Integrin αIIb-mediated PI3K/Akt activation in platelets.
Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E(724)AERKFERKFE(734), but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.
Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Haixia
H
Niu
Xue
X
Chen
Ralph A
RA
Gruppo
Ding
D
Li
Yanhua
Y
Wang
Lin
L
Zhang
Kemin
K
Wang
Weiran
W
Chai
Yueping
Y
Sun
Zhongren
Z
Ding
T Kent
TK
Gartner
Junling
J
Liu
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D016466:CHO Cells; D002465:Cell Movement / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005340:Fibrinogen / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013928:Thromboxane A2 / Q000096:biosynthesis; D019061:src-Family Kinases / Q000378:metabolism
D010455:Peptides; D040201:Platelet Membrane Glycoprotein IIb; D018179:Receptors, Thrombin; D013928:Thromboxane A2; D005340:Fibrinogen; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; C112908:protease-activated receptor 4