Abstract Number: PB1752
Meeting: ISTH 2020 Congress
Background: The spleen tyrosine kinase Syk plays an essential role in platelet proximal signaling mediated by ITAM-dependent receptor activation. Recently, we showed that the crosstalk between GPIbα and platelet inhibitory pathways (cAMP/PKA, cGMP/PKG) results in significant inhibition of platelet aggregation but surprisingly induces prolonged Syk activation mediated by hyperphosphorylation of SykY525/526 (kinase domain) and SykY352 (interdomain-B) but downregulation of SykS297(interdomain-B).
Aims: To investigate the role of SykS297 phosphorylation in relation to Syk activation and its regulation by serine/threonine (S/T) protein kinases and phosphatases.
Methods: Echicetin beads (EB) and convulxin (cvx) were used as specific GPIbα and GPVI agonists, respectively. Platelet aggregation was performed by light transmission aggregometry. Phosphorylated proteins were analyzed by immunoblotting using standard and phos-tag SDS-PAGE. Intracellular messengers inositol monophosphate (InsP1) and Ca2+i were quantified by ELISA/IPOne assay and flow cytometry, respectively.
Results: Mass spectrometry analysis revealed upregulation of SykS297 induced by ADP, thrombin and Ca2+-ionophore, which was significantly downregulated by cAMP/cGMP-elevating agents. Upon ITAM-mediated platelet signaling induced by EB or cvx, SykS297 was significantly upregulated in a transient, SFK and Syk-dependent manner. Interestingly, the cAMP-elevating agent iloprost and the global PKC inhibitor GF109203X reduced EB/cvx-induced SykS297, but mediated SykY525/526, Y352 hyperphosphorylation and prolonged Syk activation. The hyperphosphorylation profiles of Syk Y-sites led to a significant increase of InsP1/InsP3 production and Ca2+i-mobilization, but only partial inhibition of platelet aggregation in GPIbα/GPVI-activated platelets. Furthermore, inhibition of the S/T-protein phosphatase 1 and 2A by calyculin A and okadaic acid resulted in time-dependent stoichiometric increase of global Syk phosphorylation and increase of SykS297 but not of SykY525/526, Y352.
Conclusions: Activated PKC stimulates SykS297 phosphorylation, which is also observed when S/T-phosphatases are inhibited. Reduced SykS297 phosphorylation coincides with enhanced Syk activation, suggesting that Syk phosphorylation at S297 represents a mechanism for feedback inhibition in human platelets and a potential diagnostic/therapeutic target.
To cite this abstract in AMA style:Makhoul S, Dorschel S, Gambaryan S, Pagel O, Zahedi RP, Walter U, Jurk K. Feedback Inhibition of Human Platelet Syk by PKC-caused Syk S297 Phosphorylation [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/feedback-inhibition-of-human-platelet-syk-by-pkc-caused-syk-s297-phosphorylation/. Accessed November 30, 2021.
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