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.

Effect of estrogen-progestin hormonal replacement therapy on blood coagulation and fibrinolysis in postmenopausal women

OBJECTIVE: To evaluate antithrombin III (AT), thrombin (Fragment 1+2 [F1+2] and thrombin-antithrombin [TAT]) generation markers, as well as other coagulation parameters, such as prothrombin time, partial activated thromboplastin time, thrombin time, fibrinogen, euglobulin lysis time, and platelet count, in postmenopausal women after hormonal therapy. STUDY DESIGN: Forty-five patients who received either 0.625 mg/day unopposed oral conjugated equine estrogen (CEE), 0.625 mg/day oral CEE plus medroxyprogesterone acetate (MP), or 50 µg/day transdermal 17beta-estradiol plus MP, were included. Tests were performed before (T0) and after 3 (T3), 6 (T6) and 12 (T12) months of treatment. AT was determined by an amidolytic method, whereas F1+2 and TAT complex were measured by ELISA. RESULTS: There was a significant reduction in the AT level of patients who received oral CEE plus MP at T3. There was no AT reduction in patients taking either oral CEE alone or transdermal 17beta-estradiol plus MP. F1+2 increased in all patients, but it reached statistical significance only in patients receiving transdermal 17beta-estradiol MP at T3. CONCLUSIONS: The CEE associated with MP treatment may reduce AT levels, whereas unopposed CEE or transdermal 17beta-estradiol plus MP does not change AT. These changes might not be clinically relevant in the general population; however, hormonal replacement therapy may increase the risk of thrombosis in women with congenital or acquired thrombophilia.

OBJETIVO: Avaliar os marcadores antitrombina III (AT), fragmento 1 + 2 da trombina (F1+2) e complexo trombina-antitrombina (TAT), bem como outros parâmetros da coagulação, como tempo de pró-trombina, tempo parcial de tromboplastina ativado, tempo de trombina, fibrinogênio e tempo de lise da euglobulina em mulheres na pós-menopausa após terapia hormonal. DESENHO DO ESTUDO: Foram incluídas 45 voluntárias que receberam estrogênios conjugados eqüinos (ECE) 0,625 mg/dia, isoladamente ou associado ao acetato de medroxiprogesterona (AMP) ou usaram o 17beta-estradiol (50 µg/dia) transdérmico com AMP. Os exames foram realizados antes do tratamento (T0) e após três (T3), seis (T6) e doze (T12) meses após o início do tratamento. AT foi avaliada pelo método amidolítico, enquanto que o F1+2 e o complexo TAT por ELISA. RESULTADOS: Houve redução significante nos níveis de AT em pacientes que receberam ECE associado ao AMP no T3. Não houve redução na AT em mulheres que usaram ECE isoladamente ou aquelas com 17beta-estradiol transdérmico e AMP. O F1+2 aumentou em todos os grupos, mas apenas o grupo com 17beta-estradiol transdérmico e AMP apresentou diferença significante durante o T3. CONCLUSÕES: A associação de ECE e AMP pode reduzir os níveis de AT, enquanto ECE isoladamente ou 17beta-estradiol transdérmico com AMP não modificam-o acentuadamente. Essas alterações poderiam ser mais relevantes clinicamente na análise populacional. Todavia, a terapia de reposição hormonal aumentaria o risco de trombose em mulheres com trombofilia prévia congênita ou adquirida.

Proteína C reactiva y fibrilación auricular: evidencia de un proceso inflamatorio en la iniciación y perpetuación de la arritmia / C-reactive protein and atrial fibrillation: evidence for an inflammatory process in the initiation and perpetuation of the arrhythmia

Propósitos del estudio: Determinar la existencia de un proceso inflamatorio en los pacientes (ptes) con fibrilación auricular (FA) y su eventual relación con la trombogénesis. Métodos: Se incluyeron 109 pts con valvular, tanto crónica (n =40) como paroxística (n =69) sin tratamiento anticoagulante. Se determinaron niveles de proteína C –reactiva (PCR), niveles de complejo trombina-antitrombina (TAT) y parámetros clínicos y ecocardiográficos predictores de embolia y, exámenes generales de laboratorio. Resultados: La edad promedio del grupo fue 67 ± 14 años. Los niveles de PCR fueron de 1,0 ± 1,4 mg/dl en los FA paroxística y 1,1 ± 2,4 mg/dl en los con FA crónica versus 20 controles). Los niveles de TAT confirmaron la existencia de un estado protrombótico, pero se demostró asociación entre PCR y TAT. En el análisis multivariado, la PCR se relacionó a otros marcadores de inflamación sistémica (VHS y recuento de glóbulos blancos) y la presencia de disfución VI (p =0,02). A 30 días y 1 año, se constató una caída significativa de los niveles de PCR en el grupo FA paroxística. Finalmente, los niveles de PCR resultaron ser predictores de la mantención de ritmo sinusal a 1 año (PCR =1,2 ± 1,8 mg/dl en ptes con FA versus 0,5 ± en los con ritmo sinusal a 1 año, p = 0,048). Conclusiones: Existe evidencia de un estado inflamatorio en los ptes con FA no valvular. Su persistencia se asocia a la matención de la arritmia en la evolución alejada a 1 año.

The evaluation of clotting time in bovine thrombin, reptilase©, and thrombin-like fraction of Crotalus durissus terrificus venom using bovine, equine, ovine, bubaline and human cryoprecipitates

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase©, and bovine thrombin of fibrinogen polls on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows: 12 samples from humans, 9 from bovine, 10 from equine, 10 from ovine and 7 from bubaline. The results obtained showed average levels of 375.50 mg per cent for humans, 218.33 mg per cent for bovine, 240.80 mg per cent for equine, 267.70 mg per cent for ovine and 664.00 mg per cent for bubaline. Upon the formation of pools of human and animals fibrinogens, the following results were obtained: 435 mg per cent for humans, 444 mg per cent for bovine, 337 mg per cent por equine, 390 mg per cent for ovine and 530 mg per cent for bubaline. Statistical analysis (using the analysis of variance for entirely randomized experiment for the calculation of F statistics) demonstrated that the bubaline fibrinogen level was higher than that of human, and both were higher than those of ovine, equine, and bovine. Clotting times were determined using different dilutions of bovine thrombin, thrombin-like fraction of Crotalus durissus terrificus venom, and Reptilase©. Comparing these clotting times, results for human and bovine were found to be very similar, whereas using equine, ovine and bubaline the results above a dilution of 1:3 were markedly different. The results obtained permitted the following conclusions to be drawn show that: 1) bovine thrombin presented better interactivity with fibrinogen extracted both from human and bovine cryoprecipitates; 2) there was similar behavior when bovine thrombin was substituted for Reptilase© and for the thrombin-like fraction of Crotalus durissus terrificus venom; 3) cryoprecipitate from bovine can, in special circumstances, substitute human cryoprecipitate in medical practice; 4) human and bovine cryoprecipitates can be used with both Reptilase© and Crotalus durissus terrificus fractions using a dilution up to 1:5; 5) the use of bovine cryoprecipitate can be recomended using either bovine thrombin, Reptilase©, or thrombin-like fraction of Crotalus durissus terrificus venom.

Presencia selectiva de trombina en pulmones de pacientes con metástasis pulmonar / Selective presence of thrombin in the lungs of patients with pulmonar metastasis

La trombina, componente clave de la cascada de la coagulación, podría participar en el desarrollo de la metástasis pulmonar, al incrementar la adhesión: plaquetas-células tumorales in vitro y el número de metástasis in vivo. Se midieron cromogénicamente (Sustrato específico de trombina, S-2238) las concentraciones de trombina en lavados broncoalveolares de 20 pacientes con metástasis pulmonares y se compararon con lavados broncoalveolares de 20 pacientes con cáncer de pulmón y 20 testigos. La mediana de la concentració de trombina en los lavados broncoalveolares de los pacientes con metástasis pulmonar fue 5.4 x 10-9 M 82.5 x 10-9 M-13 x 10-9 M). Esto representó un aumento de 10 y 100 veces en las concentraciones de trombina, en comparación con los de los lavados de pacientes con cáncer de pulmón (0.6 x 10-9 M; 0.2 x 10-9 M-2.2 x 10-9 M) y los testigos (0.6 x 10-9 M; 0.02 x 10-9 M-0.4 x 10-9 M) respectivamente (p<0.02). Estos resultados demuestran que la trombina está presente, en forma selectiva, en los pulmones de pacientes con metástasis pulmonar, y que podría estar involucrada en su desarrollo

La trombina: ¿Un nuevo factor útil en el diagnóstico temprano de metástasis pulmonar? / The trombin: Anew useful factor in the early diagnosis of pulmonary metastasis?

Los eventos celulares tempranos que inducen una metástasis pulmonar son desconocidos. La trombina, componente clave de las cascada de la coagulación, podría estar involucrada en el desarrollo de este padecimiento, ya que incrementa la adhesión plaquetas-células tumorales in vitro y el número de metástasis in vivo. Para evaluar si la trombina podría ser considerada como un marcador en su diagnóstico, mediamos los niveles de trombina en elevados broncoalveolares (LBA) de 20 pacientes con dicho padecimiento y los comparamos frente a los de 20 pacientes con cáncer de pulmón y 20 controles. La mediana de la concentración de trombina en los lavados de los pacientes con metástasis fue 5.4 x 10-9 M (2.5 x 10-9 M - 13 x 10-9 M). Esto presentó un incremento de 10 y 100 vece en los niveles de trombina, en comparación con los encontrados en los lavados de pacientes con cáncer de pulmón (0.6 x 10-9 M; 0.2 x 10-9 M-2.2 x 10-9 M) y los controles (0.06 x 10-9 M; 0.02 x 10-9 M - 0.4 x 10-9 M), respectivamente (p < 0.02). Lavados de pacientes con metástasis y cáncer de pulmón incrementaron la proliferación celular en un 34.9 por ciento (1.5 - 58.3 por ciento) (p< 0.02). La hurudina, un inhibidor de la trombina, disminuyó únicamente la proliferación inducida por los lavados brincoalveolares de pacientes con metástasis en un 63.8 por ciento (p < 0.05). Estos resultados demuestran que la trombina está presente en forma selectiva en los pulmones de pacientes con metástasis pulmonar, y que podría estar involucrada en el desarrollo de este padecimiento

Fibrinopéptido A y cardiopatía isquémica aguda: valor diagnóstico y pronóstico: comunicación preliminar / Fibrinepectide A and acute ischaemic heart: diagnostic and prognostic value: preliminary communication

Debido a que la cardiopatía isquémica continúa siendo una de las primeras causas de mortalidad en nuestro país y a nivel mundial, es lógico entender el auge que han tenido los trabajos que involucren marcadores paraclínicos del fenómeno de la coagulación, debido al papel fundamental de ésta, en la fisopatología de dicha enfermedad, de allí nuestro interés en la investigación del rol que desempeña el fibrinopéptido A en el diagnóstico y pronóstico de la cardiopatía isquémica aguda. Por lo cual se realizó un estudio prospectivo, en donde se estudiaron 17 pacientes con cardiopatía isquémica aguda (13 angor y 4 IM), que ingresaron a la UCC del Hospital "Domingo Luciani", durante el lapso agosto-septiembre del año 94, a todos ellos se les tomó muestra al ingreso, a las 6 y a las 12 horas de su llegada al hospital, para derteminar los niveles plasmáticos de Fibrinopéptido A (marcador de la actividad de la trombina), así como también estudios paraclínicos convencionales (ECG, Enzimas Cardíacas, Rx de Tórax, ematología completa, Urea Creatinina, PTT, PT, Plaquetas). Analizando las relaciones existentes entre las diferentes variables mediante la técnica de Anova y T. Test; se evidencia que existe diferencia estadísticamente significativa entre el grupo control y los pacientes con cardiopatía isquémica aguda (IM y Angor) en los niveles de fibrinopéptido A, dando valores de P=0.036 demostrando de esta manera, la utilidad de este marcador en la cardiopatía isquémica aguda

Thrombin-antithrombin activity and C1-inactivator in type I diabetics

Previous studies on Diabetes mellitus Type 1 ended in a controversy as to whether there was an increased or decreased fibrinolysis. Also whether fibrinolysis if present was primary or secondary to a hypercoagulable state. The results of screening tests of fibrinolysis are frequently indecisive. C[1]-Inactivator [C[1]-1] [%] as inhibitor of fibrinolysis and thrombin anti-throbmin [TA T] [ug/ml] complex as a sensitive index of the coagulation cascade were determined in 41 male patients with type I diabetes mellitus without complications and in 25 patients of the same disease with microvascular complications [retinopathy, nephropathy and/or neuropathy]. The effect of duration of the disease and the response of the disease to control, were studied. In spite of the fact that screening results of fibrinolysis were not decisive, C[1] -1 and TA T were specific and indicative. TA T was higher in complicated cases [m 9.7 +/- 2.1 SD] than in non-complicated ones [m 5.6 +/- 2.7 SD]; and in uncontrolled complicated cases [m 11.3 +/- 3.0 SD] than in controlled ones [m 9.7 +/- 2.1 SD]. The effect of control was evident also in non-complicated cases where TAT was higher in uncontrolled [m 6.2 +/- 1.9 SD] versus controlled ones [5.6 +/- 2.7 SD]. The longer the duration of the disease the higher the level of TA T, where it was [m 7.2 +/- 2.1 SD] in 1-2 yrs duration and reached m 10.2 +/- 3.1 SD in 5-9 yrs duration. C[1]-l was also higher in complicated diabetes [m 118.6. +/- 18.5 SD] than in non-complicated cases [m 107.5 +/- 16.0 SD] and in both complicated uncontrolled cases [116.3 +/- 15.0 SD] than in complicated controlled ones [118.6 +/- 18.5 SD] also in non-complicated uncontrolled cases [m 115.0 +/- 18.8 SD] than in controlled ones [m 107.5 +/- 160 SD]. The results point to an increased rate of fibrinolysis in response to increased hypercoagulable state in type I Diabetes Mellitus and this is more accentuated the longer the duration of the diseases and that both improve on a better control of the disease

Fibrinopeptide A: an indicator of increased thrombin activity in diabetes mellitus

To determine the role of Fibrinopeptide [FPA] in vascular complication in diabetics, it is estimated by a sensitive RIA technique in7 control subjects and 28 diabetics classified according to their line of treatment into IDD and NIDD who were further subdivided into those with vascular complications and those without vascular complications. Results showed that diabetics as a whole had a significantly higher mean values of both fasting and postprandial FPA compared to controls. A significant difference for fasting FPA was noticed between non complicated IDD and NIDD [P < .05], between complicated IDD and non complicated NIDD [P < 0.01] and between non complicated NIDD and complicated NIDD [P < 0.01]. Postprandial FPA was significantly higher in non complicated IDD, non complicated NIDD and complicated NIDD compared to controls [P > 0.05, P > 0.01 and P < 0.05 respectively]. Uncomplicated diabetics had a significantly higher mean fasting FPA level [P < 0.05] compared to complicated but not regarding to postprandial FPA. Finally a significant positive correlation was observed between fasting FPA versus fasting blood glucose in non complicated NIDD [r = 0.85, P < 0.05] and complicated NIDD [r = 0.86, P < 0.05], also between postprandial FPA and both fasting FPA [r = 0.94, P < 0.01] and fasting blood glucose [r = 0.91, P < 0.01] in complicated NIDD. It may be concluded that elevated FPA level in diabetes is considered as a sensitive specific parameter of in vivo thrombin activation in diabetes [Hypercoagulable state]. This elevation is associated with diabetes more than being associated with vascular complication