I. Alberca

The c.−1639G > A polymorphism of the VKORC1 gene is a major determinant of the response to acenocoumarol in anticoagulated patients

Much of the variability in the sensitivity to warfarin in anticoagulated patients is associated with the c.−1639G > A polymorphism of the vitaminK‐epoxide reductase (VKORC1) gene. However, its association with the acenocoumarol dose in patients under anticoagulant therapy has not been studied. The c.−1639G > A genotype of VKORC1 was determined in 113 patients on stable anticoagulation requiring low (n = 42), medium (n = 42) or high (n = 21) acenocoumarol doses. To evaluate the association between acenocoumarol requirements and the c.−1639G > A variant, multivariate logistic regression models were fitted, adjusting for age, gender, and the c.430C > T and c.1075A > C variants of cytochrome P450 2C9 (CYP2C9). A total of 90·5% of the patients in the low acenocoumarol dose group carried the A allele of VKORC1:c.−1639G > A. The A allele independently increased the odds of requiring a low acenocoumarol dose [odds ratio (OR) 9·4; 95% confidence interval (CI) 1·9–46·4; P = 0·006], especially when the homozygous form was present (OR 44·2; 95% CI 5·5–354·6; P < 0·001). The A allele was less frequent in the high dose group showing an inverse association with the requirement for high doses (OR 0·04; 95% CI 0·01–0·22; P < 0·001). The A allele of the c.−1639G > A polymorphism of VKORC1 is therefore associated with a low‐dose requirement for acenocoumarol in patients receiving anticoagulant therapy.

Mesenchymal stem cells are functionally abnormal in patients with immune thrombocytopenic purpura

BACKGROUND AIMS Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by an accelerated destruction of platelets as a result of the presence of autoreactive antibodies. Patients with ITP also display activated platelet-autoreactive T cells. Mesenchymal stem cells (MSC) inhibit both T- and B-cell activation and may have functional impairments in autoimmune disorders. METHODS We analyzed the potential role of MSC in the pathogenesis of ITP. RESULTS MSC from ITP showed an impaired proliferative capacity and a lower capability of inhibiting activated T-cell proliferation compared with healthy donors. While MSC from controls showed a decreased expression of p27 after stimulation with platelet-derived growth factor, this effect was not observed in MSC from patients. Furthermore, MSC from healthy donors down-regulated p16 upon exposure to platelet-released supernatant, while this effect was not observed for ITP. Interestingly, caspase 9 expression was higher in MSC from ITP. CONCLUSIONS These abnormalities suggest a role of MSC malfunction in the physiopathology of the disease and may have therapeutic implications.

Risk of recurrent venous thrombosis in patients with G20210a mutation in the prothrombin gene or factor V Leiden mutation

The impact of the G20210A prothrombin mutation, factor V Leiden and 677T mutation of methylene tetrahydrofalate reductase (MTHFR) in recurrent deep venous thrombosis (DVT) is not so clear. We have prospectively monitored 259 patients following a first episode of DVT in order to determine which factors influence the development of a recurrent event. Several clinical and biological factors together with the genetic polymorphisms of factor V Leiden, G20210A prothrombin and 677T MTHFR were assessed. During a median follow-up of 786 patient-years, 27 patients (14%) developed one objective episode of recurrent venous thrombosis. The carriers of a double defect, homozygous or double heterozygous for factor V Leiden and G20210A, had an increased risk after a first episode of DVT, while patients who were isolated heterozygous for factor V Leiden or G20210 had a risk of recurrent DVT similar to patients who had neither mutation (annual incidence of 12.1, 3.1, 2.9 and 2.8%). The 677T MTHFR mutation alone or combined with hyperhomocysteinemia was not associated with an increased risk of recurrent events. The development of proximal DVT (P = 0.01) and the presence of a double defect (P = 0.01) were the only two risk factors independently associated with a high recurrence ratio in the multivariate analysis. Thus, the annual incidence of DVT recurrence in patients without any of these two risk factors was only 0.6% (95% confidence interval, 0.2–0.9). We have identified a group of patients with DVT but at very low risk of re-thrombosis in whom an extended secondary thromboprophylaxis should be carefully considered.

Prothrombin A19911G and G20210A polymorphisms' role in thrombosis

Summary. The prothrombin G20210A polymorphism, which correlates with the plasmatic prothombin levels, is the second genetic risk factor for deep venous thrombosis (DVT), although its prothrombotic role is mild. Recently, the prothrombin A19911G polymorphism, also associated with slight variations of the prothrombin level, has been suggested to modulate the thrombotic risk of the G20210A polymorphism in a preliminary study including few patients and controls. Our study evaluated the effect of the A19911G polymorphism in the arterial and venous thrombotic risk of the prothrombin 20210G/A genotype, analysing 204 consecutive DVT patients and 204 matched controls. Moreover, we analysed 213 carriers of the 20210G/A genotype (152 with DVT, 26 with arterial thrombosis and 35 healthy subjects) and 10 homozygous 20210 A/A carriers. We developed a simple method to simultaneously determine the genotype of both polymorphisms. In accordance with our case/control study, the A19911G polymorphism did not play a significant role in the development of DVT. Analysis of 120 20210 A alleles demonstrated a complete linkage disequilibrium with the 19911 A allele. These polymorphisms (alone or combined) did not modify the risk of arterial thrombosis. However, the 19911A/G genotype slightly increased the risk of developing DVT in carriers of the 20210G/A genotype (OR 3·34 vs 5·86), supporting that the prothrombin 19911 polymorphism could modulate the risk of the G20210A polymorphism in developing DVT.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol‐anchored proteins (GPI‐APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG‐A) gene.

AB0 blood group and risk of venous or arterial thrombosis in carriers of factor V Leiden or prothrombin G20210A polymorphisms

Several studies have shown an effect of AB0 blood group on hemostasis. Findings of this study indicate that non-00 group increases the risk and severity of venous thrombosis in carriers of prothrombotic polysmorphisms. See related perspective article on page 649. Background Routine analyses for thrombophilia include determination of the presence of factor V Leiden and prothrombin 20210A polymorphisms. However, the usefulness of these determinations is controversial and the clinical benefit remains questioned because of the moderate risk of associated thrombosis in carriers. In the search for clusters of thrombotic risk factors to estimate individual risk better, we studied the effect of AB0 blood group, a highly prevalent factor with mild prothrombotic features, on the risk and severity of venous and arterial thromboses in carriers of these polymorphisms. Design and Methods We genotyped the AB0 blood group in 981 carriers of factor V Leiden or prothrombin 20210A polymorphisms. In order to avoid the over-representation of a particular genotype and to suppress confounding factors, we included only non-related heterozygous carriers without additional genetic risk factors. We studied 609 patients with venous thromboembolism (287 with factor V Leiden, and 322 with prothrombin 20210A), 174 patients with myocardial infarction (78 with factor V Leiden, and 96 with prothrombin 20210A), and 198 controls (96 with factor V Leiden, and 102 with prothrombin 20210A). Results Non-OO blood group did not increase the risk of myocardial infarction in carriers of factor V Leiden or prothrombin 20210A. However, non-OO blood group contributed significantly to the expression of venous thrombosis associated with both factor V Leiden (OR: 1.76; 95%CI: 1.06–2.91) and prothrombin 20210A (OR: 2.17; 95%CI: 1.33–3.53). Exclusion of A2A2 and A2O from the non-00 blood group (because factor VIII-von Willebrand factor levels are similar in these and the 00 blood group) increased the thrombotic risk. Finally, non-OO blood group was associated with an earlier onset in symptomatic carriers of these polymorphisms. Conclusions Our study suggests that non-OO blood group increases the risk and severity of venous thrombosis in carriers of prothrombotic polymorphisms. Thus, AB0 phenotyping or genotyping analyses may be valuable components in assessing future thrombophilic risk profiles and might have implications for the policy of thrombosis prophylaxis and treatment.

Protein C levels in late pregnancy, postpartum and in women on oral contraceptives

4 groups of 30 women participated in a study designed to examine the protein C level during late normal pregnancy, the puerperal period, and oral contraceptive (OC) use. The results were compared with those obtained with another important inhibitor, i.e., antithrombin III. In pregnant women, protein C levels during the 3rd trimester of normal pregnancy were the same as those in control nonpregnant women. Delivery did not induce any variations in protein. Antithrombin III was slightly decreased in late pregnancy, the decrease being more important at 24 hours following delivery. The women using OCs showed a significant increase in protein C compared with the control group; antithrombin was similar in both groups. The results show that protein C levels were not altered in the 3rd trimester of normal pregnancy and in the immediate postpartum period and make it difficult to assign an important role to protein C in the mechanism responsible for the increased rick of thrombosis observed in these situations. The data also eliminate the possibility that the risk of thrombosis observed in OC users could be due to a decrease in protein C.

Association of the Thrombomodulin Gene c.1418C>T Polymorphism With Thrombomodulin Levels and With Venous Thrombosis Risk

Objective—To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. Approach and Results—The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. Conclusions—The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane–bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

The role of immature platelet fraction in acute coronary syndrome

Dear Sir, Activated platelets play a crucial role in the pathogenesis of acute coronary syndrome (ACS) (1), and platelet size can be a determinant of platelet function. Thus, large platelets are metabolically more active than small platelets and have greater pro-thrombotic potential (2). However, the clinical influence of platelet size, measured as mean platelet volume (MPV), is controversial (3–6). An alternative to MPV measurement is the analysis of immature platelet fraction (IPF). Newly formed platelets can be distinguished from mature platelets that are poor in RNA by flow cytometric quantification using fluorescent dyes that bind RNA (7). However, this method produces high intra-assay reproducibility coefficients of variation (8) and requires specialist personnel to perform it. A new, rapid and fully automated method for the measurement of reticulated platelets, expressed as the immature platelet fraction (IPF), using the Sysmex XE-2100 blood cell counter has been under development. Recently, Grove et al showed that measurements of IPF made with the Sysmex XE-2100 were significantly higher in patients with ACS than in healthy subjects (9), although further studies were required. Therefore, the aim of this study is to examine the relationship between IPF and ACS in a case-control study. A total of 404 individuals were enrolled in the study. A total of 202 patients with a documented first episode of ACS were prospectively recruited upon admission to the Coronary Unit. The diagnosis of ACS included ST segment elevation myocardial infarction (STEMI, n = 129) and non-ST segment elevation ACS (NST ACS, n = 73). The latter group included non-ST segment elevation myocardial infarction (NSTEMI) and unstable angina (UA) patients. Diagnosis of STEMI, NSTEMI and UA was made according to the new ESC/ACC consensus definition (10). The control group for our study included 202 unrelated healthy persons without a history of vascular or thromboembolic disease and a normal electrocardiogram. All subjects gave their informed consent to enter the study. Blood was collected from the antecubital vein at 08:00 a.m. on day 1 or 2 after admission. IPF was calculated as the ratio of immature platelets to the total number of platelets in a fully automated haematology analysis system (Sysmex XE-2100; Sysmex,

Effect of Hemodialysis on Protein C Levels

The immunological functions of antithrombin III and protein C were studied in 8 patients before, during and after hemodialysis. After dialysis a significant decrease in protein C and antithrombin III levels was seen. The changes observed in both proteins after hemodialysis should be considered as a component in the genesis of the hypercoagulation state in these patients.