Hugo Guglielmone

Procoagulant factors in patients with cancer

Abstract 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.

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.

Response to the DDAVP test in a patient with combined deficiency of factor V and factor VIII

Combined factor V and factor VIII deficiency (F5F8D) was first recognized in two young brothers by Oeri et al. [1] as a rare, recessive coagulation disorder. Individuals affected with this disease present with a moderate bleeding tendency and plasma levels of factor V and VIII in the range of 5–30% of normal. Extensive genetic analysis of F5F8D patients linked the disorder to causative mutations in the two genes LMAN1 and MCFD [2,3]. Subsequent studies suggest that LMAN1, a mannose-specific membrane lectin, operates as a cargo receptor for the transport of glycoproteins from the endoplasmic reticulum (ER) to Golgi intermediate compartment (ERGIC) indicating that F5F8D could result from a defect in the ER-to-Golgi transport machinery of both coagulation factors. However, in approximately 30% of individuals with this dual deficiency no LMAN1 mutations could be detected, suggesting that another gene may also be involved in this disease [4]. Recently, Zhang et al. [5] reported that mutations in a novel gene named MCFD2 (multiple coagulation factor deficiency gene 2) are also responsible for F5F8D. MCFD2 is an EF-hand domain protein localized to the ERGIC through a direct, calciumdependent interaction with LMAN1. LMAN1 and MCFD2 form a protein complex that directly interacts with FVIII [6]. Desmopressin (DDAVP) is a synthetic derivate of the antidiuretic hormone (AVP, l-arginine vasopressin) that has a prolonged and increased antidiuretic effect compared with AVP. On account of these properties, DDAVP is registered for the treatment of diabetes insipidus and enuresis nocturna. In addition, DDAVP increases the concentrations of factor VIII and von Willebrand factor in plasma [7]. Hence, DDAVP is prescribed in patients with mild haemophilia A and von Willebrand disease type 1 prior to minor surgical procedures and for non-life threatening bleedings. In this report, we performed a DDAVP test in a 11-year-old Argentinean (pre-pubertal) girl prior to her beginning menarche. The girl, at 8 years of age, had presented with moderate epistaxis and increased bruising in the legs. At 7 years of age she suffered profuse bleeding following dental trauma. Fortunately, it stopped with local compression alone. Both parents, who are not consanguineous, and the two older brothers are asymptomatic, but the paternal grandmother and a maternal uncle were complained with easy bruising and epistaxis. Laboratory tests, in the girl, revealed a prolongation of both the prothrombin time and the activated partial thromboplastin time in comparison to normal (17.8 ± 0.9 vs. 12.1 ± 0.2 seg. and 56.3 ± 2.1 vs. 29.8 ± 1.1 seg., respectively) with normal fibrinogen and thrombin time. The levels of factor V and factor VIII detected by one-stage procedure in the plasma of the proband were 23.2 ± 3.1% and 18.6 ± 6.1%, respectively (mean of two determinations assayed in three occasions >3 months apart). Furthermore, the presence of weak and strong inhibitors against factor V and factor VIII were analysed by incubation of plasma patient with normal plasma at 37 C during 2 h and the results were negative. To elucidate possible molecular defects in LMAN1 and/or MCFD2 gene, genomic DNA was purified and the smaller MCFD2 gene was sequenced and no mutations were identified. Instead, the patient was found to be compound heterozygous in exon 12 of the LMAN1 (1423T fi C) with no mutation detected on the second allele. The missense mutation Correspondence: Hugo A. Guglielmone, Servicio de Hematologı́a, Fundación Onco-Hematologı́a (FUNDONHEM) 7mo Piso, Sanatorio Allende, Obispo Oro 42, (X5000IUP) Córdoba, Argentina. Tel.: +54 351 4269273; fax: +54 351 4269273; e-mail: hgugli@bioclin.fcq.unc.edu.ar

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Identification and characterization of novel mutations implicated in congenital fibrinogen disorders

Essentials Fibrinogen Disorders are characterized by variable expressivity. Patients with fibrinogen disorders can present with bleeding, thrombosis, or both. As previously reported, genotype‐phenotype correlations are difficult to establish. Molecular modeling may help to further understand the effects of mutations on the mature fibrinogen protein.

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.

Platelet abnormalities in a family with von Willebrand disease type 2B (V1316M) and association with bleeding score.

ing for contemporary developing nations, particularly a country with the lowest GNI compared to other developing countries that have published their experience on surgeries in haemophiliacs [2]. Future directions should be towards raising awareness among public and health care professionals, gathering attention from national and international bodies and organizations, eliciting more evidence regarding the gravity of problem in the poor nations and the global attempts to increase supply of non-outdated factors in the poor nations, with the aim of equity in level of health care for Haemophiliacs globally. We are presently supported by the World Federation of Haemophilia through a twining programme with a developed Western hemophilia treatment center (Rochester NY USA) and look forward through this twinning by expanding our perioperative expertize and increased factor allotment to offer to more haemophiliacs the opportunity when indicated to undergo surgery effectively and safely.

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.