A hierarchical network of Src, Syk, Btk and PKC controls GPVI-dependent human platelet activation

Introduction Src family kinases (SFKs), spleen tyrosine kinase (Syk) and Bruton´s tyrosine kinase (Btk) play central roles in the activation of immune cells and platelets. Glycoprotein VI (GPVI) stimulation activates platelet SFKs, Syk and Btk resulting in phospholipase Cγ (PLCγ) and protein kinase C (PKC) activation. However, their functional hierarchy and cross-talk in platelets are not well understood. Recently, selective Syk and Btk inhibitors showed potency to treat thrombosis, cancers and immuno-inflammatory diseases. Using such inhibitors, we investigated hierarchy of Syk, Btk and PKC with respect to their downstream effectors in GPVI-stimulated human platelets.

Method Aggregation of washed human platelets was monitored by light transmission aggregometry in response to the GPVI agonist convulxin (cvx). Syk, Btk and PKC were inhibited by PRT, acalabrutinib and GFX, respectively. Site-specific antibodies were used to quantify phosphosites of Syk (S297, Y352, Y525/526), Btk (S180, Y223, Y551), PLCγ2 (Y759, Y1217), LAT (Y220), Akt (T308, S473) and MAPKs (Erk T202/Y204, p38 T180/Y182) in platelets, which were time-dependently (10-300s) stimulated by cvx under stirring.

Results Cvx induced a strong platelet aggregation which was abolished by 1 µM PRT or 5 µM acalabrutinib. Cvx induced a rapid, transient upregulation of multisite Y/S-phosphorylation with a clear kinetic hierarchy of Syk, Btk, LAT, PLCγ2, PKC, MAPKs and Akt. Tyrosine phosphorylation (pY) of Syk, Btk, PLCγ2, LAT preceded serine phosphorylation (pS) of Syk and Btk, Erk, p38 and Akt. PRT did not affect Syk pY352, but inhibited all other cvx-induced Y-phosphosites studied. Acalabrutinib inhibited cvx-induced Btk pY223 (autophosphorylation), PLCγ2 pY759/pY1217, Erk pT202/Y204, p38 pT180/Y182, Akt pT308/S473, Syk pS297 and Btk pS180, but not Syk pY352, pY525/526 and LAT pY220. GFX did not reduce, often enhanced pY of Syk, Btk, LAT and PLCγ2, abolished Syk and Btk pS and inhibited Erk but not p38.

Conclusion This kinetic analysis of cvx-induced phosphorylation in combination with the inhibitor effects indicate a hierarchical order of Y-/S-/T- protein kinases phosphorylation during GPVI-induced platelet activation. Stimulation of SFKs, Syk, Btk results in a strictly Btk-dependent activation of PLCγ2, PKC and Erk, whereas p38 and Akt activation are Btk- but not PKC-dependent. There is substantial cross-talk, such as the feedback inhibition of Syk and Btk by PKC-mediated phosphorylation (Syk pS297, Btk pS180). Importantly, specific phosphosites can be well used as markers for certain kinase activities: Syk pY352 as SFK marker; Syk pY525/526, LAT pY220, Btk pY551 as Syk marker; Btk pY223, PLCγ2 pY759/ p1217 as Btk marker; Syk pS297/Btk pS180 as PKC marker. The functional implications of differential inhibition of the Syk-Btk-system and their downstream effectors in human platelets are currently investigated [1] [2] [3] [4].

 

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Conflict of Interest

The authors declare no conflicts of interest.

• References

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Publication History

Article published online:
20 February 2023