全反式维甲酸及依托泊苷对急性早幼粒细胞白血病细胞磷脂酰丝氨酸暴露及促凝血活性的影响

目的：探讨磷脂酰丝氨酸（PS）暴露在急性早幼粒细胞白血病（APL）细胞促凝血活性中的作用及不同药物对其产生的影响。方法：实验共分为4组：新采集APL细胞组、APL细胞单纯培养组、APL细胞全反式维甲酸（ATRA）处理组及APL细胞依托泊苷（VP16）处理组。提取10名初发APL患者的骨髓APL细胞进行实验,提取10名健康成人外周血单个核细胞作为凝血实验的正常对照。分别用1μmol·L-1ATRA和1μmol·L-1VP16处理APL细胞24 h,利用共聚焦显微镜及流式细胞术检测各组细胞PS暴露情况。利用凝血实验检测各组细胞总的促凝活性及细胞表面磷脂的促凝血活性。利用PS特异结合蛋白乳粘素对各组细胞进行凝血抑制实验。结果：新采集的APL细胞存在一定量的PS外翻,并且与外周血单个核细胞相比,存在更高的促凝血活性（P〈0.05）,ATRA对APL细胞的PS外翻及促凝活性有抑制作用（P〈0.05）,VP16则对其有显著的促进作用（P〈0.001）。乳粘素可以拮抗APL细胞至少70%的促FXa和FIIa生成活性。结论：PS暴露在APL细胞促凝血过程中发挥着重要作用。分化治疗药物ATRA和化疗药物VP16分别通过减少和增加APL细胞表面PS的暴露来减轻和加重凝血紊乱。乳粘素通过与PS特异结合可以有效地阻断暴露的PS的促凝活性,是一种潜在的治疗APL凝血紊乱的抗凝剂。

The Effect of All-trans Retinoic Acid and Etoposide on Phosphatidylserine Exposure and Procoagulant Activity of Acute Promyelocytic Leukemia Cells

Objective： To investigate the effect of phosphatidylserine （PS） exposed on the procoagulant activity of acute promyelocytic leukemia （APL） cells and the effects of different drugs on it. Methods： APL cells were isolated from bone marrow of 10 newly diagnosed APL patients and peripheral blood mononuclear cells, used as control in coagulation assays, were collected from 10 healthy volunteers. Four groups of cells： fresh APL cells, APL cells cultured for 24 h, APL cells treated with 1 μmol. L^-1 all-trans retinoic acid （ATRA）for 24 h and APL cells treated with 1 μmol.L^-1 etoposide （VP16） for 24 h were included in the study. PS exposure on cell surface was measured by confocal microscopy and flow cytometry. Total procoagnlant activity of APL cells and the procoagulant activity derived from phospholipid at the outer membrane surface of APL cells were detected by coagulation assays. The role that PS exposure played in the procoagulant activity of APL cells was evaluated by coagulation inhibition assays. Results： Fresh APL cells showed some amount of PS exposure and it showed a higher procoagulant activity （P〈0.05） compared with peripheral blood mononuclear cells, ATRA had an inhibitory action on PS exposure and procoagulant activity of APL cells （P〈0.05）, while VP16 had a striking promoting effect （P〈0.001）. Lactadherin, a PS-speeific binding protein, could reduce at least 70% of the FXa and FIIa formation-promoting activity of APL cell. Conclusions： Exposed PS on the membrane surface played an important role in the procoagulant activity of APL cells. The differentiation-inducing agent ATRA could reduce the procoagulant activity by decreasing the PS exposure of APL cell, while the chemotherapy agent VP16 had a complete opposite effect. Through specific binding to PS, lactadherin could effectively block the procoagulant activity of exposed PS, and likely provide a new way to treat APL coagulopathy.