Performance and reference intervals of thrombin generation test: results from the Brazilian longitudinal study of adult health (ELSA-Brasil). A cross-sectional study

ABSTRACT BACKGROUND: The thrombin generation test (TGT) has shown promise for investigation of hemorrhagic and thrombotic diseases. However, despite its potential, it still needs standardization. Moreover, few studies have established reference values for TGT parameters. In Brazil, these values have not yet been established. OBJECTIVE: To determine TGT performance and reference intervals for TGT parameters in healthy individuals. DESIGN AND SETTING: Cross-sectional study conducted among participants in the Brazilian Longitudinal Study of Adult Health (Estudo Longitudinal de Saúde do Adulto, ELSA-Brasil). METHODS: The reference sample consisted of 620 healthy individuals. The calibrated automated thrombogram (CAT) method, under low and high tissue factor (TF) conditions, was used to assess thrombin generation. Test performance was analyzed using intra and interassay coefficients of variation (CV) and reference intervals were calculated using the nonparametric method proposed by the International Federation of Clinical Chemistry and the Clinical and Laboratory Standards Institute. RESULTS: The intraassay CV ranged from 1.4% to 2.2% and the interassay CV, 6.8% to 14.7%. The reference intervals for TGT parameters under low and high TF conditions were, respectively: lagtime: 3.0-10.3 and 1.4-3.7 min; endogenous thrombin potential (ETP): 1134.6-2517.9 and 1413.6-2658.0 nM.min; normalized ETP: 0.6-1.3 and 0.7-1.4; peak: 103.2-397.7 and 256.4-479.0 nM; normalized peak: 0.3-1.3 and 0.7-1.2; and time-to-peak: 5.6-16.0 and 3.4-6.7 min. These parameters were categorized relative to sex. Conclusion: TGT performance was adequate and the proposed reference intervals were similar to those of other studies. Our findings may be useful for consolidating the TGT, through contributing to its standardization and validation.

Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology / 中国实验血液学杂志

OBJECTIVE@#To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane.@*METHODS@#The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet.@*RESULTS@#The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05).@*CONCLUSION@#A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

Main Factors Influencing the Platelet Spreading / 中国实验血液学杂志

OBJECTIVE@#To explore the main factors of platelet spreading and provide the foundation for related research.@*METHODS@#Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists.@*RESULTS@#Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing.@*CONCLUSION@#The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.

Coagulación intravascular diseminada / Disseminated intravascular coagulation

Resumen La coagulación intravascular diseminada es un estado alterado de la coagulación secundario a cuadros inflamatorios locales o sistémicos. Se caracteriza por un aumento de la coagulación y una inadecuada fibrinólisis endógena que da como resultado la formación de fibrina intravascular, trombosis microvascular multiorgánica y un excesivo consumo de los factores de coagulación sanguínea, esto desencadena sangrados severos y trombosis, los cuales conducen a falla orgánica y circulatoria como principal manifestación clínica. Este artículo tiene como objetivo realizar una revisión del estado del arte describiendo su definición, etiología, fisiopatología, manifestaciones clínicas y actualidades en su diagnóstico y tratamiento. Para esto, se realizó una búsqueda bibliográfica en PubMed utilizando el término MeSH "Disseminated Intravascular Coagulation", seleccionándose 63 artículos por la relevancia y pertinencia de su información al objetivo de la investigación. De esta manera, se espera brindar al clínico las herramientas necesarias para responder adecuadamente frente a esta patología. MÉD.UIS.2020;33(2):75-84.

Abstract Disseminated Intravascular Coagulation is an altered state of coagulation secondary to local or systemic inflammatory symptoms. It is characterized by increased coagulation and inadequate endogenous fibrinolysis resulting in intravascular fibrin formation, multi-organ microvascular thrombosis, and excessive consumption of blood coagulation factors, this triggers severe bleeding and thrombosis, which lead to organic and circulatory failure as the main clinical manifestation.This article aims to review the state of the art describing its definition, etiology, pathophysiology, clinical manifestations and news in its diagnosis and treatment. Accordingly, a bibliographic search was carried out in PubMed using the MeSH term "Disseminated Intravascular Coagulation", selecting 63 articles for the relevance and pertinence of their information to the objective of the investigation. Therefore, it is expected to provide the clinician with the necessary tools to adequately respond to this pathology. MÉD.UIS.2020;33(2):75-84.

Thrombin generation and thromboelastometry tests in dogs with chronic kidney disease / Testes de geração de trombina e tromboelastometria em cães com doença renal crônica

Patients with chronic kidney disease (CKD) have paradoxical hemostatic potential because they have bleeding episodes but are also prone to thrombosis. Few studies have evaluated blood viscoelastic properties in dogs with kidney disease; on the other hand, hypercoagulability has been observed in these patients. It is also emphasized that the platelet function and its participation in this process have not yet been fully understood. The objective of this study was to evaluate and compare the Thrombin Generation Test (TGT) and also viscoelastic properties of the blood measured by thromboelastometry (TEM) in dogs with proteinuria in CKD. Twenty healthy dogs (Control Group) and 19 dogs with CKD in stage III or IV, classified according to International Renal Interest Society - IRIS, were selected, and the reference test of urine protein:creatinine ratio (UPCR) should be greater than one (CKD group). Blood samples for TEM, thrombin generation, Prothrombin Time (PT), activated Partial Thromboplastin Time (aPTT), and fibrinogen concentration was collected at a single time for both groups after inclusion criteria being confirmed. Statistical analysis was performed according to the distribution of variables at 5% significance level. Differences were observed between healthy dogs and those with proteinuria in CKD noted in TEM. The TGT was unable to differentiate between sick and healthy groups. However, when the nephropathy was stratified, increases in TTP and peak thrombin concentration by TGT were observed in females and dogs over 30 days of diagnosis of CKD. Both tests signaled a discrete state of hypercoagulability. In fact, TEM is more sensitive to detect hypercoagulability in dogs with CKD. However, the TGT has potential clinical application by allowing long-term sample storage.(AU)

Os pacientes com doença renal crônica (DRC) apresentam um potencial hemostático paradoxal, pois apresentam episódios de sangramento, mas também são propensos à trombose. Poucos estudos avaliaram as propriedades viscoelásticas sanguíneas em cães com doenças renais, entretanto, a hipercoagulabilidade já foi observada nestes pacientes. Ressalta-se ainda que a função plaquetária e sua participação neste processo ainda não foram totalmente esclarecidas. O objetivo foi avaliar e comparar o teste de geração de trombina (TGT) e as propriedades viscoelásticas sanguíneas medidas pela tromboelastometria (TEM) em cães com DRC proteinúrica. Foram selecionados 20 cães saudáveis (grupo controle) e 19 cães com DRC em estágios III ou IV classificados segundo o IRIS e a relação proteína/creatinina urinária maior que um (grupo DRC). As amostras de sangue para a realização da tromboelastometria (TEM), geração de trombina, tempo de protrombina (TP), tempo de tromboplastina parcial ativada (TTPA) e concentração de fibrinogênio foram colhidas em momento único para ambos os grupos após os critérios de inclusão confirmados. A análise estatística foi realizada de acordo com a distribuição das variáveis, ao nível de 5% de significância. Foi observada diferença entre os cães saudáveis e os com DRC proteinúrica observados na TEM. O teste de geração de trombina não foi capaz de diferenciar os grupos doente e saudável. Entretanto, quando os nefropatas foram analisados de forma estratificada, foram observados aumentos do ETP e da concentração máxima de trombina (peak) pelo TGT em fêmeas e em cães com mais de 30 dias de diagnóstico da DRC. Ambos os testes sinalizando para um discreto estado de hipercoagulabiliade. A tromboelastometria é mais sensível para detectar a hipercoagulabilidade em cães com DRC. Entretanto, o teste de geração de trombina tem melhor aplicabilidade por permitir o armazenamento da amostra em longo prazo.(AU)

Effect of Protein Kinase A Activation on Aggregation Function of Platelets / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro.@*METHODS@#The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator.@*RESULTS@#Compared with the controls, 5 μmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P＜0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P＜0.05). 2.5-10 μmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P＜0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P＞0.05).@*CONCLUSION@#PKA activation inhibits agonists-induced platelet aggregation.

Eficácia e segurança de anticoagulantes orais diretos comparados à anticoagulação tradicional com heparina e varfarina para tratamento de tromboembolismo pulmonar e trombose venosa profunda / Effectiveness and safety of direct oral anticoagulants compared to traditional anticoagulation with heparin and varfarine to treat pulmonary thromboembolism and deep venous thrombosis

Tecnologia: Inibidores Diretos do Fator Xa (IDFXa) ­ Rivaroxabana, Apixabana, Edoxabana ­ e Inibidores Diretos da Trombina (IDT) ­ Dabigatrana ­ todos são anticoagulantes orais diretos (DOAC). Indicação: tratamento e prevenção de fenômenos tromboembólicos. Pergunta: Para tratamento de tromboembolismo pulmonar (TEP) e trombose venosa profunda (TVP), os DOAC são mais eficazes e seguros que a anticoagulação tradicional com heparina e varfarina? Métodos: Levantamento bibliográfico na base de dados Pubmed seguindo estratégias de buscas predefinidas. Avaliação da qualidade metodológica das revisões sistemáticas com a ferramenta Assessing the Methodological Quality of Systematic Reviews (AMSTAR). Resultados: Foram selecionadas e incluídas 4 revisões sistemáticas. Conclusão: Na maioria dos estudos incluídos, os DOAC demonstraram eficácia e segurança similar à anticoagulação tradicional com heparina e varfarina para tratamento de TEP e TVP. Em um estudo, o risco de TVP recorrente foi menor no tratamento de IDFXa (por menos 3 meses de tratamento) e de episódios de sangramento maior foi menor no tratamento de IDT e IDFXa (por mais 3 meses de tratamento)

Technology: Direct Factor Xa Inhibitors (DFXaI) - Rivaroxaban, Apixaban, Edoxaban ­ and Direct Thrombin Inhibitors (DTI) - Dabigatran - all are direct oral anticoagulants (DOAC). Indication: treatment and prevention of thromboembolic phenomena. Question: For treatment of pulmonary thromboembolism (PTE) and deep vein thrombosis (DVT), are DOACs more effective and safer than traditional anticoagulation with heparin and warfarin? Methods: Bibliographic survey in the Pubmed database following predefined search strategies. Evaluation of the methodological quality of systematic reviews with the tool Assessing the Methodological Quality of Systematic Reviews (AMSTAR). Results: 4 systematic reviews were selected and included. Conclusion: In most of the included studies, DOAC demonstrated similar efficacy and safety to traditional anticoagulation with heparina and warfarin for the treatment of PTE and DVT. In one study, the risk of recurrent DVT was lower in the treatment of DFXaI (for at least 3 months of treatment) and of major bleeding episodes was lower in the treatment of DTI and DFXaI (for another 3 months of treatment)

Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions

ABSTRACT Background: In order to standardize a thrombin generation() protocol, we analyzed the analytical variables and sensitivity of this test to hypo/hypercoagulability states. Methods: The effect of the tissue factor concentration and the intra- and interassay precision were analyzed. To evaluate the hypercoagulability status, the plasma of women under an oral contraceptive was tested, while plasma from hemophilia A patients at 1, 3 and 7 days after recombinant FVIII infusion, and lyophilized plasma deficient in FVII or FVIII were used for the evaluation of hypocoagulability. Results: The intra-assay coefficient of variation was <10% with 1 and 5 pM of low and high TF. The oral contraceptive users showed increased thrombin generation in comparison to non-users, which was more pronounced with low TF (endogenous thrombin potential ETP) p = 0.0009; peak p = 0.0009; lagtime p = 0.0008). In relation to the FVIII-deficient plasma, a higher TG was observed as FVIII levels were increased and a better discrimination was obtained for different concentrations of FVIII with low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0004). Using low TF, plasma from hemophilia A patients showed higher TG values after 1 day of recombinant FVIII infusion vs after 3 days (ETP p < 0.0001; peak p < 0.0001; lagtime p = 0.0407), while the lowest values were observed after 7 days. With FVII-deficient plasma, thrombin generation was lower than normal plasma and a more pronounced difference was observed with high TF compared to low TF (ETP p < 0.0001; peak p < 0.0001; lagtime p < 0.0001). Conclusion: Under our conditions the thrombin generation test seems to be sensitive to evaluation of hyper/hypocoagulability states. Standardization of the thrombin generation test may have an application in the evaluation of bleeding and thrombotic disorders.

Hemorragia posparto: nuevas evidencias / Postpartum hemorrhage: new evidence

Introducción: la hemorragia posparto (HPP) es toda pérdida sanguínea mayor de 500 ml luego de un parto vaginal o mayor de 1000 mililitros luego de una cesárea, o cualquier pérdida sanguínea luego del parto que provoque inestabilidad hemodinámica, independiente del tipo de parto. Puede provocar signos y síntomas de choque que si no se maneja adecuadamente podría comprometer la vida de la paciente. Objetivo: realizar una revisión bibliográfica sobre HPP. Métodos: se realizó una exhaustiva revisión bibliográfica de la literatura médica más actualizada sobre HPP. Resultados y discusión: 32 artículos médicos en idioma español y en idioma inglés de los últimos años se incluyen en el presente trabajo, lo que incluye guías clínicas. La HPP se clasifica en hemorragia postparto primaria, temprana o inmediata y en hemorragia postparto secundaria o tardía. Dentro de las principales causas de hemorragia postparto primaria tenemos como nemotecnia las 4 T que incluyen: Tono, Trauma, Tejido y Trombina, siendo la más frecuente la relacionada con el tono que representa el 70% de los casos. La clave del manejo de la HPP es reconocer el sangrado excesivo en el posparto, identificar la causa, e iniciar una intervención adecuada según el caso. Conclusión: la HPP es la segunda causa de muerte materna a nivel mundial, por lo tanto, es muy importante conocer sus causas, manejo y la prevención de la misma.

Introduction: postpartum hemorrhage (PPH) is any blood loss greater than 500 ml after a vaginal birth or more than 1000 milliliters after a cesarean, or any blood loss after delivery that causes hemodynamic instability regardless of the type of delivery. It can cause signs and symptoms of shock, which if not properly handled could compromise the patient life. Objective: we performed an exhaustive review of the most recent medical literature about PPH. Methods: an exhaustive bibliographic review of the most recent medical literature on PHP was carried out. Results and discussion: 32 medical articles in Spanish and in English language of the last years were included in the present work including clinical guidelines. PPH is classified as primary, early or immediate postpartum hemorrhage and secondary or late postpartum hemorrhage. Among the main causes of primary postpartum hemorrhage, we have as mnemonic the 4 T that include: Tone, Trauma, Tissue and Thrombin, the most frequent being the one related to the tone that represents 70% of the cases. The key of the management is to recognize hemorrhage after the delivery, identify the cause and start an opportune and adequate treatment. Conclusion: PPH is the second cause of maternal death worldwide, therefore, it is very important to know its causes, management and prevention.

Molecular characteristics of polysulfated fractions from the green seaweed Caulerpa cupressoides and actions on thrombin generation in vitro

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel / 浙江大学学报·医学版

OBJECTIVE@#To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.@*METHODS@#After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected.@*RESULTS@#The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h.@*CONCLUSIONS@#Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

Wound healing effect of regenerated oxidized cellulose versus fibrin sealant patch: An in vivo study

BACKGROUND: Topical hemostatic agents are used when ligation, electrocauterization, or other conventional hemostatic methods are impractical. Because a hemostatic agent is a foreign body, it can cause foreign body reactions, inflammation, and infections that can interfere with the wound healing process. Therefore, we should select hemostatic agents after considering their effects on wound healing. Here, we compared the effects of hemostatic agents on wound healing in a rectus abdominis muscle defect in rats. METHODS: Twelve Sprague Dawley rats were subjected to creation of a 6 × 6 mm defect in the rectus abdominis muscle and divided into four groups: control group; group A, Tachosil fibrin sealant patch; group B, Surgicel Fibrillar oxidized regenerated cellulose; and group C, Surgicel Snow oxidized regenerated cellulose. For the histologic analysis, biopsies were performed on the 3rd, 7th, and 27th days. RESULTS: The foreign body reaction was the weakest in group A and most significant in group C. The inflammatory cell infiltration was the weakest in group A and similar in groups B and C. Muscle regeneration differed among periods. The rats in group A were the most active initially, while those in group C showed prolonged activity. CONCLUSION: Tachosil and Surgicel administration increased inflammation via foreign body reactions, but the overall wound healing process was not significantly affected. The increased inflammation in the Surgicel groups was due to a low pH. We recommend using Tachosil, because it results in less intense foreign body reactions than Surgicel and faster wound healing due to the fibrin action.

Characterization of the distinct mechanism of agonist-induced canine platelet activation

Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled receptors (GPCRs) which transduce signals through various G proteins. Recent studies have elucidated the role of GPCRs and their corresponding G proteins in the regulation of events involved in platelet activation. However, agonist-induced platelet activation in companion animals has not been elucidated. This study was designed to characterize the platelet response to various agonists in dog platelets. We found that 2-methylthio-ADP-induced dog platelet aggregation was blocked in the presence of either P2Y₁ receptor antagonist MRS2179 or P2Y₁₂ receptor antagonist AR-C69931MX, suggesting that co-activation of both the P2Y₁ and P2Y₁₂ receptors is required for ADP-induced platelet aggregation. Thrombin-induced dog platelet aggregation was inhibited in the presence of either AR-C69931MX or the PKC inhibitor GF109203X, suggesting that thrombin requires secreted ADP to induce platelet aggregation in dog platelets. In addition, thrombin-mediated Akt phosphorylation was inhibited in the presence of GF109203X or AR-C69931MX, indicating that thrombin causes Gi stimulation through the P2Y₁₂ receptor by secreted ADP in dog platelets. Unlike human and murine platelets, protease-activated receptor 4 (PAR4)-activating peptide AYPGKF failed to cause dog platelet aggregation. Moreover, PAR1-activating peptide SFLLRN or co-stimulation of SFLLRN and AYPGKF failed to induce dog platelet aggregation. We conclude that ADP induces platelet aggregation through the P2Y₁ and P2Y₁₂ receptors in dogs. Unlike human and murine platelets, selective activation of the PAR4 receptor may be insufficient to cause platelet aggregation in dog platelets.

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as "fibrin glue" or "fibrin sealants", are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was "heterologous fibrin sealant". The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.(AU)

Anticoagulantes orais diretos para o tratamento da trombose venosa profunda: revisão de revisões sistemáticas / Direct oral anticoagulants for treatment of deep vein thrombosis: overview of systematic reviews

A number of limitations of standard therapy with warfarin for deep vein thrombosis (DVT) have been established. This overview of systematic reviews presents the baseline results for efficacy and safety of the new direct oral anticoagulants (DOACs) thrombin inhibitors, and activated factor X (Xa) inhibitors in patients with DVT. Searches were run on PubMed and the Cochrane Database of Systematic Reviews. Twenty-three studies were retrieved, and one systematic review was judged eligible. This review scored maximum according to AMSTAR criteria and included 7,596 patients for analysis of thrombin inhibitors and 16,356 patients for analysis of factor Xa inhibitors. The results of the meta-analysis indicate that DOACs are similar for DVT treatment when compared to standard treatment with warfarin. The incidence of major bleeding is somewhat lower in patients treated with factor Xa inhibitors and similar to standard therapy when treated with direct thrombin inhibitors

A terapia padrão com varfarina para a trombose venosa profunda (TVP) tem uma série de limitações já estabelecidas. Essa revisão de revisões sistemáticas elenca os principais resultados de eficácia e segurança dos anticoagulantes orais diretos (DOACs), inibidores da trombina e do fator X ativado (Xa), em pacientes com TVP. A pesquisa foi realizada nas bases PubMed e Cochrane Database of Systematic Reviews. Foram recuperados 23 estudos, e uma revisão sistemática foi considerada elegível. Essa revisão atingiu escore máximo no AMSTAR e incluiu 7.596 pacientes para análise dos inibidores da trombina e 16.356 pacientes para a análise dos inibidores do fator Xa. Os resultados da metanálise indicam que os DOACs apresentam eficácia similar à terapia padrão no tratamento da TVP. A incidência de sangramento maior é um pouco menor nos pacientes tratados com os inibidores do fator Xa e similar à terapia padrão no tratamento com inibidores diretos da trombina

Extraction, physical-chemical characterization and in vitro inhibitorypotential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds / Extração, caracterização físico-química e potencial inibitório in vitro da geração detrombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras

The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) wereanalyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p 100 kDa. These procedures,combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU(Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. Withrespect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TGby both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crudeSPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinctcrude SPs from Brazilian seaweeds.

O valor biotecnológico das macroalgas para ensaios de varredura de geração de trombina-GT pouco tem sido explorado usando polissacarídeos sulfatados-PSs como substitutos à heparina. Foramanalisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às característicasfísico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, aotempo de protrombina-TP e a GT. A extração com papaína rendeu (p 100 kDa. Esses procedimentos,combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. OsTTPAs foram dependentes da sulfatação dos PSs brutos e variaram de 2,81 (A. muscoides) a 21,30 UI (Halymenia sp.) vs. heparina (193 UI). O TP não foi alterado. Com respeito ao ensaio de GT, os PSs brutos modificaram, dependente de concentração e independentemente de massa molecular, GT pelas viasintrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca total usando PSs brutosde A. muscoides em paralelo à heparina (p < 0,05). A trombose in vitro é modulada diferencialmente porPSs brutos distintos de algas marinhas brasileiras.

Structural and physical-chemical analyses of sulfated polysaccharidesfrom the sea lettuce Ulva lactuca and their effects on thrombin generation / Análises estrutural e físico-química de polissacarídeos sulfatados da alface do mar Ulva lactuca e seus efeitos sobre geração de trombina

Ulva lactuca (Chlorophyceae) has biotechnologically-important sulfated-polysaccharides (Ul-SPs), but their potentials on thrombin generation (TG) are unknown. This study analyzed the structural and physicalchemical features of the Ul-SPs as modulators of TG. Proteolytic digestion yielded (13.13%) extract containing sulfate (20.43%) and total sugars (65.72%), besides ulvan consisting of rhamnose, xylose, glucose, glucuronic acid and α-/β-types glycosidic linkages as characterized by one-/two-dimensions nuclear magnetic resonance (NMR) experiments. Fractionation of the Ul-SPs by DEAE-cellulose chromatography yielded Ul-SP1 and Ul-SP2 (0.50 and 0.75 M NaCl, respectively) showing sulfation (15.72-18.04%) and total sugars (59.73-60.58%) consistent with the charge density pattern by combination of agarose/polyacrylamide gel eletrophoresis using sequential staining with toluidine blue and stains-all, although with slight differences in their sizes (40 and >100 kDa, respectively). By both activated partial thromboplastin time (APTT) and prothrombin time (PT) tests, anticoagulation of the fractions was virtually detected by APTT (0.39 and 0.43 IU, respectively) against heparin (193 IU). Fractions acted differently on both intrinsic/extrinsic pathways in TG using 60-fold diluted human plasma, with 50% efficacies up to 8.3 μg, whereas at high concentrations suggested intrinsic hypercoagulability since heparin abolished both systems at low amounts. Ul-SPs block TG, but predicting thrombosis in increasing doses.

Clorofícea Ulva lactuca possui polissacarídeos sulfatados (Ul-PSs) importantes biotecnologicamente, porém são desconhecidos seus potenciais sobre geração de trombina (GT). Analisaram-se as características estruturais e físico-químicas dos Ul-PSs como moduladores de GT. Digestão proteolítica rendeu (13,13%) extrato contendo sulfato (20,43%) e açúcares totais (65,72%), além de ulvana, como caracterizada por experimentos de ressonância magnética nuclear uni-/bi-dimensionais, consistindo de ramnose, xilose, glucose, ácido glucurônico e ligações glicosídicas tipos-α/-β. Fracionamento dos Ul-PSs por cromatografia de DEAE-celulose rendeu Ul-PS1 e Ul-PS2 (0,50 e 0,75 M de NaCl, respectivamente) mostrando sulfatação (15,72-18,04%) e açúcares totais (59,73- 60,58%) consistentes com o grau de densidade de carga por combinação de eletroforese em gel de agarose/poliacrilamida usando coramento sequencial com azul de toluidina e "stains-all", embora com diferenças quanto aos seus tamanhos (40 e >100 kDa, respectivamente). Por ambos os testes do tempo de tromboplastina parcial ativada (TTPA) e do tempo de protrombina, anticoagulação das frações foi detectada virtualmente pelo TTPA (0,39 e 0,43 UI, respectivamente) frente heparina (193 UI). Frações atuaram diferentemente sobre ambas as vias intrínsica/extrínsica na GT usando plasma humano diluído 60 vezes, com eficácias de 50% até 8,3 μg, enquanto em concentrações maiores sugeriram hipercoagulabilidade intrínsica visto que heparina aboliu ambos os sistemas em quantidades baixas. Ul-PSs bloqueiam GT, porém prevendo trombose em doses crescentes.

Endovascular treatment of pancreatitis-related gastrointestinal bleeding

Hemorrhage following pancreatitis may become complicated during the disease course and with possibly fatal consequences. Radiologic findings are as follows: the presence of hematomas, hemorrhagic pseudocysts, extravasation of contrast media or the formation of arterial pseudoaneurysms. The digital subtraction angiography findings are as follows: contrast extravasation due to arterial rupture; pseudoaneurysm formation; and luminal irregularity. A pseudoaneurysm is considered to be treated as soon as detected due to its risk of rupture. Endovascular management, which includes embolization using coils, N-butyl cyanoacrylate, gelatin sponge, stents, and thrombin, is an effective option for the treatment for pancreatitis-related bleeding.