Diannexin 在抗磷脂综合征中的抗凝保护作用及机制研究

Disruption of the anticoagulant annexin A5 two-dimensional crystal lattice and elevation of the circulating microparticles (MPs) are the major mechanisms of the hypercoagulability state of antiphospholipid syndrome (APS). Diannexin, a homodimer of annexin A5 produced by recombinant DNA technology, may display potential anticoagulation. Our previous study showed that APS patients exhibited significantly higher plasma levels of MPs and annexin A5 anticoagulant resistance, whereas diannexin could correct this anticoagulant resistance. Therefore, we hypothesize that diannexin is able to reduces the release of MPs by reestablishing the two-dimensional crystal lattice. In this research project, we will employ PS-expressing cell membrane models to demonstrate whether diannexin is competent to rebuild the anticoagulant crystal shield by binding PS and prevent MPs generation by endocytosis of PS. In addition, we will constitute APS animal models to show whether diannexin can protect against vascular thrombosis and pregnancy morbidities. Our project is helpful to elucidate the anticoagulant role and mechanism of diannexin in APS and provide evidences for the application of diannexin in APS treatment.

细胞膜表面Annexin A5二维晶格抗凝屏障损坏和血循环中微粒（MPs）升高是抗磷脂综合征（APS）高凝状态的重要机制。重组技术合成的 Annexin A5同型二聚体Diannexin具有潜在抗凝活性。本项目前期工作显示：APS患者血浆MPs显著升高并存在Annexin A5抗凝抵抗现象，而Diannexin可纠正这种抵抗现象。因此，本项目研究假说是：Diannexin通过在细胞膜表面重建二维晶格抗凝结构而减少MPs释放。本项目将利用细胞膜磷脂酰丝氨酸（PS）暴露模型，借助共聚焦扫描激光和原子力显微镜探索Diannexin与PS结合是否可在细胞表面形成二维晶格结构，并通过胞饮PS减少MPs产生；利用APS动物模型确定Diannexin是否可抑制血栓形成和减少胚胎丢失。本项目将有助于阐明Diannexin对APS的抗凝作用及机制，并为Diannexin作为治疗APS的新型抗凝药物提供依据。

抗磷脂综合征（APS）最常见的产科并发症是以血栓炎症反应为特征的子痫前期（PE）。而细胞膜表面磷脂酰丝氨酸（PS）暴露和血循环中微粒（MPs）升高是APS发生血栓高凝状态的重要机制。已知Annexin A5同型二聚体Diannex可与PS高亲和性结合，并具有更长半衰期。所以本项目进行了以下工作：（1）通过蛋白重组技术合成了Diannex，并通过共聚焦扫描激光显微镜证明了其可特异的与纯PS微球和血小板膜表面暴露的PS高亲和性结合。（2）通过构建MPs流式细胞检测技术和收集健康志愿者、APS、PE患者的血浆和胎盘组织，证明了APS和PE患者血浆MPs水平显著升高，并且PE患者胎盘组织的炎症小体蛋白水平显著升高。（3）通过流式细胞术，在细胞水平证明了Diannex可以减少细胞MPs释放，抑制血栓炎症反应。（4）通过构建PE小鼠模型，证明了Diannex可通过抑制MPs产生和炎症小体活化，抑制MPs诱导的PE表型出现。以上研究结果证明了：Diannexin遮蔽细胞膜表面暴露的PS，可通过减少MPs释放和炎症小体活化抑制血栓炎症反应发生。所以本项目阐明了Diannexin抑制APS介导血栓炎症反应的机制，为其是否可用于APS患者PE预防提供了证据。