-15 with 0.05% wt onto the polymer matrix could be useful in biomedical applications for bone tissue engineering.

Preparation, deproteinization, characterization, and antioxidant activity of polysaccharide from Medemia argun fruit

In this study, the crude polysaccharide from argun palm (Medemia argun) fruit was extracted with hot water and deproteinized by four different methods. Its structure characterization, thermal property and antioxidant activity were investigated by FT-IR spectroscopy, Scanning electron microscope, Congo-red, I2-KI, DPPH• assay, ABTS•+ assay, TGA and DSC analysis. It indicated that the NaCl method was the best method in deproteinization. The argun fruit polysaccharide linked by the β-glycosidic bonds. The molecular weight of polysaccharide deproteinized with NaCl was 4.46 × 105 Da. It showed that the polysaccharide from argun palm fruit consisted of fructose, arabinose, rhamnose, galactose, glucose, xylose, and galacturonic acid. The result of TGA and DSC assay indicated that argun fruit polysaccharide has a favorable thermal stability. It was proved that, the argun fruit polysaccharide possessed high antioxidant activity and would be a new natural antioxidant.

Structure characterization, physicochemical property and immunomodulatory activity on RAW264.7 cells of a novel triple-helix polysaccharide from Craterellus cornucopioides

In the study, a new triple-helix polysaccharide with favorable stability was purified from C. cornucopioides. Its structural characterization, stability and solution behavior were investigated by the GC–MS, periodate oxidation-smith degradation, FT-IR, 1D and 2D NMR spectroscopy, methylation analysis, Scanning electron microscope, Congo-red, CD, TGA and DSC analysis. The results showed that Craterellus cornucopioide polysaccharide (CCP) possessed the molecular weight of 1.97 × 103 kDa, is mainly composed of mannose (48.73%), galactose (17.37%), glucose (15.97%) and xylose (17.93%), respectively. It was a heteroglycan with (1 → 3)‑linked‑β‑d‑Manp‑(1 → 6)‑linked α‑d‑Galp backbone distributed by (1 → 4)‑linked‑α‑d‑Xylp‑t‑α‑d‑Manp and t‑β‑d‑Glup units at O-6. The result of TGA and DSC assay indicated that CCP has a favorable thermal stability. MTT and Scanning electro microscopy (SEM) assay showed that CCP could significantly improve the proliferation activity and induce cells activation of RAW264.7 in a certain range of concentrations and period.

Bovine Heparin Compared to Porcine Heparin to Demonstrate Bioequivalence

Introduction: Heparin prevents blood clots from forming in patients undergoing heart surgeries, dialysis, multiple other procedures, and for medical treatment of thrombosis such as associated with cancer. Currently, all heparin products in the U.S. are derived from the intestinal mucosa of pigs. Seventy-five percent of the crude porcine heparin used to make the active pharmaceutical ingredient (API) comes from outside the U.S., with a majority originating from China. Reintroduction of bovine heparin into the U.S. market would expand sources for this critical drug, thus addressing concerns about potential shortages and product adulteration. This study was undertaken to determine the bioequivalence of bovine and porcine heparins using assays relevant to the clinical setting. The assays used in this study were chosen because they overcome limitations of the conventional APTT for heparin monitoring. Selected TEG and ACT assays use whole blood which better simulates physiologic conditions and assesses the full in vitro anticoagulation potential of heparin; these assays are also routinely used clinically for heparin monitoring. The thrombin generation assay is state-of-the-art for hemostatic clinical lab testing, and it provides a more sensitive endpoint of the final generation of thrombin once coagulation is activated. Heparins were studied at concentrations used clinically and tested by assays sensitive to these concentrations.Methods: Bovine heparin API (BH; 7 lots) and US clinical grade porcine heparin (PH: 3 lots) were tested in parallel using recalcified whole blood thromboelastography (TEG; Haemonetics), celite activated clotting time (ACT; Hemochron), and thrombin generation (TGA; Diapharma). Fresh blood was obtained from healthy volunteers under an IRB approved protocol (n=6 per group). Previous work from our lab (Jeske W, Thrombosis & Hemostasis Societies of North America, P57, 2018) identified a weaker potency of BH than PH when compared on an equigravimetric basis in pharmacopeial assays; however, equivalent potency could be obtained when BH was standardized against PH on a unitage basis. In this study, heparins were studied on both a gravimetric and a unitage basis for a comprehensive evaluation. The potency of the BH was determined by pharmacopeial compliant anti-Xa and anti-IIa chromogenic assays cross-referenced to the porcine USP Heparin Reference Standard.Results: All results are depicted in Table 1. For the TEG, the R value, time of latency from start to initial fibrin formation, and the K value, time to achieve a defined clot strength due to thrombin generation and platelet activation, bovine heparin and porcine heparin did not demonstrate a significant difference in anticoagulant activity. The TEG angle, a measure of the speed of fibrin build-up and cross-linking (clot strengthening or rate of clot formation), and the maximum amplitude (MA) value, a function of the maximum of fibrin and platelet binding representing the strongest point of fibrin clot formation, also revealed no significant difference between bovine and porcine heparin anticoagulant activity. For the ACT, at concentrations of 10 µg/mL and 25 µg/mL, there were no statistically significant differences between BH and PH. For the TGA, measuring the time delay until the initiation of thrombin generation following tissue factor (TF) activation, the highest amount of thrombin generated after TF activation, and the total amount of thrombin generated after TF activation (AUC), showed a trend that PH was more potent than BH, but wide variation in the results did not allow for statistical differences to be identified.Conclusion: The results of this investigation demonstrate that bovine heparin produces an equivalent anticoagulant activity as porcine heparin in the TEG, ACT, and TGA assay systems. The equivalent ACT results, in particular, were unexpected since gravimetric amounts of heparins were evaluated. As for all heparins, standardization of bovine heparin in accordance with the process used for porcine heparin will assure equivalent anticoagulant activity among bovine and porcine heparins in whole blood, plasma-based, and pharmacopeial assays. This study further demonstrates that the current assays used to monitor porcine heparin can be similarly used to monitor bovine heparin. [Display omitted] DisclosuresWalenga:Eurofarma: Research Funding.

Nanofiltration to remove microparticles and decrease the thrombogenicity of plasma: in vitro feasibility assessment

As plasma contains procoagulant microparticles (MPs), removing MPs by 75-nm nanofiltration may decrease plasma in vitro thrombogenicity while maintaining the hemostatic activity from coagulation factors. We defined conditions to nanofilter leukoreduced plasma on a 75-nm hollow-fiber membrane filter. Plasma quality was assessed by coagulation, immunochemical, and electrophoretic assays. MP removal was evaluated by biophysical (flow cytometry, dynamic light scattering, nanoparticle tracking analysis, and tunable resistive pulse sensing) and functional (thrombin generation assay [TGA; Technothrombin], prothrombinase [Zymuphen MP-activity], tissue factor [Zymuphen MP-TF], and procoagulant phospholipid-dependent clotting time [STA-Procoag-PPL] assays) methods. Spiking experiments using platelet MPs were performed to determine extent of removal by nanofiltration. Freshly collected leukoreduced, but not previously frozen, plasma could be readily nanofiltered on a 0.01-m2 75-nm nanofilter under conditions preserving protein and lipoprotein profile, coagulation factor content, and global coagulation activity (prothrombin time, activated partial thromboplastin time). Biophysical methods confirmed an extensive removal of MPs during nanofiltration. All functional assays indicated a marked reduction of plasma in vitro thrombogenicity. There was no thrombin generation in nanofiltered plasma tested by TGA assay with "RC-low phospholipid concentration" reagent, while it was similar to that of starting and leukoreduced plasma samples when using "RC-high phospholipid concentration" reagent. More than 9 log of MPs were removed by nanofiltration. Nanofiltration of 75 nm efficiently removes MPs and decreases in vitro thrombogenicity of plasma without affecting the protein content or the hemostatic activity of coagulation factors. Studies are needed to evaluate the impact of MP removal on in vivo thrombogenic risks and hemostatic efficacy.

Thrombin generation and low-molecular-weight heparin prophylaxis in pregnant women with thrombophilia.

Pregnancy is associated with increased risk of venous thromboembolism, especially in the presence of thrombophilia. However, there is no consensus on the optimal approach for thromboprophylaxis in this population. Recent evidence suggests that thrombin generation correlates with the overall procoagulant state of the plasma. Our aim was to evaluate thrombin generation in a prospective cohort of thrombophilic pregnant women, and investigate the effectiveness of low-molecular-weight heparin (LMWH) prophylaxis in pregnancy. Women with severe (n=8), mild (n=47) and no (n=15) thrombophilia were followed throughout their pregnancies. Thrombin generation was evaluated in each trimester as well as five days and eight weeks postpartum (as a reference category). In women undergoing LMWH prophylaxis, thrombin generation and anti-Factor-Xa activity were measured just before and 4 hours after administration (peak effect). Thrombin generation was determined using Technothrombin TGA assay system. For the analysis, median peak thrombin and endogenous thrombin potential were used. Peak thrombin and endogenous thrombin potential were increased during pregnancy compared to the non-pregnant state with the highest results in the severe thrombophilia group. In women receiving LMWH prophylaxis a decrease was observed in thrombin generation at peak effect but over the progression of pregnancy the extent of this decrease reduced in a stepwise fashion. Our results show that thrombin generation demonstrates the hypercoagulable state in thrombophilic pregnancies. In addition, we found the effect of LMWH prophylaxis to progressively decrease with advancing stages of pregnancy.

Thrombin Generation and Low Molecular Weight Heparin Prophylaxis In Pregnant Women With Thrombophilia

IntroductionPregnancy is associated with an increased risk of venous thromboembolism (VTE) which is even more pronounced in the presence of inherited thrombophilia. Despite the well-established relation between thrombophilic pregnancies and VTE, there is no consensus on the optimal approach for thromboprophylaxis in this population. There is growing evidence that thrombin generation correlates with the overall procoagulant state of the plasma leading to elevated thrombosis risk. The aim of this study was to evaluate thrombin generation over the course of pregnancy in women with inherited thrombophilia compared to healthy pregnant women. We also aimed to investigate the effectiveness of low molecular weight heparin (LMWH) prophylaxis in pregnancy by measuring thrombin generation and anti-Factor-Xa activity. MethodsIn this cohort study women with severe (n=8) and mild (n=47) thrombophilia were followed throughout their pregnancies. In addition, healthy pregnant women (n=15) were recruited as control subjects. Thrombin generation was evaluated in each trimester as well as 5 days after delivery and 8 weeks postpartum (baseline). In order to assess the effect of LMWH therapy at each stage of the pregnancy, thrombin generation and anti-Factor-Xa activity were measured just before and 4 hours after administration of the drug (peak and trough effect). Thrombin generation was determined using Technothrombin TGA assay system, and the results were evaluated with the provided software. For analysis, the median peak thrombin and endogenous thrombin potential were determined. The anti-Factor-Xa activity was measured by a commercially available chromogenic assay. ResultsIn all the 3 study groups, both peak thrombin and endogenous thrombin potential were increased over the course of pregnancy compared to the non-pregnant state. Peak thrombin and endogenous thrombin potential were higher in the severe thrombophilia group than in the other 2 groups in every trimester and also after delivery. There was no distinct difference in thrombin generation between the mild group and the controls. In women undergoing LMWH prophylaxis a decrease was observed in both the peak thrombin and endogenous thrombin potential after the drug was administered. Over the course of pregnancy the extent of this decrease reduced in a stepwise fashion. The difference between the 2 measurements (before and after LMWH) was smallest in the third trimester and increased again after delivery. Anti-Factor-Xa activity appeared to be enhanced at the peak effect of LMWH. However, the difference between the anti-Factor-Xa activity before and after LMWH administration remained unchanged over the progression of pregnancy. None of the women suffered from VTE or had any bleeding complications during the observation period. ConclusionsOur results show that thrombin generation is increased during pregnancy, with higher levels in women with severe thrombophilic defects. In addition, our data demonstrate a decrease in the effect of LMWH with advancing stages of pregnancy. Until now no studies evaluated prospectively the effect of LMWH prophylaxis on thrombin generation over pregnancy. Our findings contribute to the ongoing debate about the necessity and intensity of thromboprophylaxis during pregnancy, providing new information about the coagulable state of mild and severe thrombophilic pregnant women. The decreasing antithrombotic effect of LMWH over pregnancy suggests a need for dose adjustment in late gestational weeks. Furthermore, our results imply that thrombin generation as a global coagulation test may demonstrate more sensitively the effect of LMWH than the anti-Factor-Xa activity assay. Disclosures:No relevant conflicts of interest to declare.

Enhanced thrombin generation in women with a history of oral contraception-related venous thrombosis

In women who suffer venous thrombosis (VT) during oral contraceptive (OC) use, a transient risk factor (OC) is removed during the acute event, while most co-existing forms of thrombophilia persist and presumably continue to maintain hypercoagulability. The aim of this study was to establish if hypercoagulability persists long after OC-related VT and if it could be attributed to thrombophilia. 60 women (age 33.0 ± 8.5 years) were investigated 5 - 64 (median 33) months after OC-related VT (patients) and compared to 63 apparently healthy women (controls). All women were tested for thrombophilia, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, P-selectin and C-reactive protein. Thrombin generation was measured by Technothrombin® TGA assay. Overall haemostasis potential (OHP) assay with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) as supplementary parameters were measured by repeated fibrin formation and degradation registration. In patients increased endogenous thrombin potential (4,205 ± 440 nM x min vs 4,015 ± 421 nM x min, p=0.017), increased OCP (22.6 ± 4.6 Abs-sum vs 20.8 ± 4.1 Abs-sum, p=0.025), shorter APTT (30.9 ± 3.8s vs 33.4 ± 3.6s, p<0.001) and lower antithrombin activity (99, 93-105% vs 104, 100-109%, p<0.05) were observed. Thrombophilia was observed in 22/60 (36%) patients and in 5/63 (7.9%, p<0.001) controls. The only significant difference between thrombophilic and non-thrombophilic patients was higher soluble P-selectin in the former subgroup (22, 20-33 μg/L vs 17, 12-22 μg/L, p=0.012). In women with a history of OC-related VT persistent hypercoagulability was observed, which, however was not augmented by the presence of thrombophilia.

Abstract 051: Common Single Nucleotide Polymorphisms in the Coagulation Factor XII Gene (F12) are Associated with Endogenous Thrombin Potential via In Situ Activation of the Intrinsic System of Coagulation: The Cardiovascular Health Study

Background: Currently, a majority of evidence suggests that the tissue factor-mediated extrinsic coagulation pathway is most important in generating thrombin in vivo , although accumulating evidence implicates the coagulation factor XII/factor XI-mediated intrinsic system as well. The endogenous thrombin potential (ETP), a measure of thrombin generation, is gaining importance in assessing risk of hemorrhage and thrombosis, but genetic determinants of ETP have not been investigated in population-based studies. Methods: Total ETP (ETP T ) was measured in citrated plasma by TechnoClone TGA assay in a random subset of Cardiovascular Health Study (CHS) participants ( n =1,000). Extrinsic pathway ETP (ETP EX ) was measured by the addition of anti-FXIa antibody to the assay; Intrinsic pathway ETP (ETP IN ) was calculated by subtracting ETP EX from ETP T . Genotyping was performed in the NHLBI’s Candidate-gene Association Resource (CARe) Study on 49,320 SNPs using Illumina’s custom IBCv2 genotyping array in 866 participants. Associations of SNPs with age- and sex-adjusted ETP phenotypes were evaluated by linear regression using PLINK; results were considered statistically significant if p≤ 2x 10 -6 . Results: Two SNPs on the IBC array were significantly associated with lower ETP T : rs2545801 (β=-45.87 nM ± 7.3 nM; p=5.7x10 -10 ; minor allele frequency (MAF) =0.24), and rs1801020 (β=-45.76 nM ± 7.3 nM; p=7.4x10 -10 ; MAF= 0.23). Both SNPs are located in the coagulation factor XII gene ( F12 ); rs2545801is located in a 5’ region upstream of the transcriptional start site, and rs1801020 in a 5’ untranslated region; the SNPs are in strong linkage disequilibrium (LD) (r 2 = 0.92). Each SNP had a significant effect on ETP thrombin peak height. The mean (SD) thrombin peak heights were 408 nM (152nM) vs.523 nM (123 nM) and 404 nM (147 nM) vs. 523 nM (123 nM) for those homozygous for the minor and major alleles of rs2545801 and rs1801020, respectively. Addition of corn trypsin inhibitor (CTI; an inhibitor of activated FXII (FXIIa)) had no effect on ETP, suggesting that the F12 SNPs may affect the FXII-dependent generation of activated FXI (FXIa) in vivo. Therefore, we inhibited FXIa by the addition of anti-FXIa antibody, which resulted in a large and variable effect on ETP thrombin peak height. Consistent with these observations, analyses of SNPs associated with ETP IN revealed increased significance of the p-values for the F12 SNPs (p=1.40 x 10 -15 and p=1.60 x 10 -14 for rs1801020 and rs254580, respectively). Conclusion: These data support the importance of F12 SNPs on thrombin generation, and suggest that these SNPs affect the amount of FXIa generated in vivo . Additional work is needed to confirm this hypothesis, and to examine relationships of these biomarkers and SNPs with thrombotic diseases.

Low Concentrations of rhFVIIa or FEIBA Significantly and Rapidly Reverse the Anticoagulant Effects of Supratherapeutic Edoxaban

Abstract 1252 Introduction:Edoxaban is an oral, direct factor Xa (FXa) inhibitor currently in phase 3 clinical development for stroke prevention in nonvalvular atrial fibrillation (AF) and the treatment and secondary prevention of venous thromboembolism (VTE). Guidance for the emergency management of serious bleeding and strategies for reversing the anticoagulant effects edoxaban are needed. Both recombinant human FVIIa (rhFVIIa; NovoSeven®) and a human plasma fraction containing nonactivated forms of FII, FIX, and FX/proteins C and S and activated FVII (FEIBA; factor eight inhibitor bypassing activity) have been identified as potential reversal agents for edoxaban. The minimum effective doses and the time course required for either rhFVIIa or FEIBA to reverse supratherapeutic concentrations of edoxaban in an anticoagulated patient have yet to be established. This ex vivo study was conducted to assess the effects and establish the time course of various concentrations of rhFVIIa and FEIBA on the reversal of edoxaban’s anticoagulant effects. Methods:Edoxaban at supratherapeutic concentrations of 500 and 1000 ng/mL (therapeutic level is ∼250 ng/mL for Cmax of a 60-mg dose) was added to fresh blood drawn from healthy subjects; control samples were diluent alone. After 60 min of incubation, rhFVIIa, FEIBA, or control was added to the samples. rhFVIIa was added to produce final concentrations of 0.8 and 1.8 μg/mL, which correspond to known maximal observed human plasma concentrations at doses of 40 and 90 μg/kg. FEIBA was added to produce concentrations of 0.75 and 1.4 U/mL, which correspond to human plasma concentrations at the therapeutic doses of FEIBA of 50 to 100 U/kg. In order to determine reversal of anticoagulant effects over time, coagulation assays (PT, aPTT, anti-Xa, intrinsic FXa activity, thrombin generation assay [TGA], D-dimer, and FVIIa activity) were measured at times 0, 0.25, 0.50, 1, 2, and 4 h after incubation with rhFVIIa, FEIBA, or control. Results:In edoxaban alone and control samples, PT, aPTT, and anti-Xa results reflected previously established anticoagulant activity. Both 500 and 1000 ng/mL of edoxaban completely inhibited intrinsic FXa and thrombin generation as measured by TGA (peak). Measures of FVIIa activity indicated that the concentrations of rhFVIIa utilized were consistent with previously reported responses. With the exception of thrombin levels, reversal of edoxaban anticoagulant activity by both rhFVIIa and FEIBA was observed for PT, aPTT, and anti-Xa beginning at 0.25 h and was maintained across the experimental period. At 1000 ng/mL edoxaban, the observed decrease at 0.25 h post-reversal of anticoagulant activity was 82% for anti-Xa, 72% for PT, and 58% for aPTT. Compared with baseline, the maximum reversal of intrinsic FXa activity for edoxaban 500 ng/mL was ∼30% and for 1000 ng/mL was 15% across all concentrations of rhFVIIa and FEIBA. The TGA assay indicated both rhFVIIa and FEIBA reversed ∼45% and 20% of the effect of edoxaban at 500 ng/mL and 1000 ng/mL, respectively, 4 h after adding the reversal agents. With the exception of 1 baseline sample, levels of D-dimer did not show significant changes with the addition of edoxaban or with its subsequent reversal by either rhFVIIa or FEIBA. Conclusion:Low therapeutic concentrations of rhFVIIa and FEIBA showed significant and rapid reversal of supratherapeutic concentrations of the anticoagulant activity induced by edoxaban based on PT, aPTT, and anti-Xa activity. Disclosures:Halim:Daiichi Sankyo Pharma Development: Employment. Li:Daiichi Sankyo Pharma Development: Employment. Stein:Daiichi Sankyo: Consultancy, Research Funding; AACC: Consultancy, Honoraria, Research Funding; Abbott: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; FDA: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; ISIS: Consultancy, Honoraria, Research Funding; Merck & Co: Consultancy, Honoraria, Research Funding; National Lipid Association: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Reliant: Consultancy, Honoraria, Research Funding; Regeneron: Consultancy, Honoraria, Research Funding; SanofiAventis: Consultancy, Honoraria, Research Funding; Schering-Plough: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Wyeth: Consultancy, Honoraria, Research Funding. Mendell:Daiichi Sankyo Pharma Development: Employment.

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Abstract 4324 Introduction:Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods:Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results:A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions:These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures:Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

Low Generation Polypropylenimine Dendrimer Graft β-cyclodextrin: An Efficient Vector for Gene Delivery System

The purpose of this study was to evaluate the characterization of a new cationic vector. Low generation polypropylenimine (DAB-8) was conjugated to beta-cyclodextrin (beta-CyD), and the chemical characters of the vector were investigated and confirmed by 1H NMR, FT-IR, TGA, TEM, particle size, and zeta potential assay. The biological property was identified by MTT assay and gene transfer efficiency was performed in cell lines. The results showed that the new vector had low cytotoxicity and high transfection efficiency in vitro. This suggested that beta-CyD-DAB-8 has a potential ability to act as a non-viral vector in gene delivery.

Synthesis and application as polymer electrolyte of hyperbranched copolyethers derived from cationic ring-opening polymerization of 3-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}methyl- and 3-hydroxymethyl-3′-methyloxetane

A kind of novel hyperbranched copolyethers intending for the solid polymer electrolyte was synthesized via the cationic ring-opening polymerization of 3-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}methyl-3′-methyloxetane (MEMO) and 3-hydroxymethyl-3′-methyloxetane (HMO) in the presence of BF 3·Et 2O as an initiator. Herein HMO was employed to create the hyperbranched structure, whereas MEMO was responsible for the ionic transportation of the resulting copolymers. The terminal structure featured by a cyclic fragment was definitely detected by MALDI-TOF measurement. The degree of branching of the copolymers was calculated by means of 13C NMR spectra. The DSC analysis implied that they hold the excellent segment motion performance and perfectly amorphous state beneficial for the ionic transportation. The ionic conductivity measurements showed that the sample HMO 30 reaches a maximum ionic conductivity of 8.0 × 10 −5 S/cm at 30 °C and 7.4 × 10 −4 S/cm at 80 °C, respectively, after doping with lithium salt LiTFSI. Moreover, the TGA assay exhibited that these hyperbranched copolymers possess the higher thermostability as compared with their liquid counterparts.