Platelet CLEC-2: a molecule with 2 faces

In this issue of Blood, Haining et al reveal an unexpected role for platelet C-type lectin-like receptor-2 (CLEC-2) in thrombosis, which is independent of its hemi-immunoreceptor tyrosine-based activation motif (hemITAM) signaling.¹ 

CLEC-2 is a highly expressed platelet receptor.^1,2  Its intracellular domain contains a unique single YxxL motif (hemITAM) that is capable of activating platelets through downstream signaling effectors Syk and SLP-76 upon ligand engagement of a CLEC-2 dimer. Platelet CLEC-2 is involved in 2 important biological processes: blood/lymphatic vessel separation and thrombosis. The former function has been well characterized in mice lacking platelet CLEC-2 or its intracellular signaling molecules Syk or SLP-76 because these mutant mouse models share a distinct blood-lymphatic mixing vascular phenotype. This indicates a critical function of hemITAM signaling for CLEC-2: to regulate blood-lymphatic separation during lymphatic vascular development.³  The blood-lymphatic mixing phenotype is also seen in mice lacking the O-glycoprotein podoplanin,⁴  the only known physiological ligand of CLEC-2. Podoplanin is specifically expressed on lymphatic endothelial cells. Therefore, these data indicate that platelet activation mediated by CLEC-2 hemITAM signaling upon binding to lymphatic endothelial podoplanin is essential for forming an independent lymphatic vascular system. Other than this well-defined role in vascular development, mice with CLEC-2–deficient platelets also exhibit impaired platelet aggregate formation and lower susceptibility to arterial thrombus formation relative to wild-type controls.^5,6  Podoplanin does not seem to be involved in platelet CLEC-2–mediated thrombus formation as it is expressed neither on blood endothelial cells nor on other blood cells under physiological conditions.⁷  But, whether hemITAM-mediated signaling is required in this biological process remains to be determined.

In this study, Haining et al generated a novel knock-in mouse model that expresses a mutant form of CLEC-2 with its cytoplasmic hemITAM YxxL motif mutated (Y7A KI). The Y7A KI platelets express normal surface levels of CLEC-2 as well as other platelet surface receptors. Y7A KI platelets display normal responses to classic agonists such as thrombin and collagen, but have a defective response to the CLEC-2 agonist rhodocytin. By binding to CLEC-2, both rhodocytin and podoplanin activate platelets through hemITAM-dependent signaling. Like CLEC-2 knockout (KO) mice, Y7A KI mice have the blood-lymphatic mixing phenotype, confirming the critical role of the hemITAM-dependent signaling of CLEC-2 in promoting separation of lymphatic vessels from the blood vessel during development. However, unlike CLEC-2 KO mice, Y7A KI mice have normal bleeding time and thrombus formation, in 2 different arterial thrombosis models, similar to that of wild-type controls. Importantly, treatment of Y7A KI mice with the Fab′ fragments of the anti-CLEC-2 antibody, INU1, which does not cross-link CLEC-2 to induce hemITAM signaling, reduces thrombus formation, revealing a hemITAM signaling–independent role for CLEC-2 in thrombosis.

Haining et al show that CLEC-2 is required for platelets to interact with other platelets adherent to sites of vascular injury within the vessel to form thrombus, which again raises the often-asked question: how does platelet CLEC-2 contribute to thrombus formation (see figure)? Podoplanin is not known to be present on platelets or other blood cells under normal conditions.⁷  In addition, the INU1 Fab′ fragments block aggregate formation of Y7A KI platelets on collagen under flow conditions in vitro. These data are suggestive of additional endogenous ligand(s) for platelet CLEC-2. There have been several attempts to identify new ligands/counterreceptors for platelet CLEC-2, but none of the studies appear to be conclusive. Thus, future studies are required to reveal the mysterious blood-borne CLEC-2–interacting molecule(s) and its binding site on CLEC-2. Podoplanin and CLEC-2–mediated platelet activation is also critical for protecting vascular integrity in many important organs, such as lymph nodes and the brain.^7,8  This hemITAM signaling–independent platelet CLEC-2 function is neither required for vascular development/integrity nor involved in hemostasis, that is, initial platelet adhesion. Therefore, it represents an attractive therapeutic target for development of an effective and safe antithrombosis therapy.