title: The dual kinase and scaffold function of Btk downstream of GPVI and CLEC-2 in platelets
Creators: name: Nock, Sophie
Publisher: name: University of Reading
Year: 2021

description: Background Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase found in platelets and B-cells. In platelets, Btk has demonstrated a role in facilitating both ITAM and hemITAM-mediated platelet activation. Downstream of the B-cell receptor, Btk has been shown to induce signalling independently of its kinase domain, suggesting it may have a role as a scaffold protein. Aims To determine if Btk can act as an adaptor protein in platelets and to identify if there are differential roles of Btk downstream of GPVI and CLEC-2. Methods Indicators of platelet activity such as aggregometry, granule release and Ca2+ mobilisation, along with western blotting were used to study the effect of acalabrutinib, an inhibitor of Btk, downstream of GPVI and CLEC-2 mediated platelet activation. To identify a role for Btk kinase function downstream of GPVI and CLEC-2 signalling an NFAT-Luciferase reporter assay was used, expressing wild type or a kinase dead Btk mutant in Btk-deficient cells. Total Internal Reflection Fluorescence Microscopy (TIRFM) and Stochastic optical reconstruction microscopy (STORM) was used to investigate the co-localisation of Btk with other signalling proteins. Results At specific Btk inhibiting concentrations of acalabrutinib, platelet aggregation downstream of GPVI is normal despite loss of Btk pY223, an indicator of Btk activity and PLCγ2 pY759 (PLCγ2 active site) phosphorylation. In a cell line model, GPVI mediated signalling is maintained despite inhibition with acalabrutinib or expression of a kinase dead mutant of Btk. However, Btk kinase function is essential for CLEC-2 mediated signalling at submaximal concentrations of rhodocytin in platelets and in a cell line model but can be ii overcome at high concentrations of rhodocytin. Btk colocalised more with the transmembrane receptor LAT than either GPVI or CLEC-2 on collagen or rhodocytin, respectively. A proportion of Btk colocalised with GPVI, with the localisation and clustering of Btk unaltered in the presence of acalabrutinib. However, Btk inhibition altered the clustering of GPVI suggesting a kinase dependent role of Btk in the clustering of GPVI. Conclusions Platelets can aggregate without Btk pY223 phosphorylation downstream of GPVI showing a scaffolding role for Btk. Furthermore, Btk does not require kinase activity to signal downstream of GPVI in a cell line model. Localisation of Btk does not change when its kinase activity is inhibited with a Btk-specific dose of acalabrutinib, suggesting kinase independent recruitment of Btk, providing further evidence of adaptor protein function downstream of GPVI. However, Btk kinase activity is required for signalling downstream of CLEC-2 at submaximal concentrations, as platelets fail to aggregate when Btk pY223 is lost. At high concentrations of rhodocytin when Btk is inhibited, platelets can still aggregate. Secondary feedback is likely mediating the aggregation of platelets. This demonstrates a novel difference between ITAM and hemITAM signalling.