Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization

Abstract

Key Points• CLEC-2 can be downregulated from circulating platelets by anti-CLEC-2 antibodies through Src-family kinasedependent internalization.• Platelet-specific Syk deficiency abrogates anti-CLEC-2 antibodies-induced thrombocytopenia, but not CLEC-2 internalization.Platelet aggregation at sites of vascular injury is not only essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemi-immunoreceptor tyrosine-based activation motif-containing C-type lectinlike receptor 2 (CLEC-2) mediates powerful platelet activation through a Src-and spleen tyrosine kinase (Syk)-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 not only contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis, and prevention of inflammatory bleeding, making it a potential pharmacologic target to modulate these processes.We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immunodepletion occurs through Srcfamily kinase-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with platelet-specific Syk deficiency, INU1-induced CLEC-2 internalization/degradation was fully preserved whereas the associated thrombocytopenia was largely prevented. These results show for the first time that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia. (Blood. 2015;125(26):4069-4077)

C‐type lectin‐like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor‐bearing mice

“…showed that CLEC‐2 platelet levels can be decreased in vivo by administering anti‐CLEC‐2 monoclonal antibody, INU1 . INU1‐induced CLEC‐2 immunodepletion is mediated by Src family kinase‐dependent receptor internalization in vitro and in vivo , presumably followed by intracellular degradation . We generated rat anti‐mouse CLEC‐2 monoclonal antibody, 2A2B10.…”

The ins and outs of endocytic trafficking in platelet functions

“…Endocytosis of purinergic receptors e.g ., P2Y 1 and P2Y 12 , mediates their resensitization [11]. Endocytosis of C-type Lectin Receptor-2 (CLEC-2) and Thrombopoietin receptor (Mpl/TPOR) regulates their surface levels and downstream signaling [12, 13]. These data clearly show that endocytosis has significant importance in platelet physiology.…”

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…We used outbred strain CD1 or the inbred strain C57BL/6 as well as the transgenic strains Gp6 −/− (Bender et al, 2011), Itga2 −/− (Holtkotter et al, 2002), Lat −/− and Syk fl/fl, Pf4−Cre+/− (Lorenz et al, 2015) (all C57BL/6 background) as indicated. Male and female mice of 6 to 12 weeks were used in equal numbers for all experiments, unless stated otherwise.…”