Functional significance of the platelet immune receptors GPVI and CLEC-2

Abstract

Although platelets are best known for their role in hemostasis, they are also crucial in development, host defense, inflammation, and tissue repair. Many of these roles are regulated by the immune-like receptors glycoprotein VI (GPVI) and C-type lectin receptor 2 (CLEC-2), which signal through an immunoreceptor tyrosine-based activation motif (ITAM). GPVI is activated by collagen in the subendothelial matrix, by fibrin and fibrinogen in the thrombus, and by a remarkable number of other ligands. CLEC-2 is activated by the transmembrane protein podoplanin, which is found outside of the vasculature and is upregulated in development, inflammation, and cancer, but there is also evidence for additional ligands. In this Review, we discuss the physiological and pathological roles of CLEC-2 and GPVI and their potential as targets in thrombosis and thrombo-inflammatory disorders (i.e., disorders in which inflammation plays a critical role in the ensuing thrombosis) relative to current antiplatelet drugs.

Figure 1. ITAM receptors activate platelets via a (hem)ITAM phosphotyrosine signaling cascade.

(A) GPVI activation initiates Src family kinase–mediated (SFK-mediated) phosphorylation of two conserved tyrosines in the associated FcRγ ITAM and allows the recruitment and docking of Syk’s SH2 domain. GPVI signaling is regulated through shedding by ADAM10/17, with G6B-b preventing shedding in circulating platelets via the tyrosine phosphatases SHP-1 and SHP-2. (B) CLEC-2 activation induces phosphorylation on the hem(ITAM), allowing binding of Syk. (A and B) Once recruited, Syk undergoes a series of auto- and trans-phosphorylations by SFK, leading to recruitment and phosphorylation of the adaptor protein LAT and recruitment of this pathway’s effectors, such as GRB2, GADS, SLP76, VAV1/3, PIP3, and BTK/TEC. The signaling cascade culminates in the phosphorylation and activation of PLCγ2, which binds to phosphorylated LAT and PIP3 on the plasma membrane. PIP3 also recruits BTK and TEC, which phosphorylate PLCγ2. Activation of PLCγ2 induces formation of the secondary messengers inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), resulting in the mobilization of intracellular Ca²⁺ stores and activation of PKC, respectively. These changes result in the secretion of intracellular α-granules and dense granules and inside-out activation of platelet integrins, including α_IIbβ₃, leading to platelet aggregation. CLEC-2 signaling in human platelets depends on actin polymerization and the secondary mediators, ADP and TxA₂.

Figure 2. CLEC-2 and GPVI contribute to α_IIbβ₃-dependent and -independent hemostasis.

(A) At the site of arterial injury, platelets roll, adhere, and aggregate to arrest bleeding through integrin α_IIbβ₃. The activation of GPVI by collagen drives integrin activation. Activated platelets release secondary mediators such as ADP and TxA₂, leading to recruitment of circulating platelets and formation of a platelet-fibrin-fibrinogen plug. The resulting thrombus is composed of a densely packed, fibrin-rich core and a looser outer shell of platelets. The diffusion of ligands from the core to the outer shell determines the size of the thrombus. Fibrin and fibrinogen bind to and activate GPVI, which may serve to propagate and stabilize the thrombus. (B) At the site of inflammation, platelets prevent hemorrhaging independent of α_IIbβ₃. The contribution of platelet receptors to the inflammatory hemostasis is stimulus- and organ-dependent. During immune complex–mediated dermatitis, endothelial and stromal cell activation promotes recruitment, activation, and transmigration of neutrophils to the inflamed skin. Single platelets rapidly adhere and seal the neutrophil-mediated vascular damage through GPVI. The bleeding in GPVI-deficient mice is limited by the interaction of CLEC-2 with podoplanin on macrophages and stromal cells. In the absence of both receptors, a severe loss of vascular integrity is observed, with increased bleeding in the inflamed skin.

Figure 3. Multiple roles of CLEC-2 and GPVI in thrombo-inflammation and thrombosis.

During thrombo-inflammatory diseases, platelets regulate immune cell recruitment at the site of inflammation. Deletion of GPVI (Gp6^–/–) decreases the inflammatory reaction as a consequence of a reduction in neutrophil recruitment and the number of inflammatory macrophages. Conversely, deletion of CLEC2 (Clec1b^–/–) potentiates the inflammatory reaction through an increase in neutrophil infiltration and inducing a proinflammatory macrophage phenotype. In addition, GPVI promotes the underlying inflammation that drives plaque formation and drives thrombus formation upon plaque rupture. During venous thrombosis, CLEC-2 promotes thrombosis at sites of breach in the vessel wall by upregulating podoplanin on stromal cells and inflammatory macrophages.