Institute of Transfusion Medicine and Haemotherapy, University of Wuerzburg, Oberduerrbacher Straße 6, D-97080 Wuerzburg, Germany.
Institute of Transfusion Medicine and Haemotherapy, University of Wuerzburg, Oberduerrbacher Straße 6, D-97080 Wuerzburg, Germany.
Cell Signal. 2019 Oct;62:109351. doi: 10.1016/j.cellsig.2019.109351. Epub 2019 Jun 28.
Platelets express key proteins of the proteasome system, but its functional role in the regulation of platelet integrity, however, is not fully understood yet. Therefore, this study evaluated activating and inhibitory platelet signalling pathways using the potent and selective proteasome inhibitor bortezomib. In washed platelets, the effect of bortezomib on viability and on aggregation was assessed. In addition, fibrinogen binding and CD62P expression were determined. The influence on activating and inhibitory signalling was detected by phosphorylation levels of essential messenger molecules. Platelet viability was maintained after incubation with 0.01 μM to 1 μM bortezomib, but tampered with 100 μM bortezomib. Agonist-induced aggregation was only reduced under 100 μM bortezomib and with weak induction by 10 μM adenosine diphosphate. Similarly, phosphorylated kinase levels of the activating signalling pathways were not affected by 0.01 μM to 1 μM bortezomib. In contrast, proteasome inhibition resulted in the reduction of inhibitor-induced vasodilator-stimulated phosphoprotein phosphorylation, accompanied with the partial decrease of induced inhibition of fibrinogen binding and CD62P expression. In conclusion, platelet activation and aggregation are not dependent on proteasome activity. Instead, inhibitory signalling is partially attenuated under proteasome inhibition. Supramaximal inhibitory concentrations of bortezomib (above 1 μM) lead to heterogeneous effects on activating or inhibitory systems, probably caused by decreasing platelet viability.
血小板表达蛋白酶体系统的关键蛋白，但它在调节血小板完整性中的功能作用尚未完全了解。因此，本研究使用强效和选择性蛋白酶体抑制剂硼替佐米评估激活和抑制血小板信号通路。在洗涤血小板中，评估硼替佐米对活力和聚集的影响。此外，还测定了纤维蛋白原结合和 CD62P 表达。通过关键信使分子的磷酸化水平检测对激活和抑制信号的影响。在孵育 0.01 μM 至 1 μM 硼替佐米后，血小板活力得以维持，但在 100 μM 硼替佐米孵育时受到干扰。激动剂诱导的聚集仅在 100 μM 硼替佐米和 10 μM 二磷酸腺苷弱诱导下减少。同样，激活信号通路的磷酸化激酶水平不受 0.01 μM 至 1 μM 硼替佐米的影响。相比之下，蛋白酶体抑制导致抑制剂诱导的血管扩张刺激磷酸蛋白磷酸化减少，同时诱导的纤维蛋白原结合和 CD62P 表达抑制部分减少。总之，血小板激活和聚集不依赖于蛋白酶体活性。相反，在蛋白酶体抑制下，抑制性信号部分减弱。硼替佐米的超最大抑制浓度（高于 1 μM）对激活或抑制系统产生不均一的影响，可能是由于血小板活力降低所致。
