Platelet depletion enhances lethal, hemorrhagic and myotoxic activities of Bothrops asper snake venom in a murine model.

Platelets play key roles in hemostasis, inflammation, immune response, and tissue repair. Although it is known that viperid snake venoms induce thrombocytopenia and platelet hypoaggregation, the roles of these effects in the overall outcome of envenoming are poorly known. This study aimed to assess the effect of platelet depletion on several toxic activities induced by the venom of the Central American viperid snake Bothrops asper in a mouse model. A profound thrombocytopenia was induced in mice by the administration of aspercetin, a C-type lectin-like protein that induces platelet agglutination and drop in platelet counts, while a control group was treated with saline solution instead. Upon envenoming, animals rendered thrombocytopenic developed a higher extent of local and systemic hemorrhage and local myonecrosis, as compared to control envenomed mice. In addition, the median lethal dose (LD50), determined by the intraperitoneal route, was significantly lower in thrombocytopenic mice, underscoring a higher toxicity of venom in these conditions. No difference in the value of LD50 between the two groups was observed when using the intravenous route of injection, and no difference was observed in the magnitude and time-course of footpad edema. Skeletal muscle regeneration was assessed 14 days after venom injection in muscle. Both experimental groups showed a similarly poor regeneration, suggesting that platelets do not play a key role in the regenerative process in these experimental conditions. Results indicate that depletion of platelets increases hemorrhagic and myotoxic effects, as well as overall toxicity, of B. asper venom, implying that platelets play a protective hemostatic role in this model of envenoming.

Highly resilient, biocompatible, and antibacterial carbon nanotube/hydroxybutyl chitosan sponge dressing for rapid and effective hemostasis.

Uncontrolled hemorrhage is the leading cause of trauma death. The development of safe and efficient hemostatic agents that can rapidly and effectively control bleeding is of great significance to rescue the injured. However, the mechanical, absorptive, and antibacterial properties of conventional two-dimensional hemostatic agents are not satisfactory. Herein, a series of effective three-dimensional hemostatic dressings (JWCNT/HBC sponges) are developed by chemical modification of joint-welded carbon nanotube (JWCNT) sponges with hydroxybutyl chitosan (HBC) for hemorrhage hemostasis. The JWCNT/HBC sponges exhibit high elasticity, porous structure, and suitable blood-absorption and blood-maintaining performance. Moreover, the introduction of HBC endows the JWCNT/HBC sponges with favorable blood compatibility and good antibacterial activity. The sponge treated with 0.5% HBC (JWCNT/0.5%HBC sponge) displays better antiseptic capability, faster blood clotting ability in vitro and shorter hemostasis time in vivo than the commercial gelatin sponge. The JWCNT/HBC sponges combine the advantages of JWCNT sponges and HBC in the adhesion and activation of platelets and red blood cells, thus becoming a good medical material for trauma hemostasis.

Associations between welding fume exposure and blood hemostatic parameters among workers exposed to welding fumes in confined space in Chonburi, Thailand.

BACKGROUND: Occupational welding fumes contain varieties of toxic metal particles and may affect cardiovascular system like the Particulate Matters (PM). Few studies have focused on the effects of toxic metals on the hemodynamic balance; however, the reporting results were not consistent. This study aimed to investigate the association between toxic metals exposure (Chromium (Cr), Manganese (Mn) and Lead (Pb)) and blood hemostatic parameters status after a 3-week exposure cessation among workers exposed to welding fumes. METHODOLOGY: Structured interviews and biological samplings were conducted for 86 male workers without a history of Anemia and Cardiovascular diseases (CVDs) and working in a confined space to construct crude oil tanks. Metal levels of Cr, Mn and Pb in urine were measured during the working days using Inductively Coupled Plasma Mass Spectrometer (ICP-MS) method. The concentrations of hemostatic proteins in blood (White blood cell counts (WBC), Lymphocytes, Monocyte, Eosinophil, Neutrophil, Hematocrit (Hct) were assessed after a 3 weeks exposure cessation. Workers were divided into groups based on occupation type (welder group and non-welder group), and based on metal levels (high and low exposure groups) for comparison. Linear regression models were used to explore the association between metal exposure and multiple blood hemostatic parameters adjusted for age, Body Mass Index (BMI), and smoking status. RESULTS: Urine Mn and Cr level of the welder group was significantly higher than the non-welder group (Mn: 0.96 VS 0.22 ug/g creatinine, p < 0.001; Cr: 0.63 VS 0.22 ug/g creatinine, p < 0.01). The mean value of Hct in the welder group was 44.58 ± 2.84 vol%, significantly higher than the non-welder group (43.07 ± 3.31 vol%, p = 0.026). The median value of WBC in the high Mn-exposed group (6.93 ± 1.59 X 106 Cell/ml) was significantly lower than the low Mn-exposed group (7.90 ± 2.13 X 106 Cell/ml, p = 0.018). The linear regression analyses showed that there was a significantly negative association between log transformed WBC value and the Mn exposure groups (high and low) after adjusting for age, BMI, and smoking status (ß = - 0.049, p = 0.045), but no significant result was found between WBC and occupation types (welder and non-welder) (p > 0.05). Multiple linear regression analysis also showed positive association between Hct and occupational types (welder and non-welders) (ß = 0.014, p = 0.055). The other hemostatic parameters were not different from controls when divided by occupation type or metal level groups. CONCLUSIONS: Our results showed that welders were exposed to about 3 to 4 times higher Mn and Cr concentrations than non-welders. Moreover, one third of the non-welders were exposed to high-exposure groups of Mn and Cr metals. Regression models revealed a significant association of the WBC counts with the Mn exposure group. Therefore, we infer that Mn exposure may play a significant role on the blood hemostatic parameters of workers in the confined space. Hazard identification for non-welders should also be conducted in the confined space.

The effect of chemical and structural modifiers on the haemostatic process and cytotoxicity of the beta-chitin patch.

Beta-chitin patch has previously been proven to be an effective haemostat, but whether modifying the patch affects its efficacy and safety, remains unanswered. In this study, the patch was modified using polyethylene oxide, Pluronic-F127, calcium, increased thickness or polyphosphate, and their effect on the process of haemostasis and cytotoxicity was tested and compared with standard-of-care, Surgicel and FloSeal. Whole blood collected from volunteers was applied to the patches to test their whole blood clotting and thrombin generation capacities, whilst platelet isolates were used to test their platelet aggregation ability. The fluid absorption capacity of the patches was tested using simulated body fluid. Cytotoxicity of the patches was tested using AlamarBlue assays and PC12 cells and the results were compared with the standard-of-care. In this study, beta-chitin patch modifications failed to improve its whole blood clotting, platelet aggregation and thrombin generation capacity. Compared to non-modified patch, modifications with polyethylene oxide or calcium reduced platelet aggregation and thrombin generation capacity, while increasing the thickness or adding polyphosphate decreased platelet aggregation capacity. The cytotoxicity assays demonstrated that the beta-chitin patches were non-toxic to cells. In vivo research is required to evaluate the safety and efficacy of the beta-chitin patches in a clinical setting.

Inherent Antibacterial and Instant Swelling ε-Poly-Lysine/ Poly(ethylene glycol) Diglycidyl Ether Superabsorbent for Rapid Hemostasis and Bacterially Infected Wound Healing.

Severe traumatic bleeding control and wound-related anti-infection play a crucial role in saving lives and promoting wound healing for both the military and the clinic. In this contribution, an inherent antibacterial and instant swelling ε-poly-lysine/poly (ethylene glycol) diglycidyl ether (EPPE) superabsorbent was developed by a simple mild ring-opening reaction. The as-prepared EPPE1 displayed a porous structure and rough surface and exhibited instant water-triggered expansion with approximately 6300% swelling ratio in deionized water. Moreover, EPPE1 presented efficient pro-coagulation capacity by hemadsorption that can facilitate blood cell gathering and activation in vitro and exhibited a shorter in vivo hemostasis time than that of commercial gelatin sponge and CELOX in both rat tail amputation and noncompressible rat liver lethal defect model. Also, EPPE1 showed excellent antibacterial capacity, prominent biocompatibility, and great biodegradability. Additionally, EPPE1 significantly promotes in vivo wound healing in a full-thickness skin defect model due to its great hemostasis behavior and remarkable bactericidal performance. Hence, EPPE has great potential for serving as an extensively applied hemostatic agent under varied clinical conditions.

Gelatin-based adhesive hydrogel with self-healing, hemostasis, and electrical conductivity.

As a kind of natural protein derived material, gelatin has been widely used in the preparation of medical hydrogels due to its good biocompatibility, non-immunogenicity and the ability of promoting cell adhesion. Functionalization of gelatin-based hydrogels is a hot topic in research and its clinic application. Herein, a novel gelatin-based adhesive hydrogel was prepared via mussel-inspired chemistry. Gelatin was firstly functionalized by dopamine to form dopamine grafted gelatin (GelDA). After the mixture with 1,4-phenylenebisboronic acid and graphene oxide (GO), the GelDA/GO hydrogels were obtained by H2O2/HRP (horseradish peroxidase) catalytic system. Based on the self-healing and tissue adhesion of the hydrogels, the hemostatic property has been exhibited in the rat hepatic hemorrhage model. Additionally, the incorporation of GO endowed conductivity and enhanced the mechanical property of GelDA/GO hydrogels. The electromyography (EMG) signals of finger movement were successfully monitored by using hydrogel as the adhesive electrodes of EMG monitor. L929 cell experiments showed that the hydrogels had good cytocompatibility. The results indicated the potential application of GelDA/GO hydrogels in tissue adhesives, wound dressings, and wearable devices.

Rapid Hemostatic Biomaterial from a Natural Bath Sponge Skeleton.

Uncontrolled bleeding is the main cause of mortality from trauma. Collagen has been developed as an important hemostatic material due to its platelet affinity function. A bath sponge skeleton is rich in collagen, also known as spongin. To understand the hemostatic effect of spongin, spongin materials, SX, SFM and SR were prepared from the bath sponge Spongia officinalis, and hemostatic experiments were performed. The SX, SFM and SR were significantly better than the positive control, type I collagen, in shortening the whole blood clotting time in vitro and hemostasis upon rat tail amputation. In a hemostatic experiment of rabbit common carotid artery injury, the hemostatic time and 3 h survival rate of the SFM group were 3.00 ± 1.53 min and 100%, respectively, which are significantly better than those of the commercial hemostat CELOX-A (10.33 ± 1.37 min and 67%, respectively). Additionally, the SFM showed good coagulation effects in platelet-deficient blood and defibrinated blood, while also showing good biocompatibility. Through a variety of tests, we speculated that the hemostatic activity of the SFM is mainly caused by its hyperabsorbency, high affinity to platelets and high effective concentration. Overall, the SFM and spongin derivates could be potential hemostatic agents for uncontrolled bleeding and hemorrhagic diseases caused by deficiency or dysfunction of coagulation factors.

Phase 1b Study to Evaluate Safety, Tolerability, and Maximum Tolerated Dose of PF-05230907 for Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: This study aimed to determine the maximum tolerated dose and to evaluate the overall safety and tolerability of single doses of PF-05230907 in subjects with acute intracerebral hemorrhage. METHODS: Individuals presenting with intracerebral hemorrhage were enrolled in a phase 1, multicenter, open-label clinical trial. A Bayesian modified continual reassessment method design based on treatment-emergent thromboembolic or ischemic events was adopted. Sequential dosing, an external data monitoring committee, and prespecified stopping rules were incorporated as safeguards. RESULTS: Twenty-one subjects received PF-05230907. The mean (±SD) age in years and intracerebral hemorrhage volume in mL at baseline were 62 (±9) and 18 (±11), respectively. Two treatment-emergent thromboembolic or ischemic events occurred (deep vein thrombosis and cerebral ischemia), in the 30 µg/kg dose group. There were no other clear drug-related toxicities at dose levels ranging from 5 to 30 µg/kg. At the time of study termination, the maximum tolerated dose was estimated to be 24 µg/kg, with a mean fitted dose-toxicity estimate of 11.9% (95% CI, 1.2%-27.4%). CONCLUSIONS: Single doses of PF-05230907 appeared to be tolerated across a range of doses in the intracerebral hemorrhage population, with thrombotic events observed only at the highest dose level tested. Recruitment within the recommended therapeutic window of opportunity remains a challenge. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02687191.

Comparison of bone wax and chitosan usage on post-sternotomy bone healing.

BACKGROUND: Sternotomy is a standard approach performed in almost every surgical procedure on the heart and mediastinum. Effective hemostasis of the sternum is required to keep the operative field dry, avoid excessive blood transfusions during surgery, and prevent reoperation due to massive postoperative bleeding, which can further increase morbidity and mortality in patients. Bone wax is a mechanical hemostat commonly used after sternotomy and has been known to affect bone healing, trigger chronic inflammatory reactions, and increase the rate of infection. The application of chitosan, which has intrinsic hemostat ability, as hemostatic material is believed to improve bone healing following sternotomy. This study aimed to compare the effectiveness of bone wax and chitosan on bone healing after sternotomy. METHODS: Median sternotomies were performed on 2 groups of New Zealand White rabbits. Each group of 16 animals received either bone wax or chitosan powder as hemostatic material. The degree of bone healing, the number of foreign-body giant cells, and the number of osteoblasts were evaluated after 6 weeks. RESULTS: Radiographs showed that significantly more animals in the chitosan group had total sternal healing (p = 0.033). Histopathology revealed that the number of foreign-body giant cells was significantly less (p = 0.036) and the number of osteoblasts was significantly greater (p < 0.0001) in the group of animals that received chitosan. CONCLUSION: The use of chitosan as hemostatic material can promote better bone healing compared to bone wax.

A novel injectable starch-based tissue adhesive for hemostasis.

Hemorrhage remains one of the direct causes of high mortality. The development of ideal hemostatic materials with sound ability to deal with severe wound is urgent needed. Although starch-based hemostatic powder has been widely used, hydrous physiological environments severely hamper its binding to the target tissue, thereby limiting the effectiveness in hemostasis. Herein, inspired by mussel adhesive protein, a novel injectable tissue-adhesive hydrogel (St-Dopa hydrogel) composed of starch, succinic anhydride and dopamine was developed in situ by enzymatic crosslinking. The results show that St-Dopa hydrogels were intimately integrated with biological tissue and formed robust barriers to reduce blood loss. St-Dopa hydrogels exhibited superior capacity for in vitro and in vivo hemostasis as compared with chitin hydrogels. In addition to the ease of operation, St-Dopa hydrogels exhibited rapid sol-gel transition, porous microscopic morphology, good swelling ratio and biodegradability, tissue-like elastomeric mechanical properties and excellent cyto/hemo-compatibility. These results suggest that this newly developed St-Dopa hydrogel is a promising biological adhesive and hemostatic material.

Antibacterial and Hemostatic Thiol-Modified Chitosan-Immobilized AgNPs Composite Sponges.

Wound bleeding and infection are two of the major threats to patients' lives, but developing safe materials with high hemostasis efficiency and antibacterial activity remains a major challenge. Silver nanoparticles (AgNPs) are suitable as antibacterial agents in the hemostatic process, but the application is hampered because of easy accumulation of toxicity. Herein, thiol-modified chitosan (TMC) was prepared by modifying with mercaptosuccinic acid and then was used to immobilize AgNPs to obtain composite sponges (TMC/AgNPs) for stemming the bleeding and preventing infection. TMC/AgNPs sponges had complex interlaced tubular porous structure with high porosity (99.42%), indicating high absorption. TMC had high immobilization efficiency for AgNPs-the release rate of AgNPs was 14.35% after 14 days-but the TMC/AgNPs sponge still had excellent antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In vitro and in vivo experiments confirm that the TMC/AgNPs sponge had fast and efficient hemostatic performance in comparison with the PVF sponge, and its possible mechanism was the synergistic effect of high blood absorption capacity and the interaction between amino, sulfydryl, and blood cells. Furthermore, the TMC/AgNPs sponge can promote wound healing by preventing wound infection, while the PVF sponge cannot. More importantly, the sponges had good safety due to the immobilization of TMC for AgNPs.

Analysing the blood-stemming effect of Ankaferd Blood Stopper in medulla spinalis surgery

Background/aim: The aim of this study was to investigate the possible toxicity of the Ankaferd Blood Stopper (ABS) on the neural system. Materials and methods: Thirty Sprague Dawley rats were randomized into ABS (n: 15) and control (n: 15) groups. Following the anaesthetic induction, total laminectomy was performed to the lower thoracic, and upper lumbar areas in both groups and medulla spinalis was exposed. Two myelotomies were performed on the medulla spinalis. One millilitre ABS was applied to the incision site in the ABS group, and one millilitre 0.9% saline solution was applied in the control group. Rats were observed for 15 days regarding general behaviour, neurological signs, mobility, and signs of infection. Sixteen days later, all rats were decapitated under anaesthesia. Medulla spinalis was removed en bloc from all rats and was stained with Heamatoxylin & Eosin and luxol fast blue. Results: There was no significant difference between the ABS group and the control group regarding oedema, gliosis, the intensity of inflammatory cells, the presence of neuronal degeneration, neuron counts, and myelin degeneration. Conclusion: No clinical or histopathological evidence for the neurotoxic effect of the ABS was observed in the present study. Our findings might precipitate the use of ABS on human subjects regarding medulla spinalis surgery.

Hydroxybutyl chitosan/diatom-biosilica composite sponge for hemorrhage control.

Effective bleeding control is critical first step in current civilian and military trauma treatment, however commercially available hemostatic materials are difficult to achieve expected effects. In this study, a composite sponge (H-D) based on hydroxybutyl chitosan (HBC) and diatom-biosilica (DB) was designed for hemorrhage control. H-D exhibited hierarchical porous structure, favorable biocompatibility (hemolysis ratio < 5 %, no cytotoxicity), along with high and fast fluid absorbability (11-16 times than that of weight), given effective hemostasis effect (clotting time shortened by 70 % than that of control). In vitro coagulation tests demonstrated that H-D could provide strong interface effect to induce erythrocyte absorption and aggregation, as well as activating the intrinsic coagulation pathway and thus accelerated blood coagulation. These results proved that H-D composite sponge has great potential for hemorrhage control.

Fabricating antimicrobial peptide-immobilized starch sponges for hemorrhage control and antibacterial treatment.

It is important to control immediate hemorrhage and prevent infection simultaneously in the wound management. However, most of hemostatic materials are associated with low efficiency of hemostasis, poor biocompatibility and lack of antimicrobial properties. A kind of starch-based macroporous sponges (KR-Sps) immobilized covalently with antimicrobial peptide KR12 using highly efficient thiol-ene photo click reaction were developed. The physical properties of these sponges could be fine-tuned by varying the ratio of modified starch/HS-PEG-SH and the polymer concentration. The in vitro and vivo results demonstrated that KR-Sps induced thrombosis, shortened clotting time and reduced the blood loss at bleeding site. Besides, KR12 immobilized sponge exhibited inherent antimicrobial properties against Gram (+) and (-) bacteria and methicillin-resistant Staphylococcus aureus (MRSA), which could maintain at least 5 days. Therefore, KR-Sps were believed to be an excellent candidate as hemostatic and antimicrobial product for the intraoperative wound management.

In situ synthesis of poly (γ- glutamic acid)/alginate/AgNP composite microspheres with antibacterial and hemostatic properties.

In the present work, a poly(γ-glutamic acid)/alginate/silver nanoparticle (PGA/Alg/AgNP) composite microsphere with excellent antibacterial and hemostatic properties was prepared by the in situ UV reduction and emulsion internal gelation method, and its potential application for antibacterial hemostatic dressing was explored. Well dispersed AgNPs were in situ synthesized by a UV reduction method with alginate as stabilizer and reductant. The AgNPs showed excellent antibacterial activities against both gram-negative and gram-positive bacteria. Additionally, the AgNPs prepared by the in-situ UV reduction exhibited better biocompatibility and antibacterial effects than those prepared by the conventional chemical reduction method. PGA/Alg/AgNP composite microspheres were then prepared with the AgNPs by an emulsion internal gelation method. Such microspheres were found to be a porous and hollow network with pH-sensitive swelling properties and excellent hemostatic performance, indicating its application potentials as an advanced antibacterial hemostatic material.

Administration of recombinant FVIIa (rFVIIa) to concizumab-dosed monkeys is safe, and concizumab does not affect the potency of rFVIIa in hemophilic rabbits.

Essentials Hemophilia patients on concizumab prophylaxis may need rFVIIa to treat breakthrough bleeds. Effect and safety of concizumab + rFVIIa were tested in vitro and in vivo. Concizumab + rFVIIa had no additive effects on bleeding in hemophilic rabbits. High steady-state levels of concizumab did not affect the safety of rFVIIa in cynomolgus monkeys. SUMMARY: Background Concizumab is a monoclonal antibody (mAb) against tissue factor pathway inhibitor (TFPI), currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A/B with and without inhibitors. In patients with inhibitors, the treatment choice for breakthrough bleeding will comprise bypassing agents, e.g. activated recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrates. Objectives To explore the effect and safety of concizumab and rFVIIa when they are simultaneously present. Methods Human blood made hemophilic with a FVIII antibody was spiked with increasing concentrations of concizumab, rFVIIa, or concizumab and rFVIIa in combination, and this was followed by thrombin generation test or thromboelastography. Blood loss in hemophilic rabbits was measured when concizumab, rFVIIa or concizumab + rFVIIa was administered either before or during cuticle bleeding. In a safety study, cynomolgus monkeys were exposed to high steady-state concizumab concentrations and given three doses of rFVIIa, and then subjected to full necropsy and histopathological examination. Results In human blood, concizumab + rFVIIa had more pronounced procoagulant effects under hemophilic conditions than the sum of individual responses. In contrast, concizumab + rFVIIa had no additional effects on blood loss in hemophilic rabbits as compared with rFVIIa or concizumab alone. In cynomolgus monkeys, the macroscopic and microscopic pathological examinations revealed no thrombi or other signs of excessive coagulation activation. Both rFVIIa and concizumab caused increases in thrombin-antithrombin and D-dimer concentrations; this effect tended to be additive with concomitant administration. Conclusions Concizumab did not affect the potency or safety of rFVIIa in vivo. These results support a clinical evaluation of rFVIIa at standard dose (90 µg kg-1 ) to treat breakthrough bleeds in concizumab clinical trials.

Biodegradable N, O-carboxymethyl chitosan/oxidized regenerated cellulose composite gauze as a barrier for preventing postoperative adhesion.

Tissue adhesion is one of the most common complications after surgery (especially after abdominal surgery), causing notable influences after the damaged tissue has healed. A physical barrier placed between the wound site and the adjacent tissues is a convenient and highly effective technique to minimize or prevent abdominal adhesions. In this work, the N, O-carboxymethyl chitosan/oxidized regenerated cellulose (N, O-CS/ORC) composite gauze was prepared. The N, O-CS/ORC composite gauze is degradable; in addition, the gauze exhibits excellent antimicrobial functionality against S. aureus and E. coli bacteria. Moreover, the notable hemostatic efficacy of the N, O-CS/ORC composite gauze was confirmed in rabbit livers/ears as models. The results showed that the N, O-CS/ORC composite gauze is nontoxic toward normal cells and can restrain the adhesion of fibroblast cells, thereby indicating its potential use in preventing tissue adhesion. In addition, the rat models for abdominal defect-cecum abrasion were used to evaluate the efficacy of N, O-CS/ORC composite gauze in preventing tissue adhesions after surgery. The results indicated that the N, O-CS/ORC composite gauze can significantly prevent postsurgical peritoneal adhesions. Finally, the potential anti-adhesion mechanism of the N, O-CS/ORC composite gauze, which may attribute to the combination of barrier function and instinct activity of N, O-CS and ORC, was investigated.

Oxidative and haemostatic effects of copper, manganese and mercury, alone and in combination at physiologically relevant levels: An ex vivo study.

Water contamination with metals due to anthropogenic activity is increasing and subsequent exposure increases the risk of associated toxicity. Exposure is not limited to a single metal but usually involves mixtures of different metals at different concentrations. Little is known about the contribution of this type of exposure, in humans, to the development of non-communicable diseases such as cardiovascular disease, and an increased risk to thrombosis. The World Health Organization has established limits for metal levels in drinking water and this includes levels for copper (Cu), manganese (Mn) and mercury (Hg). In this study, at 100X these limits, the ability of the metals' oxidative effects as catalysts of the Fenton reaction and/or ability to bind glutathione (GSH) were determined. The haemostatic effects of these metals, alone and in combination, at the World Health Organization limit were then evaluated. The ultrastructural and viscoelastic alterations of exposed ex vivo whole blood were also evaluated using scanning electron microscopy and thromboelastography® (TEG), respectively. Cu, alone and in combination with Mn and/or Hg, induced hydroxyl radical formation and reduced GSH levels. Ex vivo exposure caused deformation of erythrocytes and accelerated platelet activation especially for Cu, alone and in combination, with Mn. Reduction in the lysis potential of the clot was also observed for all combinations, especially Cu in combination with Hg as well as Mn alone. Although the TEG findings were not statistically significant, the trends indicate that the exposure to these metals, alone and in combination, adversely affects thrombus formation in ex vivo blood, thereby potentially increasing the risk in exposed individuals for thrombosis.

Fabrication of photo-crosslinkable glycol chitosan hydrogel as a tissue adhesive.

In this work, an in situ gelling system composed of glycol chitosan (GC) was fabricated and evaluated regarding its tissue-adhesive, anti-bacterial and hemostatic properties. GC conjugated with 3-(4-hydroxyphenyl) propionic acid gelled immediately after illumination with blue light in the presence of ruthenium complex. The phenolic GC hydrogel was investigated regarding its mechanical property, hydration, degradation rate, cytotoxicity, tissue adhesiveness, and hemostatic ability. The hydrogel was shown to glue two pieces of tissues tightly in an egg-membrane model. The antibiotic-incorporated hydrogel killed bacteria effectively. When the hydrogel was applied to a wound in a mouse liver model, bleeding was reduced quickly and greatly. All the promising results show that the photo-chemically crosslinkable GC hydrogel could be used as a tissue adhesive, controlled drug release, and a hemostat.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.