Abstract
Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen.
Highlights
Platelets are essential for primary haemostasis, and play a major role in the development of cardiovascular disease
There are a number of Ig-like immunoreceptor tyrosine-based inhibitory motifs (ITIM)/immunoreceptor tyrosine-based switch motifs (ITSM) receptors expressed on the platelet surface, including G6B-b, platelet endothelial cell adhesion molecule-1 (PECAM-1), carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) and TREMlike transcript-1 (TLT-1)
SHP-2 has been demonstrated to associate with G6B-b, but this has not been demonstrated in human platelets
Summary
Platelets are essential for primary haemostasis, and play a major role in the development of cardiovascular disease. Healthy endothelium releases soluble factors, such as prostacyclin (PGI2) and nitric oxide (NO), which act to inhibit platelet activation In addition to these soluble factors, platelets express a number of immunoglobulin (Ig)-like receptors at the platelet surface that relay a negative signal to the cell to either maintain the resting state or limit the extent of activation. These receptors elicit their effects via the immunoreceptor tyrosine-based inhibitory motifs (ITIM) or immunoreceptor tyrosine-based switch motifs (ITSM) in their intracellular tails, which can interact with Src homology (SH) 2 domain-containing proteins. There are a number of Ig-like ITIM/ITSM receptors expressed on the platelet surface, including G6B-b, platelet endothelial cell adhesion molecule-1 (PECAM-1), carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) and TREMlike transcript-1 (TLT-1)
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