Giovanna D'Andrea

Increased Risk for Venous Thrombosis in Carriers of the Prothrombin G→A20210 Gene Variant

Venous thrombosis is the third most common cardiovascular disorder after ischemic heart disease and stroke [1]. In addition to circumstantial risk factors (such as surgery, pregnancy, or immobilization), genetic abnormalities leading to hypercoagulability and to thrombophilia have been found in patients with thromboembolic disease [2]. Among patients from different ethnic populations, a common mutation in the gene of factor V (factor V Leiden mutation) [3] has been found in up to 60% of cases of familial thrombophilia [4]. Although the factor V Leiden mutation is a major risk factor for the development of venous thrombosis, many thrombotic events have an unclear pathogenesis. A common mutation, a GA transition at nucleotide position 20210 in the prothrombin gene locus, has recently been described [5]. The A allele is associated with higher plasma prothrombin levels, and in one study [5], carriers of this allele had a 2.8-fold increased risk for venous thrombosis compared with persons homozygous for the G allele. We sought to determine the presence of this mutation in 281 patients with venous thrombosis and investigated whether the presence of the prothrombin A (20210) allele was an independent, inherited risk factor for venous thrombosis. Methods After approval by the local ethics committees, the study was conducted according to the principles of the Declaration of Helsinki. All participants provided informed consent. Patients Persons with documented venous thrombosis who had been referred to one of two thrombosis centers (the IRCCS Casa Sollievo della Sofferenza in San Giovanni Rotondo, Italy, and the Ospedale A. Cardarelli in Naples, Italy) were investigated. Deep venous thrombosis was confirmed by phlebography or ultrasonography. Pulmonary embolism was diagnosed by angiography or ventilation-perfusion lung scanning. By using a previously validated questionnaire based on the World Health Organization questionnaire for cardiovascular disease [6], specially trained staff obtained a complete clinical summary from all patients, with emphasis on personal history of circumstantial risk factors for venous thromboembolism (surgery, immobilization, pregnancy, postpartum, trauma, use of oral contraceptives, varicose veins, and cancer) and for arterial thrombosis. Controls While patients were being recruited, apparently healthy persons were also randomly selected from a healthy population in southern Italy. None of these persons had been exposed to circumstantial risk factors for 8 weeks before the visit or had a history of venous thromboembolism, as determined by using a structured questionnaire validated for the retrospective diagnosis of venous thrombosis [7]. As was done with patients, trained staff obtained a detailed clinical summary from all controls, with emphasis on personal and family history for angina pectoris, myocardial infarction, ischemic stroke, and peripheral arterial disease [6]. Blood Collection and Coagulation Tests Blood samples were collected into vacuum tubes that contained 3.8% trisodium citrate as an anticoagulant. Samples were subjected to centrifugation at 2000 g for 15 minutes to obtain platelet-poor plasma, which was frozen and stored at 70C until assays were performed. Antiphospholipid antibodies (lupus anticoagulant and IgG anticardiolipin antibodies [measured by enzyme-linked immunosorbent assay, Byk Gulden, Italy]); antithrombin III, protein C, and amidolytic and immunologic protein S antigen (Behering, Marburg, Germany); and total and free protein S antigen (measured by enzyme-linked immunosorbent assay [Diagnostica Stago, Asnieres, France]) were measured in all patients [8, 9]. A thromboplastin-based assay on factor II-deficient plasma (Diagnostica Stago) was performed to measure prothrombin activity in 157 controls (72 men and 85 women) who were indistinguishable from other controls in age, sex, and social status (occupation). Results were expressed as the percentage of the amount of prothrombin in pooled normal plasma (arbitrarily designated as 100%). Clotting assays were performed on a KC4 Amelung coagulometer (Amelung, Austria). Extraction and Analysis of DNA Standard protocols were used to extract DNA from peripheral blood leukocytes [6]. The presence of factor V Leiden mutation was detected as described elsewhere [10], with some modifications [11]. The presence of the GA mutation of the prothrombin gene was tested according to the method of Poort and colleagues [5]. Statistical Analysis All analyses were done by using the Statistical Package for Social Science, version 6.1 for Macintosh (SPS, Inc., Chicago, Illinois). Differences in means and proportions were tested by using the Mann-Whitney U-test or the Kruskal-Wallis one-way analysis of variance and chi-square statistic or the Fisher exact test, as appropriate. We performed all analyses after grouping homozygous and heterozygous carriers of the prothrombin mutation and factor V Leiden mutation. Prevalence odds ratios (ORs), considered as the prevalence of existing disease, and 95% CIs were calculated by the normal approximation. If np(1 p) was small (<5), we used the exact method to calculate CIs (n = total number of participants, p = proportion of participants with a specific condition, and 1 p = proportion of participants without that condition). Adjusted odds ratios and their 95% CIs were calculated by logistic regression models that controlled for age, sex, presence of arterial thrombotic episodes, and factor V Leiden mutation. For all tests, a P value of 0.05 or less was considered statistically significant. Results Between May 1996 and December 1997, 348 patients (151 men and 197 women) with documented venous thrombosis were investigated. Sixty-seven patients (19.25%) had had at least one previous thrombotic events. Because these 67 patients are likely to have a greater susceptibility to thromboembolic episodes, we excluded them from the analysis. Thus, we analyzed 281 patients (128 men and 153 women; age range, 3 to 81 years). The median age at the time of the first thrombotic episode was 38.0 years (range, 16 to 81 years) for men and 35.0 years (range, 3 to 77 years) for women. The presenting thrombotic episode was deep venous thrombosis in one leg in 234 patients (106 men and 128 women; age range, 16 to 81 years), thrombosis of the upper extremities in 27 patients (13 men and 14 women; age range, 3 to 72 years), and isolated mesenteric vein thrombosis in 20 patients (age range, 17 to 70 years; 9 men and 11 women). Forty-five patients (17 men and 28 women) who had had deep venous thrombosis had also experienced an episode of pulmonary embolism. The control group consisted of 850 randomly selected, apparently healthy persons (388 men and 462 women; median age, 36.0 years [range, 22 to 66 years]). All patients and controls were white and were from the same region. The clinical characteristics of patients and controls are shown in Table 1. Table 1. Clinical Characteristics of Study Participants Forty patients (14.23% [95% CI, 10.14% to 18.32%]) carried the prothrombin GA20210 mutation; 35 were heterozygotes and 5 were homozygotes. Thirty-nine controls (4.59% [CI, 3.17% to 6.01%]) (P < 0.001) were heterozygotes, and none were homozygotes. The A20210 allele was noted in 8.01% of patients (CI, 5.77% to 10.25%) and 2.29% of controls (CI, 1.58% to 3.00%) (P < 0.001). The distribution of genotypes did not significantly differ from distributions predicted from the Hardy-Weinberg equilibrium in patients (P = 0.151) or controls (P > 0.2) and closely resembled those reported elsewhere [5]. Mean (SD) prothrombin activity was 99.33% 16.30% in the 150 patients without the prothrombin A20210 allele and 125.20% 7.69% in the 7 controls with the allele (Mann-Whitney U-test, P < 0.001). Fifty-one patients carried the factor V Leiden mutation (18.15% [CI, 13.64% to 22.66%]); 50 were heterozygotes and 1 was a homozygote. Forty-three controls carried this mutation (5.06% [CI, 3.59% to 6.53%]; P < 0.001); all were heterozygotes. Crude odds ratios for venous thrombosis associated with the presence of the prothrombin GA20210 or factor V Leiden mutation are reported in Table 1. We analyzed the association between the two gene variants and a history of venous thrombosis by stratifying patients and controls according to the presence of one or two gene variants (Table 2). The increased risk (odds ratio, 2.51) associated with the prothrombin GA20210 mutation was clearly unrelated to the overrepresentation of the factor V Leiden mutation; the coexistence of both mutations was seen only in patients (n = 14; 4.98% [CI, 2.44% to 7.52%]). The estimated risk associated with the prothrombin GA20210 mutation was similar when we excluded carriers of inherited defects of natural anticoagulants, patients with antiphospholipid antibodies, and patients exposed to circumstantial risk factors for venous thromboembolism. However, the risk for venous thrombosis associated with the prothrombin GA20210 mutation approached statistical significance only in the subset of patients with additional risk factors (Table 2). The distribution of the prothrombin GA20210 gene variant was similar in patients with and those without additional risk factors (P = 0.171). The prevalence of the factor V Leiden mutation did not differ between patients with and those without risk factors. The median age at the time of the first thrombotic episode was 38.0 years (range, 10 to 67 years) in the 26 patients with the prothrombin GA20210 mutation and 39.0 years (range, 16 to 65 years) in the 37 patients carrying the factor V Leiden mutation. The median age was 37.0 years (range, 3 to 81 years) in the 204 patients without either mutation and 31.5 years (range, 16 to 49 years) in the 14 patients carrying both mutations (Kruskal-Wallis test, P = 0.129). Table 2. Risk Estimate in All Study Participants and after Stratification by Risk Factors for Venous Thrombosis according to Presence of Prothro

High prevalence of thrombophilic genotypes in patients with acute mesenteric vein thrombosis

OBJECTIVES:Mesenteric vein thrombosis is a rare but severe abdominal emergency, often requiring intestinal resection. New genetic prothrombotic defects such as factor V Leiden, the prothrombin transition G20210A, and the methylenetetrahydrofolate reductase TT677 genotype have been described in association with venous thrombosis. Our goal was to assess prevalence and clinical significance of genetic thrombophilia in mesenteric vein thrombosis.METHODS:Twelve patients with acute mesenteric vein thrombosis were compared with 431 healthy people from the same geographical area. The factor V Leiden, the prothrombin transition G20210A, and the methylenetetrahydrofolate reductase TT677 genotype were identified by polymerase chain reaction and restriction analysis.RESULTS:A thrombophilic genotype was present in 9 patients (75%): the methylenetetrahydrofolate reductase TT677 genotype was present in 6 (50%), the factor V Leiden in 3 (25%), and the prothrombin transition G20210A in 3 (25%). Combined mutations were present in 4 (33%) patients.CONCLUSIONS:The factor V Leiden, the prothrombin transition G20210A, and the methylenetetrahydrofolate reductase TT677 genotype are important predisposing factors in the pathogenesis of mesenteric vein thrombosis. Their identification bears strong clinical implications for management of patients with mesenteric vein thrombosis.

Inherited platelet disorders: thrombocytopenias and thrombocytopathies.

Platelets play an important role in normal haemostasis halting blood flow immediately after injuries through four fundamental different mechanisms: adhesion, aggregation, secretion, and expression of procoagulant activity. First, in the presence of vascular damage, platelets adhere to the connective tissue and, particularly when the damage occurs in vessels with a low shear rate, to subendothelial collagen, fibronectin and laminin. On the other hand, when damage occurs in regions with a high shear rate, platelet adhesion requires the presence of subendothelial von Willebrand factor (VWF) and specific platelet receptors, such as the glycoprotein Ib/IX/V (GPIb/IX/V) complex. In any case, following initial adhesion, platelets aggregate to complete the formation of a solid haemostatic plug. Platelet aggregation requires stimulation by agonists such as ADP, thrombin, collagen, or epinephrine, as well as the presence of calcium or magnesium ions and specific plasma proteins (fibrinogen, VWF and the glycoprotein IIb/IIIa (GPIIb/IIIa) complex). Platelet stimulation results in the generation of intracellular second messengers that convey the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or VWF) then binds to GPIIb/IIIa and cross-links adjacent platelets to produce platelet aggregates, following platelet secretion and the elaboration of platelet procoagulant activity. In platelet disorders caused by a reduction in the number of platelets (thrombocytopenia) (Table I) or by platelet function defects (thrombocytopathies) (Table II), bleeding generally occurs immediately after injury, primarily in the skin, mucous membranes, nose, and gastrointestinal and urinary tracts and, generally, does not involve joints and muscles. Congenital disorders are uncommon and sometimes may be misdiagnosed with acquired disorders, which are much more frequently encountered in clinical practice. The differential diagnosis between congenital or acquired platelet disorders is often very complex and requires medical experience and a careful assessment of the patient’s personal and family history of bleeding episodes. A lifelong bleeding diathesis may suggest a congenital platelet disorder, which is often first recognised during childhood, but an onset in adulthood does not exclude a congenital defect. Because technological progress in laboratory practices and greater information about platelet functions have increased the specificity and accuracy of diagnosis of platelet disorders, a large number of cases has been identified recently. In this review, we describe current knowledge on congenital platelets disorders with regards to granule packaging, signalling and platelet coagulant function, and hereditary thrombocytopenias in order to improve information available for research and clinical practice. Table I Classification of thrombocytopathies