Abstract
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 R724KEFAKFEEER734. 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 R724KEFAKFEEER734, 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 αIIbβ3E724AERKFERKFE734, 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.
Highlights
Integrins are a and b heterodimeric receptors required for numerous essential biological processes [1]
Src Family Kinase (SFK) and PI3K are Key Factors Involved in aIIb-mediated Platelet Activation
We showed that the treatment of human b3D724 platelets in presence of fibrinogen with LIBS-specific mAb D3 and PT25-2, respectively caused aggregation, thromboxane A2 (TxA2) production and granule secretion [23]
Summary
Integrins are a and b heterodimeric receptors required for numerous essential biological processes [1]. Transformation from the resting state to the active or high-affinity state typically results from integrin-mediated inside-out signaling initiated indirectly by activation of other receptors [1]. This transformation induced by inside-out signaling is controlled by the interaction between the membrane proximal, highly conserved regions of the cytoplasmic domains of the a and b subunits [3,4,5,6]. Agonist-induced physiologic disruption of this interaction appears to be caused by the binding of talin [9], Kindlin [10] or other proteins [1,11] to the cytoplasmic domain of b3
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