Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.
Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.
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DOI：
10.1161/atvbaha.115.306054
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发表时间：
2015-12
期刊：
影响因子：
--
通讯作者：
McIntyre TM
中科院分区：
文献类型：
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作者：
Gupta N;Li W;McIntyre TM
血小板表达功能性泛素-蛋白酶体系统。质谱分析显示血小板含有几种去泛素化酶，但这些酶是否具有功能、调节蛋白质组或影响血小板反应性尚不清楚。血小板裂解物含有泛素-蛋白质去泛素化酶活性，其水解Lys 48和Lys 63聚泛素缀合物，其被化学上不相关的去泛素化酶抑制剂PYR 41和PR 619抑制。这些抑制剂急性和显着增加单和多泛素化的静息血小板的蛋白质组。PYR 41（静脉内，15分钟）显著损害FeCl 3损伤的颈动脉中的闭塞性血栓形成，并且去泛素酶抑制降低了全血通过涂覆有胶原的微流体室的高剪切流期间的血小板粘附和滞留。全内反射显微镜显示，在没有流动的情况下，粘附和铺展被这些抑制剂强烈地抑制，稳定的过程扩展失败，并且形成的凝块的收缩减少。去泛素化酶抑制也急剧减少同型血小板聚集反应的不完全激动剂ADP和胶原蛋白通过GPVI的作用，但也完全激动剂凝血酶。抑制的聚集伴随着PAC-1与活化的IIb/IIIa的结合减少和P-选择素易位至血小板表面的抑制。去泛素化酶抑制作用可消除激动剂诱导的细胞内钙离子峰值，抑制Akt磷酸化，并减少激动剂刺激的PTEN磷酸酶磷酸化。血小板表达蛋白酶体相关的去泛素化酶USP 14和UCHL 5，并且通过b-AP 15对这些酶的选择性抑制再现了一般去泛素化酶抑制剂对离体血小板功能的抑制作用。去泛素化酶对泛素化血小板蛋白质组的重塑促进激动剂刺激的细胞内信号转导和血小板反应性。
Platelets express a functional ubiquitin-proteasome system. Mass spectrometry shows platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown. Platelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 poly-ubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased mono- and poly-ubiquitination of the proteome of resting platelets. PYR41 (i.v., 15 min) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed adhesion and spreading in the absence of flow was strongly curtailed by these inhibitors with failure of stable process extension, and reduced retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to the incomplete agonists ADP and collagen acting through GPVI, but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed PAC-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated PTEN phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function. Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.