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Cyclin Y is expressed in platelets and modulates integrin outside-in signaling

ABSTRACT: Diabetes is associated with platelet hyperactivation and enhanced risk of thrombosis development. We have previously shown that calpain inhibition stabilizes the platelet proteome in diabetic patients. However, a global proteome analysis comparing platelets from healthy donors and from diabetic patients is missing. Therefore, we performed comparative proteomic studies to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analysis identified cyclin Y in platelets and levels were found to be reduced in platelets from diabetic patients. In the latter, cyclin Y was degraded by the protease calpain. In order to assess the function of cylcin Y in platelets, mice globally lacking cyclin Y were generated using CRISPR/Cas9 technology by inserting a premature Stop codon in exon 1 of Ccny gene. Cyclin Y deletion resulted in a reduction in the circulating platelet number but the agonist-induced platelet aggregation was comparable in platelets from wild-type and Ccny-/- mice, as was agonist-induced α and dense granule secretion. However, cyclin Y-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in “outside in” integrin signalling. The phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of the β3 integrin in Ccny-/- platelets. Stimulation of human platelets or platelets from wildtype mice with thrombin led to the membrane translocation of cyclin Y where it associates with the tyrosine kinase Fyn suggesting that cyclin Y might play a role as an adaptor protein for the tyrosine kinase to phosphorylate the cytoplasmic tail of the β3 integrin. Thus, cyclin Y is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Diabetes Mellitus

SUBMITTER: Fiorella Andrea Solari

LAB HEAD: Albert Sickmann

PROVIDER: PXD021271 | Pride | 2021-05-31

REPOSITORIES: Pride

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Project description:Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin Alejandro Zimman, Bjoern Titz, Evangelia Komisopoulou, Sudipta Biswas, Thomas G. Graeber, and Eugene A. Podrez ABSTRACT Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have now employed mass spectrometry-based tyrosine, serine, and threonine global phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide a new resource to study platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCy2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple novel insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. Methods Human platelets isolated by gel filtration (2.7 x 108 / mL) were incubated in Tyrode's buffer with 50 uM KODA-PC or PLPC (as control) for 30 min at 37 degrees C. Platelets were then centrifuged at 3,700 g (10 min, 35 degrees C) and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonists were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, and label-free quantitation as described in the Supplemental Materials and Methods of the publication. The same protocol was applied when studying platelet activation by thrombin, where gel-filtered platelets were incubated in Tyrode's buffer with 0.05 U/mL thrombin or in buffer alone (Resting) for 3 min at 37 degrees C.

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Cyclin Y is expressed in platelets and modulates integrin outside-in signaling

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