Platelet FcγRIIA Signaling Results in Ubiquitination and Cellular Translocation of Activated Syk

Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for α_IIbβ₃ outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunotyrosine activation motif (ITAM) signaling, such as FcγRIIA, GPVI and CLEC-2. We previously reported Syk ubiquitination following GPVI stimulation, and the role of c-cbl as the E3 ubiquitin ligase (Dangelmaier, Blood 2005). Ubiquitination is an important post-translational modification that modulates signal transduction by regulating the activity, subcellular localization or stability of proteins. We tested the hypothesis that ubiquitination participates in signaling, and examined ubiquitination of Syk downstream of platelet FcγRIIA activation.

Using both washed human platelets and HEL cells, we observed ubiquitination of Syk upon FcγRIIA engagement by cross-linking IV.3 mAb (10 ug/ml) with goat anti-mouse Fab’₂ (30 ug/ml). Both tyrosine phosphorylation and ubiquitination of Syk occurred within 15 sec, peaked by 1-3 min and decreased thereafter. The pattern of ubiquitination was consistent with 1 to 3 Ub molecules per Syk molecule. Ubiquitinated-Syk (Ub-Syk) was increased in the presence of PR-619, a deubiquitinating enzyme inhibitor, confirming ubiquitination of Syk. Ub-Syk associates with the cytoskeletal-rich platelet fraction, membrane skeleton fraction and with cytosolic fraction in detergent lysed platelets that were fractionated by lower g-forces (15,500 x g) and higher g-forces (100,000 x g). This suggests that Ub-Syk is translocated into all cellular compartments upon platelet activation. Ub-Syk was absent upon pre-treatment with Src-family kinase inhibitor PP2 (10 uM), but minimally affected in the presence of Syk inhibitor PRT318 (1 uM), in both platelets and HEL cells, as compared to DMSO treated control cells. Further, phosphorylation of c-cbl was inhibited strongly by PP2, but only slightly inhibited by PRT318, suggesting that ubiquitination of Syk depends on Src kinase activity. Of note, Ub-Syk was not degraded by the proteasome, since no accumulation of Ub-Syk was observed by pretreatment with proteasome inhibitors MG132 or Epoxomicin in either platelets or HEL cells, compared to control cells.

In conclusion, Syk is ubiquitinated upon cross-linking platelet FcγRIIA and is translocated to all major subcellular compartments. Since ubiquitinated Syk is activation-dependent and not subject to proteasomal degradation, it likely serves as a novel adapter molecule for protein-protein interactions in mediating platelet activation via FcγRIIA.