Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.

Epsilon-aminocaproic acid prevents high glucose and insulin induced-invasiveness in MDA-MB-231 breast cancer cells, modulating the plasminogen activator system.

Obesity and type II diabetes mellitus have contributed to the increase of breast cancer incidence worldwide. High glucose concentration promotes the proliferation of metastatic cells, favoring the activation of the plasminogen/plasmin system, thus contributing to tumor progression. The efficient formation of plasmin is dependent on the binding of plasminogen to the cell surface. We studied the effect of ε-aminocaproic acid (EACA), an inhibitor of the binding of plasminogen to cell surface, on proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and plasminogen activation system, in metastatic MDA-MB-231 breast cancer cells grown in a high glucose microenvironment and treated with insulin. MDA-MB-231 cells were treated with EACA 12.5 mmol/L under high glucose 30 mmol/L (HG) and high glucose and insulin 80 nmol/L (HG-I) conditions, evaluating: cell population growth, % of viability, migratory, and invasive abilities, as well as the expression of uPA, its receptor (uPAR), and its inhibitor (PAI-1), by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, MMP-2 and MMP-9 mRNAs were evaluated by RT-PCR. Markers of EMT were evaluated by Western blot. Additionally, the presence of active uPA was studied by gel zymography, using casein-plasminogen as substrates. EACA prevented the increase in cell population, migration and invasion induced by HG and insulin, which was associated with the inhibition of EMT and the attenuation of HG- and insulin-dependent expression of uPA, uPAR, PAI-1, MMP-2, MMP-9, α-enolase (ENO A), and HCAM. The interaction of plasminogen to the cell surface and plasmin formation are mediators of the prometastasic action of hyperglycemia and insulin, potentially, EACA can be employed in the prevention and as adjuvant treatment of breast tumorigenesis promoted by hyperglycemia and insulin.

Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degradation.

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate d-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Effects of Tissucol and epsilon aminocaproic acid in the healing process following dental extraction in dehydrated rats.

A histological study was conducted of the alveolar bone healing process following tooth extraction of dehydrated rats after the implantation of fibrin adhesive (TISSUCOL) associated to previous irrigation of the wound with a 5% epsilon aminocaproic acid solution (EACA). Seventy two rats were used, divided into three groups receiving different treatments after the surgical procedure. In group I, the gingival mucosa was sutured after extraction of the right upper incisor. In groups II and III, chronic dehydration was produced by water deprivation for 9 days (3 days in the preoperative period and 6 days in the postoperative period). In the animals of Group II, after tooth extraction, the gingival mucosa was sutured in the same way as performed in group I. In group III, after extraction, the dental socket was irrigated with 5% EACA, followed by implantation of the fibrin adhesive (TISSUCOL). The mucosa was sutured in the same way as performed in the other groups. At 3, 7, 15 and 21 postoperative days, the animals were sacrificed in number of 6 for each group. Specimens containing the dental socket were removed and fixed in 10% formalin and decalcified in an equal part formic acid and sodium citrate solution. After routine processing, the specimens were embedded in paraffin for microtomy. We obtained 6 microm semi-serial slices that were stained with hematoxylin and eosin for histological evaluation. The results showed that the water deprivation in the pre- and postoperative periods caused a delay in the alveolar bone healing process. The use of the fibrin adhesive (TISSUCOL) produced an improvement in the fibrinolytic picture caused by dehydration.

Uso del ácido épsilon-aminocaproico para disminuir el sangrado en la cirugía de escoliosis: ensayo clínico / Use of epsilon-aminocaproic acid to reduce bleeding in scoliosis surgery: clinical trial

Introducción: La anestesia para la corrección de las deformidades del raquis ha sido motivo de preocupación debido a la gran agresión al paciente, fundamentalmente por las cuantiosas pérdidas sanguíneas que se producen. Actualmente, el enfoque farmacológico para la prevención del sangrado ha cobrado gran interés para muchos autores, empleándose fármacos antifibrinolíticos como el ácido épsilon-aminocaproico. Objetivos: Evaluar el efecto de este fármaco como agente fibrinolítico en la corrección quirúrgica de la escoliosis y su influencia sobre los resultados de los factores que regulan la hemostasia en el período perioperatorio. Material y método: Se realizó un ensayo clínico en 42 pacientes subdivididos secuencialmente en dos grupos. A los pacientes del Grupo I se les administró una dosis de 5 g de EACA previo a la incisión de la piel, seguido por la infusión continua de 1 g/h hasta el cierre de la piel. El Grupo II no fue tratado con EACA. Se realizó coagulograma durante el preoperatorio, el intraoperatorio y el postoperatorio inmediato. Se cuantificó el sangrado intraoperatorio (ml) por aspiración del campo operatorio y por el pesaje de las compresas. Durante la estancia en la sala de recuperación se evaluó el sangrado postoperatorio a las 2 y a las 24 horas, clasificándolo en escaso, moderado y severo. Resultados: En todos los momentos el sangrado fue mayor en el Grupo II. El coagulograma fue normal en un mayor porcentaje de pacientes del Grupo I, a la vez que los valores de hematocrito fueron mayores y menor el número de transfusiones. Existieron diferencias significativas entre ambos grupos (p < 0,05). Conclusiones: En todos los momentos los valores anormales de coagulograma y de sangrado fueron mayores en el Grupo II; el ácido épsilon-aminocaproico resultó ser un fármaco eficaz en la prevención del sangrado intraoperatorio en pacientes operados de escoliosis. No fueron encontrados efectos adversos con el uso de esta droga. (AU)

Acido Epsilon-Aminocaproico y sangrado postoperatorio en cirugía cardiovascular con CEC : Premio Segundo Congreso Latinoamericano de tecnología extracorpórea / Epsilon-aminocaproic acid and posoperative bleeding in cardiovascular surgery with CPB

Varios trabajos han señalado la importancia de la prevención de la activación fibrinolítica por la CEC para reducir el sangrado en el postoperatorio de la cirugía de corazón. En este estudio se analizó una población de 135 pacientes coronarios. En 68 pacientes coronarios consecutivos, se administró 6 gramos de EACA endovenoso en bolo, luego de la inyección de heparina en la aurícula derecha (300 U por Kg) y previo al bypass cardiopulmonar. Esta población tratada con EACA llamada grupo T, se comparó en forma retrospectiva con una población no tratada con esta droga, llamada grupo NT, significativamente similar en el tipo de patología así como en los procedimientos realizados. En este grupo de pacientes (grupo T) se confirma la disminución del sangrado postoperatorio y del requerimiento transfusional. Se evaluó la influencia de esta terapéutica en el costo de la cirugía, teniendo en cuenta el elevado precio de la aprotinina analizado por Hardy y col. (EACA $ 5.85 vs Aprotinina $ 239,54 a $ 717,42), el costo de los productos de la sangre ya puesto en evidencia por Arom y Emery, y el tiempo total de internación (tiempo desde el día de cirugía hasta el día de alta del hospital) que fueron las variables consideradas en este estudio. En el grupo T se observó una reducción del sangrado promedio del 46 por ciento sobre los valores del grupo NT (grupo NT 797,87 ml ñ 553,06 ml vs grupo T 425,02 ml ñ 262,42 ml para una p = 0,0001). Asimismo, los requerimientos transfusionales disminuyeron en un 31 por ciento promedio para el grupo T (grupo NT 1261,19 ml ñ 555,43 ml vs grupo T 864,10 ml ñ 299,99 ml para una p = 0,0001). El tiempo de internación postoperatorio fue un 20 por ciento menor para el grupo T (grupo NT 11,22 días ñ 3,67 días vs grupo T 8,95 días ñ 3,07 días para una p = 0,0001). Conclusiones: la profilaxis de la activación fibrinolítica en la cirugía de corazón disminuye el sangrado postoperatorio, el requerimiento transfusional y el tiempo total de internación. En ese sentido el EACA es tan útil como la aprotinina, y más económica, para reducir el costo global de la cirugía de revascularización miocárdica. (AU)

Osseous regeneration in the presence of fibrin adhesive material (Tissucol) and epsilon-aminocaproic acid (EACA).

The effects of Tissucol and Tissucol/EACA on bone healing were evaluated histologically. Experimental defects were made in both tibias of 25 rats. Test materials were placed in defects in right tibias and left tibias served as control. Five animals in each group were killed at 1, 3, 7, 14 and 21 days after surgery. Results showed that: a) Tissucol did not interfere with connective and osseous tissue formation; b) Tissucol allowed new bone formation; c) Tissue residues in Tissucol groups in sections of 21-day specimens did not impair healing; d) Tissucol/EACA was usually completely resorbed and healing was complete 21 days after surgery in the Tissucol/EACA group.

Effects of epsilon aminocaproic acid on primary hemostasis.

These studies examined the effect of an oral dose of epsilon-aminocaproic acid (EACA) on primary hemostasis. Bleeding time tests (with and without the use of a blood pressure cuff) were measured before and 2 h following EACA in 56 patients with mild bleeding disorders and/or thrombocytopenia. Preliminary studies evaluated the reproducibility of these tests in 13 patients who had bleeding times (cuff) ranging from 8.0 to greater than 20 min. Their replicate bleeding time values with cuff agreed within 2.5 min and those without cuff within 3 min. Therefore, the 56 study patients were considered to have had no change in their bleeding times after EACA, if their bleeding time with cuff was +/- 2.5 min and/or their nonoccluded value was +/- 3 min of their baseline values, respectively. An isolated increase in bleeding times was observed in 6 of 56 (11%) patients. All 6 had myelodysplasia associated with long bleeding times; their nonoccluded values increased by 5-14 min. Of the 56 study patients, 54% showed a decrease in their bleeding times following EACA. The changes were evident with venostasis in 18 of 30 (60%) and without venostasis in 12 (40%) patients. These studies suggest that EACA may improve primary hemostasis in some patients with prolonged bleeding times.