A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2

Abstract

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

CLEC‐2 signaling via Syk in myeloid cells can regulate inflammatory responses

“…3). These findings are in line with reports in platelets showing that CLEC-2 signals lead to PLCg activation, an enzyme crucial for intracellular calcium increase [18,19]. The use of mAbs to selective trigger surface receptors has provided important insight into the signaling and function of different CLRs [40][41][42].…”

“…However, its signaling and function in myeloid cells has been difficult to address due to the dearth of agonists that selectively trigger CLEC-2. Rhodocytin acts as a CLEC-2 agonist in platelets [19] and has been shown to activate myeloid cells [29]. However, rhodocytin, in our assays, activated DCs primarily via a MyD88-dependent mechanism, possibly due to contamination with TLR agonists, which obscured the putative CLEC-2/Syk-dependent response.…”

“…CLEC-2 was first identified in a screen for natural killer (NK) cell receptors and its mRNA was found in myeloid cells and in some NK-cell clones [17]. More recently, platelets were found to also express CLEC-2 and this receptor was shown to be a target of rhodocytin, a snake venom toxin that induces platelet aggregation [18][19][20]. In addition to rhodocytin, the mucin-like glycoprotein podoplanin (also known as GP38) has been identified as an endogenous ligand for CLEC-2 [21].…”

Spleen tyrosine kinase inhibition in the treatment of autoimmune, allergic and autoinflammatory diseases

“…(b) Glycoprotein VI (GPVI), a major collagen receptor of platelets, is an FcγR-related receptor and is closely associated with FcαRs [24]. Collagen activates the FcγR-associated receptor GPVI on platelets and triggers Syk activation in an ITAM-dependent way [24,28]. SLP76, a Syk substrate, is required for arterial thrombus formation [28].…”

“…Collagen activates the FcγR-associated receptor GPVI on platelets and triggers Syk activation in an ITAM-dependent way [24,28]. SLP76, a Syk substrate, is required for arterial thrombus formation [28]. (c) Platelet agonists like rhodocytin and podoplanine activate the receptor C-type lectin-like receptor 2 (CLEC2), which recruits Syk to the phosphorylated tyrosine in the CLEC2 ITAMs [4,28].…”