Syk-dependent Phosphorylation of CLEC-2: A Novel Mechanism of Hem-Immunoreceptor Tyrosine-Based Activation Motif Signaling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the haematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other haematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that is dependent on CLEC-2 and Syk. These studies demonstrate that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behaviour in vitro

Lymphatic blood filling in CLEC-2-deficient mouse models

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization,secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2)activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM).Here, we demonstrate using sucrose gradient ultracentrifugation and methyl--cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization,Rac1 activation, and release of ADP and thromboxane A2 (TxA2). The role of ADP and TxA2 in mediating hosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast,tyrosine phosphorylation of the GPVIFcR -chain ITAM, which has 2 YxxL motifs,is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators,and Rac activation

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: a novel mechanism of haemostasis

The Podoplanin-CLEC-2 axis is critical in mice for prevention of haemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of haemostasis in which podoplanin supporting platelet capture and activation at arteriolar rates of shear

Low dose Btk inhibitors selectively block platelet activation by CLEC-2

Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinaemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at ‘low dose’ in patients to target CLEC-2 in thrombo-inflammatory disease

Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs

Characterisation of the C-type lectin receptor CLEC-2: expression, ligands and functions

Myeloid cells express a plethora of C-type lectin receptors (CLR) that can regulate inflammatory responses. Dectin-1 belongs to a sub-family of CLRs that possesses an extracellular C-type lectin domain (CTLD) and a single YxxL intracellular motif (hemITAM) that allows signalling via Syk kinase and induction of downstream functions. Based on consensus sequences for the CTLD and hemITAM, we identified CLEC-2 as a dectin-1-like receptor. CLEC-2 was previously characterised as a Syk-coupled platelet receptor able to induce platelet aggregation when targeted by the snake venom rhodocytin and by cells expressing the endogenous protein podoplanin. I generated monoclonal antibodies against mouse CLEC-2 and found that CLEC-2 is also expressed on lymphoid and myeloid cells, including dendritic cells (DC). Notably, treatment with LPS increases CLEC-2 expression by myeloid cells and synergises with CLEC-2 signaling to induce increased secretion of IL-10 but not IL-12. This increased IL-10 production is also observed in the serum of mice administered with anti-CLEC-2 mAb and LPS, and is dependent on the presence of macrophages and DCs. Furthermore, I generated a CLEC-2 conditional KO mouse line that will provide a tool to study CLEC-2 function in myeloid cells in vivo. Collectively, these data indicate that CLEC-2 expression is not restricted to platelets and that it plays a role on the vascular development and modulation of TLR responses

The roles of platelet clec-2 and podoplanin in skin wound healing

Platelet-expressed C-type lectin-like receptor-2 (CLEC-2) and glycoprotein (GP)VI play important roles in inflammation, in particular inflammatory haemostasis in the skin. The CLEC-2-ligand, podoplanin, is upregulated in the inflamed and wounded skin, but the role of the CLEC-2-podoplanin interaction and the signalling downstream of podoplanin in the repair process is unclear. I have addressed these questions by investigating skin wound healing in wild-type (WT) mice, transgenic mice that lack platelet GPVI or CLEC-2 or both receptors (double knockout; DKO), and podoplanin cytoplasmic tail-deficient (PdpnCyto) mice. Deletion of both CLEC-2 and GPVI impairs vascular integrity in the skin resulting in accelerated wound healing. The beneficial effect was due to increased plasma leakage in the tissue that promoted fibrin generation, enhanced re-epithelialisation and angiogenesis, and decreased immune cell infiltration. Accelerated wound healing also led to smaller scar formation. This healing phenotype is not due to developmental defects in DKO animals as similar results were obtained in podoplanin-blocking antibody-injected GPVI-deficient mice. Wound healing is independent of the signalling downstream of podoplanin as PdpnCyto mice had similar healing kinetics compared to WT mice. PdpnCyto mice were however capable of upregulating podoplanin during wound healing, suggesting further application of this model in inflammatory settings. Alongside wound repair, the PdpnCyto mice were characterised. I have shown that the cytoplasmic tail is dispensable for the separation of blood and lymphatic vessels. In addition, I have used a metabolomics approach to reveal an increase in M1 pro-inflammatory metabolites, i.e. glycolysis and inducible nitric oxide synthase (iNOS)-mediated arginine pathway, in bone marrow-derived podoplanin-deficient macrophages, which possibly support the anti-inflammatory activity of podoplanin in macrophages

Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β

BACKGROUND: The C‐type lectin‐like receptor 2 (CLEC‐2) and the collagen receptor glycoprotein (GP)VI activate platelets through Src and Syk tyrosine kinases, and phospholipase Cγ2. The initial events in the two signaling cascades, however, are distinct, and there are quantitative differences in the roles of proteins downstream of Syk activation. The activation of Akt and mitogen‐activated protein kinases (MAPKs) has been shown to enhance platelet activation by GPVI, but their role in CLEC‐2 signaling is not known. OBJECTIVES: We sought to investigate the role of the Akt and MAPK pathways in platelet activation by CLEC‐2. RESULTS: The CLEC‐2 agonist rhodocytin stimulated phosphorylation of Akt and p38 and extracellular signal‐related kinase (ERK) MAPKs, but with a delay relative to Syk. Phosphorylation of these proteins was markedly inhibited in the combined presence of apyrase and indomethacin, consistent with the reported feedback action of ADP and thromboxane A(2) in CLEC‐2 signaling. Phosphorylation of Akt and phosphorylation of ERK were blocked by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin and the protein kinase C (PKC) inhibitor Ro31‐8220, respectively, whereas Syk phosphorylation was not altered. On the other hand, both inhibitors reduced phosphorylation of the Akt substrate glycogen synthase kinase 3α/β (GSK3α/β). Phosphorylation of GSK3α/β was also blocked by the Akt inhibitor MK2206, and reduced at late, but not early, times by the MEK inhibitor PD0325901. MK2206 and PD0325901 inhibited aggregation and secretion in response to a low concentration of rhodocytin, which was restored by GSK3α/β inhibitors. CONCLUSIONS: These results demonstrate that CLEC‐2 regulates Akt and MAPK downstream of PI3K and PKC, leading to phosphorylation and inhibition of GSK3α/β, and enhanced platelet aggregation and secretion