SHARPIN (Src homology 3 and multiple ankyrin repeat domains protein (SHANK)-associated RH domain-interacting protein) as part of the linear ubiquitin chain assembly complex (LUBAC) catalyses the addition of linear (Met1-linked) ubiquitin chains to substrates. As part of this complex SHARPIN acts as a multi-functional modulator of immune/inflammatory responses through regulation of NfkB activation. In addition, SHARPIN can act as a negative regulator of integrin function. Despite platelets being anucleate cells several studies have determined potential roles for both ubiquitination and NfkB in regulating platelet function. However, little is known about either linear ubiquitination and/or SHARPIN in mouse platelets. In this study, we evaluated platelet function in mice with impaired SHARPIN expression. We confirmed that SHARPIN was expressed in platelets from wild-type mice but not in mice homozygous for SHARPINcpdm allele (cpdm/cpdm) and that this correlated with a reduction in linear ubiquitination. Platelet function in response to thrombin was unaffected. In contrast, CRP-XL-and U46619-mediated platelet responses and thrombus formation under flow on a collagen-coated surface were significantly reduced in the cpdm/cpdm mice. This was associated with impaired U46619-mediated intracellular signalling as well as a reduction in CRP-mediated ERK phosphorylation. Despite the reported role for NfκB in regulating platelet function, inhibiting IκBα phosphorylation did not recapitulate the cpdm/cpdm phenotype. Together, these data indicate that the lack of SHARPIN and linear ubiquitination results in impaired thrombosis and platelet functional responses to CRP and U46619. This phenotype is independent of NfκB pathway inhibition but may involve alternative signalling pathways regulated by linear ubiquitination.

中文翻译：

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SHARPIN在血小板线性蛋白泛素化和功能中的作用

作为线性泛素链装配复合体（LUBAC）的一部分的SHARPIN（Src同源性3和多个锚蛋白重复域蛋白（SHANK）相关的RH域相互作用蛋白）催化线性（Met1连接）泛素链向底物的添加。作为这种复合物的一部分，SHARPIN通过调节NfkB激活而充当免疫/炎症反应的多功能调节剂。此外，SHARPIN可以作为整合素功能的负调节剂。尽管血小板是无核细胞，但已有几项研究确定了泛素化和NfkB在调节血小板功能中的潜在作用。然而，对于小鼠血小板中的线性泛素化和/或SHARPIN知之甚少。在这项研究中，我们评估了SHARPIN表达受损的小鼠的血小板功能。我们证实，SHARPIN在野生型小鼠的血小板中表达，但在SHARPINcpdm等位基因（cpdm / cpdm）纯合子的小鼠中不表达，并且这与线性泛素化的降低有关。响应凝血酶的血小板功能不受影响。相反，在cpdm / cpdm小鼠中，CRP-XL和U46619介导的血小板反应以及在胶原包被的表面上流动下的血栓形成显着减少。这与受损的U46619介导的细胞内信号传导以及CRP介导的ERK磷酸化减少有关。尽管报道了NfκB在调节血小板功能中的作用，但抑制IκBα磷酸化并不能概括cpdm / cpdm表型。一起，这些数据表明，缺少SHARPIN和线性泛素化会导致血栓形成和对CRP和U46619的血小板功能反应受损。该表型独立于NfκB途径抑制，但可能涉及线性泛素化调节的替代信号途径。