Persistent antiphospholipid antibody (aPL) in asymptomatic carriers as a risk factor for future thrombotic events: a nationwide prospective study.

OBJECTIVES: The long-term prognosis of individuals fulfilling the laboratory criteria, but not clinical criteria, of antiphospholipid syndrome (APS) has not been widely investigated. The primary aim of this study was to evaluate the incidence of first thrombotic event (deep venous thrombosis (DVT), pulmonary embolism (PE), myocardial infarction (MI), stroke or transient ischaemic attack (TIA) in a nationwide antiphospholipid antibody (aPL) carrier cohort. DESIGN: We conducted a prospective nationwide cohort study. SETTING: The aPL profile of participants was recorded from the laboratory database. Information was collected about thrombotic and pregnancy complications, subsequent medical history, other risk factors for thrombosis, use of prophylactic antithrombotic medication and general health. PARTICIPANTS: Participants included adult asymptomatic aPL carriers recognized in Finland during 1971-2009. MAIN OUTCOME MEASURE: The main outcome measure was incidence of first thrombotic event. RESULTS: A total of 119 (89% female) aPL carriers were followed for mean (SD) of 9.1 (7.5) years (range 3-41 years). Sixty-one per cent of the study participants had autoimmune disease, most often systemic lupus erythematosus (SLE). Thirty-six of 119 (30%) were either double or triple positive, 56% single lupus anticoagulant (LA) positive, and 8% and 5% single anticardiolipin antibodies (aCL) and anti-ß2glycoprotein I antibodies (aß2GPI) positive, respectively. Nine (7.6%) study patients experienced a first thrombotic event (five DVT, one PE, two MI, one TIA) mean (SD) 7.2 (8.3) years (range 1-26 years) after aPL detection (annual incidence rate 0.8%). All individuals who developed thrombotic complications had autoimmune disease. Annual rate of first thrombotic event in carriers of single positivity (0.65%) was equal to the known risk of thrombosis in the healthy Caucasian population, whereas the rate was two times higher in carriers of double or triple positivity (1.27%). Sixteen of 79 (20%) women experienced pregnancy complications. CONCLUSIONS: Double or triple positivity for aPL is a risk factor for future thrombotic events, especially in individuals with an underlying autoimmune disease, whereas single positivity does not seem to carry an elevated risk of thrombosis.

Type II antithrombin deficiency caused by a founder mutation Pro73Leu in the Finnish population: clinical picture.

BACKGROUND: It has been shown that some antithrombin (AT) activity assays do not correctly detect inherited type II AT deficiency, but erroneously classify these patients as normal. OBJECTIVES: Our aim was to investigate the mutations causing type II AT deficiency and to correlate the AT activity results with the genetic findings. PATIENTS/METHODS: A large population (n = 104; 42 families) of Finnish patients with known AT type II deficiency were interviewed for clinical data. Their AT activity was measured with five commercially available methods, and the SERPINC1 gene was genotyped. RESULTS: The mutations detected in type II AT-deficient patients were as follows: p.Pro73Leu (AT Basel) in 37 of 42 (88.1%) families; and p.Val30Glu, p.Arg425Cys and p.Pro439Ala in one family each. In two families, no mutation was detected. In the carriers of AT Basel two AT activity assays correctly identified most of the patients as AT-deficient, whereas three assays misclassified almost all of these patients as normal. Carriers of the founder mutation had, in addition to an elevated risk of venous thrombosis, a high risk of arterial thrombosis at young age, especially stroke. CONCLUSION: In Finland, a population with a strong founder effect, AT type II deficiency is caused predominantly by a single point mutation, p.Pro73Leu. The mutation is associated with a significant thrombotic risk. Reduced AT activity caused by this mutation cannot be detected by all available screening methods. This must be taken into account in the choice of laboratory method used for screening.

Costs and practicability of clean feeding of dairy cattle during radioactive contamination of grasslands.

Both the farm-specific and regional costs of clean feeding as a countermeasure to reduce ingestion of contaminated grass when there is insufficient supply of other types of roughage were estimated for dairy farming in Finland in the first year after contamination. The cost estimation considered expenditures and revenues associated with milk production and were calculated using farm models developed for economic planning. A hypothetical contamination scenario was designed using RODOS models for atmospheric dispersion and transfer in terrestrial food chains. Costs for intervention after two similar hypothetical atmospheric dispersion and deposition scenarios in early June and in July were estimated. As a reference, the cost of complete replacement of fodder throughout the area was also calculated. Feed substitution costs were higher in June than in July, due to the availability of some harvested silage in the later scenario. In the first case, the additional costs of clean feeding amounted to one-fifth of the normal production costs. Effective advisory/support services, available to farmers, can substantially improve the implementation of countermeasures. However, high costs and insufficient sources of clean feed would restrict the use of clean feeding as the sole countermeasure after serious contamination during the growing season.

Immunologic and hematologic properties of antibodies to prothrombin and plasminogen in a mouse model.

Antibodies to prothrombin have been associated with venous and arterial thrombosis, and they cross-react with a structurally closely related protein plasminogen. We immunised 16 mice with human prothrombin and 15 mice with human plasminogen. Mice immunised with prothrombin developed cross-reactive antibodies to plasminogen (12/16), beta2-glycoprotein I (4/16), tissue-type plasminogen activator (6/16) and cardiolipin (11/16). Mice immunised with plasminogen developed cross-reactive antibodies to prothrombin (8/15), tissue-type plasminogen activator (2/12) and cardiolipin (5/12). Functional effects of antibodies were examined. Immunisation with prothrombin induced lupus anticoagulant activity in 9/14 mice. In mice immunised with plasminogen, radial fibrinolysis was inhibited in 8/10 and plasminogen activation in the chromogenic assay was inhibited in 9/11. No cross-functionality was observed. In conclusion, antibodies to prothrombin and plasminogen cross-react in vivo. Antibodies to prothrombin and plasminogen have different functional profiles, immunisation with prothrombin leads to prolonged blood clotting time, and immunisation with plasminogen induces antibodies interfering with fibrinolysis.

Antibodies to prothrombin crossreact with plasminogen in patients developing myocardial infarction.

In a prospective study on healthy middle-aged men, high level of antibodies to prothrombin implied a risk of myocardial infarction. The possible mechanism(s) of these antibodies in coronary thrombosis are not known. Because prothrombin belongs to the kringle proteins and shares structural homology with a fibrinolytic kringle protein plasminogen, we studied whether antibodies to prothrombin crossreact with plasminogen. Sera from 17 healthy middle-aged men who later developed myocardial infarction were studied. Binding of antibodies to immobilized prothrombin (EIA) was inhibited by using soluble prothrombin, plasminogen and synthetic peptides of 20 amino acids from plasminogen kringle 5 (P304, P305) and from prothrombin kringle 2 (P302) as inhibitors. The peptides contained the conserved pentapeptide CRNPD of the kringle proteins. Soluble prothrombin inhibited up to 50% the binding of antibodies to immobilized prothrombin in all sera. Plasminogen inhibited binding in 9/17 (53%) sera (a decrease of at least 20%). P305 inhibited binding to prothrombin in 8/17 (47%), P304 in 4/17 (23%) and P302 in 6/17 (35%) sera. In structural analysis, presentation of the pentapeptide was conformationally different between the peptides. We conclude that crossreactive antibodies binding to prothrombin and plasminogen occur in sera of patients later developing myocardial infarction. The crossreactive epitope seems to be conformational and include the conserved pentapeptide of the kringle proteins. These antibodies may interfere with the fibrinolytic function of plasminogen and contribute to the development of myocardial infarction.

High antibody levels to prothrombin imply a risk of deep venous thrombosis and pulmonary embolism in middle-aged men--a nested case-control study.

Antibodies against phospholipid-binding plasma proteins, such as beta2-glycoprotein I (beta2-GPI) and prothrombin, are associated with thromboembolic events in patients with systemic lupus erythematosus and also in subjects with no evident underlying diseases. We wanted to examine whether increased levels of antibodies to negatively-charged phospholipids (cardiolipin), to phospholipid-binding plasma proteins beta2-GPI and prothrombin and to oxidised low-density lipoprotein (LDL) were associated with risk of deep venous thrombosis or pulmonary embolism in subjects with no previous thrombosis. The antibodies were measured in stored serum samples from 265 cases of deep venous thrombosis of the lower extremity or pulmonary embolism occurring during a median follow-up of about 7 years and from 265 individually matched controls. The study subjects were middle-aged men participating in a cancer prevention trial of alpha-tocopherol and beta-carotene and the cases of thromboembolic events were identified from nationwide Hospital Discharge Register. The risk for thrombotic events was significantly increased only in relation to antiprothrombin antibodies. As adjusted for body mass index, number of daily cigarettes and history of chronic bronchitis, myocardial infarction and heart failure at baseline, the odds ratio per one unit of antibody was 6.56 (95% confidence interval 1.73-25.0). The seven highest individual optical density-unit values of antiprothrombin antibodies were all confined to subjects with thromboembolic episodes. In conclusion, the present nested case-control study showed that high autoantibody levels against prothrombin implied a risk of deep venous thrombosis and pulmonary embolism and could be involved in the development of the thrombotic processes.

Antibodies to phospholipid-binding plasma proteins and occurrence of thrombosis in patients with systemic lupus erythematosus.

There is accumulating evidence that anti-phospholipid (aPL) antibodies in the sera of patients with autoimmune diseases bind to a complex of anionic phospholipids and plasma phospholipid-binding proteins, namely beta 2-glycoprotein I (beta 2-GPI) and prothrombin. It has been suggested that a conformational change in beta 2-GPI, induced by binding either to anionic phospholipids or to the oxygen molecules on the irradiated microtiter plate, reveals cryptic antigenic epitope(s) in the native protein. We used an enzyme-linked immunoassay for measuring antibodies against two phospholipid-binding proteins, i.e., beta 2-GPI and prothrombin, absorbed to an irradiated plate in an unselected series of 139 patients with systemic lupus erythematosus (SLE). Elevated levels of antibodies against beta 2-GPI were found in 49% of patients and antibodies against prothrombin in 34% of patients. Both antibodies were significantly associated with deep venous thrombosis in patients with SLE (P = 0.009 for both antibodies). Accordingly, testing of these antibodies seems to be clinically useful in evaluating the risk of thrombosis.

Affinity-purified cardiolipin-binding antibodies show heterogeneity in their binding to oxidized low-density lipoprotein.

Antiphospholipid antibodies in autoimmune sera have been shown to react with a complex of phospholipids (cardiolipin) and a plasma phospholipid-binding protein, beta 2-glycoprotein I (apolipoprotein H). The binding of these antibodies was inhibited by oxidized low-density lipoprotein (LDL) in sera from patients with systemic lupus erythematosus (SLE), suggesting cross-reactivity between antiphospholipid antibodies and antibodies binding to oxidized LDL. We purified antiphospholipid antibodies by cardiolipin-polyacrylamide column from seven SLE sera and studied the reactivity of eluted fractions with cardiolipin-beta 2-glycoprotein I complex and oxidized LDL (malondialdehyde-conjugated LDL) in solid-phase enzyme immunoassay. In four sera the binding of IgG antibodies to cardiolipin-beta 2-glycoprotein I complex and to oxidized LDL appeared in the same fractions, whereas in three sera reactivities against cardiolipin and oxidized LDL were observed, at least in part, in separate fractions. The binding to solid-phase cardiolipin was dependent on the presence of exogenous beta 2-glycoprotein I in all fractions. Our findings show that antiphospholipid antibodies are heterogeneous in their binding to oxidized LDL, indicating that these two antibodies may have different subspecificities. Some eluted fractions reacted only with oxidized LDL, and did not show binding to cardiolipin-beta 2-glycoprotein I complex, suggesting that the lipid part in the antigenic complex might be responsible for the cross-reactivity of these antibodies. Accordingly, the biological functions of antibodies against phospholipid-beta 2-glycoprotein I complex and antibodies against oxidized LDL may also be different.

Antibodies to prothrombin imply a risk of myocardial infarction in middle-aged men.

Antiphospholipid antibodies in patients with "antiphospholipid syndrome" may be directed at least in part against plasma phospholipid-binding proteins, such as beta 2-glycoprotein I or prothrombin, which are involved in the control of thrombosis and haemostasis. IgG-class antibodies against prothrombin and beta 2-glycoprotein I were measured by enzyme-linked immunoassay in initially healthy middle-aged dyslipidaemic men (non-high-density lipoprotein > 5.2 mml/l). Serum samples had been drawn at entry to a 5-year coronary primary-prevention trial with gemfibrozil from 106 subjects who experienced either a non-fatal myocardial infarction or cardiac death during the follow-up and from 106 subjects without coronary episodes, matched for treatment group (gemfibrozil/placebo) and geographical area. The antiprothrombin antibody level, as expressed in optical density units, was significantly higher in patients than in controls (0.26 +/- 0.17 versus 0.22 +/- 0.09; p < 0.02). A high level of antiprothrombin antibodies (highest tertile of distribution) predicted a 2.5-fold increase in the risk (95% confidence interval 1.2-5.3) of myocardial infarction or cardiac death. The distribution of IgG-class antibodies against beta 2-glycoprotein I did not differ significantly between cases and controls. The joint effect of antiprothrombin antibodies and other factors associated with hypercoagulative state: triglyceride level, lipoprotein(a) and smoking, was multiplicative for the risk. Antiprothrombin antibodies are a new immunological predictor of myocardial infarction and the effect of these antibodies may be mediated by hypercoagulative mechanisms.

Lack of functional similarity between complement factor H and anticardiolipin cofactor, beta 2-glycoprotein I (apolipoprotein H).

Beta 2-glycoprotein I (beta 2-GPI) is a 50 kDa protein in human plasma composed of five repeating complement control protein modules thereby closely resembling complement factor H which has 20 such units. Both beta 2-GPI and factor H (150 kDa) have binding sites for negatively charged polyions. beta 2-GPI has been shown to act as a cofactor for antiphospholipid antibodies upon their binding to anionic phospholipids. In factor H the polyanion recognition site participates in the discrimination between alternative pathway activating and non-activating surfaces. In light of the structural similarity between beta 2-GPI and factor H we have examined whether beta 2-GPI has a role in the alternative complement pathway recognition process. Both activators (zymosan) and non-activators (sheep erythrocytes) of the alternative complement pathway were coated with C3b. Radiolabelled factor H was observed to recognize C3b on both surfaces, whereas beta 2-GPI bound to neither. In competition experiments beta 2-GPI could not prevent the association of 125I-H with either non-activator or activator bound C3b. Conversely, factor H could not replace beta 2-GPI as a cofactor for antiphospholipid antibodies upon their binding to anionic phospholipids. It is concluded that beta 2-GPI and factor H, despite similarities in structure, exhibit distinct, non-overlapping functions.

Anti-cardiolipin antibodies and risk of myocardial infarction in a prospective cohort of middle-aged men.

BACKGROUND: Data concerning the relation between antiphospholipid (aPL) antibodies and myocardial infarction in subjects without evidence of overt autoimmune disease are conflicting. All published studies have been performed on survivors of myocardial infarction or in patients with established coronary heart disease. The purpose of the present study was to determine whether the presence of aPL antibodies, namely, anti-cardiolipin (aCL) antibodies, carries a risk for myocardial infarction in a prospective cohort. METHODS AND RESULTS: The sera to be studied were drawn at entry from middle-aged dyslipidemic men (non-high-density lipoprotein cholesterol, > or = 5.2 mmol/L) participating in the Helsinki Heart Study, a 5-year coronary primary prevention trial with gemfibrozil. Samples were tested for IgG-class antibodies to cardiolipin by an ELISA. The risk was estimated with logistic regression analysis using a nested case-control design with 133 patients (myocardial infarction or cardiac death) and 133 control subjects, matched for treatment (gemfibrozil/placebo) and geographical area. The aCL antibody level, as expressed in optical density units, was significantly higher in patients than in control subjects (0.417 versus 0.361; P < .005). Subjects with the antibody level in the highest quartile of distribution had a relative risk for myocardial infarction of 2.0 (95% confidence interval, 1.1 to 3.5) compared with the remainder of the population. This risk was independent of confounding factors, such as age, smoking, systolic blood pressure, low-density lipoprotein (LDL), and high-density lipoprotein. There was a correlation between the levels of aCL antibodies and antibodies to oxidized LDL (r = .40, P < .001), and their joint effect was additive for the risk. CONCLUSIONS: In a prospective cohort of healthy middle-aged men, the presence of a high aCL antibody level is an independent risk factor for myocardial infarction or cardiac death. Antibodies to cardiolipin and oxidized LDL may, at least in part, represent cross-reactive antibody populations.

Antibody against oxidized low-density lipoprotein predicting myocardial infarction.

BACKGROUND: Oxidation of low-density lipoprotein is believed to be an important step in the pathogenesis of atherosclerosis. The purpose of the present study was to determine whether antibody against oxidized low-density lipoprotein, reported to be associated with the progression of carotid atherosclerosis, is predictive of cardiac death and nonfatal myocardial infarction. METHODS: Serum samples from 135 cases and their controls, drawn at entry from middle-aged dyslipidemic men participating in the Helsinki Heart Study, a 5-year coronary primary prevention trial with gemfibrozil, were tested for immunoglobulin G class antibodies against oxidized low-density lipoprotein by enzyme-linked immunosorbent assay. RESULTS: The mean antibody level, expressed in optical density units, was significantly higher in cases than in controls (0.412 vs 0.356, P = .002). After adjustment for age, smoking, blood pressure, and high-density lipoprotein cholesterol level, there was a 2.5-fold increased risk (95% confidence interval, 1.3 to 4.9) of a cardiac end point in the highest tertile of antibody level vs the lowest tertile (P = .005 for trend). CONCLUSIONS: Elevated levels of antibodies against oxidized low-density lipoprotein were predictive of myocardial infarction. The effect was independent of low-density lipoprotein cholesterol levels, and the joint effect was additive. Elevated antibody levels modified the effects of classic coronary risk factors.