Borut Božič

Antiphospholipid antibodies as non-traditional risk factors in atherosclerosis based cardiovascular diseases without overt autoimmunity. A critical updated review

The role of antiphospholipid antibodies (aPL) associated with cardiovascular diseases has been extensively studied in autoimmune patients, however it was largely unknown whether and how aPL associate with coronary artery disease (CAD), ishemic stroke (IS) and peripheral artery disease (PAD) in non-autoimmune patients. The current review attempts to prioritize for the first time clinical studies based on cause-outcome and strengths relationships in reference to aPL and CAD/PAD, in addition to supplementing Breys comprehensive review on IS with other, additional studies. Our overview indicates that all case-control and cross-sectional studies found an aPL association with CAD, PAD and IS, while cohort and nested case-control studies reported a prevailing negative risk association between aPL and IS (confirming Brey), with an unclear/unresolved risk association between aPL and CAD. The only cohort, follow-up study found in PAD reported on positive risk association between aPL and disease. The most frequently associated aPL in all studies reported, irrespective of disease, was aCL, with a less frequent association reported for LA, aβ2GPI and other aPL.

Prevalence of antiphospholipid antibodies in deep vein thrombosis and their relationship to blood coagulation and fibrinolysis.

Elevated levels of antiphospholipid antibodies are associated with an increased risk of thrombosis. To establish the prevalence of these antibodies in deep vein thrombosis (DVT), IgG and IgM antibodies to cardiolipin (aCL) and phosphatidylserine (aPS) were determined by enzyme-linked immunosorbent assay in 118 patients with DVT either during an acute episode (N = 53) or at least 2 months after acute DVT (N = 65). Most patients (76%) had proximal leg DVT and no one had evident autoimmune disorder. aCL and aPS values higher than 4 standard deviations above the mean value of the control group (147 blood donors) were considered increased. Increased IgG aCL were observed in 10% of DVT patients (controls: 5%, not significant), increased IgG aPS in 16% of DVT patients (controls: 5%, p less than 0.005) and both types in 4% of DVT patients (controls: 3%, not significant). In the subgroup of 41 patients with previous idiopathic DVT, prevalence of increased IgG aPS was the highest: 27% (p less than 0.001). Increased antibodies of IgM isotype were observed in 3% (aCL) and 2% (aPS) of all DVT patients (controls: 8% and 4%, respectively, not significant). Elevated IgG aCL or aPS were not associated with significant changes in platelet count, antithrombin III and protein C. However, in patients with increased IgG aPS deficient fibrinolysis due to high plasminogen activator inhibitor activity was observed before and after 20 min upper arm venous occlusion. DVT patients with increased IgG aPS might be exposed to a greater risk of rethrombosis due to deficient fibrinolysis than DVT patients without these antibodies.

Influence of degraded phosphatidylserine on binding of antiphospholipid antibodies

BACKGROUND Antiphospholipid antibodies (aPL) show great heterogeneity. Different phospholipids, with or without protein cofactor(s), and phospholipid binding proteins alone have been proposed as the target molecules for aPL. In order to determine the influence of phospholipid degradation products on the binding of aPL, sera from 6 patients with the antiphospholipid syndrome were studied. METHODS Fresh and aged phosphatidylserine and cardiolipin were used as coating reagents in solid-phase immunoassay procedures. Antibody reactivity was tested by enzyme-linked immunosorbent assay in the sera and in eluates from columns packed with polystyrene scrapings coated with either cardiolipin or phoshatidylserine. RESULTS Three reaction patterns of affinity-purified antibodies were seen: (1) reactivity with phosphatidylserine but not with cardiolipin or degraded phosphatidylserine, (2) reactivity with cardiolipin and degraded phosphatidlyserine, and (3) reactivity with all three phospholipid antigens. CONCLUSIONS Striking differences in the antiphospholipid antibody reactivity with cardiolipin, phosphatidylserine and degraded phosphatidylserine in the presence of serum proteins were observed among patients with venous thromboembolism. The analyses showed that the degradation of phosphatidylserine influences the binding of aPL in in vitro assays.

β2-glycoprotein I and annexin A5 phospholipid interactions: Artificial and cell membranes

The aim of this report was an overview of beta2-glycoprotein I (beta2-GPI)- and annexin A5 (AnxA5)-phospholipid interactions including candidate beta2-GPI receptors, and their relevance to the investigation of physiological/pathological processes. Both beta2-GPI and AnxA5 have thrombomodulatory functions in vivo and their antigenicity is particularly important for thrombotic manifestations and pregnancy complications in antiphospholipid syndrome. Specific elements of beta2-GPI- and AnxA5-phospholipid interactions are different. The crucial elements for beta2-GPI are conformational change and dimerization, both of which are also required and necessary for receptor signaling, leading to the prothombotic state. AnxA5 differs in its ability to crystallize into a protective shield, the disruption of which seems to be the major prothrombotic mechanism. These differences may explain some of the functional consequences of both molecules seen in the pathological conditions. Future alternative therapies of antiphospholipid syndrome are proposed to be based on the expanding knowledge of beta2-GPI- and AnxA5-phospholipid interactions, specifically antagonizing beta2-GPI receptors, as well as inhibiting their signaling pathways.

Detection of antiphosphatidylserine/prothrombin antibodies and their potential diagnostic value.

Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, β 2-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.

Modified phosphatidylserine-dependent antithrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies

Abstract Background: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. Methods: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. Results: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. Conclusions: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Avidity of anti-β2-glycoprotein i antibodies in patients with antiphospholipid syndrome

Antibodies against β2-glycoprotein I (anti-β2GPI) are one of the hallmarks of the antiphospholipid syndrome (APS). However, they are heterogenic regarding their epitope specificity, pathogenic mechanisms and their avidity. In the current study we present some outstanding issues about avidity of anti-β2GPI antibodies. Our results confirmed that high avidity anti-β2GPI are associated with thrombosis and APS, while in low avidity anti-β2GPI group non-APS (predominantly systemic lupus erythematosus) patients prevailed.

Anti-Phosphatidylserine/Prothrombin Antibodies Are Associated with Adverse Pregnancy Outcomes

Objective. To determine the prevalence and clinical association of anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in patients with a history of pregnancy complications relevant to antiphospholipid syndrome (APS). Materials and Methods. Two hundred and eleven patients with a history of (a) three or more consecutive miscarriages before 10th week of gestation (WG) (n = 64), (b) death of a morphologically normal fetus beyond 10th WG (n = 72), (c) premature birth of a morphologically normal neonate before 34th WG due to eclampsia, preeclamsia and placental insufficiency (n = 33), and (d) less than three unexplained consecutive miscarriages before 10th WG (n = 42). Subjects sera were analyzed for lupus anticoagulant (LA), anti-cardiolipin (aCL), anti-β 2-glycoprotein I (anti-β 2GPI), and aPS/PT antibodies. Results. 41/169 (24.3%) of patients were positive for at least one measured aPL. The highest prevalence was found for aPS/PT and aCL (13.0% and 12.4%, resp.) followed by LA (7.7%) and anti-β 2GPI (7.1%). 11/169 with APS-related obstetric manifestations had only aPS/PT. 17.8% of patients were positive for LA or aCL and/or anti-β 2GPI; however when adding the aPS/PT results, an additional 7% of patients could be evaluated for APS. Conclusion. aPS/PT are associated with recurrent early or late abortions and with premature delivery irrespective of other aPL.

In vitro model of annexin A5 crystallization on natural phospholipid bilayers observed by atomic force microscopy

Annexin A5 is a potent anticoagulant protein with a thrombomodulatory function. It is frequently mentioned with systemic inflammatory autoimmune disease, which share higher vulnerability to cardiovascular diseases. The protein has the ability to bind to membranes containing negatively charged phospholipids in a calcium-dependent manner. The potent anticoagulant properties of the protein are a consequence of this crystallization, which forms the lattice of annexin A5 over phospholipid surface, blocking its availability for coagulation reactions. Crystallization of annexin A5 has been proven on homogeneous synthetic phospholipids. However, the crystallization of annexin A5 on inhomogeneous, naturally derived phospholipid surfaces, in p3 and p6 crystal form, has now been reported for the first time. Atomic force microscopy was chosen for the observation of the crystallization of annexin A5 on different solid supported phospholipid bilayers. In this study model, the optimal results were obtained by using: 0.5 mg/ml lipid vesicles suspension (70% phosphatidylcholine, 30% phosphatidylserine) in HEPES buffer saline (HBS) with 2 mM CaCl2, large unilamellar vesicles with sizes around 200 nm, 41°C of phase transition temperature and 21 μg/ml of native annexin A5 in HBS with 2 or 20 mM CaCl2. Results were evaluated by imaging and force measurements. Demonstration that native annexin A5 is able to spontaneously crystallize on naturally derived, inhomogeneous phospholipids is supporting the putative role of annexin A5 crystal structures as possible antithrombotic shield. This in vitro system is probably more appropriate for studying the pathogenetic role of antiphospholipid antibodies.

Antibodies to phosphatidylserine/prothrombin complex as an additional diagnostic marker of APS?

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low (n = 9), heterogeneous (n = 31) and high (n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p < 0.05). No SLE patients had high avidity antiprothrombin antibodies.