Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by arterial/venous thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies that mainly recognize ␤2 glycoprotein I (␤2GPI). To investigate potential platelet ligands of ␤2GPI, platelet membrane proteins from healthy persons and patients with APS were passed through a ␤2GPI-affinity column. By using mass spectrometry, platelet factor 4 (PF4) appeared as the dominant ␤2GPI binding protein. PF4 could bind in vitro, with high-affinity, recombinant ␤2GPI, and the binding was abrogated by soluble ␤2GPI. Coprecipitation experiments further confirmed this interaction. In silico molecular docking showed that PF4 tetramers can bind 2 ␤2GPI molecules simultaneously. Size exclusion chromatography confirmed that anti-␤2GPI antibodies selectively interact with complexes composed of (␤2GPI) 2 -(PF4) 4 . In addition, as shown by the ␤2GPI antigenicity evaluation, the reactivity of APS sera was higher against PF4-␤2GPI complex than against ␤2GPI alone. On complex formation, anti-␤2GPI-␤2GPI-PF4 significantly induced platelet p38MAPK phosphorylation and TXB2 production, mainly through F(ab) 2 fragments of antibodies. In summary, this study makes evident that ␤2GPI forms stable complexes with PF4, leading to the stabilization of ␤2GPI dimeric structure that facilitates the antibody recognition. This interaction can probably be involved in the procoagulant tendency of APS. (Blood. 2010;115:713-723)

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Antiphospholipid antibodies (aPL) constitute a heterogeneous group of autoantibodies that share the ability to bind phospholipids (PL) alone, protein-PL complexes, or PL-binding proteins. They have been detected in isolation, in association with autoimmune diseases such as systemic lupus erythematosus (SLE), and during the course of different infections. aPL have been associated with an array of clinical manifestations in virtually every organ, although deep vein and arterial thrombosis as well as pregnancy morbidity are predominant. The co-occurrence of these clinical findings with aPL constitutes the so-called antiphospholipid syndrome (APS). aPL can be detected by immunological methods [e.g., anticardiolipin antibodies (aCL)] or by functional methods that exploit the effect of aPL on blood coagulation [lupus anticoagulant (LA)]. Since aPL are heterogeneous, numerous immunological and coagulation assays have been developed. These assays have not been fully standardized, and, therefore, problems such as high interlaboratory variation are relatively frequent. Recently, recommendations have been published regarding LA and aCL testing. Not all aPL are pathogenic. However, when they are not associated with infections, they have a role in the pathogenesis of APS. Clinical and experimental data have shown that aPL exert their pathogenic activity by interfering with the function of coagulation factors, such as thrombin and factors X, XI and XII, and with the function of anticoagulant proteins of the protein C system. In addition, aPL interaction with platelets and endothelial cells induces a pro-adhesive activated phenotype.

Background Platelet derived chemokines, such as PF-4 and a recently isolated protein product of its nonallelic variant gene PF-4var, are implicated in several aspects of vascular thrombosis and inflammation. The above chemokines present only 4.3% aminoacid divergence in the mature proteins; however they exhibit distinct platelet secretion mode and function. The precise role of PF-4var regarding the haemostatic balance is not yet studied. Previous study from our group demonstrates a novel interaction between β2-glycoprotein I (β2GPI), the major autoantigen in APS, and PF-4 or PF-4var. This complex formation leads in the stabilization of β2GPI dimeric structure which facilitates the antibody recognition and platelet activation, as indicated by p38MAPK phosphorylation and thromboxane production. Objectives To determine PF-4var plasma levels in patients with APS and evaluate the correlation with clinical and laboratory parameters of the disease. Methods From 70 patients, who fulfill the revised diagnostic criteria for APS, blood samples were taken and separate samples of serum, plasma and platelets were isolated. Complete blood count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), aPTT, anti-cardiolipin (anti-CL) and anti-β2GPI antibodies were measured. A healthy control (n=20) and a disease control group (SLE, n=10) were included in the study (study in progress to increase the number of both groups). Plasma levels of PF-4var were determined using a commercially available ELISA, which absolutely discriminates PF-4var from PF-4 protein. Statistical analyses were evaluated by Mann-Whitney t-test and Kruskal-Wallis test. Results APS patients showed higher levels of PF-4var compared to healthy individuals (median 137 pg/ml; intarquartile range 66.4-200.5 pg/ml versus 79.03 [40.3-99.2] pg/ml, p=0.0052). PF-4var levels were significantly elevated in patients suffering from primary APS (PAPS) than those with APS secondary to SLE (SAPS), (197.7 [113.3-304.8] pg/ml versus 126.6 [49.94-170] pg/ml, p=0.0086). Regarding the clinical presentation of the disease, patients who experienced thrombotic events versus pregnancy morbidity or arterial versus venous thrombotic events do not show statistically significant difference in PF-4var levels. A positive correlation was also revealed between the presence of thrombocytopenia and the elongation of aPTT with the higher PF-4var levels (p= 0.0048 and p=0.0195, respectively). Conclusions Preliminary results suggest that higher PF-4var levels are present in plasma of APS patients and especially in those with PAPS and these are associated with laboratory characteristics indicative for higher risk of thrombotic complications. Disclosure of Interest: None Declared