TY - JOUR A1 - Pollitt, Alice Y. A1 - Poulter, Natalie S. A1 - Gitz, Eelo A1 - Navarro-Nuñez, Leyre A1 - Wang, Ying-Jie A1 - Hughes, Craig E. A1 - Thomas, Steven G. A1 - Nieswandt, Bernhard A1 - Douglas, Michael R. A1 - Owen, Dylan M. A1 - Jackson, David G. A1 - Dustin, Michael L. A1 - Watson, Steve P. T1 - Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells* JF - The Journal of Biological Chemistry N2 - The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development. KW - endothelial cell KW - lipid bilayer KW - platelet receptor KW - tyrosine-protein kinase KW - CLEC-2 ITAM KW - podoplanin KW - Src family KW - kinase Syk Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120770 VL - 289 IS - 52 ER - TY - JOUR A1 - Perrella, Gina A1 - Montague, Samantha J. A1 - Brown, Helena C. A1 - Garcia Quintanilla, Lourdes A1 - Slater, Alexandre A1 - Stegner, David A1 - Thomas, Mark A1 - Heemskerk, Johan W. M. A1 - Watson, Steve P. T1 - Role of tyrosine kinase Syk in thrombus stabilisation at high shear JF - International Journal of Molecular Sciences N2 - Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s\(^{−1}\)). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A\(_2\) (TxA\(_2\)), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen. KW - disaggregation KW - platelet KW - Syk KW - thrombus KW - tyrosine kinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284243 SN - 1422-0067 VL - 23 IS - 1 ER - TY - JOUR A1 - Neagoe, Raluca A. I. A1 - Gardiner, Elizabeth E. A1 - Stegner, David A1 - Nieswandt, Bernhard A1 - Watson, Steve P. A1 - Poulter, Natalie S. T1 - Rac inhibition causes impaired GPVI signalling in human platelets through GPVI shedding and reduction in PLCγ2 phosphorylation JF - International Journal of Molecular Sciences N2 - Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species. KW - platelets KW - Rac1 KW - glycoprotein VI KW - EHT1864 KW - GPVI shedding KW - phospholipase C gamma 2 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284350 SN - 1422-0067 VL - 23 IS - 7 ER -