ANTIPHOSPHOLIPID ANTIBODIES AND ENDOTHELIAL CELLS

抗磷脂综合征（APS）是一种复发性血栓形成的疾病， 妊娠丢失和血小板减少与持续阳性 抗磷脂（aPL）和狼疮抗凝（LA）试验。是否aPL 是致病性的，如果是的话，是什么机制尚不清楚。体外研究 已经证明aPL可以抑制蛋白C激活， 通过活化蛋白C（APC）或调节抗凝剂 某些磷脂结合蛋白的功能，如 β/2/糖蛋白1（β/2/GP 1）或胎盘抗凝蛋白（PAP 1）。最近，通过使用血栓形成的小鼠模型，我们的中心已经表明， aPL抗体具有直接的血栓形成特性。但 发生这种情况的机制尚不清楚。本研究建议， 确定来自APS的人多克隆和单克隆抗体是否具有 对磷脂或磷脂结合蛋白的不同特异性 在体外激活内皮细胞。EC激活将由 粘附分子：I-CAM-1、V-CAM-1和E-选择素的表达 HUVEC。此外，无论β/2/GP 1、PAP 1、TADL（一种肽衍生物） 来自人腺病毒II，其模拟了 β/2/GP 1）和β-20（磷脂结合位点的肽类似物 载脂蛋白A1）调节aPL对EC的激活作用， 测定单克隆和多克隆aPL抗体对人肝癌细胞的作用 EC在体内的活化，也将通过测量 小鼠提睾肌微循环中白细胞对EC的影响 注射了aPL抗体白细胞粘附于 血管壁将是EC激活的指示。为了确定 无论粘附分子如I-CAM-1、V-CAM-1、E-选择素或P- 选择素介导的EC体内活化，将使用两种方法： 1)小鼠将被注射aPL抗体和两个提睾肌 将通过手术暴露出来一个提睾肌将保持不治疗， 另一方面，特异性单克隆抗粘附分子（anti-I-CAM-1）， 1、抗V-CAM-1、E-选择素、P-选择素）抗体 以确定这些抗体是否消除了增强的白细胞 aPL对EC的粘附; 2）EC在体内的活化（白细胞 aPL的粘附）也将在P-选择素/E-选择素中测定 缺陷小鼠可变区中的特定氨基酸基序是否 的aPL抗体与特异性、功能和体内 还将确定这些抗体的作用。

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.