Luigi Iannaccone

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

Prognostic factors in noncirrhotic patients with splanchnic vein thromboses.

OBJECTIVES AND METHODS:Splanchnic vein thrombosis (SVT), not associated with cancer or liver cirrhosis, is a rare event and scanty data are available on its natural history, long-term prognosis, and treatment. In this study 121 SVT patients consecutively seen from January 1998 to December 2005 were included and 95 of them were followed up for a median time of 41 months. Screening for thrombophilic factors was performed in 104 patients. New thrombotic or bleeding episodes were registered and anticoagulant therapy was performed according to preestablished criteria.RESULTS:SVT was an incidental finding in 34 (28.1%) patients; 34 (28.1%) presented with abdominal infarction; 39 (32.2%) had bowel ischemia or acute portal vein thrombosis; 14 (11.6%) had bleeding from portal hypertensive sources. Survival rates at 1, 3, and 7 yr were 95%, 93.3%, and 89.6%, respectively; 87.5% of deaths occurred at onset of SVT as complications of intestinal infarction. Patients with isolated portal vein thromboses had symptoms and intestinal infarction in 16/41 (39%) and 0/41 (0%) of the cases, respectively, whereas superior mesenteric vein thromboses, isolated or not, were associated with symptoms and intestinal infarction in 69/75 (92%) and 34/75 (45%), respectively.During the follow-up 14 (14.7%) suffered from 39 episodes of gastrointestinal bleeding with no deaths. A previous gastrointestinal bleed was associated with new hemorrhagic events during follow-up.New venous thrombotic episodes occurred in 10 of 95 patients (10.5%), of which 73% were in the splanchnic area. Seven out of these 10 patients had a chronic myeloproliferative disease (MPD) and none was on anticoagulation.CONCLUSIONS:Anticoagulant therapy was effective to obtain recanalization of acute SVT in 45.4% of patients and preserved patients from recurrent thrombosis when given lifelong.

Anticardiolipin antibody titre and plasma homocysteine level independently predict intima media thickness of carotid arteries in subjects with idiopathic antiphospholipid antibodies

This study evaluated whether IgG anticardiolipinantibody (aCL) titre and traditional risk factors for atherosclerosis bore any relationship to the intima media thickness (IMT) of carotid arteries of patients with idiopathic antiphospholipid antibodies (aPL). IMT was assessed by high-resolution sonography at the common carotid, carotid bifurcation and internal carotid in 42 (13 male, 29 female, mean age 31§ 10 years) aPL subjects, 29 with primary thrombotic antiphospholipid syndrome and 13 with persistence of aPL in the absence of any underlying disorder. In the same subjects the following were measured: plasma fibrinogen (FNG), von Willebrand factor (vWF), plasminogen activator inhibitor (PAI), homocysteine (HC), total cholesterol (CHO), triglycerides (TG), high density and low density lipoprotein (HDL and LDL), platelet numbers and aCL of IgG and IgM isotype. IMT of the internal carotid was greater in males than females (0.48±0.03 vs 0.39±0.01 mm, P = 0.02). IMT of the carotid bifurcation was greater in thrombotic than nonthrombotic subjects (0.50§ 0.02 vs 0.42§ 0.02 mm, P = 0.04). By simple regression, IMT of the common carotids correlated with age (P < 0.0001) IgG aCL titre (P = 0.001), FNG (P = 0.006), LDL (0.01), CHO (0.02) and PAI (P = 0.02). IMT of the carotid bifurcation correlated with age (P = 0.002), IgG aCL titre (P = 0.0002), FNG (P = 0.0001), HC (P = 0.009), CHO (P = 0.02), vWF (P = 0.01) and number of thrombotic events (P = 0.03). IMT of the internal carotids correlated with age (P = 0.002), IgG aCL titre (P = 0.0001), FNG (P = 0.0008), PAI (P = 0.002) and HC (P = 0.01). By stepwise multiple regression analysis, IgG aCL titre independently predicted IMT at all carotid segments examined (P always < 0.005). In addition, plasma FNG and HC also resulted independent predictors of IMT at the carotid bifurcation (P = 0.001 and P < 0.0001, respectively) and internal carotid (P = 0.03 and P < 0.0001, respectively). These data strongly support an atherogenic role for IgG aCL in patients with aPL. Measurement of plasma HC and FNG may help define aPL subjects at higher vascular risk who may require lowering of HC and FNG by vitamin and/or pharmacologic intervention. Lupus (2002) 11, 208–214.

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.

Gain of function gene mutations and venous thromboembolism: Distinct roles in different clinical settings

Objective: To calculate the prevalence of common gain of function gene mutations in patients with different clinical manifestations of venous thromboembolism. Design and setting: Case–control study in two hospitals in Italy. Participants: 387 patients with venous thromboembolism and 286 controls. Main measures: Factor V (FV) Leiden, factor II (FII) A20210 and JAK2 V617F mutations. Results: Among patients with deep vein thrombosis in one leg, 23 (20.9%) carried FV Leiden and FII A20210 mutations. Similar figures were observed in patients with cerebral vein thrombosis (CVT; n = 9; 20.0%) and in patients presenting with splanchnic vein thrombosis (SVT; n = 26; 18.7%). A lower prevalence was obtained in patients with retinal vein thrombosis (n = 11; 11.8%). The JAK2 F617 mutant allele was found in 27 (21.1%) patients with SVT, but in none of the patients presenting with a thrombotic event from different districts. 13 of the 27 JAK2 V617F-positive subjects with SVT were previously known to have a myeloproliferative disease (MPD). Three other patients had a diagnosis of MPD after the occurrence of the thrombotic event. Conclusion: Carriership of FV Leiden or FII A20210 mutations identifies an at-risk condition for venous thrombosis in the lower extremities, SVT or CVT. In patients with SVT, screening for the JAK2 V617F mutation may be useful in recognising patients who should be carefully observed for the subsequent development of overt MPD. Thus, genetic tests may play a different role, various clinical manifestations of venous thromboembolism being associated with distinct risk profiles.

High-density lipoprotein inversely relates to its specific autoantibody favoring oxidation in thrombotic primary antiphospholipid syndrome.

Abnormalities of the lipid profile partly explain the atherogenic tendency of systemic lupus erythematosus but the picture is unclear in thrombotic primary antiphospholipid syndrome (PAPS). Herein we compare the lipid profile, high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein A (ApoA-I), apolipoprotein B (ApoB), tryglicerides (TRY)), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-ß2GPI) and C-reactive protein (CRP) from thrombotic PAPS (n = 34), thrombotic patients with inherited thrombophilia (IT; n = 36), subjects persistently positive for antiphospholipid antibodies (aPL, n = 18) with no underlying autoimmune or non-autoimmune disorders and healthy controls (n = 28) and determined the reciprocal effects of anti-lipoprotein antibodies, the lipid profile, oxLDL-ß2GPI and CRP. Average concentrations of HDL (p < 0.0001), LDL (p < 0.0001), CHO (p = 0.0002), ApoA-I (p = 0.002) were lower in PAPS whereas average TRY was higher (p = 0.01) than other groups. Moreover, the aPL and PAPS group showed higher levels of IgG anti-HDL (p = 0.01) and IgG anti-ApoA-I (p < 0.0001) whereas the PAPS group showed greater average oxLDL-ß2GPI (p = 0.001) and CRP (p = 0.003). Within the PAPS group, IgG anti-HDL correlated negatively to HDL (p = 0.004) and was an independent predictor of oxLDL-ß2GPI (p = 0.009). HDL and ApoA-I correlated negatively with CRP (p = 0.001 and p = 0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favoring low-grade inflammation and enhanced oxidation in thrombotic PAPS. Lupus (2010) 19, 711—716.

Subclinical atherosclerosis in primary antiphospholipid syndrome

Abstract:  To test the atherosclerosis hypothesis in primary antiphospholipid syndrome (PAPS) we measured intima media thickness (IMT) of carotid arteries and other cardiovascular risk factors in 44 patients with PAPS (mean age 35 ± 12 years), in 25 patients with inherited thrombophilia (mean age 40 ± 10 years), and in 34 normal controls (mean age 38 ± 11 years). The frequency of smoking, hypertension, and dyslipidemia was similar across groups. IMT was almost similar across groups at age groups below 40 years but IMT was greater in PAPS than controls at the common carotid (P= 0.01), at the bifurcation (P= 0.003), and at the internal carotid (P= 0.005) in the age group over 40 years. Atherosclerosis is a possibility in PAPS patients in their fourth decade of life or older.

Bleeding and re-thrombosis in primary antiphospholipid syndrome on oral anticoagulation An 8-year longitudinal comparison with mitral valve replacement and inherited thrombophilia

The aim of this study was to compare bleeding and re-thrombosis in primary antiphospholipid syndrome (PAPS), mitral valve replacement (MVR) and inherited thrombophilia (IT) at different oral anticoagulation intensities. It entailed a prospective 8-year follow-up on 67 patients with PAPS, 89 with IT and 24 with MVR. Anticardiolipin (aCL) antibodies detected by Elisa and lupus anticoagulant by clotting assays. At INR 2-3 minor bleeding rate was higher in MVR (33.3) than PAPS (10.9) and IT (4.2)(p<0.0001). At INR 3-4 minor bleeding rate was higher in PAPS (142) than IT (33.3) and MVR (5.8)(p<0.0001). At either INR major bleeding rate were not significantly different across the three groups, but in PAPS major and minor bleeding rates were superior at INR 3-4 than INR 2-3 (p=0.02 and p<0.0001). Re-thrombosis rate was higher in PAPS than IT at INR 2-3 (4.0 vs 0.35) (p=0.01) and at INR 3-4 (10.5 vs. nil). The hazard ratio for re-thrombosis between PAPS and IT was 13 (95% IC 1.6-102.2, p=0.015). By regression analysis, baseline IgG aCL titre (>80 GPL) p=0.001) and male sex (p=0.03) independently predicted re-thrombosis. In conclusion, in PAPS, high intensity oral anticoagulation was not superior to conventional intensity in preventing re-thrombosis but was offset by greater bleeding rates. Male sex and elevated baseline IgG aCL predicted rethrombosis in PAPS that is 13-fold more re-thrombogenic than IT.

A rapid screen for lupus anticoagulant with good discrimination from oral anticoagulants, congenital factor deficiency and heparin, is provided by comparing a sensitive and an insensitive APTT reagent

Lupus anticoagulants (LA) are associated with an increased risk of thrombosis and laboratory detection is of major importance. Various tests are available for LA screening and confirmation, but they differ in sensitivity and specificity, frequently lacking the ability to discriminate between the presence of LA, heparin and oral anticoagulants. We noticed that a patient with LA who had a prolonged activated partial thromboplastin time (APTT) by our routine method, gave a normal result with a different APTT reagent. This latter reagent, which contained soy bean phosphatides (SBP), was compared with a reagent containing rabbit brain phospholipids complexed with kaolin (RBK), for APTT measurement in a variety of patients. There was no significant difference in APTT ratio between the two reagents in plasma samples from healthy normal subjects. In LA samples, SBP gave consistently lower APTT ratios than RBK (mean × SEM, 1.04 × 0.05 and 2.08 × 0.19 for SBP and RBK respectively; P< 0.001). In LA patients receiving oral anticoagulants for antithrombotic prophylaxis or treatment, the APTT ratio was again significantly shorter with SBP (1.60 × 0.17 and 3.40 × 0.67; P< 0.05). In LA negative patients receiving oral anticoagulants, the relationship was reversed, and a higher APTT ratio was obtained with SBP than RBK (1.61 × 0.13 and 1.31 × 0.12; P< 0.001). In addition, there were no significant differences in APTT ratios for the two reagents when samples from patients receiving heparin therapy, or patients with acquired factor VIII deficiency or inherited deficiency of factor VIII or IX were studied. The use of the SBP reagent alongside a LA sensitive APTT reagent allows a rapid screening for LA, as well as a confirmation of the phospholipid dependency of the inhibitor.

Occurrence of the JAK2 V617F mutation in the Budd-Chiari syndrome.

Myeloproliferative diseases represent a major risk factor for Budd–Chiari syndrome. In 32 patients with Budd–Chiari syndrome, the JAK2 V617F mutation was detected, in heterozygous state, in 11 individuals (34.4%; 95% confidence interval: 18.6–53.2). Eight patients with (72.7%; 95% confidence interval: 39.0–94.0) and six without (28.6%; 95% confidence interval: 11.3–52.2) the JAK2 V617F mutation had a diagnosis of myeloproliferative diseases before or at the occurrence of the venous thrombotic event. In three patients carrying the JAK2 V617F mutation, a myeloproliferative disease was not detected. Determination of the JAK2 V617F mutation may be useful to recognize patients with Budd–Chiari syndrome with or at risk for the subsequent development of overt myeloproliferative diseases.