Maria Aparecida Eiko Noguti

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech‐derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2×104 mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1–P4′ positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI‐2T (K8R, I9V, S10, K11W, P12A), LDTI‐5T (K8R, I9V, S10, K11S, P12L) and LDTI‐10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI‐2T and ‐5T, prolong the blood clotting time, inhibit thrombin (K i 302 nM and 28 nM) and trypsin (K i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI‐10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1′ (I9V). The data indicate further that LDTI‐5T might be a model candidate for generation of active‐site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme‐inhibitor interactions.

The role of IL-6, IL-8 and MCP-1 and their promoter polymorphisms IL-6-174GC, IL-8-251AT and MCP-1-2518AG in the risk of venous thromboembolism: A case-control study

INTRODUCTION Cytokines increased the risk of venous thromboembolism (VTE) in some case-control studies, but not in a prospective study. Data concerning the role of cytokines in the risk of VTE are limited. We examined in a case-control study the association of VTE and levels of interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) and assessed whether promoter polymorphisms (IL-6 -174GC, IL-8 -251AT, MCP-1 -2518AG) would affect the thrombotic risk and cytokine levels. MATERIALS AND METHODS The study included 119 patients (94 women) with a first event of VTE aged between 18-60 years, and 126 healthy controls (100 women) matched for age (±5 years). Blood was collected >7 months after the thrombotic event. Odds ratios (ORs) were calculated per increase of cytokines levels by 1 pg/mL. RESULTS ORs adjusted for age and sex were 1.520 [95% Confidence Interval (CI) 1.177 - 1.962] for IL-6, 1.095 (95% CI 1.002 - 1.196) for IL-8 and 1.000 (0.988 - 1.012) for MCP-1. With additional adjustment for ethnic composition, body mass index (BMI) and high sensitive C-reactive protein (hs-CRP), risk estimates remained significant for IL-6 and became of borderline statistical significance for IL-8. Polymorphisms did not influence the thrombotic risk and the cytokine levels in study participants. CONCLUSION VTE was associated with IL-6 and IL-8 levels, and for IL-6 this association was independent of BMI and hs-CRP. Thus far, a causal relationship between inflammation and VTE remains to be clarified and more prospective data are warranted.

Effect of estrogen-progestin hormonal replacement therapy on plasma antithrombin III of postmenopausal women

BACKGROUND This study was performed to evaluate antithrombin III levels in postmenopausal women receiving hormonal replacement treatment. METHODS It is a prospective randomized study concerning 19 postmenopausal patients, aged 40 to 65 years, who received either continuous daily oral equine conjugated estrogen 0.625 mg (group A, N=10) or daily transdermal 17beta-estradiol 50 microg (group B, N=9). Medroxyprogesterone acetate (5 mg/day, 14 days monthly) was given to all patients. Blood samples were obtained before and after 3, 6, 9 and 12 months of treatment. Coagulation tests included Antithrombin III (functional method), prothrombin time, partial activated prothrombin time, thrombin time, factor V, fibrinogen, platelet count and euglobulin lysis time. Friedman analysis of variance and Mann-Whitney test were used for statistical analysis. RESULTS Antithrombin III level was reduced (p<0.05) in group A but not in group B, although it remained within normal range. No changes were detected in the other coagulation tests. CONCLUSIONS These data suggest that oral conjugated estrogen replacement reduces functional ATIII, whereas transdermal estradiol replacement therapy does not modify it.

The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

Sickle cell disease (SCD) is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden), the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women), 29 with SS (sickle cell anemia; 28 years, range: 13-52 years) and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years) hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8%) and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8%) and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease), a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, beta-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

N-RAS and K-RAS gene mutations in Brazilian patients with multiple myeloma.

Point mutations affecting codons 12, 13 (exon 1) and 61 (exon 2) of the N-RAS gene and codons 12 and 13 (exon 1) of the K-RAS gene are identified in approximately 30.0% and 10.0%, respectively, of multiple myeloma (MM) patients living in the northern hemisphere. To date, there are no reports about the prevalence of RAS gene mutations in MM Brazilian patients, and this comprised the aim of the present study. DNA from bone marrow aspirates of 252 patients with MM (139 males and 113 females; aged 59.33 ± 11.95 years) were investigated for whole exons 1 and 2 of the N-RAS gene and whole exon 1 of the K-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Fifty-three out of 252 (21.03%) MM patients presented RAS mutations. Heterozygous mutations at codons 4, 10 (exon 1), 61 and 65 (exon 2) of the N-RAS gene were identified in seven out of 252 (2.78%) patients. K-RAS heterozygous mutations at codons 7, 12, 13 (exon 1) were seen in 46 out of 252 (18.25%) patients. To the best of our knowledge, the mutation at codon 7 of K-RAS gene is reported for the first time in MM. Taken together, these results suggest that Brazilian MM patients are characterized by: (i) a low prevalence of RAS mutation and (ii) RAS mutations located at distinct regions of the critical codons of the N-RAS and K-RAS genes.

Hemorrhagic and thrombotic complications in patients with myeloproliferative diseases

OBJECTIVE To correlate the incidence of hemorrhage and thrombosis to bleeding time (BT) and platelet aggregation in 27 consecutive patients with myeloproliferative diseases (MPD). DESIGN Retrospective study. SETTING Public tertiary referral center. PATIENTS Eighteen patients with chronic myelogenous leukemia (CML), 5 with polycytemia vera (PV), 2 with essential thrombocytemia (ET) and 2 with idiopathic myelofibrosis (MF). Dukes BT and epinephrine-induced platelet aggregation were performed on the patients and on 10 healthy individuals. RESULTS Eleven patients presented symptoms (41%):9 with hemorrhage (33%) and 5 with thrombosis (19%). There were less symptomatic patients in the CML group (28%) than in the other MPD (67%), without statistical significance (Fisher, p = 0.06). Dukes BT was longer in symptomatic patients (Mann-Whitney, p < 0.05). Platelet aggregation was abnormal in 7 patients (26%) and 71% of them were symptomatic (Fisher, p = 0.07). CONCLUSIONS The high incidence of bleeding and thrombosis in patients with MPD was related to prolonged BT, but not to platelet aggregation abnormalities.

Association between early glycemic control and improvements in markers of coagulation and fibrinolysis in patients with septic shock-induced stress hyperglycemia.

PURPOSE The purpose of this study is to evaluate the coagulation and inflammatory profiles in septic shock patients with baseline hyperglycemia under glycemic control. METHODS Prospective, observational study conducted in an intensive care unit of a university hospital, including 41 septic shock nondiabetic patients with hyperglycemia (n = 21) or normoglycemia (n = 20) profiles at baseline. Hyperglycemic patients received a glucose control protocol (target glycemia, <150 mg/dL). Metabolic, inflammatory, and coagulation markers were measured at baseline and after 24 hours. RESULTS Median glycemic values between groups were different at baseline but not after 24 hours. Baseline coagulation profile was similar in both groups with elevated levels of coagulation markers, reduced factor VII, protein C, and antithrombin activities and fibrinolysis impairment. Normoglycemic patients had unchanged coagulation markers after 24 hours. After treatment, previously hyperglycemic patients exhibited increased plasminogen concentrations (P = .03) and reduced levels of plasminogen activator inhibitor 1 (P = .01) and tissue plasminogen activator (P = .03) as compared with baseline. They also had higher factor VII (P = .03), protein C (P = .04), and antithrombin (P = .04) activities than normoglycemic patients. Inflammatory markers were elevated in both groups and improved after 24 hours, independently of the glycemic profile. CONCLUSIONS Glycemic control during septic shock is associated with improvements in coagulation and fibrinolysis parameters compared with baseline and normoglycemic patients.

Associação entre a evolução da disfunção orgânica e as concentrações de citocinas na fase inicial do choque séptico

OBJETIVO: Analisar o comportamento das disfuncoes orgânicas e sua correlacao com a resposta inflamatoria, avaliada pelas concentracoes basais de citocinas e pela evolucao dessas concentracoes, na fase precoce do choque septico. METODOS: Foram avaliados pacientes com idade acima de 18 anos e diagnostico de choque septico com menos de 48 horas de inicio das disfuncoes orgânicas. Foram mensuradas interleucina 6 (IL-6), interleucina 8 (IL-8), interleucina 10 (IL-10) e proteina C reativa na inclusao e apos 24 horas, sendo calculado o delta desses valores. A evolucao das disfuncoes orgânicas foi avaliada atraves do escore Sequential Organ Failure Assessment (SOFA) na admissao e apos 24 horas para determinacao do delta SOFA, posteriormente categorizado como piora ou melhora. Os resultados foram expressos como media ± desvio padrao ou mediana (percentil 25%-75%). Consideraram-se significativos resultados com valor descritivo de p menor que 0,05. RESULTADOS: Foram incluidos 41 pacientes com mediana do SOFA de 8,0(6,5 -10,0) e 8,0(6,0-10,0) na admissao (T0) e apos 24 horas (T1). Piora, melhora ou ausencia de alteracao do SOFA foram encontradas respectivamente em 11 (Grupo 1), 17 (Grupo 2) e 13 pacientes (Grupo 3). No grupo 1 os valores basais de IL-6, IL-8 e IL-10 foram mais elevados. No Grupo 1 houve aumento significativo de IL-8 apos 24 horas. A variacao do SOFA apos 24 horas mostrou correlacao significativa, embora fraca, com as concentracoes basais de IL-6 e IL-8. CONCLUSAO: As concentracoes basais mais elevadas de IL-6, IL-8 e IL-10 associam-se a evolucao desfavoravel da disfuncao orgânica. A elevacao das concentracoes de IL-8 nas primeiras 24 horas mostrou-se correlacionada a piora dessa disfuncao.

Performance of Platelin LS and dilute Russell's viper venom for the screening of lupus anticoagulant in patients with venous thromboembolism.

Lupus anticoagulant is associated with thrombosis and pregnancy morbidity, and its detection is of major clinical importance. The nature and concentration of phospholipids strongly influence the sensitivity of activated partial thromboplastin time (aPTT) reagents to lupus anticoagulant. We investigated the ability of Platelin LS, an aPTT reagent, to screen lupus anticoagulant among 94 patients with venous thromboembolism by comparing its performance with the dilute Russell viper venom time (dRVVT). Twenty-four patients had an abnormal aPTT and dRVVT, whereas 37 only had a prolonged dRVVT. In users of oral anticoagulants (n = 56), the dRVVT prolonged more frequently than the aPTT (98.2 vs 39.3%, P < 0.0001). After the mixing study, seven patients maintained abnormal aPTT and dRVVT ratios, five of whom had prolonged mixture with both tests. The agreement in the mixing study between aPTT and dRVVT was substantial (κ = 0.78, 95% confidence interval = 0.48–1.00). Except for one patient, the aPTT screened all cases that demonstrated phospholipid dependency of their inhibitor during the confirmatory procedure with the dRVVT. In conclusion, the aPTT using Platelin LS was highly associated with the presence of lupus anticoagulant detected by the dRVVT among patients with venous thromboembolism, and could be reliably employed as a screening assay for lupus anticoagulant.

Possible influence of clinical stage and type of treatment in the persistence of residual circulating t(14;18)-positive cells in follicular lymphoma patients.

Many patients with follicular lymphoma (FL) achieve response after treatment but complete remission (CR) rates are very low. Thus the majority of them will relapse, mainly those in advanced stage disease, due to the persistence of residual disease. Therefore, this study had the following aims: to determine the presence of bcl-2/IgH rearrangement in peripheral blood of early and advanced stage FL patients after treatment and to correlate it with their clinical situation at the same moment. We obtained 100 consecutive peripheral blood samples from 30 FL cases and conducted molecular studies using two separate semi-nested PCRs for MBR and mcr rearrangements. These semi-nested PCRs for bcl-2/IgH rearrangement were able to detect one positive cell among 10,000 normal cells. Clinical and molecular evolution of patients diagnosed as early stage disease suggested that molecular response could be obtained even with conventional chemotherapy or radiotherapy. In this group of patients, 64% achieved molecular response in some point during follow-up. However, only 23% of patients diagnosed as advanced stage disease reached molecular response when treated with chemotherapy (with or without radiotherapy). Due to the low number of subjects assessed in this study, we only found a tendency to significance when clinical stage at the diagnosis was associated to molecular response (P = 0.095). We observed 100% of concordance between clinical remission and molecular response in patients after bone marrow transplantation or in those cases treated with monoclonal antibody anti-CD20. This retrospective study, performed in a restricted number of patients, suggests that molecular response can be obtained in FL patients diagnosed at early stage disease, even with conventional chemotherapy and radiotherapy. In advanced stage disease, concordance between clinical remission and molecular response was observed in the majority of patients after bone marrow transplantation or in those cases treated with monoclonal antibody anti-CD20. The prognostic significance of this data should be confirmed with extended follow-up and in a larger number of patients.