"Disfibrinogenemia Jujuy", alteración asociada a trastornos hemorrágicos / "Dysfibrinogenemia Jujuy", associated to bleeding disorders / "Disfibrinogenemia Jujuy", alteração associada a distúrbios hemorrágicos

El objetivo del presente trabajo fue estudiar a una joven paciente con manifestaciones hemorrágicas, caracterizar su fibrina plasmática e identificar la posible alteración molecular del fibrinógeno de la paciente y sus familiares directos. Se diagnosticó una disfibrinogenemia en la paciente, su madre y el medio-hermano, ambos asintomáticos. En estos individuos, la formación y lisis de la fibrina plasmática difirieron de los controles. La fase lag prolongada indicó la liberación lenta y defectuosa de los fibrinopéptidos; la pendiente menor que la del control sugirió una polimerización dificultada. La DOMax inferior mostró una fibrina compuesta por fibras delgadas. La fibrinolisis inducida por estreptoquinasa resultó más rápida que la correspondiente control. La secuenciación del ADN reveló una deleción homocigota que condujo a la ausencia de la glicina 14 de la cadena Aa del fibrinógeno (AaGly14del según nomenclatura de http://www.geht.org/databaseang/fibrinogen). La madre y el medio hermano resultaron heterocigotas para la misma mutación. Esta alteración no descripta previamente, que se ha denominado fibrinógeno Jujuy, podría no interaccionar correctamente con el sitio activo de la trombina, provocando que los fibrinopéptidos se hidrolicen y liberen lentamente, originando fibras de fibrina más delgadas y degradables que las del control. Este mecanismo explicaría el sangrado moderado de la paciente.

The aim of this work was to study a young female with moderate bleeding symptoms, to characterize the plasma fibrin and to identify the possible molecular alteration in the fibrinogen of the patient and her family. A dysfibrinogenemia was diagnosed in the patient, the mother and the half-brother, both the latter asymptomatic. Kinetic parameters obtained from fibrin formation and lysis assays of the patient’s plasma samples were significantly different compared to the ones obtained with control plasma. A prolonged lag phase indicated slow and defective fibrinopeptide releases, whereas a minor slope suggested an impaired fibrin assembly. A lower ODMax revealed a fibrin network composed of thinner fibers. Fibrinolysis induced by streptokinase resulted faster than control. DNA sequencing showed a homozygous deletion leading to AaGly14del (according to http://www.geht.org/databaseang/fibrinogen). The mother and the half-brother resulted heterozygous for the same mutation. This previously undescribed alteration was named fibrinogen Jujuy. The mutate fibrinogen might not be correctly fixed to the active site of thrombin resulting in slow cleavage and release of fibrinopeptides, rendering thinner fibers, more susceptible to lysis than control. This mechanism may explain the moderate bleeding symptoms of the patient.

O objetivo do presente trabalho foi estudar uma jovem paciente com manifestações hemorrágicas, caracterizar sua fibrina plasmática e identificar a possível alteração molecular do fibrinogênio da paciente e seus familiares diretos. Foi diagnosticada uma disfibrinogenemia na paciente, sua mãe e o meio-irmão, ambos assintomáticos. Nestes indivíduos, a formação e lise da fibrina plasmática diferiram dos controles. A fase lag prolongada indicou a liberação lenta e defeituosa dos fibrinopeptídeos; a pendente menor que a do controle sugeriu uma polimerização dificultada. A DOMax inferior mostrou uma fibrina composta por fibras delgadas. A fibrinólise induzida por estreptoquinase resultou mais rápida que a correspondente controle. A sequenciação do DNA revelou uma deleção homozigótica que conduziu à ausência da glicina 14 da cadeia Aa do fibrinogênio (AaGly14del conforme nomenclatura de http://www.geht.org/databaseang/fibrinogen). A mãe e o meio irmão resultaram heterozigotos para a mesma mutação. Esta alteração não descrita previamente, que denominamos fibrinogênio Jujuy, poderia não interagir corretamente com o sítio ativo da trombina, provocando que os fibrinopeptídeos se hidrolisem e liberem lentamente, originando fibras de fibrina mais finas e degradáveis que as do controle. Este mecanismo explicaria o sangramento moderado da paciente.

Impact of homocysteine-thiolactone on plasma fibrin networks.

Epidemiologic studies have shown that hyperhomocysteinemia is an independent risk factor for vascular disease. Homocysteine (Hcy) circulates as different species, mostly protein bound, and approximately 1% as its reduced form and the cyclic thioester homocysteine-thiolactone (HTL). Despite the level of plasma thiolactone being markedly low, detrimental effects are related to its high reactivity. HTL reacts with proteins by acylation of free basic amino groups; in particular, the epsilon-amino group of lysine residues forms adducts and induces structural and functional changes in plasma proteins. In order to assess the effects of HTL on plasma fibrin networks, a pool of normal plasma incubated with HTL (100, 500 and 1,000 µmol/L, respectively) was evaluated by global coagulation tests and fibrin formation kinetic assays, and the resulting fibrin was observed by scanning electron microscopy. HTL significantly prolonged global coagulation tests in a concentration-dependent manner with respect to control, and increases were up to 14.5%. Fibrin formation kinetic parameters displayed statistically significant differences between HTL-treated plasma and control in a concentration-dependent way, showing higher lag phase and lower maximum reaction velocity and final network optical density. Electron microscopy analysis of HTL plasma networks revealed a compact architecture, with more branches and shorter fibers than control. We can conclude that HTL induced a slower coagulation process, rendering more tightly packed fibrin clots. Since these features of the networks have been related to impaired fibrinolysis, the N-homocysteinylation reactions would be involved in the prothrombotic effects associated to hyperhomocysteinemia.

Characterization of N-homocysteinylated albumin adducts.

Increased plasma homocysteine levels are considered an important risk factor for vascular disease. Homocysteine, an intermediate compound in methionine metabolism, is an amino acid that includes a thiol group, and circulates as different species. One of them, Homocysteine thiolactone (HTL) forms adducts through irreversible reactions with epsilon-NH2 groups of lysine residues. These processes can alter the structure and biological function of diverse proteins that may be involved in the detrimental effects of homocysteine. Particularly, in this work we evaluated HTL-mediated molecular changes in human serum albumin (HSA) through electrophoretic techniques. Albumin and HTL were incubated (37 °C, 6 h) at HSA:HTL molar ratios of 1:25, 1:50 and 1:100. Polyacrylamid gel electrophoresis showed that electrophoretic mobility was increased in the treated HSA respect to control, in an HTL-concentration-dependent manner. That anodic shift of the treated samples was also observed in crossed immunoelectrophoresis profiles. As expected, a decrease in the isoelectric point of the homocysteinylated albumin (pI 4.7) in comparison to that of control (pI 4.8) was shown by the isoelectric focusing technique. Moreover, the electropherogram acquired by capillary zone electrophoresis indicated that migration times and full width at half height were enhanced with the rise of HTL concentration. We propose that the in vitro structural changes of albumin described in the present work would be involved in the harmful effects of the N-homocysteinylation process.

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.

Homocysteine modifies fibrin clot deformability: another possible explanation of harm.

Several studies have associated elevated plasma homocysteine (Hcy) levels with higher risk of thrombosis. In order to evaluate possible changes in fibrin strength and deformability mediated by Hcy, the effect of the amino acid on plasma fibrin clot was studied, measuring the viscoelastic clots response as a function of Hcy concentration added to plasma (final concentrations: 0, 50, 100, 250 and 500 microM). Storage (G') and loss (G'') moduli were significantly higher than control at all Hcy concentrations evaluated in a dose-independent way (G'50 microM Hcy=254+/-10 Pa vs. 178 +/- 30 Pa, p=0.012; G''50 microM Hcy=32+/-1 Pa vs. 24 +/- 2 Pa, p=0.012). The tangent of the phase shift angles tan delta obtained from Hcy-clots with respect to control system proved to be unchanged (tan delta50 microM Hcy=0.130+/-0.007 Pa vs. 0.150 +/- 0.020 Pa, NS). Increases observed in G' and G'' values allowed us to conclude that Hcy action led to the stiffening of the clots formed in a dose independent way. The higher crosslinking of the fibrin network (higher G') contributed both to this structural behavior and to a higher compartmentalization and viscosity of the fluid phase (higher G'') of the gel. The lower deformability of the clots formed after Hcy addition was also detected through deformation sweep assays. These material's characteristics may lead to pathological behavior, increasing the chances of obstruction in the blood vessels.

Variabilidad de las redes de fibrina / Fibrin network variability

La fibrina (polímero del fibrinógeno) es una malla proteica constituyente del tapón hemostático. Esta red se caracteriza principalmente por su estructura espacial, las dimensiones de sus fibras, el grado de ramificación, porosidad, elasticidad y rigidez, propiedades que dependen de factores como temperatura, concentración de iones y otras sustancias plasmáticas, pero principalmente de fibrinógeno, trombina y factor XIII. Cuando aumentan las concentraciones de fibrinógeno o trombina, las redes de fibrina son más densas, de menor porosidad y formadas por fibras más cortas. Las alteraciones de la molécula de fibrinógeno pueden producir fibrina anormal, de diferente disolución, ya sea que permanezca tiempos prolongados en circulación induciendo la formación de otros trombos, o bien que se degrade anticipadamente produciendo riesgo hemorrágico. El aumento de calcio incrementa la rigidez y porosidad de la red. La fibrina tiene una importante función como cofactor de la fibrinolisis. La activación del plasminógeno y la acción de la plasmina sobre la fibrina dependen de la estructura de la red. Las redes compactas son lisadas más lentamente, mientras que dentro del mismo gel, las fibras delgadas son disueltas antes que las gruesas. Por otra parte, fibras gruesas resultan mejores cofactores de la activación del plasminógeno. Conocer estos factores facilita la comprensión de situaciones fisiopatológicas que producen diferente respuesta fibrinolítica fisiológica o inducida por fármacos.

Fibrin is a protein network present in the clot. It can be characterized by its structure, fibers dimensions, ramification degree, porosity, viscoelasticity and deformability. All these properties depend on temperature, ions concentrations and on other plasmatic substances, mainly fibrinogen, thrombin and factor XIII. Greater fibrinogen or thrombin concentrations produce more dense, less porous and shorter fibered-fibrin networks. Fibrinogen alterations can generate abnormal fibrin, with different susceptibility to lysis, while calcium ions increase the fibrin networks' rigidity and porosity. Plasminogen activation and fibrinolysis rate are strongly dependent on the size and the fibrin network architecture. Tight networks are lysed more slowly while, within the same gel, thin fibers are lysed before thick ones. Otherwise, thicker fibers proved to be a better cofactor of plasminogen activation. Taking into account these factors helps understand the different fibrinolytic response, mainly when it is not explained by laboratory studies.

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.

Influence of homocysteine on fibrin network lysis.

To elucidate some of the links between homocysteine and vascular disease, we have evaluated the effect of the amino acid on the formation (by kinetics studies), structure (by electron microscopy) and lysis of the fibrin network, using tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have studied whether homocysteine could alter the activity of the components involved in fibrinolysis (by amidolytic and thrombolytic methods). The results showed that homocysteine-associated networks were more compact and branched than controls (52 +/- 6 vs 44 +/- 5 fibers/field, P = 0.008), and were formed by shorter and thicker fibers. This clot proved to be more resistant to fibrinolysis with u-PA than control [lysis time 50%: 257 +/- 16 (homocysteine) vs 187 +/- 6 min (control); P < 0.004], but there were no differences with t-PA. Homocysteine did not affect the biological activities of plasmin, or plasminogen activation by t-PA and u-PA. Defective fibrinolysis with u-PA was therefore associated with homocysteine-fibrin structural alterations rather than the homocysteine effect on the biological activities of the fibrinolytic components evaluated. Results suggest that hyperhomocysteinemic patients could produce tight clots, were more resistant to lysis, and generated a procoagulant environment in situ. We believe that our findings may contribute to understanding the mechanisms involved in the homocysteine harmful effect.

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)

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