R. H. W. M. Derksen

Patients with antiphospholipid antibodies and venous thrombosis should receive long term anticoagulant treatment.

OBJECTIVE--To determine whether the finding of antiphospholipid antibodies in patients with venous thromboembolic episodes should influence the duration of treatment with anticoagulant drugs by mouth. METHODS--A retrospective study was carried out in 19 patients with antiphospholipid antibodies and a history of venous thromboembolic episodes. The median follow up from the first venous thromboembolic episode was 93 months and the median age at this episode was 26 years. The patients had in total 34 venous thromboembolic episodes. The total follow up period comprised 32 periods with and 23 periods without anticoagulant drugs. RESULTS--The probability of being free of recurrent venous thromboembolic episodes, calculated by the Kaplan-Meier method, was significantly influenced by the use of anticoagulant drugs. Patients receiving oral anticoagulants had at eight years a 100% probability of survival without recurrence, whereas patients in whom anticoagulant drugs were stopped had a 50% probability of a recurrent venous thromboembolic episode at two years, and a 78% probability of recurrence at eight years. CONCLUSION--Patients with venous thromboembolic episodes and antiphospholipid antibodies have a high risk for recurrent venous thromboembolic episodes and long term treatment with anticoagulant drugs by mouth is an effective prophylaxis.

Coagulation screen is more specific than the anticardiolipin antibody ELISA in defining a thrombotic subset of lupus patients.

In 111 lupus patients we compared the potential of the IgG and IgM anticardiolipin antibody (ACA) enzyme linked immunosorbent assay (ELISA) and four different lupus anticoagulant (LAC) assays (partial thromboplastin time (PTT) of a 1:1 mixture of patient and control plasma with phospholipids from animal (PTT-st) or human brain (PTT-HB); PTT with dilutions of human brain phospholipids (PL dilution); and kaolin clotting time of mixtures of patient and control plasma (KCT] to identify patients with thrombosis (26/111), fetal loss (19/46), and/or thrombocytopenia (11/106). The highest specificity for thrombosis (87%) was found with PTT-HB and PL dilution (sensitivity 65%, detection rate 61%); for fetal loss (93%) with PL dilution (sensitivity 47%; detection rate 82%), and for thrombocytopenia (83%) with KCT (sensitivity 82%; detection rate 36%). Compared with LAC assays, the sensitivity of ACA-ELISA was high (greater than or equal to 77%), but specificity (less than or equal to 51%) and detection rate (less than or equal to 52%) were low. So, a panel of three LAC assays (PTT-HB, PL dilution, and KCT) can identify lupus patients apparently at risk for thrombosis, fetal loss, and/or thrombocytopenia, whereas the ACA-ELISA is insufficiently specific.

Platelet adhesion to dimeric β2‐glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibα and apolipoprotein E receptor 2′

Summary.  Background: The major antigen implicated in the antiphospholipid syndrome is beta2‐glycoprotein I (β2GPI). Dimerized β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized β2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized β2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized β2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized β2GPI was completely inhibited by the addition of soluble forms of both apoER2′ and GPIbα, and the addition of receptor‐associated protein and the removal of GPIbα from the platelet surface. GPIbα co‐precipitated with apoER2′, suggesting the presence of complexes between GPIbα and apoER2′ on platelet membranes. The interaction between GPIbα and dimeric β2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+‐dependent. Deletion of domain V from dimeric β2GPI strongly reduced its binding to both GPIbα and apoER2′. Antibodies that inhibit the binding of thrombin to GPIbα inhibited platelet adhesion to dimeric β2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbα had no effect. Dimeric β2GPI showed reduced binding to low‐sulfated GPIbα compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric β2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbα and apoER2′. These receptors are present in a complex on the platelet surface.

Interaction of ß2-glycoprotein I with members of the low density lipoprotein receptor family

Summary.  The antiphospholipid syndrome (APS) is a non‐inflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2‐glycoprotein I (β2GPI) bound to phospholipids. We have previously demonstrated that dimerization of β2GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2’ (apoER2′) a receptor belonging to the low‐density lipoprotein receptor (LDL‐R) family. Here, we show that dimeric β2GPI, but not monomeric β2GPI, interacts with four other LDL‐R family members: the LDL‐R related protein (LRP), megalin, the LDL‐R and the very‐low density lipoprotein receptor (VLDL‐R). Interaction between dimeric β2GPI and LDL‐R, apoER2′ and VLDL‐R was best described with a one‐site binding model (half‐maximal binding; ∼20 nm for apoER2′ and VLDL‐R and ∼300 nm for LDL‐R), whereas the interaction between dimeric β2GPI and LRP or megalin was best described with a two‐site binding model, representing a high‐ (∼3 nm) and a low‐affinity site (∼0.2 μm). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of β2GPI, which is known to disrupt the phospholipid binding site of β2GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric β2GPI can interact with different LDL‐R family members. This interaction is dependent on a binding site within domain V of β2GPI, which does not overlap with the phospholipid‐binding site within domain V.

PROTEIN C AND OTHER COFACTORS INVOLVED IN THE BINDING OF ANTIPHOSPHOLIPID ANTIBODIES : RELATION TO THE PATHOGENESIS OF THROMBOSIS

In this review we will discuss the possible interference of antiphospholipid antibodies with the protein C system. Antiphospholipid antibodies can interfere with the protein C system in different ways: (i) via inhibiting the formation of thrombin; (ii) via interference with the activation of protein C by the thrombomodulin-thrombin complex; (iii) via inhibition of the assembly of the protein C complex; (iv) via inhibition of the activity of protein C, directly or via its cofactor protein S, and (v) via antibodies directed against the substrates of APC, factors Va and VIIIa, thereby protecting them for inactivation. The experimental and theoretical indications that one of these mechanisms will explain the pathogenesis of the antiphospholipid syndrome is critically examined.

Risk factors for thrombosis in lupus patients

Lupus anticoagulant, concentrations of anticardiolipin antibodies, antithrombin III, plasminogen, (free) protein S, protein C, prothrombin, platelet counts, and bleeding times were determined in 74 lupus patients (58 with systemic lupus erythematosus; 16 with lupus-like disease) to establish the presence of risk factors for thrombosis in these patients. Of the variables evaluated, lupus anticoagulant had the strongest association with a history of thrombosis. Both positive anticardiolipin antibody concentrations and the presence of (mild) thrombocytopenia were significantly associated with a history of thrombosis and the presence of lupus anticoagulant. Reduced concentrations of antithrombin III, plasminogen, (free) protein S, and protein C were found in some patients but were not associated with either thrombosis or lupus anticoagulant. Mean concentrations of total protein S were significantly lower in patients with thrombosis than in those without and in patients with lupus anticoagulant than in those without. The antigenic concentration of prothrombin was reduced in 3/74 (4%) lupus patients. These three patients had lupus anticoagulant but no history of thrombosis, which suggests that a low prothrombin concentration protects patients with lupus anticoagulant from the development of thrombosis. A prolonged bleeding time was associated with the presence of lupus anticoagulant but not with a history of thrombosis. Analysis by stepwise logistic regression did not disclose additional risk factors for thrombosis in lupus patients with lupus anticoagulant. Increased antithrombin III concentrations and decreased free protein S concentrations are often found in lupus patients, unrelated to lupus anticoagulant or thrombosis.

Annexin A5 polymorphism (−1C→T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome

Background: Annexin A5 is thought to have a role in the pathophysiology of the antiphospholipid syndrome (APS)—a syndrome characterised by recurrent thrombosis and pregnancy morbidity. Objective: To investigate whether anti-annexin A5 immunoglobulin (Ig)M or IgG antibodies, or the −1C→T polymorphism of annexin A5, is a risk factor for thrombosis or miscarriage, and whether the −1C→T polymorphism is correlated with APS. Methods: A cohort study was carried out with a population of 198 patients with primary APS, systemic lupus erythematosus or lupus-like disease. For the detection of anti-annexin A5 antibodies and the measurement of annexin A5 plasma levels, ELISA-type methods were used. The annexin A5 −1C→T mutation was detected by restriction fragment length polymorphism. Results: 71 patients were positive for annexin A5 IgM or IgG antibodies, of whom 53 patients were positive for anti-annexin A5 IgG antibodies and 27 of 198 patients were positive for anti-annexin A5 IgM antibodies. The prevalence of IgM or IgG anti-annexin A5 antibodies was not significantly associated with thrombosis or miscarriage on multivariate analysis. The prevalence of the −1C→T mutation in the annexin A5 gene (46/198 patients) was significantly associated with miscarriage (odds ratio 2.7, 95% confidence interval 1.1 to 6.7, independent risk factor). Conclusion: The detection of anti-annexin A5 antibodies does not seem relevant for estimating the risk for thrombosis or miscarriage in APS. The −1C→T mutation was an independent risk factor for miscarriage, which is independent of APS.

Anti-prothrombin antibodies and their relation with thrombosis and lupus anticoagulant

Antiphospholipid antibodies are a heterogeneous group of antibodies, comprising antibodies with different antigen specificity. Prothrombin is one of the antigens which can be detected by antiphospholipid antibodies and therefore anti-prothrombin antibodies belong to the antiphospholipid antibody family. The presence of antiphospholipid antibodies correlates strongly with thromboembolic complications; however a mechanism by which these autoantibodies induce a thrombotic complication in vivo is not understood. The classic assays for the detection of antiphospholipid antibodies (LAC and anticardiolipin ELISAs) aim to measure all the antiphospholipid antibodies present in the samples without making a distinction between the different subspecificities of the antibodies present in one single sample. Moreover, most of the in-vitro studies performed were carried out with total IgGs, which contain a mixture of antibodies. The absence of an accurate characterization of the plasma samples and the lack of specificity of the IgGs used in in-vitro tests makes it difficult to determine the contribution of antiprothrombin antibodies to the thrombotic complications. Here we review and critically analyse the literature regarding the clinical relevance of the presence of antiprothrombin antibodies and the possible participation of these antibodies in the pathogenesis of the thrombotic complications.

Fluctuations of anticardiolipin antibody levels in patients with systemic lupus erythematosus: a prospective study.

In 53 patients with systemic lupus erythematosus sequential blood samples obtained during a four year period (range 6-47 months) were screened for anticardiolipin antibodies (ACAs). Disease activity and treatment with prednisone were also assessed and related to ACA concentrations. During follow up only 21 patients for ACA IgG (40%) and 25 for ACA IgM (47%) remained in the ACA category (negative, low positive, high positive) found at the first sample taken at entrance. Marked increases from negative to high positive concentrations were sometimes seen and were not accompanied by typical events such as thrombosis or thrombocytopenia (the ACA syndrome). Shifts in ACA concentrations could not always be explained by changes in prednisone dose. Also, in patients with low dose prednisone treatment or none at all (n = 22) 10 patients (45%) changed ACA IgG category and 12 patients (55%) fluctuated in ACA IgM categories during follow up. As a consequence of the variability in ACA titres relations of ACAs with the ACA syndrome depended on the blood sample studied. In the second sample, randomly taken half way through follow up, no significant relations with the ACA syndrome could be found. Anticardiolipin antibody IgG was significantly associated with disease activity in 11/47 patients (23%) and in the group as a whole. During remission ACA IgG was significantly associated with the ACA syndrome, whereas during moderate/severe disease activity in the same patients that correlation was not significant. Anticardiolipin antibody IgM was much less influenced by disease activity, and in only 4/47 patients (9%) could a significant relation with disease activity be shown. Associations of ACA IgM with the ACA syndrome were significant during both lupus flares and remission.

Prospective study of fluctuations of lupus anticoagulant activity and anticardiolipin antibody titre in patients with systemic lupus erythematosus.

Fluctuations of lupus anticoagulant activity and anticardiolipin antibody titres were studied in 53 patients with systemic lupus erythematosus (SLE). The median study time was 26 months with a median number of 12 samples. Lupus anticoagulant was measured by the kaolin clotting time (KCT) and dilute Russell viper venom time (dRVVT) assays; anticardiolipin antibodies were assayed by an enzyme linked immunosorbent assay (ELISA). Normal and increased KCTs or dRVVTs were seen during follow up in 13 and 12 patients, respectively. IgG anticardiolipin antibodies changed from negative to positive or positive to negative in 26 patients and IgM anticardiolipin antibodies in 16 patients. Disease activity and treatment with prednisone could account for these fluctuations in the kaolin clotting time (KCT) in 7 of 13 patients and in the dRVVT in 2 of 12 patients. Whole group analysis showed that the KCT, dRVVT, and IgM anticardiolipin antibodies were not associated with disease activity, in contrast with IgG anticardiolipin antibodies. During treatment with prednisone normal KCT and dRVVT results were obtained more easily than normal anticardiolipin antibody levels. It is recommended that lupus patients should not be classified as antiphospholipid antibody positive or negative on the basis of only one sample.