INVOLVEMENT OF ANNEXIN V IN THE ENDOTHELIAL APOPTOSIS INDUCED BY AUTOANTIBODIES.

狼疮抗凝剂（LAC）与动脉和静脉血栓形成、血小板减少症和复发性胎儿丢失有关。我们之前报道过，具有LAC活性的血浆诱导内皮细胞凋亡并结合膜联蛋白V。在本研究中，我们从10例LAC患者中分离出两个IgG抗体，一个与膜联蛋白V有亲和力，一个不与膜联蛋白V有亲和力。在所有10例患者中，LAC和凋亡活性均局限于膜联蛋白v结合部分。DNA断裂与人脐静脉内皮细胞培养基中IgG的添加量呈剂量依赖性；用磷脂脂质体去除患者IgG中的抗磷脂抗体并没有消除诱导凋亡的活性或与膜联蛋白v的结合。这些结果表明，LAC患者通常具有不结合磷脂的抗体，并负责诱导内皮细胞凋亡。核小体间DNA断裂通常被认为是细胞凋亡的特征之一，其中大多数是由依赖ATP的半胱天冬酶激活驱动的。另一方面，有报道称ATP耗竭可诱导细胞凋亡并伴随DNA断裂。为了解决这个明显的矛盾，我们分析了DNA：在atp耗尽的细胞中产生的分裂活动。在低霉素无糖培养基中培养的HL-60早幼粒细胞白血病细胞中，核小体间DNA片段作为早期事件发生。丝氨酸蛋白酶抑制剂能阻断DNA的断裂，而半胱天冬酶抑制剂则不能。与此一致的是，ICAD/DFF45不能抑制无细胞系统中ATP耗尽的细胞质的dna片段化活性。当向无细胞实验提供ATP时，80%的dna片段化活性丧失。然后蛋白酶体抑制剂恢复了降低的活性，这表明蛋白酶体的作用是保护细胞免受atp耗竭引起的损伤

Lupus anticoagulant (LAC) is associated with arterial and venous thrombosis, thrombocytopenia, and recurrent fetal loss. We have reported previously that plasma with LAC activity induces apoptosis in endothelial cells and binds annexin V. In this study, we separated two IgG antibody fractions, one with and one without affinity for annexin V, from 10 patients with LAC. LAC and apoptotic activities were localized in the annexin V-binding fraction in all 10 patients. DNA fragmentation was dose-dependeub paralleling the amount of IgG added to the human umbilical vein endothelial cell culture medium, and was inhibited by preincubation with annexin V. Removal of the antiphospholipid antibodies from patient IgG with phospholipid liposomes did not abolish the apoptosis-inducing activities or binding to annexin V. These results imply that patients with LAC often have antibodies that do not bind phospholipids and are responsible for the induction of apoptosis in endothelial cells.Internucleosomal DNA fragmentation is generally perceived as one of the characteristic features of apoptosis, most of which are driven by caspase activation dependent upon ATP. On the other hand, ATP depletion has been reported to induce apoptosis accompanying DNA fragmentation. To address this apparent paradox, we analyzed the DNA: fragmenting activity generated in ATP-depleted cells. In HL-60 promyelocytic leukemia cells cultured in glucose-free medium with oligomycin, internucleosomal DNA fragmentation occurred as an early event. The DNA fragmentation was blocked by serine protease inhibitors but not by caspase inhibitors. Consistently, ICAD/DFF45 could not inhibit the DNA-fragmenting activity of the ATP depleted cytosol in a cell-free system. When ATP was supplied to the cell-free assay, 80% of the DNA-fragmenting activity was lost. The reduced activity was then restored by proteasome inhibitors, suggesting a role of proteasome to protect from a cellular insult derived from ATP-depletion