Desarrollo de un nuevo ensayo de actividad fibrinolítica
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Fecha
2010
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
El estudio de la Fibrinólisis se ha centrado principalmente en la medición de niveles plasmáticos de proteínas pro-fibrinolíticas y de sus inhibidores. Los ensayos de medición global de la actividad fibrinólitica que actualmente se utilizan (tiempo de lisis de euglobulinas, lisis del coagulo en solución salina· u otros), son laboriosos, de baja sensibilidad y no toman en cuenta el rol que juegan las plaquetas en el proceso fibrinólitico. Basado en lo anterior, nuestro objetivo fue desarrollar un nuevo ensayo de medición global de la fibrinólisis, que incluya la participación de plaquetas. Metodológicamente nos basamos en tiempo de lisis del coagulo en plasma (TLC) desarrollado por Lisman T (2005).
El TLC se modificó incorporando plasma rico en plaquetas (PRP) estimulado con agonistas plaquetarios Para inducir la coagulación se agregó cloruro de calcio y tP A para acelerar la lisis, y se determino el cambio en la turbidez a 405 nm por 180min. El tiempo de lisis del coagulo se definió como el tiempo en que el máximo de la absorbancia cae al 50% de su valor.
Se determinó el coeficiente de variación intra e inter ensayo, el rango nom1al en controles sanos y evaluó en estados de hiperfibrinolisis como la Enfermedad de Von Willebrand tipo-1 (EVW tipo-1 ), patología que presenta buena respuesta clínica a agentes anti-fibrinolíticos (ej. Ácido Tranexamico ).
Nuestros resultados mostraron que los tiempos de lisis con PRP (TLC-PRP) son significativamente más largos que los TLC, lo que denota estabilización del coagulo por las plaquetas.
TLC-PRP estimulados con Ristocetina (TLC-PRP-Ris) y TRAP (TLC-PRPTRAP) correlacionan positivamente con niveles plasmáticos de: LDL-Colesterol (r = 0.521, p = <0.0001, n =71), Fibrinógeno (r = 0.602, p = <0.0001, n = 21) y negativamente con HDL-colesterol (r = -0.346, p = 0.005, n = 21), pero sólo TLC-PRP-Ris correlaciona con PAI-1 plasmático (p= <0.007, r= 0.394) y Factor Von Willebrand (p= <0.012, r= 0.340).
Los pacientes con EVW-tipo-1 obtuvieron significativamente tiempo más cortos de TLC-PRP-Ris y TLC-PRP-TRAP que controles sanos.
The study of fibrinolysis has been focused mainly on the measurement of plasma levels of pro-fibrinolytic proteins and their inhibitors. The overall measurements of fibrinolytic activity currently used ( euglobulin lysis time, clot lysis in saline or other), is laborious, of low sensitivity and do not take into account the role played by platelets in the fibrinolytic process. Based on the abovementioned, our goal was to develop a new global test for the measurement of fibrinolysis, which includes the participation of platelets. Methodologically, we rely on the clot lysis time (TLC), described by Lisman T (2005) introducing slight modifications. The TLC was modified by adding platelet-rich plasma (PRP) stirnulated with platelet agonists and calcium chloride to induce coagulation. tP A was exogenously added to accelerate the clot lysis, and the turbidity changes were measured at 405 nm during 180min. The clot lysis time was defined as the time when the maximum absorbance falls to 50% of its value. To validate the new assay, we determined the coefficient of variation intra and in ter assay, the normal range in healthy controls and evaluated hiperfibrinolytic states such as Von Willebrand disease type 1 (type-1 VWD), a pathology that has a good clinical response to anti-fibrinolytic agents (eg. Tranexamic Acid). Our results showed that PRP lysis time (TLC-PRP) is significantly longer than TLC, reflecting stabilization of the clot by platelets. TLC-PRP stimulated with Ristocetin (TLC-PRP-Ris) and TRAP (TLC-PRP-TRAP) levels correlated positively with plasma of LDL-cholesterol (r = 0.521 , p = <0.0001 , n = 71), fibrinogen (r = 0.602, p = <0.0001, n = 21) and negatively with HDL-cholesterol (r =- 0.346, p = 0.005, n = 21). Only TLC-PRP-Ris correlated positively with PAI-1 plasma levels (p = 0.007, r = 0.394) and Von Willebrand factor (p = 0.012, r = 0.340). Patients with VWD-type-1 showed significantly shorter TLC-PRP-Ris and TLCPRP- TRAP than healthy controls. These results suggest that TLC-PRP-Ris is sensitive to VWF levels and PAI-1 , showing a shorter time oflysis in patients with VWD. It remains to establish whether increased fibrinolisis, as evidence by this test, in patients with VWF disease correlates with clinically manifestation ofhigher bleeding frequency.
The study of fibrinolysis has been focused mainly on the measurement of plasma levels of pro-fibrinolytic proteins and their inhibitors. The overall measurements of fibrinolytic activity currently used ( euglobulin lysis time, clot lysis in saline or other), is laborious, of low sensitivity and do not take into account the role played by platelets in the fibrinolytic process. Based on the abovementioned, our goal was to develop a new global test for the measurement of fibrinolysis, which includes the participation of platelets. Methodologically, we rely on the clot lysis time (TLC), described by Lisman T (2005) introducing slight modifications. The TLC was modified by adding platelet-rich plasma (PRP) stirnulated with platelet agonists and calcium chloride to induce coagulation. tP A was exogenously added to accelerate the clot lysis, and the turbidity changes were measured at 405 nm during 180min. The clot lysis time was defined as the time when the maximum absorbance falls to 50% of its value. To validate the new assay, we determined the coefficient of variation intra and in ter assay, the normal range in healthy controls and evaluated hiperfibrinolytic states such as Von Willebrand disease type 1 (type-1 VWD), a pathology that has a good clinical response to anti-fibrinolytic agents (eg. Tranexamic Acid). Our results showed that PRP lysis time (TLC-PRP) is significantly longer than TLC, reflecting stabilization of the clot by platelets. TLC-PRP stimulated with Ristocetin (TLC-PRP-Ris) and TRAP (TLC-PRP-TRAP) levels correlated positively with plasma of LDL-cholesterol (r = 0.521 , p = <0.0001 , n = 71), fibrinogen (r = 0.602, p = <0.0001, n = 21) and negatively with HDL-cholesterol (r =- 0.346, p = 0.005, n = 21). Only TLC-PRP-Ris correlated positively with PAI-1 plasma levels (p = 0.007, r = 0.394) and Von Willebrand factor (p = 0.012, r = 0.340). Patients with VWD-type-1 showed significantly shorter TLC-PRP-Ris and TLCPRP- TRAP than healthy controls. These results suggest that TLC-PRP-Ris is sensitive to VWF levels and PAI-1 , showing a shorter time oflysis in patients with VWD. It remains to establish whether increased fibrinolisis, as evidence by this test, in patients with VWF disease correlates with clinically manifestation ofhigher bleeding frequency.
Notas
Tesis (Tecnólogo Médico con especialidad en Bioanálisis Clínico, Inmunohematología y Banco de Sangre)
Palabras clave
Fibrinólisis