Nevzorova Tatiana A, Mordakhanova Elmira R, Daminova Amina G, Ponomareva Anastasia A, Andrianova Izabella A, Le Minh Giang, Rauova Lubica, Litvinov Rustem I, Weisel John W
1Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., Kazan, Russian Federation 420008 Russia.
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2/31 Lobachevsky str., Kazan, Russian Federation 420111 Russia.
Cell Death Discov. 2019 Jun 24;5:106. doi: 10.1038/s41420-019-0188-0. eCollection 2019.
Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.
肝素诱导的血小板减少症（HIT）是肝素治疗的一种并发症，有时与血栓形成有关。HIT的标志是针对肝素/血小板因子4（PF4）复合物的抗体，该抗体通过血小板激活导致血小板减少和血栓形成。尽管具有临床重要性，但免疫性血小板激活的分子机制和后期后果尚未完全了解。在此，我们研究了人PF4与HIT样单克隆小鼠抗人PF4/肝素IgG抗体（命名为KKO）形成的复合物对体外分离的人血小板的即时和延迟影响。KKO/PF4复合物对血小板的直接激活作用通过血小板表面磷脂酰丝氨酸（PS）和P-选择素的过表达得到证实。免疫性血小板激活伴随着线粒体跨膜电位（ΔΨm）的降低，同时血小板中ATP含量显著逐渐减少，表明能量代谢受到破坏。在相当一部分血小板中观察到含PF4的免疫复合物诱导的PS表达和线粒体去极化的组合被认为是正在发生的血小板死亡的标志，这与质膜上有PS但ΔΨm正常的活化活血小板亚群相反。用KKO/PF4处理的活化和死亡血小板均形成表面带有PS的促凝细胞外微泡。扫描和透射电子显微镜显示KKO/PF4处理的血小板有显著的形态变化，包括其碎片化，这是细胞死亡的另一个指标。KKO/PF4的大多数作用可被抗FcγRII单克隆抗体IV.3阻止。KKO/PF4复合物诱导的血小板不良功能和结构变化与钙蛋白酶的强烈时间依赖性激活有关，但半胱天冬酶3仅有微量裂解。结果表明，致病性含PF4的HIT样免疫复合物通过FcγRIIA受体诱导直接的促血栓形成血小板激活，随后是血小板的非凋亡性钙蛋白酶依赖性死亡，这可能是HIT发生过程中血小板减少的重要机制。
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