TY - JOUR
T1 - In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVIdriven platelet function
AU - Durrant, Tom
AU - Hutchinson, Lawrence
AU - Heesom, Kate
AU - Anderson, Karen
AU - Stephens, Len
AU - Hawkins, Phillip
AU - Marshall, Aaron
AU - Moore, Samantha
AU - Hers, Ingeborg
N1 - 27-Apr-17
PY - 2017/6/13
Y1 - 2017/6/13
N2 - The Class I Phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these Class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of Class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns(3,4,5)P3 generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high resolution proteomic MS to identify the targets of PtdIns(3,4,5)P3 in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P3 interactome, including kinases, signaling adaptors and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αIIbβ3 integrin activation downstream of the collagen receptor glycoprotein VI (GPVI). Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P3 signalosome of human platelets, and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how Class I PI3K shapes platelet function.
AB - The Class I Phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these Class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of Class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns(3,4,5)P3 generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high resolution proteomic MS to identify the targets of PtdIns(3,4,5)P3 in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P3 interactome, including kinases, signaling adaptors and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αIIbβ3 integrin activation downstream of the collagen receptor glycoprotein VI (GPVI). Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P3 signalosome of human platelets, and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how Class I PI3K shapes platelet function.
U2 - 10.1182/bloodadvances.2017005173
DO - 10.1182/bloodadvances.2017005173
M3 - Article (Academic Journal)
C2 - 29242851
SN - 2473-9529
VL - 1
SP - 918
EP - 932
JO - Blood Advances
JF - Blood Advances
IS - 14
ER -