The Pathogenesis of Venous Thromboembolism: Evidence for Multiple Interrelated Causes

Context Venous thromboembolism (VTE) is thought to arise from the interaction of environmental and genetic factors in persons predisposed to VTE. Contribution The authors studied persons with protein S, protein C, or antithrombin deficiency who were first-degree relatives of persons who had VTE. They assessed each relative for environmental exposures and additional thrombophilic defects. They found that risk for VTE increased with the number of defects and with exposure to environmental risk factors. Cautions The findings were not based on the total population of relatives. The authors could not quantify risk for interactions between specific defects and environmental risk factors. Implications The risk for VTE increases with the number of thrombophilic defects and environmental risk factors in persons with a hereditary predisposition to VTE. The study provides evidence that VTE arises from interactions between environmental and genetic risk factors and quantifies the risks. The Editors Venous thromboembolism (VTE) has an incidence of 0.1 to 0.2 per 100 person-years (1, 2). It has been speculated that the development of VTE results from interactions between multiple genetic and environmental risk factors (3). Several inherited or acquired coagulation defects have been identified as VTE risk factors over the past 30 years. Known thrombophilic defects include hereditary deficiencies of protein S, protein C, and antithrombin; factor V Leiden; the prothrombin G20210A mutation; high levels of coagulant factors VIII, IX, and XI; hyperhomocysteinemia; and antiphospholipid antibodies (4). Whether patients with VTE and their relatives should be tested for all of these defects is still debatable. Clinical implications mainly depend on the absolute risk for a first or recurrent episode of VTE in persons with a single defect or in those with a combination of defects. Most common defects are mild risk factors for VTE. In contrast, hereditary deficiencies of protein S, protein C, and antithrombin are strong risk factors but are rare. Although interactions between these deficiencies and 1 or more other thrombophilic defects might increase the risk for VTE, they cannot be studied easily because the prevalence of such combinations is low. Thus far, only a few studies have reported the risk for VTE associated with coinheritance of deficiencies of protein S, protein C, or antithrombin and factor V Leiden or the prothrombin G20210A mutation (510). The results were not consistent, possibly because of small numbers of cases. We performed a retrospective study to assess the contribution of currently known hereditary thrombophilic defects and exogenous risk factors to the absolute risk for VTE in a large series of protein S, protein C, or antithrombin-deficient families. Methods We aimed to assess interactions between known thrombophilic deficiencies and defects, including hereditary deficiencies of protein S, protein C, and antithrombin. Because these deficiencies are strong risk factors for VTE, the effects and clinical impact of interactions with more prevalent and mild thrombophilic defects may be more pronounced than interactions between mild defects. To enroll sufficient numbers of affected persons needed for accurate risk estimates, we identified families with these rare deficiencies. The family cohort study design enabled us to assess the clinical impact of interactions from absolute risk estimates. Participants The study comprised 3 cohorts of families with hereditary deficiencies of protein S, protein C, or antithrombin. Probands were consecutive patients with VTE who had 1 of these deficiencies. Primary care physicians referred patients with clinically suspected VTE to 1 of 2 hospitals in our region of the Netherlands; 50% were referred to the thrombosis outpatient clinic of our university hospital. Previous clinical trials did not show differences between patients who were referred to either hospital for this reason. Because it is common practice in the Netherlands to confirm suspected VTE, the proportion of patients who were not referred was probably small. All patients who had VTE confirmed at our outpatient clinic were tested for protein S, protein C, or antithrombin deficiencies, unless they had extensive malignant disease. After a deficiency was established by repeated measurement and after causative acquired conditions were excluded, the patient's first-degree relatives who were older than age 15 years were identified by pedigree analysis and were contacted through the probands. Because the number of antithrombin-deficient probands was small, second-degree relatives with a deficient parent were also identified. Relatives were assessed for deficiency at the time of identification and then were followed for thromboembolic events from age 15 years to the time of testing. All participants provided informed consent. Physicians at our outpatient clinic collected detailed information about previous episodes of VTE, exposure to exogenous risk factors for VTE, and anticoagulant treatment using a validated questionnaire (11) and by reviewing medical records. The use of oral contraceptives and an obstetric history were documented for women. Blood samples were taken after clinical data had been collected. All relatives were tested for 10 thrombophilic deficiencies and defects in addition to their index deficiencies, including deficiencies of protein S, protein C, antithrombin, and plasminogen; factor V Leiden; prothrombin G20210A; high levels of factors VIII, IX, and XI; hyperhomocysteinemia; and lupus anticoagulant. Diagnosis of VTE Venous thromboembolism was considered established if deep venous thrombosis was confirmed by compression ultrasonography or venography, and pulmonary embolism was confirmed by ventilationperfusion lung scanning, spiral computed tomography scanning, or pulmonary angiography. When these techniques were not yet available, VTE was considered established when the patient had received full-dose unfractionated heparin and a vitamin K antagonist for at least 3 months. Venous thromboembolism was considered secondary if it had occurred at or within 3 months after exposure to exogenous risk factors, including major surgery, trauma, immobilization for more than 7 days, oral contraceptives, hormone replacement therapy, pregnancy, and malignant disease. In the absence of these risk factors, VTE was considered primary. Laboratory Studies Protein S and protein C antigen levels were measured by enzyme-linked immunosorbent assay (Dako, Glostrup, Denmark); activity of protein C (Berichrom Protein C, Dade Behring, Marburg, Germany), antithrombin (Coatest, Chromogenix AB, Mlndal, Sweden), and plasminogen (S2251, Chromogenix AB) was measured by chromogenic substrate assays. Levels of protein S, protein C, and antithrombin were expressed as a percentage of the levels measured in pooled plasma set at 100%. Normal ranges were determined in 393 healthy blood donors who had no family history of VTE, were not pregnant, and had not used oral contraceptives for at least 3 months. Protein S deficiency type I was defined by decreased free and total protein S levels, that is, below normal ranges, and protein S deficiency type III was defined by decreased free protein S levels and normal total protein S levels. After we had demonstrated that type III protein S deficiency was not a risk factor for thrombosis, families with this deficiency were excluded from the analysis (12). Protein C deficiency type I and type II were defined by decreased levels or activity of protein C antigen, and antithrombin deficiency was defined by decreased levels of antithrombin activity. Deficiencies were considered inherited if they were confirmed by measuring a second sample that was collected 3 months later and were found in at least 2 family members. Relatives with acquired conditions were excluded. If there was a discrepancy between the results of the 2 tests, a third sample was tested. A deficiency was considered acquired, through use of oral contraceptives or pregnancy, unless it was confirmed at least 3 months after withdrawal of oral contraceptives or delivery, respectively. Factor V Leiden and the prothrombin G20210A mutation were demonstrated by polymerase chain reaction (13, 14). Factors VIII:C, IX:C, and XI:C were measured by 1-stage clotting assays (Amelung GmbH, Lemgo, Germany) and were increased at levels above 150%. Lupus anticoagulant was defined by abnormal values of dilute Russell viper venom time, activated partial thromboplastin time, and tissue thromboplastin inhibition, which normalized by adding phospholipids to the participant's plasma (15). Fasting and postmethionine-loading levels of homocysteine were measured by high-performance liquid chromatography (16). Hyperhomocysteinemia was defined as a fasting homocysteine level above 18.5 mol/L, a postloading level above 58.8 mol/L, or both, as described in a Dutch population (17). Blood samples were collected from probands and relatives at least 3 months after VTE. If probands or relatives were receiving long-term treatment with vitamin K antagonists, samples were taken after treatment had been interrupted for at least 2 weeks; in the meantime, nadroparin was given subcutaneously. Additional tests were performed on plasma stored at80C from relatives who had been enrolled at a time when 1 of more of these deficiencies or defects had not yet been recognized as risk factors for VTE. Statistical Analysis We compared the absolute risk for VTE in deficient and nondeficient relatives in each of the 3 cohorts and in the pooled cohorts. Probands were excluded from the analysis to avoid selection bias. Annual incidences were calculated by dividing the number of symptomatic relatives by the total number of observation-years. Ninety-five percent CIs were calculated around the incidence rates by using the Poisson distribution assumption. Observation time was defined as the period from age 15 yea