Platelets have an established role in thrombosis and haemostasis. C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. Using a recently developed mouse model for platelet-specific deletion of platelet-CLEC-2, CLEC1bfl/flGPIb-Cre, we show that CLEC-2 contributes to thrombus stability in mice. However, CLEC-2 inhibition in human blood using monoclonal antibody, AYP1, or recombinant CLEC-2 did not translate these observations to human. This presents CLEC-2 as a promising therapeutic target in thromboinflammatory or inflammatory disease, without detriment to thrombosis and haemostasis. We show that hemin, the oxidised form of haem released post-haemolysis, is a novel CLEC-2 ligand which can activate and aggregate platelets through its hemITAM domain. Interestingly, anti-malarial drug hydroxychloroquine is able to inhibit hemin-induced mouse and human platelet activation, but not ROS generation. In a mouse model of thrombosis, the ferric chloride injury of the carotid artery, we demonstrate that hydroxychloroquine treatment presents small, non-occlusive thrombi, compared to saline-treated controls. This may present hemin-CLEC-2 as a promising therapeutic target in thromboinflammatory disease. Beyond thrombosis, platelets contribute to the development, progression and resolution of the inflammatory response. CLEC-2 is protective during mouse models of sepsis, and other inflammatory diseases. Here we show that CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from inflamed tissue during murine peritonitis through its ligand, podoplanin. Treatment using recombinant CLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. Both the thromboinflammatory and immunomodulatory functions of CLEC-2 present strategies to reduce tissue inflammation and inflammatory thrombi, and could be therapeutically exploited to limit disease progression
