Alterations of fibrin network structure mediated by dermatan sulfate.

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 µg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.

Glicosaminoglicanos en hemostasia: Acción del dermatán sulfato sobre el sistema fibrinolítico / Glycosaminoglycans on hemostasis: Dermatan sulfate action on the fibrinolytic system

El dermatán sulfato (DS) es un glicosaminoglicano endógeno, ampliamente conocido por su acción anticoagulante mediante su interacción con el cofactor II de la heparina para potenciar la inhibición de trombina. En los últimos años se ha sugerido que además el DS aumentaría la actividad fibrinolítica, aunque el mecanismo aún no ha sido completamente dilucidado. En este trabajo se presenta una revisión detallada de los resultados propios y una discusión de la bibliografía disponible respecto de la evaluación del efecto del DS sobre el sistema fibrinolítico. En estudios de activación fibrinolítica, por métodos amidolíticos y coagulométricos, el DS mostró tener efecto pro-fibrinolítico mediante potenciación de la activación de plasminógeno por t-PA y uPA, efecto independiente de su conocida acción anticoagulante. En estudios de caracterización de fibrina, las redes obtenidas en presencia de DS presentaron fibras más largas y delgadas que las redes control, mayor grado de compactación y de lisabilidad; efecto pro-fibrinolítico asociado a su acción anticoagulante. Los resultados presentados contribuyen al esclarecimiento del mecanismo de acción del DS sobre el sistema plasminógeno-plasmina y permiten plantear hipótesis sobre el rol fisiológico de este glicosaminoglicano.

Dermatan sulfate (DS) is well-known for its anticoagulant activity by binding to heparin cofactor II in order to enhance the antithrombin action. It has also been suggested that DS has a profibrinolytic effect, although the exact molecular mechanism is unknown. This review exposes the results obtained and discusses on the available literature on DS effect on the fibrynolytic system. DS exhibited a stimulating effect on the activation of plasminogen by plasminogen activators (t-PA and u-PA), by in vitro amidolytic and coagulometric methods, showing a pro-fibrinolytic effect independent of its known anticoagulant action. Studies of fibrin networks obtained in the presence of DS showed longer and thinner fibers than controls, increased degree of compaction and lisability. Thus, DS displayed a pro-fibrinolytic effect associated to its anticoagulant action. These results contribute to clarify the mechanism of DS action on the plasminogen-plasmin system and to the understanding of the physiologic role of this glycosaminoglycan.

Insight into the profibrinolytic activity of dermatan sulfate: effects on the activation of plasminogen mediated by tissue and urinary plasminogen activators.

INTRODUCTION: Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II to enhance antithrombin action. It has also been suggested that DS has a profibrinolytic effect, although the exact molecular mechanism is as yet unknown. MATERIALS AND METHODS: An in vitro amidolytic method was used to study the effect of high and low molecular weight-DS on the activation of Glu and Lys-plasminogen by tissue and urinary plasminogen activators (t-PA and u-PA). RESULTS: Both high and low molecular weight-DS exhibited a stimulating effect on the activation of plasminogen by PAs. Interestingly, high molecular weight-DS stimulated Glu and Lys-plasminogen activation by t-PA and u-PA in a way and to an extent similar to that in which fibrin(ogen) degradation products (PDF) increased the t-PA assay. Meanwhile low molecular weight-DS had a lower effect. No DS had any effect on plasmin or u-PA amidolytic activity. The facilitation of the conversion of Glu-plasminogen to plasmin in the presence of DS was confirmed by SDS-PAGE; high molecular weight-DS effect was greater than low molecular weight-DS in accordance with the chromogenic assays. Moreover, the combination of PDF and high and low molecular weight-DS, respectively, did not further stimulate t-PA activation of either Glu or Lys-plasminogen suggesting that both substances may compete for the same binding sites. CONCLUSIONS: Through in vitro assays we demonstrated that high and low molecular weight-DS enhance plasminogen activation by u-PA and t-PA, suggesting that the profibrinolytic activity of DS might be via potentiation of plasminogen conversion to plasmin.

Plasma homocysteine cutoff values for venous thrombosis.

BACKGROUND: Hyperhomocysteinemia is considered an independent risk factor for vascular occlusive diseases. To date, there is no general agreement on hyperhomocysteinemia cutoff values. METHODS: To establish a homocysteine cutoff value, we performed a case-control study in 118 patients suffering from venous thrombosis and in 115 healthy subjects. We calculated odds ratios at different cutoff points and considered hyperhomocysteinemia as homocysteine levels above which the risk of venous thrombosis was increased. RESULTS: Initially we used the 97.5th percentiles for fasting homocysteine levels in the control group to calculate odds ratios (95% CI) of 9.5 (2.6-35.3), 3.7 (0.8-17.9) and 4.5 (1.7-123.8) for the total population, women and men, respectively. When individuals with well-known thrombotic risk factors were excluded (selected population), odds ratios were 10.5 (2.7- 41.1), 6.5 (1.3-32.1) and 11.2 (1.2-103.1), respectively, confirming hyperhomocysteinemia as an independent risk factor for venous thrombosis. We did not find any association of venous thrombosis with the homozygous methylenetetrahydrofolate reductase C677T mutation. When the hyperhomocysteinemia cutoff was set at other arbitrary points, odds ratios for the selected population were statistically significant only at >12 micromol/L. CONCLUSIONS: Based on our results, we propose 12 micromol/L as the hyperhomocysteinemia cutoff value.

Es confiable determinar el fibrinógeno como derivado del tiempo de protrombina? / Is fibrinogen determination by prothrombine time derived method confidant?

Los niveles elevados de fibrógeno (Fbg) plasmático son considerados como un factor de riesgo para eventos trombóticos y enfermedad cardiovascular. El Fbg se cuantifica habitualmente utilizando el método coagulable de Clauss y el de Fbg derivado del tiempo de protrombina. A pesar de ser éste último, simple y económico, ha sido cuestionado en distintos estudios porque sobrestimaría los valores de Fbg. Para evaluar la confiabilidad de éste método, se lo comparó respecto al valor obtenido por el método de Clauss. Se evaluó, además, el efecto de la heparina (0,2 y 0,6 UI/ml) sobre las determinaciones por el método de Fbg derivado. La equivalencia entre ambos métodos se estableció por el test de Bland y Altman y el test de la Mediana. El efecto de la heparina se evaluó por regresión lineal y correlación de Pearson. Se puede concluir que los valores de Fbg por el método de Fbg derivado dentro de los rangos normales correlacionan con los obtenidos con el método de Clauss. Cuando éstos valores superan los 400 mg/dl debería determinarse el Fbg por el método de Clauss u otra metodología. Los valores obtenidos mediante el método de Fbg derivado no se modifican en muestras que contienen heparina en el rango terapéutico

Es confiable determinar el fibrinógeno como derivado del tiempo de protrombina? / Is fibrinogen determination by prothrombine time derived method confidant?

Los niveles elevados de fibrógeno (Fbg) plasmático son considerados como un factor de riesgo para eventos trombóticos y enfermedad cardiovascular. El Fbg se cuantifica habitualmente utilizando el método coagulable de Clauss y el de Fbg derivado del tiempo de protrombina. A pesar de ser éste último, simple y económico, ha sido cuestionado en distintos estudios porque sobrestimaría los valores de Fbg. Para evaluar la confiabilidad de éste método, se lo comparó respecto al valor obtenido por el método de Clauss. Se evaluó, además, el efecto de la heparina (0,2 y 0,6 UI/ml) sobre las determinaciones por el método de Fbg derivado. La equivalencia entre ambos métodos se estableció por el test de Bland y Altman y el test de la Mediana. El efecto de la heparina se evaluó por regresión lineal y correlación de Pearson. Se puede concluir que los valores de Fbg por el método de Fbg derivado dentro de los rangos normales correlacionan con los obtenidos con el método de Clauss. Cuando éstos valores superan los 400 mg/dl debería determinarse el Fbg por el método de Clauss u otra metodología. Los valores obtenidos mediante el método de Fbg derivado no se modifican en muestras que contienen heparina en el rango terapéutico (AU)