Abstract
Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2 −/− platelets treated with 2.5-μg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.
Highlights
Platelets are the primary mediators of thrombosis and hemostasis
We demonstrate that T-cell ubiquitin ligand-2 (TULA-2) is a negative regulator of signaling initiated by the hem-immunoreceptor tyrosine activation motif (hemITAM)-containing C-type lectin-like receptor 2 (CLEC-2) receptor
In terms of spleen tyrosine kinase (Syk) phosphorylation, our data agree with published data describing the effect of TULA-2 on Syk downstream of two ITAM-containing receptors, FcγRIIA and glycoprotein VI (GPVI).[22,35]
Summary
Platelets are the primary mediators of thrombosis and hemostasis. They are small anucleate cells that exist in a quiescent state, and in a discoid shape. Platelets respond to vascular damage by binding to von Willebrand factor and collagen, which initiates an intracellular signaling cascade that culminates in shape change, production of thromboxane A2 (TXA), and release of granular contents. Release of secondary mediators such as TXA and adenosine diphosphate reinforces the original excitatory signal and recruits new platelets to the growing thrombus. Just as this process is essential to maintain hemostasis, a similar process is necessary to insure lymphatic and blood vessel separation. Lymphatic endothelial cells differentiate from venous endothelial cells and, unlike venous and arterial endothelial cells, they express podoplanin, which is the only known received July 15, 2018 accepted October 10, 2018
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