ANTIPHOSPHOLIPID ANTIBODIES AND ENDOTHELIAL CELLS

抗磷脂综合征(APS)是一种反复发生的血栓形成障碍， 与持续阳性相关的妊娠丢失和血小板减少 抗磷脂(APL)和狼疮抗凝(LA)试验。APL是否 是致病的吗？如果是，是通过什么机制(S)尚不清楚。体外研究 已经证明APL可以抑制蛋白C的激活、失活 活化蛋白C(APC)激活的Va因子或调节抗凝剂 某些磷脂结合蛋白的功能，如 β/2/糖蛋白1或胎盘抗凝蛋白 1)。最近，通过使用血栓形成的小鼠模型，我们的中心展示了 APL抗体具有直接的血栓形成特性。然而， 这种情况发生的机制(S)尚不清楚。这项研究建议 确定来自APS的人多克隆和单克隆抗体是否具有 磷脂或磷脂结合蛋白的不同特异性 体外激活内皮细胞。EC激活将由以下因素决定 黏附分子I-CAM-1、V-CAM-1和E-选择素在血管内皮细胞中的表达 HUVEC。此外，无论是β/2/GP1、PAP 1、tADL(一种源于 来自人腺病毒II，模拟磷脂结合部位 β/2/GP1)和LAP-20(与磷脂结合部位类似的多肽 载脂蛋白A1)调节APL对EC的激活 下定决心。抗APL单抗和多克隆抗体对血管内皮细胞生长的影响 体内EC的激活，也将通过测量粘附性来检测 小鼠提睾肌微循环中白细胞对EC的影响 注射APL抗体。白细胞对细胞的粘附性增加 血管壁将是EC激活的标志。为了确定 无论I-CAM-1、V-CAM-1、E-选择素或P-选择素等黏附分子 选择素在体内介导EC的激活，将使用两种途径： 1)小鼠将被注射APL抗体和两块提睾肌 会在手术中暴露。一块Cremaster肌肉将不会得到治疗 另一种是特异性的抗黏附分子(抗I-CAM- 1、抗V-CAM-1、E-选择素、P-选择素)抗体 以确定这些抗体是否能消除增强的白细胞 APL与EC的黏附；2)体内EC(白细胞)的活化 APL的粘附性也将在P-选择素/E-选择素中确定 有缺陷的小鼠。可变区中是否有特定的氨基酸基序 APL抗体与特异性、功能和活体相关 这些抗体的效果也将被确定。

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.