Anti-phospholipid Syndrome (APS) is a disorder of recurrent thrombosis, pregnancy loses and thrombocytopenia associated with persistently positive anti-phospholipid (aPL) and lupus anticoagulant (LA) tests. Whether aPL are pathogenic and if so by what mechanism(s) is unclear. In vitro studies have demonstrated that aPL may inhibit protein C activation, inactivate factor Va by activated protein C (APC) or modulate the anticoagulant function of certain phospholipid binding proteins, such as Beta/2/glycoprotein 1 (Beta/2/GP1) or placental anticoagulant protein (PAP 1). Recently, by using a mouse model of thrombosis, our center has shown that aPL antibodies have direct thrombogenic properties. However, the mechanism(s) by which this occurs is unclear. This study proposes to determine whether human polyclonal and monoclonal antibodies from APS with different specificities to phospholipids or phospholipid binding proteins activate endothelial cells in vitro. EC activation will be determined by expression of adhesion molecules: I-CAM-1, V-CAM-1 and E-selectin in HUVEC. In addition, whether beta/2/GP1, PAP 1, TADL (a peptide derived from human adenovirus II, that mimics the phospholipid binding site of Beta/2/GP1), and LAP-20 (a peptide analog to the phospholipid binding site of apolipoprotein A1) modulate the activation of EC by aPL will be determined. The effects of monoclonal and polyclonal aPL antibodies on activation of EC in vivo, will also be examined by measuring adhesion of leukocytes to EC in the microcirculation of the cremaster muscle of mice injected with aPL antibodies. Increased adhesion of leukocytes to the vessel wall will be an indication of EC activation. In order to determine whether adhesion molecules such as I-CAM-1, V-CAM-1, E-selectin or P- selectin mediate EC activation in vivo, two approaches will be utilized: 1) mice will be injected with aPL antibodies and two cremaster muscles will be surgically exposed. One cremaster muscle will remain untreated and in the other the specific monoclonal anti-adhesion molecules (anti-I-CAM- 1, anti-V-CAM-1, E-selectin, P-selectin) antibodies will be administered to determine whether these antibodies abrogate the enhanced leukocyte adhesion to EC by aPL; 2) the activation of EC in vivo (leukocyte adhesion) by aPL will also be determined in P-selectin/E-selectin deficient mice. Whether specific amino-acid motifs in the variable regions of aPL antibodies correlate with specificity, function, and in vivo effects of these antibodies will also be determined.
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