Vascular Biology Program, Boston Children's Hospital, Boston, Massachusetts, USA; Harvard Medical School, Boston, Massachusetts, USA. Electronic address: https://twitter.com/NathanAsquith1.
Vascular Biology Program, Boston Children's Hospital, Boston, Massachusetts, USA; Harvard Medical School, Boston, Massachusetts, USA.
J Thromb Haemost. 2024 Nov;22(11):3235-3248. doi: 10.1016/j.jtha.2024.07.021. Epub 2024 Aug 8.
Platelets are essential for hemostasis and thrombosis and play vital roles during metastatic cancer progression and infection. Hallmarks of platelet function are activation, cytoskeletal rearrangements, and the degranulation of their cellular contents upon stimulation. While α-granules and dense granules are the most studied platelet secretory granules, the dense tubular system (DTS) also functions as a secretory system for vascular thiol isomerases. However, how DTS cargo is packaged and transported from megakaryocytes (MKs) to platelets is poorly understood.
To underpin the mechanisms responsible for DTS cargo transport and leverage those for therapeutic protein packaging into platelets.
A retroviral expression system combined with immunofluorescence confocal microscopy was employed to track protein DTS cargo protein disulfide isomerase fused to enhanced green fluorescent protein (eGFP-PDI) during platelet production. Murine bone marrow transplantation models were used to determine the release of therapeutic proteins from platelets.
We demonstrated that the endoplasmic reticulum retrieval motif Lys-Asp-Glu-Leu (KDEL) located at the C-terminus of protein disulfide isomerase was essential for the regular transport of eGFP-PDI-containing granules. eGFP-PDI, in which the retrieval signal was deleted, was aberrantly packaged, and its expression was upregulated within clathrin-coated endosomes. Finally, we found that ectopic transgenic proteins, such as tissue factor pathway inhibitor and interleukin 2, can be packaged into MKs and proplatelets by adding a KDEL retrieval sequence.
Our data corroborate the DTS as a noncanonical secretory system in platelets and demonstrate that in vitro-generated MKs and platelets may be used as a delivery system for transgenic proteins during cellular therapy.
血小板对于止血和血栓形成至关重要，在转移性癌症进展和感染过程中发挥着重要作用。血小板功能的标志是激活、细胞骨架重排以及受到刺激时细胞内容物的脱颗粒。虽然α-颗粒和致密颗粒是研究最多的血小板分泌颗粒，但致密管状系统（DTS）也作为血管硫醇异构酶的分泌系统发挥作用。然而，DTS 货物如何从巨核细胞（MKs）包装并运输到血小板尚不清楚。
为 DTS 货物运输的负责机制提供依据，并利用这些机制将治疗性蛋白包装到血小板中。
采用逆转录病毒表达系统结合免疫荧光共聚焦显微镜，跟踪血小板生成过程中融合增强型绿色荧光蛋白（eGFP-PDI）的蛋白二硫键异构酶（PDI）的 DTS 货物蛋白。使用小鼠骨髓移植模型来确定治疗性蛋白从血小板中的释放。
我们证明了位于蛋白二硫键异构酶 C 端的内质网回收基序赖氨酰-天冬氨酸-谷氨酸-亮氨酸（KDEL）对于包含 eGFP-PDI 的颗粒的正常运输是必需的。删除了回收信号的 eGFP-PDI 被异常包装，其表达在网格蛋白包被的内体中上调。最后，我们发现通过添加 KDEL 回收序列，可以将组织因子途径抑制剂和白细胞介素 2 等异位转基因蛋白包装到 MKs 和前血小板中。
我们的数据证实 DTS 是血小板中非典型的分泌系统，并表明在体外生成的 MKs 和血小板可在细胞治疗期间用作转基因蛋白的递送系统。
