Dysfunctional CLEC-2 and GPVI-Mediated Signaling in Syk Y342F Knock-in Mouse Platelets

Abstract

Platelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors like GPVI, the protease activated receptors (PARs), and C-type lectin-like receptor 2 (CLEC-2). GPVI is an ITAM-bearing receptor, while Clec-2 is a HemITAM-bearing receptor. Both ITAM and hemITAM-bearing receptors signal through spleen tyrosine kinase (Syk). Syk activity is dependent on phosphorylation of several residues including Y342, Y346, and Y519/520 (Y348, Y352, and Y525/526 in human). However, the importance of each residue on either phosphorylation of other residues or activation of Syk is unknown. Therefore, we produced a Syk Y342F knock-in mouse line via CRISPR/Cas9 to investigate the importance of Y342 on Syk-mediated signaling using a platelet system. Syk Y342F mice are viable, have no gross abnormalities, and their blood cell counts are normal. We were unable to detect any Syk Y342 phosphorylation following stimulation of Y342F platelets with the GPVI agonist collagen-related peptide (CRP) (Figure 1). Further, Platelet reactivity is reduced in response to the GPVI agonists CRP (Figure 2) and collagen, as well as the CLEC-2 agonist rhodocytin. When using CLEC-2 antibody as an agonist no response is observed in Syk Y342F mouse platelets even though WT littermate control mouse platelets responded well (Figure 3). Signaling initiated by either GPVI or CLEC-2 is also inhibited, as Syk Y519/520 phosphorylation is impaired. Furthermore, PLCg2 and SLP-76 phosphorylation are reduced in Syk Y342F platelets compared to platelets from littermate control mice following stimulation of either GPVI or CLEC-2. Although reactivity of Y342F platelets is clearly reduced compared to WT platelets, there was no difference in occlusion time following ferric chloride injury of the carotid artery. Similarly, there was no difference in mortality rate following pulmonary thromboembolism using Syk Y342F mice compared to WT littermate control mice. Nevertheless, these data demonstrate that phosphorylation of Y342 on Syk following stimulation of either ITAM or hemITAM-bearing receptors is important for Syk activation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.