Recurrent superficial venous thrombophlebitis because of mutations in the protein C and fibrinogen genes in a young Argentinian female.

: Hypodysfibrinogenemia and protein C deficiency are coagulopathies and in this report, we describe a young patient with both defects confirmed by molecular genetic tests. The patient was a 24-year-old woman referred for recurrent thrombophlebitis and finally deep venous thrombosis. Routine coagulation studies revealed mild decrease of protein C (0.49 IU, reference values 0.7-1.40 IU) and hypodysfibrinogenemia (0.88 g/l and 1.83 g/l for activity and antigen, respectively, reference values 2.0-4.0 g/l). Direct sequencing analyses were performed on FGA, FGB, and FGG genes to confirm hypodysfibrinogenemia and on the protein C gene to confirm protein C deficiency. As a result, the patient was shown to be heterozygous p.Ala82Gly in the FGG gene (Fibrinogen Dunedin) and for compound heterozygous missense mutation in protein C gene. To our knowledge, this is the first report on a case of combined dysfibrinogenemia and protein C deficiency confirmed by molecular genetic tests.

Factor X Deficiency Due to a Compound Heterozygosis Between a New Mutation (Gla72Asp) in Exon 2 and an Already Known one (Gly154Arg) in Exon 5: Factor X Mar Del Plata1).

BACKGROUND: Investigation of rare bleeding disorders in Latin-America. OBJECTIVES: The report of a new case of FX deficiency due to a compound heterozygosis. METHODS: Accepted clotting procedures were used. Sequencing of DNA was carried out by means of Applied Biosystems Instruments. RESULTS: A compound heterozygote due to the association of a new mutation (Gla72Asp) with an already known mutation (Gly154Arg) of the FX gene is reported. The proposita is a 38 year old female who had a moderate bleeding tendency (menorrhagia, epistaxis, easy bruising). The proposita has never received substitution therapy but in the occasion of a uterine biopsy. The mother was asymptomatic but was a heterozygote for the new mutation. The father was asymptomatic but had deserted the family and could not be investigated. After this abandonment the mother of the proposita re-married with an asymptomatic man and she gave birth to a son who was asymptomatic but was also heterozygous for the new mutation (Gla72Asp). As a consequence it has to be assumed that the first husband of the mother of the proposita was heterozygous for the known mutation (Gly154Arg). CONCLUSIONS: This is the third case of a new mutation in the FX gene reported, during the past few years, in Argentina.

Identification and characterization of novel mutations implicated in congenital fibrinogen disorders.

INTRODUCTION: Fibrinogen is a complex molecule comprised of two sets of Aα, Bß, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype-phenotype correlations. MATERIALS AND METHODS: DNA from 31 patients was sequenced to evaluate disease-causing mutations in the three fibrinogen genes: FGA,FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure. RESULTS: Seventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals. CONCLUSIONS: Robust genotype-phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Congenital FX Deficiency Rio Tercero: A New Heterozygous Missense Mutation (Cys241Gly) with a Potentiating Effect by a Polymorphism (c. 503-57C>T).

OBJECTIVE: The aim was to report a new family with congenital FX deficiency. PATIENTS AND METHODS: The proposita is a 41 year old female with a moderate bleeding tendency (easy bruising, menorrhagia). Parents were not consanguineous. Family history was positive for a mild bleeding tendency. RESULTS: Coagulation and genetics studies revealed that the proposita and two of her siblings were heterozygotes for a new mutation Cys241Gly in exon 6 but had different FX level (2-3% of normal in the proposita and about 50% in the two siblings. The same was true for one of her three children. The mother and the other two children of the proposita had also slightly decreased FX levels but no mutation. On the suspicion that the proposita was carrying another defect which had escaped the Sanger method, we carried out a whole exome analysis and discovered that the proposita and one of her siblings were also homozygous for a mutation of a known polymorphism (c.503-57C>T). The daughter of the proposita was instead, besides being a carrier of the missense new mutation Cys241Gly, heterozygous for the same polymorphism. The mother and two other daughters were also heterozygous for the polymorphism. There were no deletions or duplications. CONCLUSION: The polymorphism present in the family seems to be capable of potentiating the defect induced by the new mutation. This, safe for epigenetics phenomena, is the only possible explanation for the discrepancy found in the FX level between mother and daughter despite the fact that both carried the same new mutation.

A family with factor X deficiency from Argentina: a compound heterozygosis because of the combination of a new mutation (Gln138Arg) with an already known one (Glu350Lys).

The objective was to investigate a family from Argentina. The proposita was a 51-year-old woman who had a moderate bleeding tendency. Some of her children showed a mild bleeding tendency. Her mother and the husband were asymptomatic. Clotting, immunological and molecular biology techniques were used. Partial thromboplastin, prothrombin, Russell Viper venom-clotting times were moderately prolonged in the proposita, whereas they were slightly prolonged in the children and in her mother. Factor X (FX) activity was about 2-3% of normal in all assay systems. FX antigen was less than 5%. Other clotting factors and platelet were normal. Genetic analysis showed a compound heterozygosis: combination of a 'new' mutation (Gln138Arg) with an already known mutation (Glu350Lys). The children had intermediate FX levels (35-63% of normal) and were carriers of one of the two mutations present in the proposita. This is the first observation of a FX deficiency in Argentina.

Absence of in vitro Procoagulant Activity in Immunoglobulin Preparations due to Activated Coagulation Factors.

BACKGROUND: Immunoglobulin (IG) products, including intravenous (IVIG) or subcutaneous (SCIG) immunoglobulins are considered safe and effective for medical therapy; however, a sudden and unexpected increase in thromboembolic events (TE) after administration of certain batches of IVIG products has been attributed to the presence of activated coagulation factors, mainly factor XIa. Our aims were to examine the presence of enduring procoagulant activity during the manufacturing process of IGs, with special focus on monitoring factor XIa, and to evaluate the presence of in vitro procoagulant activity attributed to coagulation factors in different lots of IVIG and SCIG. METHODS: Samples of different steps of IG purification, 19 lots of IVIG and 9 of SCIG were analyzed and compared with 1 commercial preparation of IVIG and 2 of SCIG, respectively. Factors II, VII, IX, XI and XIa and non-activated partial thromboplastin time (NAPTT) were assayed. RESULTS: The levels of factors II, VII, IX, X and XI were non-quantifiable once fraction II had been re-dissolved and in all analyzed lots of IVIG and SCIG. The level of factor XIa at that point was under the detection limits of the assay, and NAPTT yielded values greater than the control during the purification process. In SCIG, we detected higher concentrations of factor XIa in the commercial products, which reached values up to 5 times higher than the average amounts found in the 9 batches produced by UNC-Hemoderivados. Factor XIa in commercial IVIG reached levels slightly higher than those of the 19 batches produced by UNC-Hemoderivados. CONCLUSION: IVIG and SCIG manufactured by UNC-Hemoderivados showed a lack of thrombogenic potential, as demonstrated not only by the laboratory data obtained in this study but also by the absence of any reports of TE registered by the post marketing pharmacovigilance department.

The old and the new in prekallikrein deficiency: historical context and a family from Argentina with PK deficiency due to a new mutation (Arg541Gln) in exon 14 associated with a common polymorphysm (Asn124Ser) in exon 5.

Prekallikrein (PK) is one of the clotting factors involved in the contact phase of blood. PK has an important historical role as its deficiency state represents the second instance of a clotting defect without bleeding manifestations, the first one being factor XII deficiency. PK deficiency is a rare clotting disorder. Moreover, only 11 patients have been investigated so far by molecular biology techniques. In this article, we briefly review some of the history around PK and also present some recent data on a newly identified family from Argentina suffering from PK deficiency. Two patients are homozygous whereas other family members are heterozygous. PK activity and antigen are 1% of normal in the homozygotes and around 60 to 70% of normal in the heterozygotes. As expected, all patients are asymptomatic of bleeding or thrombosis presentations. However, the two homozygotes showed essential hypertension. The PK deficiency in this family is due to a new mutation (Arg541Gln) in exon 14. The defect segregates together with a known polymorphism, Asn124Ser, in exon 5. The significance of the presence of hypertension in the two homozygotes is discussed in view of the extra coagulation effects of PK on vasodilation, vessel permeability, and the control of blood pressure. Structure function analysis indicates that the substitution of Arg with Gln probably impedes the transmembrane diffusion of the molecule, which therefore cannot be secreted in the homozygotes. The presence of hypertension in patients with PK deficiency has been previously reported in some but not all patients. Future research activities will probably concentrate on the effect of PK and other contact phase factors on the vascular system.

Inmunogenicidad de los concentrados de Factor VIII doblemente inactivados producidos en UNC-Hemoderivados / Immunogenecity of double inactivation FVIII concentrates manufactures by UNC-Hemoderivados

El tratamiento de elección en la hemofilia A es la administración de concentrados de factor VIII y hasta un 33 % de estos pacientes pueden desarrollar inhibidores dirigidos contra el factor infundido. Varias causas pueden estar involucradas en este proceso inmune siendo la intensidad de la terapia y tipo de concentrados empleados uno de los más estudiados. Por lo tanto, el análisis cuali / cuantitativo de diferentes proteínas que hacen a un concentrado más inmunogénico que otros cobra vital importancia. En este trabajo se evaluaron los niveles de factor VIII antígeno (FVIII:Ag), factor von Willebrand (FvW) funcional e inmunológico, factor de crecimiento transformador ß1 (TGF-ß1), fibrinógeno e inmunoglobulinas G, A y M en diferentes lotes de concentrados de factor VIII manufacturados en UNC-Hemoderivados y fueron comparados con otros productos comerciales de similares características. Se estudiaron 6 lotes de 250 UI y 2 de 500 UI producidos en UNC-Hemoderivados, dos lotes de 250 UI producidos por las firmas A y B y un lote de 500 UI comercializado por la firma C. Para las determinaciones de factor VIII/factor de von Willebrand, funcional y antigénico, se emplearon métodos estándares y para el TGF-ß1 se utilizó un ELlSA comercial. Los resultados obtenidos mostraron que las relaciones entre FVIII:C/FVIII:Ag y FVIII:C/FvW funcional fueron en promedio 0.60 y 0.52 para el producto de UNC-Hemoderivados y 0.50 y 0.38 para los productos de origen comercial. Los valores TGF­ß1 y la actividad específica arrojaron mejores resultados en el producto de UNC-Hemoderivados que los productos A, B y C y en las otras proteínas analizadas no hubo diferencias. Por lo tanto podemos concluir que los concentrados de FVIII producidos en UNC-­Hemoderivados presentan propiedades que potencialmente indicarían una menor respuesta inmune contra el FVIII infundido.

Replacement therapy using plasma factor VIII (FVIII) concentrates is currently the major mean of preventing and controlling bleeding in hemophilia A patients. However. approximately a 33% of these patients may develop inhibitors to the substituted FVIII. Several causes may be involved in the immune process being the intensity of therapy and type of concentrate used one of the most studied. Therefore. the study quali/quantitative analysis of different proteins which make a concentrated more immunogenic than other takes a vital importance. In this study we evaluated FVIII antigen (FVIII: Ag), von Willebrand factor (vWF) functional and immunological, TGF-Beta1, fibrinogen and immunoglobulins G, A and M levels in FVIII concentrates manufactured in UNC-Hemoderivados and compared with other commercial products of similar characteristics. We studied 6 lots of 250 IU and 2 lots of 500 IU manufactured in UNC-Hemoderivados, 2 lots of 250 IU produced by two pharmaceutical companies named A and B and 1 batch of 500 IU marketed by C. For the determinations of F VIII/FvW functional and antigenic we used standards methods and the TGF-ß1 was assayed by ELlSA test. The results show that the relationship between FVIII/FVIII:Ag and FVIII/vWF functional were in average 0.6 and 0.52 for the product of UNC-Hemoderivados and 0.5 and 0.38 for products from commercial sources. TGF-ß1 levels and the specific activity showed upper values in UNC-Hemoderivados compared to commercial products and none difference was observed in the other proteins assayed. Therefore we can conclude that FVIII concentrates produced at UNC-Hemoderivados have properties that indicate a potentially lower immune response against the infused FVIII.

Evaluación de neoantígenos en concentrados de factor VIII analizados por enzimoinmunoensayo / Neoantigens evaluation on F VIII concentrates analyzed by enzymeimmunoassay (EIA)

El propósito de este estudio fue evaluar la inmunogenicidad de concentrados de factor VIII producidos en UNC-Hemoderivados antes y después del tratamiento térmico empleando un enzimoinmunoensayo (EIE) desarrollado en nuestro laboratorio. Materiales y método: Para ese propósito se obtuvieron anticuerpos contra factor VIII calentado y no-calentado por inmunización de conejos y la inmunoglobulina G específica fue aislada por afinidad a Proteína A-Sepharosa. El EIE fue realizado empleando como antígeno de captura el factor VIII con o sin tratamiento térmico (0.5 ug/pocillo), los sitios inespecíficos bloqueados con albúmina al 2 por ciento y el anticuerpo revelado con un anti-IgG de conejo conjugada a peroxidasa. La presencia de neoantígenos fue estudiada por incubar durante 2 hs a 37°C y luego a 4°C toda la noche, con cantidades crecientes de factor VIII antes y después del tratamiento térmico (0.03 a 600 ug de proteínas), con cantidades adecuadas de IgG anti-factor VIII calentado y no-calentado. Luego de centrifugar la muestra para la separación de los inmunocomplejos, se valoró la presencia de anticuerpos en el sobrenadante por EIE tanto para factor VIII calentado y no calentado. Resultados: Los resultados obtenidos permitieron observar que para ambos anticuerpos (calentado y no calentado) se neutralizaba la misma cantidad de factor VIII calentado y no calentado (0.30 ug) y además, se pudo comprobar que esta conducta se repetía cuando se empleaban en el EIE como antígeno de captura, factor VIII con y sin tratamiento térmico. Conclusiones: Los datos obtenidos de este estudio nos permiten concluir que el tratamiento térmico aplicado a los concentrados de factor VIII elaborados en la UNC-Hemoderivados no producen ninguna formación de neoantígenos. A juzgar por el sensible y específico EIE desarrollado en nuestro laboratorio.

Introduction: with the purpose of improving viral security, plasma-derived products are generally subjects of solvent/detergent and heating (100°C for 30 minutes) treatment which are introduced during the manufacturing process. The formation of neoantigens in factor VIII concentrates produced after the heat treatment could trigger an immune response against the modified protein and the native protein. Objetives: the purpose of this study was to evaluate the immunogenicity of factor VIII concentrates produced in UNC-Hemoderivados before and after heat treatment, using an EIA developed in our laboratory. Materials and methods: antibodies against factor VIII with and without heating treatment were obtained by rabbit immunization. The specific IgG was isolated by protein-A-Sepharose affinity chromatograpy. Two EIA plates were coated (0.5 ug/well) one with factor VIII heated (H) and the other one with factor VIII no-heat (no-H) and the antibodies detection were performed using rabbit anti-lgG peroxidase conjugated. The neoantigens were studied by incubation of increasing concentrations of factor VIII (0.03-600 ug of proteins) before and after heating treatment during 2 hours at 37 °C and overnight at 4 °C with adequate concentrations of anti-factor VIII IgG (with and without treatment). The immunocomplexes were removed by centrifugation and the free antibodies in the supernatant were measured by EIA in plates H and no-H. Results: the EIA showed a linear behavior between antibodies dilution ranged from 1/8000 to 1/512000. With these results we can conclude that the same concentration of factor VIII heated and unheated (0.30 ug) were neutralized with either antibodies (heated and unheated). These results were similar in both EIA plates coated with factor VIII H and no-H...

Procoagulant factors in patients with cancer.

BACKGROUND: Clotting activation and thromboembolic manifestations are common features in patients with cancer. Tumor cells can directly activate the clotting through two procoagulants: tissue factor (TF) and cancer procoagulant (CP). AIMS: The aim was to evaluate the levels of TF and CP in patients with different tumors in order to: (1) establish an association between these markers and the tumor localization, (2) establish a correlation between the levels of procoagulants and the status of the disease, (3) evaluate if the treatment with chemotherapy induced some modifications on the levels of procoagulants, (4) evaluate the possibility of using procoagulants as predictors in the development of thrombosis. METHODS: Sixty-one patients with different types of cancer (lung, breast, digestive and genitourinary) and 20 normal controls were included. The activity of TF and CP was studied in serum samples. Statistical analysis of the data was performed by two-tailed Fisher exact test. RESULTS: The TF was increased in 72.5 and 0% (p < 0.01) of cancer patients and normal controls, respectively. PC was found to be increased in 88% of the cancer patients but in healthy controls it was increased in only 15% (p < 0.01). The patients with genitourinary cancer presented the highest values of both procoagulants coinciding with a major prevalence of thrombotic events. The activity CP was found in 93% of patients with stages I and II but in patients with stages II and IV disease it was found in 85% (not significant). There were no differences in the levels of both procoagulants between the patients treated with chemotherapy and those with other treatments. CONCLUSIONS: TF and CP are elevated in patients with cancer. The highest values of both procoagulants are in the genitourinary cancer group in agreement with the greater presence of thrombosis observed in this group. Clinical follow up is important in order to determine the potential value of these procoagulants and the tendency to develop thrombosis in patients with cancer.

Factores procoagulantes en pacientes con cáncer / Procoagulant factors in patients with cancer

El objetivo del presente estudio fue determinar los niveles de factor tisular (FT) y procoagulante del cáncer (PC) en pacientes con enfermedades neoplásicas para intentar establecer: 1) si existe asociación entre la presencia de estos marcadores y el origen del tumor; 2) si los niveles de estas proteínas procoagulantes se correlacionan con los estadíos I/II o III/IV de la enfermedad; 3) si los tratamientos con quimioterapia modifican los niveles séricos del FT y PC y, finalmente 4) evaluar si estos procoagulantes podrían comportarse como marcadores predictivos en el desarrollo de trombosis. Se incluyeron 61 pacientes con diferentes tipos de cáncer: pulmón (n=14), mama (n=19), digestivo (n=13), y génitourinario (n=12) y controles normales (n=20). Los resultados demostraron una sensibilidad y especificidad del 87,9% y 85%, respectivamente, para el PC y del 72,4% y 100% para el FT. Los pacientes con cáncer génitourinario presentaron los valores más altos de ambos procoagulantes coicidiendo con la mayor prevalencia de trombosis objetiva clínica y radiológicamente. Ninguno de los procoagulantes evaluados permitió difenciar estadío I-II de III-IV de la enfermedad. Por otra parte, el tratamiento con quioterapia no modificó con significancia estadística, los niveles de ambos procoagulantes. Un seguimiento clínico y de laboratorio en función del tiempo y del tratamiento sería importante para establecer el valor pronóstico de los niveles de estos procoagulantes y su propensión a desarrollar trombosis en pacientes con cáncer.

Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation.

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3',4'-tetrasulphate (QTS) and quercetin 3-acetyl-7,3',4'-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 microM, final concentration), and the platelet aggregation was induced by using adenosine 5'-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 microM) and Qc (250 microM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 microM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA2) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B2 (TxB2) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA2, as judged by TxB2 formation, in stimulated WP and a marked interference on the TxA2 receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.

Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis.

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.