Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, Kofu, Japan.
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.
J Thromb Haemost. 2018 May;16(5):960-972. doi: 10.1111/jth.13987. Epub 2018 Mar 30.
Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis.
Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.
目的 制备可诱导血小板聚集的重组 rhodocytin，通过突变验证其功能，并在此基础上开发抗 CLEC-2 药物。
方法 我们使用中国仓鼠卵巢细胞表达重组 rhodocytin（野生型 [WT]和突变型），通过透光比浊法分析血小板聚集的诱导/抑制作用、使用蓝色 native PAGE 分析多聚体的形成、以及使用流式细胞术分析与 CLEC-2 的结合。最后，我们在实验性肺转移小鼠模型中研究了突变型 rhodocytin 是否能抑制 PDPN 诱导的转移。
结果 成功获得了具有功能的 WT rhodocytin（αWTβWT），其通过共表达两个亚基获得。rhodocytin 的 α 亚基中的 Asp4 对于与 CLEC-2 的结合是必需的。αWTβWT 形成与天然 rhodocytin 相似的异源八聚体。此外，具有抑制活性的突变型 rhodocytin（αWTβK53A/R56A）形成异源四聚体，可与 CLEC-2 结合但不诱导血小板聚集，并阻断 CLEC-2-PDPN 相互作用依赖性的血小板聚集和实验性肺转移。
结论 这些发现提供了 rhodocytin 的分子特征信息，并提示突变型 rhodocytin 可作为靶向 CLEC-2 的治疗剂。
