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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 (LD 50 ), 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 LD 50 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

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.

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 (β = - 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) (β = 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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2021. The Author(s).)

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.

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 H 2 O 2 /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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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., Competing Interests: Authors declare no conflict of interest., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

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., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2018. Published by Elsevier Ltd.)

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., (© 2019 International Society on Thrombosis and Haemostasis.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Bletilla striata (Thunb.) Reichb. f. (Orchidaceae), also known as Hyacinth Orchid and Baiji (Simplified Chinese: ), not only has been widely used for the treatment of hematemesis, hemoptysis, and traumatic bleeding due to the efficacy of arresting bleeding with astringent action, but also has been applied topically to overcome ulcers, sores, swellings, and chapped skin due to the efficacy of dispersing swelling and promoting tissue regeneration. Additional medical applications include the treatment of tuberculosis, malignant ulcers, hemorrhoids, anthrax, eye diseases, and silicosis., Aim of This Review: This review aims to provide up-to-date information on the botanical characterization, medicinal uses, chemical constituents, pharmacological activities, and toxicity of B. striata. In addition, this paper also focuses on the possible exploitation of this plant for the treatment of different diseases, and uncovers opportunities for future research., Materials and Methods: The relevant information on B. striata was gathered from worldwide accepted scientific databases via an electronic search (Google Scholar, Web of Science, ScienceDirect, ACS Publications, PubMed, Wiley Online Library, SciFinder, CNKI). Information was also obtained from The Plant List, Chinese pharmacopoeia, Chinese herbal classics books, PhD and MSc dissertations, etc., Results: A comprehensive analysis of the literature obtained through the above-mentioned sources confirmed that the ethnomedical usages of B. striata have been recorded in Mongolia, Korea, Japan, and China. Phytochemical investigations revealed that the major chemical constituents of B. striata are polysaccharides, bibenzyls, phenanthrenes, triterpenoids and its saponins, steroids and its saponins, which also have been proven to be the main bioactive substances capable of exhibiting numerous pharmacological activities including wound healing, antiulcer, hemostasis, cytotoxicity, antimicrobial, anti-inflammation, anti-oxidation, immunomodulation, anti-fibrosis, antiaging, anti-allergy, and anti-itch., Conclusions: Preliminary investigations on pharmacological properties of B. striata have shown that B. striata is an outstanding astringent hemostatic medicinal, B. striata polysaccharides (BSP) as the major bioactive components not only capable of promoting wound healing, but also show good performance as a kind of promising natural biomaterial. More importantly, BSP are also reported to be excellent embolic material. However, further investigations need to be carried out to fully clarify its efficacy of dispersing swelling and promoting tissue regeneration. Moreover, this plant also needs a lot more investigations to clarify the pathways of absorption, distribution, metabolism and excretion, and to evaluate its long-term in vivo chronic toxicity before proceeding to the development of pharmaceutical formulation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The self-assembly of short peptides is a promising route to the creation of smart biomaterials. To combine peptide self-assembly with enzymatic catalysis, we design an amphiphilic short peptide I3QGK that can self-assemble into long nanoribbons in aqueous solution. Upon addition of transglutaminase (TGase), the peptide solution undergoes a distinct sol-gel transition to form a rigid hydrogel, which shows strong shear-thinning and immediate recovery properties. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements indicate the occurrence of considerable nanofibers in addition to the original nanoribbons. Liquid chromatography and mass spectrometry analyses reveal the enzymatic formation of peptide dimers from monomers through intermolecular ε-(γ-glutamyl)lysine isopeptide bonding. The dimers rapidly self-assemble into flexible and entangled nanofibers, and the coexistence of the original nanoribbons and the newly created nanofibers is responsible for hydrogelation. Factor XIII in blood is converted by thrombin to an active TGase (Factor XIIIa) during bleeding, so the peptide solution shows a more rapid and effective hemostasis via a combination of gelling blood and promoting platelet adhesion, relative to other hemostasis methods or materials. These features of I3QGK, together with its low cytotoxicity against normal mammalian cells and noninduction of nonspecific immunogenic responses, endow it with great potential for future clinical hemostasis applications.

Purpose: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here., Methods: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline., Results: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 μg/mL), (super)FVa did not (6.7 μg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 μg/mL) or saline (5.6 μg/mL)., Conclusion: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Background and Study Aims: Tissue adhesives are commonly used. The aim of this study was to assess the efficacy and hepatotoxicity of intravenous injection of N-butyl cyanoacrylate versus alpha-cyanoacrylate in a rabbit model., Materials and Methods: A total of 20 rabbits were divided into three groups: group I included four rabbits injected with lipiodol in the dorsal vein of a pinna (control group); group II included eight rabbits injected with N-butyl cyanoacrylate/lipiodol; and group III included eight rabbits injected with alpha-cyanoacrylate/lipiodol. All animals were left under normal living conditions for 1week, and then euthanised. Specimens of ear and liver were taken and fixed in 10% formalin saline for histological examination. Secondary fixation was performed using Bouin solution. Specimens of ear were decalcified in ethylenediaminetetraacetic acid (EDTA) at room temperature for 3months. Then, all specimens were processed, embedded in paraffin, sectioned, and stained with haematoxylin and eosin stains for microscopic examination., Results: Microscopic examination of all specimens of the control group revealed normal structure of pinna and liver tissue. Both test groups demonstrated a wide variability of structural changes ranging from oedema and congestion to necrosis and marked cellular inflammatory infiltration. The two groups were compared using a self-designed inflammatory score. This revealed that alpha-cyanoacrylate caused more venous sclerosis with extensive perivenous reaction and hepatotoxicity than both N-butyl cyanoacrylate and control (p<0.05 and p<0.05). N-butyl cyanoacrylate was also found to cause more venous sclerosis and hepatotoxicity than control (p<0.05)., Conclusion: This study suggested that injection of Krazy Glue, either the clinically usable N-butyl cyanoacrylate or the commercially available alpha-cyanoacrylate, caused comparable venous sclerosis. Unfortunately, both induced significant hepatotoxicity. Therefore, neither of them should be used unless all other safe options are absent. Larger studies have to be conducted and effects of these components on other organs should be investigated; however, caution must be exercised in their clinical use., (Copyright © 2016 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.)

Background: Achieving hemostasis during surgery is a common challenge across surgical specialties and procedures, including cardiovascular and peripheral vascular procedures. Although several hemostatic agents are available, they all become less effective as bleeding becomes more vigorous. Veriset™ hemostatic patch is a topical hemostatic agent that has been approved for use on solid organs and in soft tissue. The primary aim of these studies was to assess the safety of Veriset hemostatic patch during cardiovascular and peripheral vascular procedures., Methods: Adult domestic swine were given intraoperative anticoagulation and systemic antiplatelet therapy, and then underwent a variety of cardiovascular coronary artery bypass graft (CABG anastomosis and aortotomy closure) and peripheral vascular (carotid arteriotomy with patch angioplasty) procedures. Bleeding was identified or created from each anastomotic site and was brisk in many cases. Veriset hemostatic patch, Surgicel(®) Nu-Knit(®) absorbable hemostat, or standard of care treatment was applied topically to the site of bleeding, and time to hemostasis was monitored for each animal. Animals were assessed during and after the surgical procedure to determine the effects of treatment on vessel diameter, clinical laboratory parameters, and host tissue response and/or tissue integration., Results: Application of Veriset hemostatic patch had no effect on vessel diameter, similar to Surgicel Nu-Knit absorbable hemostat and standard of care treatment. Approximately 28 days after application, Veriset hemostatic patch and Surgicel Nu-Knit absorbable hemostat were completely absorbed, with no impact on serum chemistry profiles. Furthermore, Veriset hemostatic patch was as effective or more effective than Surgicel Nu-Knit absorbable hemostat when applied during carotid arteriotomy (30.0 ± 0.0 sec vs. 163.6 ± 86.9 sec, P < 0.001) and CABG anastomosis (91.9 ± 66.0 sec vs. 207.5 ± 159.2 sec, P = 0.053)., Conclusions: Veriset hemostatic patch exhibits similar safety as Surgicel Nu-Knit absorbable hemostat, with effectiveness against cardiovascular and peripheral vascular bleeding. If these preclinical results are confirmed in clinical trials, this highly effective patch with a topical application requiring less than 30 seconds is likely to prove useful in many clinical settings., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Effective hemorrhage control becomes increasingly significant in today's military and civilian trauma, while the topical hemostats currently available in market still have various disadvantages. In this study, three low molecular weight silk fibroins (LMSF) were prepared through hydrolysis of silk fibroin in a ternary solvent system of CaCl2/H2O/EtOH solution at different hydrolysis temperatures. Fourier transform infrared spectroscopy analysis showed that the content of β sheet structure in the LMSF decreased with the increase in hydrolysis temperature. The results of thromboelastographic and activated partial thromboplastin time methods showed that the LMSF hydrolyzed at 50 °C can significantly strengthen the coagulation in blood and activate the intrinsic pathway of coagulation cascade. In the murine hepatic injury model, the LMSF hydrolyzed at 50 °C can promote the blood clotting and decrease the blood loss and bleeding time. Based on these results, it can be suggested that the developed LMSF has the excellent hemostatic effect and may be a promising material in clinical hemostatic application.

Our previous studies demonstrated that thrombin is an important factor in brain injury after intracerebral and intraventricular hemorrhage. This study examined the effect of acetazolamide, a carbonic anhydrase inhibitor, on thrombin-induced hydrocephalus. There were two parts in this study. First, rats had an injection of either 50 μl saline or 3 U thrombin into the right lateral ventricle. Second, rats had an injection of 3 U thrombin into the right lateral ventricle and were treated with either vehicle or acetazolamide (30 mg/kg, intraperitoneally (IP)) at 1 h after thrombin infusion. Lateral ventricle volumes were measured in magnetic resonance imaging T2 images and the brains were used for histology analysis at 24 h later. Intraventricular injection of thrombin induced significantly larger ventricle volume (27.8 ± 3.7 vs 8.5 ± 1.3 mm(3), n = 6, p < 0.01) and more ventricular wall damage (the breakdown of the ependymal layer, 20.2 ± 3.1 vs 2.4 ± 0.8 %, n = 6, p < 0.01) compared with saline injection. Acetazolamide treatment (30 mg/kg, IP) markedly attenuated thrombin-induced hydrocephalus (16.1 ± 4.2 mm(3) vs 29.5 ± 5.3 mm(3), n = 6, p < 0.01). These results suggest decreasing CSF production by acetazolamide attenuated thrombin-induced hydrocephalus in rats.

We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n = 13) and HCRs (n = 12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 μl). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p > 0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0 ± 2.3 vs12.8 ± 1.9 mm(3) in LCR saline group; p < 0.01) and white matter lesion (9.3 ± 7.6 vs 0.6 ± 0.5 mm(3) in LCR saline group, p < 0.05). In comparison, in HCR rats thrombin induced less ventricular enlargement (17.3 ± 3.9 vs 23.0 ± 2.3 mm(3) in LCRs, p < 0.01) and smaller white matter lesions (2.6 ± 1.2 mm(3) vs 9.3 ± 7.6 mm(3) in LCRs, p < 0.05). In LCR rats, there was also upregulation of heat shock protein-32, a stress marker, and microglial activation in the periventricular white matter. These changes were significantly reduced in HCR rats. Intraventricular injection of thrombin caused more white matter damage and hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral hemorrhage with aerobic capacity.

Purpose: To evaluate in vitro cytotoxicity/biocompatibility as well as in vivo tolerability of the novel polysaccharide 4DryField® PH, certified for haemostasis and adhesion prevention., Methods: In vitro cytotoxicity/viability testing according to ISO EN 10,993 using murine and human tumour cell lines incubated with 4DryField® PH (PlantTec Medical GmbH). Using a rat model the impact of 4DryField® PH on animals viability and in vivo effects were macro- and micropathologically assessed., Results: In vitro testing revealed no cytotoxic effect of 4DryField® PH nor enhancement of viability to tumour cell lines. In vivo viability of rats was unimpaired by 4DryField® PH. Bodyweight loss in animals with abdominal injury plus treatment with 4DryField® PH was in the range of controls and less than in injured rats without treatment. At day 7 after surgery no formation of adhesions, neither macroscopic nor histological remnants nor signs of foreign body reaction were present in animals without injury. In animals with peritoneal injury and 4DryField® PH application, histopathological observation revealed minor residuals of polysaccharide in the depth of wound cavity embedded in a thickened subperitoneal layer; however, with a suggested intact neoperitoneum. The presence of mononuclear cells surrounding polysaccharide particles in varying states of degradation was observable as well., Conclusion: 4DryField® PH is not cytotoxic and does not enhance viability of tumour cell lines. High dose of 4DryField® PH of 1.09 g/kg bodyweight is well tolerated and reduces weight loss in animals with peritoneal injury. The biocompatibility of 4DryField® PH can be rated as being excellent., (© The Author(s) 2015.)

RADA16-I is a synthetic type I self-assembling peptide nanofiber scaffold (SAPNS) which may serve as a novel biocompatible hemostatic agent. Its application in neurosurgical hemostasis, however, has not been explored. Although RADA16-I is nontoxic and nonimmunogenic, its intrinsic acidity may potentially provoke inflammation in the surgically injured brain. We conducted an animal study to compare RADA16-I with fibrin sealant, a commonly used agent, with the hypothesis that the former would be a comparable alternative. Using a standardized surgical brain injury model, 30 Sprague-Dawley rats were randomized into three treatment groups: RADA16-I, fibrin sealant or gelatin sponge (control). Animals were sacrificed on day 3 and 42. Astrocytic and microglial infiltrations within the cerebral parenchyma adjacent to the operative site were significantly lower in the RADA16-I and fibrin sealant groups than control. RADA16-I did not cause more cellular inflammatory response despite its acidity when compared with fibrin sealant. Immunohistochemical studies showed infiltration by astrocytes and microglia into the fibrin sealant and RADA16-I grafts, suggesting their potential uses as tissue scaffolds. RADA16-I is a promising candidate for further translational and clinical studies that focus on its applications as a safe and effective hemostat, proregenerative nanofiber scaffold as well as drug and cell carrier., (© 2015 Wiley Periodicals, Inc.)

Introduction: In vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding., Materials and Methods: Anesthetized rabbits were treated with initial 0, 75, 200 or 450 μg kg(-1) dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15 min after the start of dabigatran administration, PCC doses of 0, 50 or 300 IU kg(-1) were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model., Results: At the suprapharmacologic dose of 300 IU kg(-1), PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75 μg kg(-1) dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300 IU kg(-1) PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p=0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding., Conclusions: In this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran., (Copyright © 2014. Published by Elsevier Ltd.)

Thrombin causes blood-brain barrier disruption, and this study examined whether thrombin can cause brain hemorrhage through protease-activated receptor-1 (PAR-1). Male wild type and PAR-1 knockout mice had an intracerebral injection of thrombin or saline. Mice then underwent serial T2 magnetic resonance imaging and were euthanized for brain hemoglobin, iron, and interleukin-1β measurements. Thrombin caused massive T2 lesions and brain hemorrhage in wild type mice. These effects were markedly reduced in PAR-1 knockout mice. Thrombin also increased brain interleukin-1β, and this was absent in PAR-1 knockout mice. In conclusion, thrombin increases interleukin-1β levels and induces intracerebral hemorrhage through PAR-1 activation.

This study characterizes a new model of thromboembolic stroke of the middle cerebral artery in C57 black/6J mice, thus offering an opportunity to use the model for studying ischemic stroke in transgenic mice. Thromboembolic stroke was induced by local injection of either 1.5 or 3.0 UI of thrombin directly into the right MCA of C57 black/6J mice. Cerebral blood flow (CBF) velocity was measured continuously by laser Doppler flowmetry, which allowed documentation of both MCA occlusion and of spontaneous recanalization. After 24 h, all animals were euthanized. Cryosections were cut at 400-μm intervals and silver stained with the high-contrast method for volumetric assessment of infarct size. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), caspase-3 and hsp 70 protein levels were investigated by immunofluorescence. Thrombin injection resulted in clot formation in all animals. Cortical infarction occurred in 63% of the mice while 37% had a spontaneous MCA recanalization during the first 20 min following thrombin injection. In cases of successful MCA occlusion with consequent infarction, the clot was stable up to 2 h after formation. Subsequently, 20% recanalized spontaneously. Infarctions were restricted to the cortex with a mean lesion volume of 36 ± 5 for 1.5 UI and 56 ± 8 for 3.0 UI thrombin. Protein levels of IL-6, TNF-α, caspase-3, and hsp 70 were significantly increased after MCAO. The results demonstrate that the mouse thromboembolic stroke model produces cortical infarctions of consistent size in C57 black/6J mice, which is dependent upon the amount of thrombin used for clot formation. Spontaneous MCA recanalization occurs after 2 h of ischemia in 20% of mice. Thus, the thromboembolic model is an applicable stroke model for C57 black/6J mice, which mimics many of the features of human stroke, including spontaneous recanalization. However, strain differences between Swiss and C57 black/6J mice must be taken into account when using the model.

Intracerebral hemorrhage (ICH) is a devastating neurological disease with a grave prognosis. We evaluated microRNA (miRNA) expression after ICH and evaluated Let7c as a therapeutic target. We harvested hemorrhagic brain 24 hours after collagenase induced ICH in the rat. Microarray analysis was performed to compare the miRNAs expression pattern between hemorrhagic hemisphere and contralateral hemisphere. An in vitro thrombin toxicity model and blood injection ICH model were also used to evaluate miRNA expression. We selected miRNA for the therapeutic target study after reviewing target gene databases and their expression. The antagonistic sequence of the selected miRNA (antagomir) was used to evaluate its therapeutic potential in the in vitro thrombin toxicity and in vivo ICH models. Among 1,088 miRNAs analyzed, let7c was induced in the thrombin and ICH models. Let7c antagomir treatment increased cell survival in the in vitro thrombin injury model and improved neurological function at 4 weeks after ICH. Let7c antagomir decreased perihematoma edema, apoptotic cell death and inflammation around hematoma. Let7c antagomir also induced insulin like growth factor receptor 1 (IGF1R) protein and phosphorylated serine threonine kinase after ICH. This study shows a distinct miRNA expression pattern after ICH. The let7c antagomir reduced cell death and edema and enhanced neurological recovery at least in part by activating the IGF1R pro-survival pathway. This suggests blocking let7c might be a potential therapeutic target in ICH.

Natural polymer-derived materials have attracted increasing interest in the biomedical field. Polysaccharides have obvious advantages over other polymers employed for biomedical applications due to their exceptional biocompatibility and biodegradability. None of the spherical embolic agents used clinically is biodegradable. In the current study, microspheres prepared from chitosan and carboxymethyl cellulose (CMC) were investigated as a biodegradable embolic agent for arterial embolization applications. Aside from the enzymatic degradability of chitosan units, the cross-linking bonds in the matrix, Schiff bases, are susceptible to hydrolytic cleavage in aqueous conditions, which would overcome the possible shortage of enzymes inside the arteries. The size distribution, morphology, water retention capacity and degradability of the microspheres were found to be affected by the modification degree of CMC. An anticancer drug, doxorubicin, was successfully incorporated into these microspheres for local release and thus for killing cancerous cells. These microspheres demonstrated controllable degradation time, variable swelling and tunable drug release profiles. Co-culture with human umbilical vein endothelial cells revealed non-cytotoxic nature of these microspheres compared to monolayer control (P>0.95). In addition, a preliminary study on the in vivo degradation of the microspheres (100-300μm) was performed in a rabbit renal embolization model, which demonstrated that the microspheres were compatible with microcatheters for delivery, capable of occluding the arteries, and biodegradable inside arteries. These microspheres with biodegradability would be promising for embolization therapies., (Copyright © 2013. Published by Elsevier Ltd.)

High molecular weight chitosan (HMWCS) is effective at hemostasis and wound healing, and will be potentially used on injured internal organs. To study its prenatal and developmental effect in vivo, forty-four ICR pregnant mice per group were singly injected intraperitoneally at 0, 125, 500 or 2000 mg/kg body weight, respectively, on gestation day 6 (GD6). Clinical signs, reproductive capacity, fetus and infant developments, and histopathological changes were then observed. The results showed that the treatment of HMWCS could decrease body weights and food consumptions, and induce diarrhea, vaginal bleeding, and some other adverse effects in F0 mice. For the emaciation and threatened abortion of pregnant mice, the numbers of live fetuses and early resorption were reduced significantly in HMWCS groups. However, the developments of F1 and F2 mice were not affected, except for lower weights of the body and some organs. In addition, the NOAEL of HMWCS in maternal toxicity was considered to be less than 125 mg/kg, and the NOAEL in developmental toxicity was 125 mg/kg., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: We describe a retinal endovascular fibrinolysis technique to directly reperfuse experimentally occluded retinal veins using a simple micropipette., Methods: Retinal vein occlusion was photochemically induced in 12 eyes of 12 minipigs: after intravenous injection of 10% fluorescein (1-mL bolus), the targeted retinal vein segment was exposed to thrombin (50 units) and to Argon laser (100-200 mW) through a pars plana approach. A beveled micropipette with a 30-μm-diameter sharp edge was used for micropuncture of the occluded vein and endovascular microinjection of tissue plasminogen activator (50 μg/mL) in 11 eyes. In one control eye, balanced salt solution was injected. The lesion site was examined histologically., Results: Retinal vein occlusion was achieved in all cases. Endovascular microinjection of tissue plasminogen activator or balanced salt solution led to reperfusion of the occluded retinal vein in all cases. Indicative of successful reperfusion were the following: continuous endovascular flow, unaffected collateral circulation, no optic disk ischemia, and no venous wall bleeding. However, balanced salt solution injection was accompanied by thrombus formation at the punctured site, whereas no thrombus was observed with tissue plasminogen activator injection., Conclusion: Retinal endovascular fibrinolysis constitutes an efficient method of micropuncture and reperfusion of an experimentally occluded retinal vein. Thrombus formation at the punctured site can be prevented by injection of tissue plasminogen activator.

The aim of this study is to investigate the effects of the Ankaferd Blood Stopper® (ABS), on cell viability, cytotoxicity, and erythrocyte numbers in in vitro cultured human blood cells. We studied the cytotoxic effects of the ABS using lactate dehydrogenase (LDH) assay, cell proliferation (WST-1) assay and hemolytic assay. The cytotoxicity increased when cells were treated with ABS dilutions of 5%, 12.5%, 25%, and 50% (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly elevated the cell number at 24 and 48 h (p < 0.05). ABS causes a significant increase (p < 0.05) in the hemolytic activity on human erythrocytes and hemolytic activity increases with increase in ABS concentrations. The red blood cell aggregation and cell membrane disruption during the coagulation process lead to induction of hemolytic activity and increase of LDH level in cell culture medium. In addition, ABS has proliferative effects on human leukocytes. Based on these results, ABS can be used as an alternative blood stopping agent safely.

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: The objective of this study was to evaluate the cytotoxicity of the plant extract ankaferd blood stopper (ABS) in vitro., Study Design: ABS was eluted with fresh Dulbecco's Modified Eagle's Medium (DMEM) without serum for 72 h, at 37 °C. The cells treated with various dilutions of ABS were seeded into 96-well microplate at 10⁴/well in triplicates. Cells without treatment served as a control group. The number of viable cells after 48 h incubation was determined by a modified 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. The relative viability of pulp cells was expressed as color intensity of the number in the experimental wells relative to that of the control group. Absorbances were read at 570 nm on a microplate reader with a background subtraction at 620 nm., Result: The results showed that ABS was cytotoxic to human pulp fibroblasts by MTT assay., Conclusions: The influence of cytotoxicity to human pulp fibroblasts depended on concentration of ABS. The more dilutions exhibited less cytotoxic characteristics compared to the more concentrated forms.

The present study was designed to evaluate the hemostatic potential of poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine]-based hemostatic dressing material on albino rabbits. In vitro cytotoxicity study of poly [ß-(1, 4)-2-amino-2-deoxy-D-glucosamine]-based hemostatic dressing samples was carried out with L929 cells, and the cytotoxic potential was evaluated at the end of 24 h. The skin irritation was carried out in albino rabbits. Extract of the material was applied topically and irritation response was evaluated up to 72 h. The hemostatic study was initiated in rabbits after general anesthesia with a mixture of ketamine and xylazine. Using a sharp surgical blade, a 1.0 cm longitudinal incision was made on the right (test) and left (control) marginal ear arteries. Through the resultant jet spray of blood, the right 1.0 cm long wound was immediately covered with a 2 × 2 cm(2) piece of test material (poly [ß-(1,4)-2-amino-2-deoxy-D glucosamine] of known weight (w1). Similarly the left wound (1.0 cm length) was covered with commercially-available bandage (control) of known weight (w2). Direct pressure was applied for 2 min and then the samples were removed and weighed immediately (w3 for test and w4 for control) after hemostasis. Blood loss (w3-w1 for the Test and w4-w2 for control) was calculated from the materials weight before and after absorbing blood. The result of the study indicated that the indigenously developed material has local biological activity in the form of hemostatic action and, together with its ability to activate macrophages, resulted in wound healing applications. Hence, the present study concluded that the poly [ß-(1,4)-2-amino-2-deoxy-D glucosamine]-based hemostatic dressing material is non-toxic, non-skin irritant, and has better hemostatic potential than a commercially available material with enhanced hemostatic capabilities for various wound dressing.

Purpose: To define the effects of hepatic artery chemoembolization with cisplatin-lipiodol suspension and gelatin sponge particles on swine liver tissue and estimate the concentration of cisplatin that would have a minimal negative effect on normal liver parenchyma., Materials and Methods: Twelve pigs were divided into four groups: group A was the control group in which hepatic arteries were embolized with lipiodol and gelatin sponge particle (n = 3); group B animals were embolized with 10 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3), group C with 20 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3), and group D with 30 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3). Pigs were euthanized 1 week after embolization, and the resected livers were cut into 10-mm-thick sections. The livers and necrotic foci were contoured in each section, and the necrosis volume ratio was calculated., Results: The necrosis volume ratios of the livers in groups A, B, C, and D were 0.832% +/- 0.334, 2.324% +/- 1.126, 8.056% +/- 3.276, and 11.82% +/- 4.921, respectively. Significant differences (P < .05) in necrosis volume ratio were found between groups A and C, groups A and D, groups B and C, and groups B and D; no significant difference was found between groups A and B., Conclusions: Hepatic artery chemoembolization with higher doses of cisplatin causes greater damage to liver tissue; 10 mg/mL cisplatin-lipiodol suspension causes minimal damage, similar to that without cisplatin, and is related to minimal negative changes in a swine model.

Background: Chitosan can inhibit fibroblastic proliferation by suppressing fibroblast cells, and has the similar physiological characteristics as normal vitreous body, so it might have the potential to become vitreous filling material and might possibly inhibit proliferative vitreous retinopathy. To investigate the possibility of chitosan as vitreous filling material, this study was designed to investigate retina, ciliary body, lens and cornea morphology changes, intraocular pressure and intraocular inflammatory factors fluctuating after chitosan intravitreous tamponade., Methods: Fifteen healthy chinchilla rabbits were chosen; three of them were a blank (negative) control group without any surgical procedure. The remaining 12 rabbits received vitrectomy on both eyes; all the right eyes (experimental group) were given 1.2-1.8 ml (average 1.5 ml) of chitosan intravitreously, while sodium hyaluronate were given in the left eyes (control group). All eyes underwent slit-lamp biomicroscope and indirect ophthalmoscope examination and intraocular pressure measurement pre- and post-op. The concentration of IL-6, IL-8 (radioimmunoassay), NO (nitrate reductase method) in aqueous humor and vitreous body were tested at day 15 and day 30 post-op. At day 30 post-op, the cornea, ciliary body, and lens were dissected for light microscopy examination, and the retinal tissues 2PD away from the optic disc on the vertical orientation of posterior pole were dissected for light- and electro-microscope examination., Results: The conjunctival congestion and slight inflammatory response in the anterior chamber disappeared within 7 days post-op. During the 30-day experiment, cornea, lens and the filling material in vitreous cavity were transparent in all animals. The retina was attached without proliferation. The intraocular pressure in the experimental group post-op fluctuated between 4.55 +/- 2.94 and 6.25 +/- 2.37 mmHg, which was not significantly different from the situation pre-op (6.18 +/- 1.19 mmHg) (P > 0.05). The intraocular pressure in the control group post-op fluctuated between 5.10 +/- 2.51 and 5.90 +/- 2.49 mmHg, which was not significantly different from the situation pre-op (6.50 +/- 0.94 mmHg) (P > 0.05). There was also no significant difference in the intraocular pressure post-op at different time points between the experimental group and control group (all P > 0.05). At day 15 post-op, IL-6 concentration in aqueous humor were 37.31 +/- 8.59 ng/ml and 39.52 +/- 9.69 ng/ml in experimental group and control group respectively, both higher than those in the blank control group (26.55 +/- 9.34 ng/ml) (P < 0.05). IL-8 concentration were 7.00 +/- 3.79 ng/ml and 6.32 +/- 3.68 ng/ml respectively, no significant difference to the blank control group (4.72 +/- 1.71 ng/ml) (P > 0.05): the concentrations of NO were 63.94 +/- 26.80 micromol/ml and 51.81 +/- 13.19 micromol/ml respectively, no significant difference to the blank control group (50.36 +/- 15.67 micromol/ml) (P > 0.05). At day 30 post-op, the concentrations of IL-6, IL-8 and NO in aqueous humor showed no significant difference among all three groups (P > 0.05). In vitreous body at day 30 post-op, the concentrations of IL-8 in experimental group and control group were 10.17 +/- 3.63 ng/ml and 10.69 +/- 3.52 ng/ml, and those of NO were 50.23 +/- 19.69 micromol/mL and 50.60 +/- 12.72 micromol/mL respectively, all higher than in the blank control group (30.37 +/- 14.63 micromol/ml) (P < 0.05); the concentrations of IL-6 were 24.51 +/- 10.71 ng/ml and 26.36 +/- 13.00 ng/ml, no significant difference to the blank control group (24.06 +/- 5.98 ng/ml) (P > 0.05). At various time points, there was no significant difference in the concentrations of IL-6, IL-8 and NO in aqueous humor and vitreous body in the experimental group and the control group (P > 0.05). There was no morphological change found under light microscopy in cornea, ciliary body and lens. The outer plexiform layer of retina was thinner, but no significant degeneration, necrosis, karyopyknosis or lysis were found under the ultrastructural microscopy., Conclusion: Chitosan intravitreous tamponade has no significant effect on the histology of the eye, doesn't cause intraocular pressure to fluctuate, and slightly increases inflammatory factors (IL-6, IL-8, NO) in comparison to the normal levels, but with no significant difference from the effects caused by sodium hyaluronate, which indicated chitosan might not lead to a clinically significant inflammatory response. It suggests that chitosan could be used as intravitreous tamponade material.

Intravenous infusion of recombinant human activated Factor VII (FVIIa) has been used for over a decade in the successful management of bleeding episodes in patients with inhibitory antibodies to Factor VIII or Factor IX. Previously, we showed that expression of murine FVIIa (mFVIIa) from an adeno-associated viral (AAV) vector corrected abnormal hemostatic parameters in hemophilia B mice. To pursue this as a therapeutic approach, we sought to define safe and effective levels of FVIIa for continuous expression. In mice transgenic for mFVIIa or injected with AAV-mFVIIa, we analyzed survival, expression levels, in vitro and in vivo coagulation tests, and histopathology for up to 16 months after birth/mFVIIa expression. We found that continuous expression of mFVIIa at levels at or below 1.5 microg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 microg/ml or higher were associated with thrombosis and early mortality, with pathologic findings in the heart and lungs that were rescued in a low-factor X (low-FX) mouse background, suggesting a FX-mediated effect. The findings from these mouse models of continuous FVIIa expression have implications for the development of a safe gene transfer approach for hemophilia and are consistent with the possibility of thromboembolic risk of continuously elevated FVIIa levels.

Recombinant human thrombin (rhThrombin) is being developed as an alternative to thrombin products purified from pooled human or bovine plasma, which are currently marketed for topical hemostasis. Preclinical studies of rhThrombin were conducted prior to its evaluation as a topical adjunct to surgical hemostasis in clinical trials. No overt clinical pathology or signs were observed in cynomolgus monkeys following implantation of a gelatin sponge containing either rhThrombin or bovine thrombin to a surgical liver wound, and similar gross and microscopic wound healing characteristics were observed over an eight-week recovery period with either compound. Repeated subcutaneous injections of rhThrombin or bovine thrombin to cynomolgus monkeys produced no treatment-related effects. Whereas no monkeys demonstrated anti-rhThrombin antibody seroconversion, specific anti-bovine antibodies were detected in all tested monkeys exposed to bovine thrombin. Addition of rhThrombin or bovine thrombin to mouse fibroblast cells resulted in expected detachment and shape change. Topical application of rhThrombin to rabbits did not cause irritation to the eye, normal skin, or abraded skin. These studies showed that topical, subcutaneous, or implanted rhThrombin was minimally immunogenic, safe, and well tolerated in nonclinical models, and supported the clinical evaluation of rhThrombin in a variety of surgical settings.

Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.

Aim: To investigate the hypothesis that different haemostatic agents could impair the histological response of human pulps capped with calcium hydroxide., Methodology: Forty-five third molars scheduled for extraction were selected. Class I cavities with pulp exposures were prepared. Three agents were used to control bleeding: 0.9% saline solution (control, n = 14), 5.25% sodium hypochlorite (n = 16) and 2% chlorhexidine digluconate (n = 15). The pulps were dressed with hard-setting calcium hydroxide cement. After 7, 30 or 90 days, teeth were extracted, formalin-fixed and prepared for histochemical techniques. The biological response was categorized using the following criteria: inflammatory response, soft tissue organization, reactionary dentine and reparative dentine. Data were submitted to statistical analysis, using nonparametric Kruskal-Wallis one-way analysis of variance on ranks. Differences amongst groups were detected using Dunn's method., Results: The statistical analysis disclosed that whilst inflammatory response decreased over time, reactionary dentine deposition and reparative dentine formation increased in the latter periods of evaluation (P < 0.05). The three agents had similar performances for all criteria evaluated. The conventional pulp response to calcium hydroxide was observed over time, and complete pulp healing was observed in 88% of the specimens after 90 days., Conclusion: The three haemostatic agents did not impair the healing process following pulp exposure and capping with calcium hydroxide at different time intervals investigated.

Thrombin has been found in neuritic plaques in Alzheimer's disease (AD). Also, traumatic brain injury, where neurons are exposed to high thrombin levels, is associated with an increased incidence of AD. Our objective was to determine the effects of thrombin administered in vivo on cognitive function and neuropathology. Rats were trained using a radial eight-arm maze and then thrombin (25 or 100 nM, 0.25 microl/h, 28 days) or vehicle was delivered via intracerebroventricular infusion. Animals that received 100 nM thrombin demonstrated cognitive impairments including deficits in reference memory and an increase in task latency. Also, significant neuropathology was detected in these animals such as enlargement of cerebral ventricles, an increased number of TUNEL-positive cells, astrogliosis, and an increase in the immunoreactivity for phosphorylated neurofilament, and apolipoprotein-E fragments. Thrombin-induced changes in cognitive function and ventricular enlargement were inhibited by hirudin. These findings demonstrate that thrombin is a mediator of neurotoxicity and cognitive deficits and suggest that inhibition of thrombin may be a treatment strategy for AD- or head trauma-associated cognitive deficits.

Oxidized celluloses (OC) containing 7, 13, and 20% carboxylic content (OC-7, OC-13, and OC-20, respectively) have been converted into aqueous colloidal dispersions and used to prepare microspheres of the antineoplastic agent camptothecin (CPT) by spray drying. Plasticizers used were glycerin, polyethylene glycol 400 (PEG-400), and polyethylene glycol 6000 (PEG-6000). Irrespective of the carboxyiic content of OC and the nature of plasticizer employed, the size of microspheres varied from 1.25+/-0.40 to 1.52+/-0.47 microm. The release studies in pH 7.4 buffer revealed the dissolution of CPT to be faster from the microsphere formulations than from physical mixtures and free CPT. The times to release 50% CPT (T-50%) from microspheres prepared using OC-7, OC-13, and OC-20 were about 31, 37, and 19 h, respectively. The in vitro cytotoxicity results indicated OC-20/CPT microspheres to be more effective than free CPT against human-derived RPMI-8402 lymphoid and THP-1 myeloid leukemia cell lines. The ED50 values for the OC-20/CPT microspheres and free CPT were 1 x 10(-5) and 0.25 x 10(-1) microg/mL, respectively, against the RPMI-8402 line and 0.5 x 10(-2) and 0.75 microg/mL, respectively, against the THP-1 line. The higher activity of OC-20/CPT microspheres compared to that of the free drug is attributed to increased dissolution of CPT from microspheres.