C-type lectin-like receptor 2 (CLEC-2) is a platelet receptor that is critical during development in blood-lymph separation and implicated in thrombus stability in thrombosis and hemostasis. It is the only known platelet activatory receptor that participates in both of these aspects of platelet function, and it is the only one to signal through a hemi-immunoreceptor tyrosine–based activation motif (hemITAM). Current investigations into the function of CLEC-2 in vivo have focused on knockout (KO) studies in which both the receptor and its signaling are deleted, making it impossible to explore the possible signaling-independent functions of the receptor, which are indicated by its only known physiological ligand, podoplanin, being an integral membrane protein. In this report, we present a novel knockin mouse model that maintains the expression of a CLEC-2 receptor that cannot signal through its hemITAM (Y7A KI). Remarkably, this mouse phenocopies the blood-lymphatic mixing and lethality of CLEC-2 KO models, but not their hemostatic/thrombotic defect. However, treatment of Y7A KI mice with Fab′ fragments of the function-blocking anti–CLEC-2 antibody, INU1, resulted in a thrombus formation defect in vivo and ex vivo, revealing a hemITAM signaling–independent role for CLEC-2 in hemostasis and thrombosis.

C型凝集素样受体2（CLEC-2）是一种血小板受体，在血淋巴分离的发展过程中至关重要，并且在血栓形成和止血过程中与血栓稳定性有关。它是唯一已知的既参与血小板功能这两个方面的血小板活化受体，又是唯一通过基于半免疫受体酪氨酸的活化基序（hemITAM）发出信号的血小板活化受体。目前对CLEC-2体内功能的研究集中在敲除（KO）研究中，在该研究中，受体及其信号均被删除，因此无法探索该受体可能与信号无关的功能，这一点由其指示只有已知的生理性配体Podoplanin是必不可少的膜蛋白。在这份报告中，我们提出了一种新型的敲入小鼠模型，该模型维持了不能通过其hemITAM（Y7A KI）发出信号的CLEC-2受体的表达。值得注意的是，该小鼠表型复制了CLEC-2 KO模型的血液-淋巴混合性和致死性，但没有表现出其止血/血栓形成缺陷。然而，用功能阻断型抗CLEC-2抗体INU1的Fab'片段处理Y7A KI小鼠会导致体内和离体血栓形成缺陷，从而揭示了CLEC-2在止血中具有不依赖hemITAM信号传导的作用和血栓形成。