Suely Meireles Rezende

Hematologic variables and venous thrombosis: red cell distribution width and blood monocyte count are associated with an increased risk

Recent studies suggest that leukocytes and erythrocytes play a role in coagulation. However, whether leukocytes, erythrocytes and other hematologic variables are associated with risk of venous thrombosis is not well known. To study this, we used data from 2473 patients with venous thrombosis and 2935 controls. The variables assessed were: total leukocytes, granulocytes, lymphocytes, monocytes, hematocrit, hemoglobin, erythrocytes and red cell indices (mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and red cell distribution width). We found a strong dose-response relation for higher red cell distribution width and monocyte count with risk of venous thrombosis, with odds ratios of 3.1 (95% confidence interval, 2.0–4.8) and 2.8 (95% confidence interval, 1.3–5.8), respectively, after adjustment for age, sex, C-reactive protein level, malignancy and co-morbidities. Monocyte count and red cell distribution width were associated with venous thrombosis even within reference ranges. A low monocyte count (<0.12×109/L) was associated with a lower risk of venous thrombosis after full adjustment (odds ratios 0.6; 95% confidence interval, 0.4–0.8). In summary, high red cell distribution width and blood monocyte count, two parameters that are inexpensive and easily obtainable, were clearly associated with an increased risk of venous thrombosis. Future studies should evaluate the underlying mechanism and the use of these variables in prediction models for first and recurrent thrombosis.

Registry of inherited coagulopathies in Brazil: first report

Summary.  Inherited coagulopathies are bleeding disorders, which require treatment for life. Keeping an updated registry on these diseases is crucial for planning care, documenting prevalence of diseases and evaluating effectiveness of resources. We have analysed data from 26 treatment centres on coagulopathies in Brazil. Information included socio‐demographic data, diagnosis of coagulopathies, severity of haemophilias A and B, presence and quantification of inhibitors in haemophilia, type of von Willebrand disease (VWD) and infection status for viral diseases. On 1 July 2007, there were 10 982 patients with inherited coagulopathies in Brazil, of which 6881 (62.7%) corresponded to haemophilia A, 1291 (11.7%) to haemophilia B, 2333 (21.2%) to VWD, 258 (2.4%) to other coagulopathies and 219 (2.0%) to undiagnosed bleeding disorders. Haemophilia A and B inhibitors were present in 9.9% and 1.9% of the patients, respectively. Human immunodeficiency virus infection was present is 6.5%, 4.8% and 1% of patients with haemophilia A, B and VWD, respectively. Hepatitis C virus infection was present in 34.9%, 29.7% and 12% of patients with haemophilia A, B and VWD, respectively. Infection by hepatitis B and human T‐cell leukemia‐lymphoma virus was also reported. This is the first report on the registry of patients with inherited coagulopathies in Brazil, supposed to be the third largest population of patients with haemophilia.

Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain.

Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S-deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, gamma-carboxylated and bound phospholipids with an apparent dissociation constant (Kd(app)) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC.

Duration of symptoms and D-dimer testing in the ruling-out of venous thromboembolism.

Within the last decade, the diagnosis of venous thromboembolism (VTE) has evolved with standard clinical riskstratification models, D-dimer testing and helical computed pulmonary tomography, among others [1–5]. The demand for diagnostic image testing in patients with suspected VTE has increased along with the costs related to this approach. To address this problem, many non-invasive management strategies have been proposed to achieve a rapid, safe and costeffective diagnosis. Due to different VTE prevalence, D-dimer performance, and resources available, local validation of these strategies is recommended [6]. Despite a few shortcomings, such as method variation, ageand setting-related sensitivity problems and a rather low specificity, D-dimer testing has been successfully employed to rule out VTE in low-risk outpatients [1,2]. Age of the clot, estimated by the duration of symptoms, is recognized as one of the potential clinical factors affecting the performance of Ddimer, but its cut-off limit has not been accurately defined [6]. In previous studies employing D-dimer, the cut-off point has ranged from 3 days to up to 43 days [6,7]. Some have proposed the duration of symptoms cut-off point to be between 7 and 15 days [8,9]. The British Committee for Standards in Haematology recommended that D-dimer testing should be used with caution if the patient had symptoms for over 2 weeks [6]. Recently, we retrospectively evaluated the role of D-dimer in a non-invasive clinical approach to VTE. The D-dimer used was a rapid quantitative ELISA method (VIDAS D-dimer; BioMerieux, Marcy l’Etoile, France) with a cut-off level of 500 ng mL. Blood samples were collected prior to anticoagulant treatment. All measurements were carried out by an operator unaware of the results of imaging tests. Imprecision of replicate determinations of samples with D-dimer concentration of 550 ng mL was 5.8%, similar to previous studies that employed this method [8,10]. We were able to review the medical records from 335 adults admitted to an outpatient emergency clinic at a hospital located in Belo Horizonte, Brazil, between August 2002 and August 2004. We also reviewed the medical records of these patients during a follow-up period of 90 days. In 205 out of 335 patients (61%) a telephone interview was also successfully undertaken. Deep vein thrombosis (DVT) and pulmonary embolism (PE) were objectively confirmed by compression ultrasonography (CUS) and lung scan or helical chest computed tomography, respectively, following standard criteria [3–5]. A patient was considered as having a VTE event when suggestive symptoms associated with positive imaging test were present at a first examination or during the 90-day follow-up period. Therefore, a false-negative case would represent a confirmed VTE event in a patient with a negative D-dimer, and a false-positive case would result from a non-confirmedVTE event in a patient with a positive D-dimer. Population age was 64 ± 14.5 years (range 22–94 years). Male to female ratio was 0.35. The prevalence of VTE was 8.7% (10 episodes of DVT and 19 episodes of PE), with 27 events at presentation and two during the follow-up period. One patient had both DVT and PE at presentation and was considered as having one PE event for analysis. Table 1 shows the sensitivity of D-dimer in relation to symptoms duration in the population studied. When we analyzed the 29 VTE events, there were 27 patients with positive D-dimer (true positives) and two false-negative results. These were related to a 48-year-old male and a 75-yearold female, who had symptoms for 30 and 60 days, respectively

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest-specific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane.

Secondary prophylaxis with warfarin for recurrent thrombosis in a patient with Glanzmann thrombasthenia and F5 G1691A

Cheson, B.D., Horning, S.J., Coiffier, B., Shipp, M.A., Fisher, R.I., Connors, J.M., Lister, T.A., Vose, J., Grillo-Lopez, A., Hagenbeek, A., Cabanillas, F., Klippensten, D., Hiddemann, W., Castellino, R., Harris, N.L., Armitage, J.O., Carter, W., Hoppe, R. & Canellos, G.P. (1999) Report of aninternational workshop to standardize response criteria for non-Hodgkin’s lymphomas. NCI Sponsored International Working Group. Journal of Clinical Oncology, 17, 1244. Cheson, B.D., Pfistner, B., Juweid, M.E., Gascoyne, R.D., Specht, L., Horning, S.J., Coiffier, B., Fisher, R.I., Hagenbeek, A., Zucca, E., Rosen, S.T., Stroobants, S., Lister, T.A., Hoppe, R.T., Dreyling, M., Tobinai, K., Vose, J.M., Connors, J.M., Federico, M. & Diehl, V. (2007) Revisedresponse criteria for malignant lymphoma. Journal of Clinical Oncology, 25, 579–586. Gisselbrecht, C., Glass, B., Mounier, N., Singh Gill, D., Linch, D.C., Trneny, M., Bosly, A., Ketterer, N., Shpilberg, O., Hagberg, H., Ma, D., Briere, J., Moskowitz, C.H. & Schmitz, N. (2010) Salvage regimens with autologous transplantation for relapsed large B-cell lymphoma in the rituximab era. Journal of Clinical Oncology, 28, 4184–4190.

Tromboprofilaxia: recomendações médicas e programas hospitalares

Venous thromboembolism (VTE) is the most preventable cause of death in hospitalized patients. Hospital-related VTE is associated with more than half of the VTE burden in a community, either in-hospital or after discharge. Selective thromboprophylaxis is recommended for patients at risk. Patient selection for thromboprophylaxis requires proper VTE risk stratification. VTE stratification may be achieved by either risk assessment models (RAM) or by models based on patients illness and associated risk factors. Whatever the model, a thromboprophylatic recommendation should be formulated for each VTE risk category. VTE thromboprophylaxis may include general measures, mechanic compression procedures, pharmacological intervention or a combined approach. After many decades of consensus statements, a large proportion of at risk patients (20% to 75%) still does not receive proper thromboprophylaxis. This study aims to alert to the relevance of thromboprophylaxis and to suggest hospital thromboprophylatic strategies in a Brazilian setting.

Epidemiology of recurrent venous thrombosis

Venous thrombosis, including deep vein thrombosis and pulmonary embolism, is a common disease that frequently recurs. Recurrence can be prevented by anticoagulants, but this comes at the risk of bleeding. Therefore, assessment of the risk of recurrence is important to balance the risks and benefits of anticoagulant treatment. This review briefly outlines what is currently known about the epidemiology of recurrent venous thrombosis, and focuses in more detail on potential new risk factors for venous recurrence. The general implications of these findings in patient management are discussed.

The influence of prothrombotic laboratory abnormalities on the risk of recurrent venous thrombosis

Venous thrombosis (VT) is a disease that occurs in approximately 1–2 per 1000 persons per year [1]. Despite adequate treatment, up to one quarter of patients with VT will experience a recurrent event within the subsequent 5 years [2]. It has been recognized that the presence of a transient or reversible provoked risk factor at the time of VT is associated with a decreased risk of recurrence after anticoagulant therapy is stopped [3]. Therefore, a relatively short period of anticoagulant treatment with vitamin K antagonists is advised for those with clear provoking risk factors for VT, such as oral contraceptive use, hospitalization, or surgery [4]. However, approximately 30%-50% of events occur without a clear provoking risk factor. Since the risk for recurrent VT is higher when associated with unprovoked event, such patients may need indefinite anticoagulant treatment after a first unprovoked event. This could, however, lead to serious side effects such as major bleeding [5]. The challenge, therefore, is to identify patient groups who suffered an unprovoked event and still have a low risk of recurrence. These patients might benefit of anticoagulants for a fixed (shorter) time. Since prothrombotic alterations can be demonstrated in at least 50% of patients with VT [6], testing patients with a first VT for thrombophilia has gained great interest. Potential advances of testing patients might be the opportunity to elucidate the cause of the thrombosis and to track unaffected family members. However, there are potential disadvantages of testing for thrombophilia. Although the presence of inherited thrombophilia is currently considered a weak predictor of recurrence in patients with a first episode of VT, [3,7,8] these results were mostly obtained in patients with any type of first VT.Whether thrombophilia has predictive value for recurrence in patients who had a first unprovoked event is less well studied. Furthermore, most studies that were published on this issue [7,9], only measured prothrombotic laboratory abnormalities (such as levels of factor VIII or homocysteine) once, increasing the chance of a false positive (high) value of these prothrombotic abnormalities which could dilute risk estimates. We performed a prospective cohort study to assess the risk of recurrence in patients with provoked and unprovoked first VT, related to the presence or absence of prothrombotic abnormalities. In addition, these abnormalities were only considered present when they were confirmed in at least two consecutive measurements. The subjects of the cohort and definitions of the study were described previously [10]. Briefly, subjects were patients with one previous venous thrombotic event that were all seen by the same clinician (i.e. DDR) from April 2000 to July 2011 at the University Hospital of Universidade Federal de Minas Gerais and at Hematológica, a specialized medical center in Hematology, Belo Horizonte, Brazil. Patients were referred from anywhere in the State of Minas Gerais by

As bases moleculares da hemofilia A

Hemophilias are bleeding disorders due to deficiency of the blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B), resulting from mutation on the gene coding for factor VIII or factor IX. Hemophilia A is more frequent than hemophilia B and affects 1:10,000 male newborns. The severity and frequency of hemorrhagic episodes is related to residual activity of factor VIII present in the plasma and relates to the type of mutation associated with the disorder. Cloning of the factor VIII gene has enabled researchers to better understand the molecular basis of hemophilia A, accounting to date, for more than 1,000 mutations associated with the disease. This comprehensive knowledge permits an improved comprehension of the genotype-phenotype relation, establishment of clinical policies when mutations related to higher risk of inhibitors development are known, identification of hemophilia carriers in case of women related to patients, implementation of a program of genetic counseling and discovery of structural-functional relationship between gene-protein. This article aims to review the molecular basis of hemophilia A, laboratory techniques used to characterize mutations and clinical implications involved in the molecular diagnosis of hemophilia A.