Diagnostic value of clot formation parameters determined by rotational thromboelastometry in 63 patients with congenital dysfibrinogenemia.

Rotational thromboelastometry (ROTEM) is a global hemostasis assay. The diagnosis added value of ROTEM in congenital dysfibrinogenemia remains to be established. The aim of this study was to analyze clot formation by ROTEM in a cohort of dysfibrinogenemic patients and to establish correlations with genotype, clinical features, and coagulation parameters. The study included genetically confirmed congenital dysfibrinogenemia cases (n = 63) and healthy controls ( n  = 50). EXTEM, INTEM, FIBTEM tests were used to measure ROTEM parameters, that is, clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF) and amplitude 10 min after CT (A10). The ISTH bleeding assessment tool was used to determine bleeding episodes. CT (INTEM) was statistically significantly shorter in congenital dysfibrinogenemia patients compared to controls while CFT (EXTEM) was prolonged. Patients's MCF in EXTEM, INTEM, and FIBTEM were similar to controls while A10 (FIBTEM) was statistically significantly lower. Fibrinogen activity was positively correlated with fibrinogen antigen, A10 and MCF in all three assays. Bleeding phenotypes were observed in 23 (36.5%) patients. Only CFT in EXTEM and CT in INTEM were statistically different in patients with bleeding phenotype versus controls. Carriers of the FGA mutation p.Arg35His had a CT (EXTEM) slightly prolonged and a reduced A10 (FIBTEM) compared to controls. Some ROTEM parameters were able to distinguish congenital dysfibrinogenemia patients from controls, and patients with a bleeding phenotype. Prolonged CFT in EXTEM were associated with congenital dysfibrinogenemia and bleeding phenotype. Bleeding episodes in most patients were generally mild and prevalence of thrombosis was very low.

Physiological correction of hereditary mild hypofibrinogenemia during pregnancy.

INTRODUCTION: Hereditary hypofibrinogenemia is a rare fibrinogen disorder characterised by decreased levels of fibrinogen. Pregnant women with hypofibrinogenemia are at risk of adverse obstetrical outcomes, depending on the fibrinogen level. AIM: We investigated how the physiological changes of hemostasis throughout the pregnancy impact the hemostatic balance in a woman with hereditary mild hypofibrinogenemia. METHODS: Fibrin clot properties were analyzed by turbidimetry and scanning electron microscopy, clot weight and red blood cells retention were measured by whole clot contraction, and in vitro thrombin generation was assessed by calibrated automated thrombogram and ex vivo by TAT. RESULTS: Throughout the pregnancy, the fibrinogen levels increased reaching normal values in the third trimester (activity 3.1 g/L, antigen 3.2 g/L). In parallel, the fibrin polymerisation increased, the fibrinolysis decreased, the fibrin clot network became denser with thicker fibrin fibers, and the fibrin clot weight and red blood cells retention increased, reaching control's value at the third trimester. Similarly, in vitro and ex vitro thrombin generation increased, reaching maximum values at the delivery. CONCLUSION: In this case of hereditary mild hypofibrinogenemia we observed a physiological increase of fibrinogen and thrombin generation. Future studies should focus on moderate and severe hypofibrinogenemia, to assess fibrinogen variation and the overall impact of increased TG on the hemostasis balance.

Impact of Fibrinogen Infusion on Thrombin Generation and Fibrin Clot Structure in Patients with Inherited Afibrinogenemia.

INTRODUCTION: Inherited afibrinogenemia is a very rare disease characterized by complete absence of fibrinogen in the circulation and an increased risk in both thrombosis and bleeding. Infusion of fibrinogen concentrate (FC) is the main approach for prevention and management of bleeding; however, it has been reported to carry a thrombotic risk. METHODS: We investigated the impact of a standard dose (40-100 mg/kg) of FC infusion on the thrombin generation (TG) parameters and the fibrin clot structure formed in plasma samples of patients with afibrinogenemia. Blood samples were collected from 20 patients before (T0) and 1 hour after infusion of FC (T1). TG was studied with calibrated automated thrombography. Fibrin clot structure was assessed with turbidimetry and scanning electron microscopy. RESULTS: FC infusions (mean Clauss fibrinogen plasma level: 1.21 g/L at T1) led to a statistically significant increase in endogenous thrombin potential (ETP) (p < 0.0001) and thrombin peaks (p = 0.02). Nevertheless, when compared with healthy controls, patients' T1 lag times were longer (p = 0.002), ETP values were lower (p = 0.0003), and thrombin peaks were lower (p < 00001). All fibrin polymerization parameters (turbidimetry) obtained at T1 were comparable to those of patients with inherited hypofibrinogenemia matched for fibrinogen plasma levels. CONCLUSION: In summary, fibrinogen infusion with a standard dose of FC increased but did not correct TG and led to formation of fibrin clots similar to those of patients with hypofibrinogenemia. All in all, our results do not support the biological evidence of hypercoagulability induced by FC in patients with afibrinogenemia.

Comparison of different activators of coagulation by turbidity analysis of hereditary dysfibrinogenemia and controls.

Turbidity analysis is widely used as a quantitative technique in hereditary dysfibrinogenemia. We aimed to compare several coagulation triggers in hereditary dysfibrinogenemia and control plasmas. We included 20 patients with hereditary dysfibrinogenemia, 19 with hotspot mutations Aα Arg35His (n = 9), Aα Arg35Cys (n = 2), γ Arg301His (n = 6), γ Arg301Cys (n = 2), and one with Aα Phe27Tyr, and a commercial pooled normal plasma. Fibrin polymerization was activated by bovine or human thrombin or tissue factor (TF), in the presence or absence of tissue type plasminogen activator. The lag time (min), slope (mOD/s), maximum absorbance (MaxAbs, mOD), and area under the curve (AUCp, OD s) were calculated from the fibrin polymerization curves and the time for 50% clot degradation (T50, min), AUCf (OD s) and the overall fibrinolytic potential from fibrinolysis curves. The lag time was significantly shorter and AUC increased in Aα Arg35His patients with bovine thrombin as compared with human thrombin. The MaxAbs and AUCp were significantly higher in γArg301His patients with bovine thrombin compared with human thrombin. Fibrin polymerization parameters of patients' samples were closer to those of control when assessed with TF compared with both human and bovine thrombin. T50 and overall fibrinolytic potential were similar in all samples regardless of the coagulation trigger used, however, with TF the AUCf of Aα Arg35His and γ Arg301His groups were significantly decreased compared with control. Bovine and human thrombin cannot be used equally for studying fibrin polymerization in hotspot hereditary dysfibrinogenemia or control plasmas.

QOLEC2: a randomized controlled trial on nutritional and respiratory counseling after esophagectomy for cancer.

BACKGROUND: Esophagectomy for cancer strongly impairs quality of life. The aim of this trial was to evaluate the effect of the nutritional and respiratory counseling on postoperative quality of life. METHODS: At hospital discharge, patients were randomized into four groups receiving respectively: nutritional and respiratory counseling, nutritional counseling alone, respiratory counseling alone, or standard care. The main endpoint was the impairment in quality of life in the first month after surgery. Linear mixed effect models were estimated to assess mean score differences (MDs) in quality of life scores. RESULTS: Patients receiving nutritional counseling reported less appetite loss (MD - 17.7, 95% CI - 32.2 to -3.3) than those not receiving nutritional counseling at 1 month after surgery. Dyspnea was similar between patients receiving vs. those not receiving respiratory counseling (MD - 3.1, 95% CI - 10.8 to 4.6). Global quality of life was clinically similar between patients receiving vs. those not receiving nutritional counseling over time (MD 0.9, 95% CI - 5.5 to 7.3), as well as in patients receiving vs. those not receiving respiratory counseling over time (MD 0.7, 95% CI - 5.9 to 7.2). CONCLUSIONS: Intensive postoperative care does not affect global quality of life even if nutritional counseling reduced appetite loss.

Fibrin clot properties to assess the bleeding phenotype in unrelated patients with hypodysfibrinogenemia due to novel fibrinogen mutations.

Congenital hypodysfibrinogenemia is a rare fibrinogen disorder, defined by decreased levels of a dysfunctional fibrinogen. We present the functional and structural characterization of two new fibrinogen variants. A duplication of 32 bases in FGA exon 5, p.Ser382GlyfsTer50 was identified in a patient (P1) with history of hemoptysis and traumatic cerebral bleeding. A missense mutation in FGG exon 8, p.Ala353Ser was identified in two siblings (P2 and P3) with tendency to bruising and menorrhagia. Fibrin polymerization was studied in plasma and in purified fibrinogen by turbidimetry. Fibrin structure was studied by a permeability assay, laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). In both plasma and purified fibrinogen samples, all patients had an abnormal polymerization characterized by a decreased maximal absorption compared to controls. The permeation constant (Ks) was markedly increased in all patients: 31 ± 9 × 10-9 cm2 in P1, and 20 ± 0.1 × 10-9 cm2 in P2 and P3, compared to 6 ± 2 × 10-9 cm2 in the control (p < 0.05). The presence of very large pores that accounts for the increased Ks was confirmed by LSCM and SEM patients' clots images. By SEM, the patients' fibrin fibers diameters were thicker: 90 ± 25 nm in P1, 162 ± 64 nm in P2 and 132 ± 46 nm in P3 compared to 74 ± 25 nm in control (p < 0.0001). In conclusion, both new causative fibrinogen mutations altered clot structure by forming thick fibers, diminishing fiber branching, and increasing pore filling space. These structural changes to clots explain the patients' bleeding phenotypes.

Fibrin γ/γ' influences the secretion of fibrinolytic components and clot structure.

BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

Purificación del fibrinógeno gamma A/gamma prima (үA/ү') por cromatografía líquida rápida de proteínas / Purification of fibrinogen gamma A/gamma prima (үA/ү') by fast protein liquid chromatography / Purificação do fibrinogênio gamma A/gamma prima (үA/ү') por cromatografía liquida rápida de proteínas

Resumen Una fracción del fibrinógeno circulante contiene una variante de la cadena ү que se origina por empalme alternativo del ARNm, denominada ү' cuya concentración en plasma se ha relacionado con un incremento en el riesgo de padecer enfermedades cardiovasculares. Por tanto, el objetivo de este trabajo fue diseñar un método de purificación del fibrinógeno үA/ү' más eficiente en relación a los descritos en la literatura, a partir de plasma humano. Se purificó el fibrinógeno үA/ү' a partir del fibrinógeno total obtenido por precipitación con β-alanina, mediante la separación por cromatografía líquida rápida de proteínas. Se confirmó la presencia de fibrinógeno үA/ү' mediante Western blot; su concentración fue determinada por ELISA. El método mostró ventajas en comparación con los métodos clásicos de separación, por ejemplo, que cantidades menores de muestra pudieron ser fraccionadas cuantitativamente en componentes puros en menor tiempo (30 min). Por tanto, se puede concluir que la técnica utilizada para la purificación de las variantes del fibrinógeno, correspondiente al Fg үA/үA y Fg үA/ү', es un método de separación eficiente que permite purificar el Fg үA/ү' libre de contaminantes principales, como lo confirma la inmunoelectroforesis.

Abstract A fraction of the circulating fibrinogen contains a variant of the y chain that is originated by mRNA alternative splicing denominated ү' whose concentration in plasma has been related to an increase in the risk of cardiovascular diseases. Thus, the objective of this work was to design a more efficient үA/ү' fibrinogen purification method in relation to those described in the literature from human plasma. The үA/ү' fibrinogen was purified from the total fibrinogen obtained by precipitation with β-alanine, using separation by fast protein liquid chromatography. Fibrinogen үA/ү', was confirmed by Western blot and its concentration was determined by ELISA. The method showed advantages compared to classical separation methods, for example, smaller amounts of sample could be fractionated quantitatively into pure components in less time (30 min). Therefore, it can be concluded that the technique used for the purification of fibrinogen variants, corresponding to Fg үA/үA and Fg үA/ү', is an efficient separation method that allows purifying the Fg үA/ү' free of main contaminants, as confirmed by immunoelectrophoresis.

Resumo Uma fracção do fibrinogênio circulante contém uma variante da cadeia y que origina-se por junção alternativa do ARNm, chamada ү' cuja concentração em plasma está asociada com um incremento no resgo de sofrer doenças cardiovasculares. Asim, o objetivo em este trabalho foi desenhar um método de purificação do fibrinogênio үA/ү' mais eficiente em relação aos que estão descritos na literatura, a partir do plasma humano. Purificou-se o fibrinogênio үA/ү' a partir do fibrinogênio total conseguido pela precipitação com β-alanina, através da separação por cromatografia líquida rápida de proteínas. Se confirmou por Western blot o fibrinogênio үA/ү'. A sua concentração foi determinada por ELISA. O método apresenta vantagens em comparação com os métodos clássicos de separação, por exemplo, quantidades menores de mostra podem ser fraccionadas quantitativamente em componentes puros, em tempos mais curtos (30 min). Entao, pode-se concluir que a técnica utilizada, para a purificação das variantes do fibrinogênio correspondente ao Fg үA/үA e Fg үA/ү', é um método de separação eficiente que permite purificar o Fg үA/ү' livre de contaminantes principais, como o confirma a imunoeletroforese.

Thrombosis-associated antifibrinogen IgG1 κ impairs fibrin polymerization and enhances platelet activation.

The present study extends our previous investigation of circulating antibody/fibrinogen/C1q complexes (FgIgC) associated with thrombosis in a heterophenotypic AαR16C proband, by focusing on the molecular and functional characteristics of the FgIgC, isolated by cryoprecipitation, FgIgC components were demonstrated by SDS-PAGE and by rotary shadowing electron microscopy. Affinity chromatography was used to isolate IgG and fibrinogen from FgIgC. Thrombin-induced clots were examined by scanning electron microscopy and turbidity measurements. IgG/fibrinogen binding was measured by ELISA. Fibrinogen Aα1-19 peptides, cleaved by thrombin from fragment N-DSK, were examined by mass spectrometry. Clot stiffness, platelet release of P-selectin, and fibrinogen self-assembly were assessed by thromboelastography, flow cytometry, and atomic force microscopy, respectively. The FgIgC effects included the following: increased P-selectin release from gel-sieved platelets, finer fiber networks and decreased stiffness of its clots, and marked inhibition of fibrinogen self-assembly. The abnormal proband fibrinogen structure displayed phosphorylated AαR16C-AαR16C homodimers and AαR16C-glutathione heterodimers. ELISA measurements disclosed pronounced binding by proband fibrinogen to proband IgG, which was blocked by the IgG's Fab fragment and by proband, but not by normal plasmic fragment E1. There was appreciable, but much weaker, binding to normal fibrinogen, to its fragments E1, and D1, and to homodimeric AαR16C fibrinogen. The antibody's primary target epitope included heterodimeric AαR16C-glutathione; a secondary epitope resided in the D region. Moreover, both the enhanced platelet activation (i.e. increased P-selectin release induced by FgIgC) and the highly phosphorylated FpA (i.e. resulting in its accelerated release by thrombin) may have contributed to the thrombotic diathesis.

Effect of von Willebrand factor on clot structure and lysis.

Von Willebrand Factor (vWF) is constitutively secreted by the endothelium and incorporated in the fibrin clots under slow clotting conditions. The aim of the present work was to study the effect of vWF on clot structure and lysis. Purified fibrinogen was mixed with vWF or Tris-buffered saline and clotted with thrombin - activated factor XIII. Fibrin polymerization was followed by turbidity at 350 nm during 2.5 h. After this time, plasmin was added on the top of the clots, and the optical density (OD) was read until baseline values. vWF effect on network[Combining Acute Accent]s porosity was evaluated by permeation using the same clotting conditions as for fibrin polymerization. Clot structure was visualized and analyzed by laser scanning confocal microscopy (LSCM). The rate of fibrin polymerization was 1.47 mOD/s in the presence of vWF and 0.5 mOD/s when vWF was not added (P < 0.05). The fibrin lysis rate was approximately four times faster when vWF was added to fibrinogen. The fibrin network porosity was (20.4 ±â€Š1.6) × 10 cm with vWF and (8.3 ±â€Š1.2) × 10 cm without external vWF (P < 0.05). The analysis of LSCM images showed that vWF increased fibrin fibers diameter and the networks[Combining Acute Accent] pores size. In conclusion, vWF covalently crosslinked to fibrin modify its structure (increases fibrin diameter and the pores filling space of the meshwork) that accelerates the fibrin lysis rate.

Study of the fibrinolytic process in a patient with antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antibodies against ß2-Glycoprotein I (aß2-GPI), cardiolipin (aCL) and lupus anticoagulant combined with venous or arterial thrombosis and/or foetal losses. A 28-year-old female was positive for aß2-GPI, aCL, aPT (antibodies against prothrombin) and lupus anticoagulant. She had two miscarriages and a deep vein thrombosis event. The patient plasma fibrinogen and IgG concentrations were two times higher than control. Fibrinolysis was induced in vitro adding tPA, before clotting plasma with 0.03 or 0.6 IU/ml thrombin, and in purified system with normal fibrinogen in the presence of 0.5 mg/ml of patient or normal IgG. The APS patient had 1.5 and 1.9 times higher clot rate formation (CRL) and maximum absorbance (MaxAbsL) at both thrombin concentrations. At 0.6 IU/ml of thrombin, the patient delay on fibrin polymerization onset was corrected. The patient Lys50MA (time needed for 50% clot dissolution) was slower (P < 0.05) at 0.03 IU/ml of thrombin; however, the lysis rate was faster at both thrombin concentrations. After adjusting the polymerization and fibrinolytic parameters according to the sample plasma fibrinogen concentration, there were almost no differences between patient and control at 0.6 IU/ml. In an IgG-fibrinogen purified system, fibrinolysis was equivalent in the presence of patient or normal IgG. In conclusion, the patient IgG fraction has no inhibitors against proteins of the fibrinolytic pathway. The differences observed between the APS patient and the control were more evident at low thrombin concentration due to the presence of aPT.

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bß Q339 into a stop codon causing hypofibrinogenemia.

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bß 339Q to stop, causing deletion of Bß chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bß, and γ chains, indicating that molecules with the truncated 37,990Da ß chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities.

Dysregulated coagulation associated with hypofibrinogenaemia and plasma hypercoagulability: implications for identifying coagulopathic mechanisms in humans.

Identifying coagulation abnormalities in patients with combined bleeding and thrombosis history is clinically challenging. Our goal was to probe the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis. The patient is a 56-year-old female with a history of haematomas, poor wound healing, and thrombosis (retinal artery occlusion and transient cerebral ischaemia). She had a normal activated partial thromboplastin time, prolonged thrombin and reptilase times, and decreased functional and antigenic fibrinogen levels, and was initially diagnosed with hypodysfibrinogenaemia. This diagnosis was supported by DNA analysis revealing a novel FGB mutation (c.656A>G) predicting a Q189R mutation in the mature chain that was present in the heterozygote state. However, turbidity analysis showed that purified fibrinogen polymerisation and degradation were indistinguishable from normal, and Bß chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Interestingly, plasma thrombin generation testing revealed the patient's thrombin generation was higher than normal and could be attributed to elevated levels of factor VIII (FVIII, 163-225%). Accordingly, in an arterial injury model, hypofibrinogenaemic mice (Fgn(+/-)) infused with factor VIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn(+/-) mice. Together, these data associate the complex bleeding and thrombotic presentation with combined hypofibrinogenaemia plus plasma hypercoagulability. These findings suggest previous cases in which fibrinogen abnormalities have been associated with thrombosis may also be complicated by co-existing plasma hypercoagulability and illustrate the importance of "global" coagulation testing in patients with compound presentations.

Structure of fibrin network of two abnormal fibrinogens with mutations in the αC domain on the human dermal microvascular endothelial cells 1.

There is evidence that clot structure can be modulated by endothelial cells, wherein the fibrinogen αC domain plays a major role in the fibrin-cell interaction. The spatial distribution of fibrin fibers from fibrinogen Caracas V and Caracas I, with heterozygous mutation in the αC domain (Aα Ser432Cys and Aα Ser466stop, respectively) on human dermal microvasculature endothelial cells (HMEC-1), was studied by laser scanning confocal microscopy. In order to assess fibrin-cell interaction and the role of the αC domain, preliminary experiments were done with inert microspheres and RGD peptide included in the clotting reaction, and forming clots with fibrinogen fragment X (fibrinogen without αC domain). Groups of stressed fibers were observed near the cell surface and were related to fibrin-cell interactions, which were abolished by the RGD peptide, and by the absence of the αC domain. The fibrin network of fibrinogen Caracas V and Caracas I was very different from that of normal fibrinogen. In general, patient's clots were characterized by very thin, tightly packed fibrin fibers, with a substantially reduced network porosity. Near the cell's surface, both abnormal fibrinogens formed a very fine meshwork, with stressed fibers 'anchored' to the cell surface, a pattern that was lost far from the cell surface. The structure of normal and patient clots performed in the absence of cells resembled that observed far from the cell surface, concluding that Caracas V and Caracas I fibrin was modulated by the presence of endothelial cells.

[Effect of antiphospholipid antibodies on the formation and lysis of fibrin network in patients with recurrent miscarriage]. / Efecto de los anticuerpos antifosfolipidicos en la formación y degradación de la malla de fibrina en pacientes con aborto recurrente.

The present work was intended to study the process of fibrin formation and lysis and plasmin generation in a group of patients with recurrent miscarriage (RM), due to the presence of antiphospholipid antibodies (N = 10); as well as in women with RM without the antiphospholipid syndrome (APS) (N = 6), compared with those of a group of healthy women (N= 8). In the group of patients with APS, nine were positive for antibodies against cardiolipin (aCL), five for anti-beta2-glycoprotein I (anti-beta2GPI), four for both antibodies, and one for antibodies against prothrombin (aPT) and lupus anticoagulant (LA). Fibrin formation and lysis was followed by turbidity and plasmin generation using chromogenic substrate S2251. The polymerization curves from RM patients without APS and the LA patient showed an increased slope and maximum turbidity compared to those of the control group. The speed of lysis was higher in the LA patient (21 +/- 0) 10(-4) deltaOD/seg and the RM patients without APS (19.6 +/- 5.7) 10(-4) deltaDO/seg, compared to that of the control group (14.5 +/- 2.8) 10(-4) deltaDO/seg. Plasmin generation increased only in RM patients without APS (85 +/- 24%) against the control group (52 +/- 3%), p = 0.005. The changes observed in the fibrin polymerization and lysis process of women with RM without APS and LA seem to be related to their higher fibrinogen levels, while the increased plasmin generation was related to the patients' morbidity.

Efecto de los anticuerpos antifosfolipídicos en la formación y degradación de la malla de fibrina en pacientes con aborto recurrente / Effect of antiphospholipid antibodies on the formation and lysis of fibrin network in patients with recurrent miscarriage

En el presente trabajo se estudió el proceso de formación y disolución de la malla de fibrina y la generación de plasmina en un grupo de pacientes con aborto recurrente (AR) debido a la presencia de anticuerpos antifosfolipídicos (N= 10), mujeres con AR sin el síndrome antifosfolipídico (SAF) (N= 6) y se comparó con un grupo de mujeres sanas (N= 8). Del grupo de pacientes estudiadas con SAF, nueve fueron positivas para anticuerpos anticardiolipina (aCL), cinco para la anti-b2-glicoproteína I (anti-b2GPI), cuatro para ambos anticuerpos, una para anticuerpos antiprotrombina (aPT) y anticoagulante lúpico (AL). El proceso de formación de la fibrina y su disolución fue estudiado por turbidimetría y la generación de plasmina mediante sustrato cromogénico S2251. Las curvas de polimerización de la(s) paciente(s) con AR sin SAF y AL presentaron un incremento en la pendiente y turbidez final, comparado con las del grupo control de mujeres sanas. La velocidad de disolución del coágulo fue mayor en la paciente con AL (21 ± 0) 10-4 DDO/seg y en las AR sin SAF (19,6 ± 5,7) 10-4 DDO/seg, comparado con el grupo control (14,5 ± 2,8) 10-4 DDO/seg. La generación de plasmina estuvo incrementada solamente en las AR sin SAF (85 ± 24%) comparado con 52 ± 3% en el grupo control, p= 0,005. Los cambios observados en el proceso de polimerización y fibrinólisis de la(s) paciente(s) con AR sin SAF y AL pudieran estar relacionados con el incremento en los niveles de fibrinógeno, mientras que los de la generación de plasmina con la entidad mórbida.

The present work was intended to study the process of fibrin formation and lysis and plasmin generation in a group of patients with recurrent miscarriage (RM), due to the presence of antiphospholipid antibodies (N= 10); as well as in women with RM without the antiphospholipid syndrome (APS) (N= 6), compared with those of a group of healthy women (N= 8). In the group of patients with APS, nine were positive for antibodies against cardiolipin (aCL), five for anti-b2-glycoprotein I (anti-b2GPI), four for both antibodies, and one for antibodies against prothrombin (aPT) and lupus anticoagulant (LA). Fibrin formation and lysis was followed by turbidity and plasmin generation using chromogenic substrate S2251. The polymerization curves from RM patients without APS and the LA patient showed an increased slope and maximum turbidity compared to those of the control group. The speed of lysis was higher in the LA patient (21 ± 0) 10-4 DOD/seg and the RM patients without APS (19.6 ± 5.7) 10-4 DDO/seg, compared to that of the control group (14.5 ± 2.8) 10-4 DDO/seg. Plasmin generation increased only in RM patients without APS (85 ± 24%) against the control group (52 ± 3%), p= 0.005. The changes observed in the fibrin polymerization and lysis process of women with RM without APS and LA seem to be related to their higher fibrinogen levels, while the increased plasmin generation was related to the patients´ morbidity.

A novel missense mutation in the FGB g. 3354 T>A (p. Y41N), fibrinogen Caracas VIII.

A novel dysfibrinogenaemia with a replacement of Tyr by Asn at Bß41 has been discovered (fibrinogen Caracas VIII). An asymptomatic 39-year-old male was diagnosed as having dysfibrinogenaemia due to a mildly prolonged thrombin time (+ 5.8 seconds); his fibrinogen concentration was in the low normal range, both by Clauss and gravimetric determination, 1.9 g/l and 2.1 g/l, respectively. The plasma polymerization process was slightly impaired, characterised by a mildly prolonged lag time and a slightly increased final turbidity. Permeation through the patients' clots was dramatically increased, with the Darcy constant around four times greater than that of the control (22 ± 2 x 10(-9) cm² compared to 6 ± 0.5 x 10(-9) cm² in controls). The plasma fibrin structure of the patient, by scanning electron microscopy, featured a mesh composed of thick fibres (148 ± 50 nm vs. 120 ± 31 nm in controls, p<0.05) and larger pores than those of the control fibrin clot. The viscoelastic properties of the clot from the patient were also altered, as the storage modulus (G', 310 ± 30) was much lower than in the control (831 ± 111) (p ≤0.005). The interaction of the fibrin clot with a monolayer of human microvascular endothelial cells, by confocal laser microscopy, revealed that the patients' fibrin network had less interaction with the cells. These results demonstrate the significance of the amino terminal end of the ß chain of fibrin in the polymerisation process and its consequences on the clot organisation on the surface of endothelial cells.

Fibrin formation and lysis studies in dengue virus infection.

Dengue virus is a mosquito-borne human viral pathogen that has recently become a major public health concern particularly in tropical and subtropical countries, predominantly in urban and periurban areas. Plasma from five patients infected by the virus was selected since they have in different degrees prolonged thrombin times: +2.1, +3.4, +5.7, +7.1 and +18.5 s, like a transitory acquired dysfibrinogenemia. The serotype could be determined in only two patients, being DEN-1 and DEN-3. The fibrinogen concentration was normal ranging from 2.5 to 3.2 g/l. In general, the fibrin degradation products of the patients were high, reaching values of 6000 ng/ml. The polymerization process was quite similar to that of the control, except in two cases where the final turbidity was almost half the control value. In one of these patients, the fibrinogen was purified and mixed with normal fibrinogen (v: v); the patients' fibrinogen impaired normal fibrin polymerization. Studies of the fibrinolytic process revealed that clots from dengue patients started to lyze before they have reached the maximum turbidity, although this was not reflected in the time needed for complete clot dissolution, which was similar to that of the control for all the patients. Fibrinolysis of clots made by mixing normal and patient purified fibrinogen (2.5: 1) was impaired. Clot images obtained by scanning electron microscopy showed that the patients' fibrin network had some degree of degradation and the fibers were thicker than those of the control (P < 0.05). This preliminary study seems to indicate that the dengue virus infection modifies the balance of coagulation-fibrinolysis toward hyperfibrinolysis and could modify the normal fibrinogen molecule.