Arritmias cardíacas na sala de emergência e UTI. Anticoagulação do paciente com FA aguda submetido a cardioversão: já podemos contar com os novos anticoagulantes orais? / Cardiac arrhythmias in the emergency room and ICU. Anticoagulation of the patient with acute af undergoing cardioversion: can we rely on the new oral antioagulants?

A fibrilação atrial (FA) é a arritmia cardíaca mais comum na população com uma prevalência de 1-2%, além disso, está associada a um risco, aproximadamente cinco vezes maior de acidente vascular cerebral do que na população em geral. A anticoagulação é a melhor maneira de prevenir os eventos tromboembólicos. A varfarina é utilizada há décadas como uma droga segura e eficaz, desde que rigorosamente controlada. Nos últimos anos, foram desenvolvidas novas classes de anticoagulantes orais: inibidores diretos da trombina e inibidores do fator Xa, conhecidos como anticoagulantes orais de ação direta (DOACs). Tanto a cardioversão elétrica quanto a cardioversão farmacológica estão associadas a um maior risco de eventos tromboembólicos durante o primeiro mês após o procedimento (5-7%). No entanto, com a utilização de nticoagulantes essa taxa é inferior a 1%. No presente artigo, faremos uma revisão das principais evidências científicas relacionadas ao uso da dabigatrana, rivaroxabana, apixabana e edoxabana durante a cardioversão e uma abordagem prática com o manejo antitrombótico em diferentes cenários clínicos (cardioversão em pacientes com uso prévio de DOACs, cardioversão em pacientes com FA com duração maior ou menor que 48 horas sem anticoagulação)

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a prevalence of 1-2%, and is associated with an almost 5-fold increase in the risk of stroke compared to the general population. Anticoagulation is the best way to prevent thromboembolic events. Warfarin has been used for decades as a safe and effective drug, provided it is strictly controlled. In recent years, new classes of oral anticoagulants have been developed: direct thrombin inhibitors and factor Xa inhibitors, known as direct oral anticoagulants (DOACs). Both electrical and pharmacological cardioversion are associated with an increased risk of thromboembolic events during the first month after the procedure (5-7%). However, with the use of anticoagulants, this rate is less than 1%. In this article, we will review the main scientific evidence related to the use of dabigatran, rivaroxaban, apixaban and edoxaban during cardioversion and a practical approach with antithrombotic management in different clinical scenarios (cardioversion of patients in previous use of DOACs, cardioversion of patients not using oral anticoagulants with episodes of AF longer or shorter than 48 h)

Uso de inhibidores directos del factor Xa en síndrome antifosfolípido: una serie de 7 casos / Use of factor Xa inhibitors in antiphospholipid antibodies syndrome: A serie of 7 cases

RESUMEN Introducción: El síndrome antifosfolípido se caracteriza por la presencia de anticuerpos contra fosfolípidos de membrana y manifestaciones clínicas, principalmente trombóticas y obstétricas. Su tratamiento se basa en la anticoagulación indefinida, generalmente con warfarina, la cual, por diversos factores, no siempre es factible por lo que es necesario el uso de terapias alternativas. Objetivo: Describirla experiencia con rivaroxabán en pacientes con síndrome antifosfolípido. Materiales y métodos: Estudio descriptivo en el que se evaluaron pacientes que cumplieron los criterios de Sydney de 2006 para síndrome antifosfolípido y que recibieron anticoagulación con rivaroxabán a dosis de 20 mg día en 2 hospitales de referencia en Medellín (Colombia), entre enero de 2012 y abril de 2015. Resultados: Se incluyeron 7 pacientes con una media de edad de 36,6 ± 10,8 arios (rango: 2355). De estos, 4 individuos tenían trombosis venosa, 5 trombosis arteriales, 5 anticuerpos anticardiolipinas positivos, 3 anticoagulante lúpico positivo, 2 pacientes tenían anti-f32 glicoproteína positivo y un paciente triple positividad de anticuerpos. La mediana de utilización de la warfarina fue de 15 meses (rango: 1-36). Las razones para el inicio de rivaroxabán fueron: sangrado (n = 2), rango subterapéutico de anticoagulación (n = 2), toxicodermia (n = 1), intolerancia gastrointestinal (n = 1) y retrombosis (n = 1). El tiempo de uso fue 17,9 ± 13,4 meses (rango: 3-34) y durante el periodo de seguimiento no se presentaron eventos adversos, pero sí 2 episodios nuevos de trombosis. Conclusión: El uso de inhibidores del factor Xa en una serie de pacientes con síndrome antifosfolípido e imposibilidad para el uso de warfarina mostró un adecuado perfil de seguridad; no obstante, hubo 2 episodios recurrentes de trombosis.

ABSTRACT Background: Antiphospholipid syndrome is an autoimmune disease with antibodies against membrane phospholipids with mainly thrombotic and/or obstetric manifestations. Its treatment is generally based on indefinite anticoagulation, usually with warfarin, and which, for various factors, is not always feasible, making it necessary to use alternative therapies. Objective: To describe the experience with rivaroxaban in patients with antiphospholipid syndrome. Materials and methods: A descriptive study was conducted on subjects that met the 2006 Sydney criteria for antiphospholipid antibodies syndrome and received anticoagulation with rivaroxaban at 20mg daily dose in 2 reference hospitals in Medellin, Colombia, between January 2012 and April 2015. Results: The study included 7 patients, with a mean age of 36±10.8 years (range 23-55). Four patients had venous thrombosis, 5 arterial, 5were positive for anticardiolipin antibodies, 3 reactive to lupus anticoagulant, 2 anti-β2 glycoprotein positive subjects, and one patient had triple antiphospholipid antibody positivity. The median time of warfarin use was 15 months (RIQ 1-36). The reasons for starting rivaroxaban were: bleeding (n = 2), sub-therapeutic coagulation ranges (n = 2), toxicoderma, gastrointestinal intolerance, and re-thrombosis (n = 1, each). The time of use was 17.9±13.4 months (range: 3-34). There were 2 recurrent cases of thrombosis during follow-up, and no adverse events. Conclusion: The use of factor Xa inhibitors in a series of patients with antiphospholipid syndrome and unable to use warfarin showed an adequate safety profile; however, 2 recurrent episodes of venous thrombosis occurred.

Evaluación de la anticoagulación con rivaroxaban, en pacientes con fibrilación auricular no valvular de reciente diagnóstico / Evaluation of oral anticoagulation with rivaroxaban, in patients with new onset non valvular atrial fibrillation

Background: Atrial fibrillation (AF) generates a hypercoagulable state with an increased thrombin generation and raised levels of thrombin-antithrombin complexes, which results in a high risk of stroke and thromboembolism. Aim: To evaluate the anticoagulant effect of rivaroxaban by anti-Xa factor activity and its correlation with thrombin-antithrombin complexes, thrombin generation and prothrombin time in patients newly diagnosed with non-valvular AF. Patients and Methods: Prospective study in patients with indication of anticoagulation. Demographic variables, cardiovascular risk factors, CHA2DS2-VASc and HAS-BLED scores were recorded. Blood samples were taken at baseline, at 3 and 24 hours after the administration of the drug and at 30 days. Rivaroxaban levels, anti-Xa activity, prothrombin time, thrombin generation and plasma levels of thrombin-antithrombin complexes were determined. Results: We studied 20 patients aged 76.3 ± 8.0 years (60% female) with a CHA2DS2-VASc score > 2 points. The anti-Xa factor activity correlated with rivaroxaban plasma levels at 3 hours (r = 0.61, p < 0.01), at 24 hours (r = 0.85, p < 0.01) and at 30 days (r = 0.99, p < 0.01), with prothrombin time at 3 hours (r = -0.86, p = 0.019) and at 30 days (r = -0.63, p = 0.02) and with a sustained decrease in thrombin generation at 30 days of follow-up (r = -0.74, p < 0.01). There was no correlation with thrombin-antithrombin complexes (r = -0.02, p = 0.83). Conclusions: Rivaroxaban consistently inhibited the mild pro-coagulant state found in newly diagnosed non-valvular AF patients through the first 24 hours and this effect was maintained at 30 days. Plasma levels of the drug correlated with anti-Xa factor activity, thrombin generation and prothrombin time

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Anticoagulant and factor Xa-like activities of Tityus discrepans scorpion venom

Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).

El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).

La importancia de la determinación del factor X activado para la anticoagulación con heparinas de bajo peso molecular: experiencia en el hospital san juan de dios, 2009-2011 (primer reporte) / The importance of the determination of factor X acivated for anticoagulation with low molecularweight heparins experience at the hospital san juan de dios. (first report) 2009-2011

La anticoagulación con heparinas de bajo peso molecular (HBPM) es una herramienta terapéutica fundamental para el tratamiento de la enfermedad tromboembólica. En el presente reporte se evidencia la importancia de cuantificar la actividad del anticuerpo antifactor X activado (Xa) para el monitoreo de la enoxaparina y analizar los grupos de pacientes en riesgo de tener niveles inferiores al terap‚utico. M‚todos: se estudiaron 34 pacientes adultos, anticoagulados con enoxaparina durante el periodo 2009-2011. Asimismo, se realizó un análisis descriptivo de las características demográficas y clínicas de todo los pacientes, en donde se indicaron las causas de la anticoagulación, la comorbilidades y el tipo de anticoagulación. Se midió la actividad anti-Xa 4 horas después de la administración de enoxaparina. Resultados: El promedio de edad de los pacientes fue de 62,3+17,7 años. Un 72,71 por ciento de los pacientes utilizaron enoxaparina como indicación para el síndrome coronario agudo. La comorbilidad más importante fue la combinación con la hipertensión arterial. El aclaramiento renal promedio fue de 62,47 ml/min, solamente tres pacientes tuvieron un aclaramiento menor a 30 ml/min; un 44,1 por ciento de los pacientes eran obesos. El 55,9 por ciento de los pacientes tuvo niveles anti-factor Xa dentro del rango terapéutico y un 35,3 por ciento tuvo valores de anti factor Xa profilácticos. Conclusión: El manejo del paciente adulto que recibe terapia anticoagulación con HBPM presenta una alta complejidad, hecho que se ve reflejado tanto a su perfil demográfico como clínico. También, se considera importante contar con la determinación del factor anti Xa para el monitoreo de HBPM para cierto grupo de pacientes vulnerables y con ello lograr el efecto deseado con esta terapia, debido a que existe un alto porcentaje de pacientes con niveles fuera del rango terapéutico.

Anticoagulation is an important therapeutic tool for patients with thromboembolic disease who receive therapy with lowmolecular weight heparins (LMWH). This report gives evidence about the importance of determining the activity of Anti-Xaactivity for monitoring enoxaparin and for identifying those patients who need this analysis based on some risk factors.Methods: We studied 34 adult patients who received enoxaparin as anticoagulation therapy during the period 2009-2011. We performed a descriptive analysis of demographic and clinical characteristics of all patients, indicating thereasons for anticoagulation, comorbidities and type of anticoagulation. We determined the anti-Xa activity 4 hours afteradministration of enoxaparin.Results: The mean age of patients was 62,3 + 17,7 years, regardless of gender. 72,7% of patients received enoxaparin astherapy for an acute coronary syndrome. The most frequent comorbidity was hypertension. The average of renal clearancewas 62,47 ml/min, only three patients had a renal clearance below 30 ml/min. 44,1% of the patients were obese. 55, 9%of patients were within therapeutic levels of anti-Xa activity and 35,3% of patients had an anti-Xa activity considered asprophylactic.Conclusion: The management of adult patients receiving anticoagulation therapy with LMWH is complex and it isreflected in their demographic and clinical characteristics. It is important to determine Anti-Xa activity to monitor the useof enoxaparin as anticoagulant therapy because of the high variability found in certain groups of patients.

Nuevos anticoagulantes orales / New oral anticoagulant drugs

Thromboembolic disease (TED) is the leading cause of morbidity and mortality worldwide. The hallmark of oral long-term anticoagulant therapy has been the use of vitamin K antagonists, whose anticoagulant effect is exerted inhibiting vitamin K epoxide reductase. Warfarin and acenocoumarol are the most commonly used. In the last five years several new drugs for long term anticoagulation have been developed, which can inhibit single clotting factors with the purpose of improving drug therapeutic range and, ideally, minimizing bleeding risks. This review addresses the state of the art on the clinical use of inhibitors of activated factor X and thrombin.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10 percent of mammalian heparin and about 5 percent as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5 percent (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4 percent, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13 percent without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Estudo comparativo entre os anticoagulantes pentassacarídeo e heparina sódica em cães (canis familiares) com insuficiência renal aguda induzida por gentamicina submetidos à hemodiálise / Comparative study between anticogulants polysaccharides and sodium heparin in dogs (canis familiares) with acute renal failure induced by gentamicin submitted renal dialysis

Como objetivo de comparar a segurança e a eficácia da anticoagulação e hemodiálise (HD) com heparina sódica e com pentassacarídeo, foram estudados 26 cães com insuficiência renal aguda induzida por gentamicina (Omg/kg/dia) submetidos a dois protocolos: Grupo 1, composto de 13 animais anticoagulados com heparina (10mg/kg, bolus i.v.), e Grupo 2, com 13 animais anticoagulados com pentassacarídeo (800UI/Kg i.v). Foi utilizado esquema diário de HD, com 60 minutos e fluxo sanguíneo de 3 a 5 ml/kg/minuto, com dialisador de polissulfona (0,4 m2), realizando-se cinco sessões por animal. Os seguintes parâmetros foram avaliados: sinais vitais (período intradialitico); níveis pré e pós dialiticos de uréia, creatinina e potássio; provas de coagulação PA, TP , TCA e Anti Xa); avaliação da eficácia dialítica, através da percentagem de remoção da uréia (PRU); volume de preenchimento do dializasor (priming); avaliação hematológica (hematócrito, contagem de plaquetas, e leucócitos pré e pós dialíticos). Os resultados mostraram que a HD reduziu significantemente os níveis de uréia e potássio em ambos os grupos e não se associou as alterações significantes nos sinais vitais durante o procedimento. Os testes específicos de coagulação (TTPA, Anti Xa) confirmaram a efetividade dos protocolos utilizados. A heparina determinou aguda e acentuada elevação do TCA para até 4 vezes o valor basal no 5° minuto de HO (T5), com redução progressiva até final das sessões. Diferentemente, o pentassacarídeo elevou de modo significante, porém níveis e aproximadamente 1,5 valor basal (T5), sem alterações significantes até o final das sessões. Os dois protocolos se associaram as medidas semelhantes a PRU e do priming. A HD induziu redução contagem de leucócitos e de plaquetas em todas as sessões sem diferenças entre grupos. Houve ainda, redução do hematócrito na 4a sessão do Grupo 2(...)

Targeting exosites on blood coagulation proteases

A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os exosítios das enzimas da coagulação. Dois exemplos são discutidos nesta curta revisão. A Botrojaracina é uma proteína derivada de veneno de serpente que se liga aos exosítios 1 e 2 da trombina. A formação do complexo impede várias atividades da trombina dependentes do exosítio 1 incluindo a clivagem do fibrinogênio e a ativação plaquetária. A Botrojaracina também interage com o proexosítio 1 da protrombina diminuindo a ativação do zimogênio pelo complexo protrombinase (FXa/FVa). O ixolaris é um inibidor com dois domínios Kunitz obtido da glândula salivar de carrapato, homólogo ao inibidor da via do fator tecidual. Recentemente foi demonstrado que o ixolaris se liga ao exosítio de ligação à heparina do FXa, impedindo o reconhecimento da protrombina pela enzima. Além disso, o ixolaris interage com o FX, possivelmente através do proexosítio de ligação à heparina. Diferentemente do FX, o complexo ixolaris-FX não é reconhecido como substrato pelo complexo tenase intrínseco (FIXa/FVIIIa). Nós concluimos que esses inibidores podem servir como ferramentas para o estudo dos exosítios da coagulação, assim como protótipos para novas drogas anticoagulantes.

Estudos estruturais e funcionais dos inibidores de proteases Bdellastasina e Ghilantina da família das antistasinas / Structural and functional studies on the antistasin family inhibitors, Bdellastasin and Ghilanten

Sanguessugas e outros animais hematófagos têm sido descritos como fonte d diversas Proteínas com propriedades anticoagulantes. Algumas destas proteína fazem parte da família Antistasina de inibidores de proteases. Ghilantina bdellastasina são membros desta família com atividade inibitória sobre fator Xa plasmina, respectivamente. O primeiro objetivo deste trabalho foi concluído com o estabelecimento do sistema de expressão em Pichia pastoris da proteína bdellastasina. O êxito obtido na expressão do inibidor demonstrou que o sistema poderia, em princípio, ser usado na expressão de outras proteínas da família. O sistema mostrou-se eficiente não somente pelo bom nível de expressão da proteína heteróloga, mas também pela facilidade de purificação. O estudo das relações estrutura-função que definem as especificidades das interações enzima-inibidor entre fator Xa e ghilantina foram alvo da segunda parte deste trabalho. Para tal, foram produzidos híbridos da bdellastasina substituindo-se regiões desta por outras da ghilantina, supostamente responsáveis pela interação com O fator Xa, com a finalidade de produzir um inibidor de fator Xa. Foram construídos cinco híbridos bdellastasina-ghilantina baseados em dados de estrutura primária e especificidade e posteriormente mais seis variantes baseados em dados da estrutura tridimensional dos inibidores antistasina e bdellastasina. A construção destes últimos teve como objetivo a introdução na bdellastasinà dos resíduos da ghilantina responsáveis pela interação com o exosítio do fator Xa, além de alterar a conformação global da bdellastasina para permitir uma interaçâo adequada. A construção dos mutantes foi feita por síntese de genes, cassete mutagênese e técnicas de PCR e a expressão realizada em Pichia pastoris com subseqüente purificação e estudos funcionais. Os híbridos produzidos, com exceção dos mutantes SRY, mantiveram a capacidade e inibição de tripsina, mas não apresentaram nenhum efeito sobre o fator Xa. Considerando-se que grandes regiões foram substituídas, é razoável pensar que a estrutura terciária final dos híbridos apresente impedimento no ajusto perfeito da enzima com o inibidor. Na última parte deste trabalho foram estudadas as funções dos domínios isolados da ghilantina na atividade global do inibidor. Com este propósito os genes da ghilantina e de seus domínios isolados foram construídos e expressos em P. pastoris....(au)