OC 78.3 - Generation and characterization of inactive Spleen tyrosine kinase mice; Evaluation of ITAM and hemITAM signaling and function in platelets

Background: The spleen tyrosine kinase (Syk) plays a central role in immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM signal transduction in numerous haematopoietic cell systems.

Aims: Evaluate the function of various tyrosine residues on the activation and activity of Syk.

Methods: To assess the mechanism of Syk regulation, the tandem tyrosines 519 and 520 in the kinase domain of mouse Syk were mutated to phenylalanines by CRISPR-Cas.

Results: The breeding of the 519/520 knockin mice did not follow Mendelian ratio as genotype analysis of the pups from heterozygous intercrosses showed only 14 in 100 were Syk Y519,520F. Because platelets have the GPVI receptor which contains an ITAM and the CLEC2 receptor which contains a hemITAM, these cells were used to study the effect of this mutation on their physiological responses and downstream signaling. There was no aggregation or dense granule secretion when Syk Y519,520F platelets were tested compared to the WT littermates at any concentration of collagen-related peptide (CRP) or a CLEC2 antibody. There was also no p-selectin exposure or alpha2beta3 activation when the mutated platelets were challenged with these agonists. No phosphorylation of the downstream signaling proteins LAT Y200 or PLCgamma2 Y1217 was detected in the mutated platelets. This phenotype was specific for ITAM- hemITAM-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to stimulate the platelets. The Syk Y591,520F mutation had a clear effect on hemostasis as demonstrated by a significant increase in tail-bleeding time although the time to thrombus formation using the ferric chloride injury model was unaltered.

Conclusion(s): We conclude that phosphorylation of Y519 and Y520 on Syk following stimulation of either GPVI or CLEC2 receptors was crucial for the ability of Syk to transduce a signal and elicit functional responses