An αIIb-Mediated Outside-in Signaling Pathway That Causes Storage Granule Secretion and Thromboxane A2 Production.

Abstract 4011

Poster Board III-947

Bidirectional signaling is an essential feature of integrin αIIbβ3 function. Outside-in signal transduction mediated by αIIbβ3 is required for normal hemostasis, clot retraction, platelet aggregation and spreading. In our previous work (Blood. 2005; 105: 4345-52), we showed that αIIb-mediated outside-in signaling is enhanced in human platelets (designated VTGβ3Δ724) lacking the terminal 39 residues of the β3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A2 (TxA2) production and granule secretion, and required ligand cross-linking of αIIbβ3 and platelet aggregation. This αIIbβ3-mediated outside-in signaling was specifically inhibited by a palmitoylated, but not a scrambled or non-palmitoylated version of a peptide corresponding to β3 cytoplasmic domain (CD) residues R724-R734. Inhibition presumably occurred because the corresponding region within the β3 CD (RKEFAKFEEER) can bind to the CD of αIIb and negatively regulate αIIb-mediated outside-in signaling. In this study, we elucidate part of the signaling pathway(s) underlying these αIIb-elicited events. The data presented here demonstrate that D3+Fg-induced TxA2 production and granule secretion by VTGβ3Δ724 platelets are Src family kinase(s) (SFK), PI3K, PKC and intracellular Ca²⁺ flux, but not ERK dependent. Further biochemical analysis demonstrated that D3+Fg treatment of control platelets ± 10U/ml apyrase and 75mM indomethacin, induced phosphorylation of ERK and slight phosphorylation of p38, but not Akt. ERK activation was not dependent on a SFK(S) or PI3K since 10mM SFK of the inhibitor PP2 and 100nM PI3K inhibitor wortmannin did not block phosphorylation of ERK. However, Akt, and p38 were intensely phosphorylated in VTGβ3Δ724 platelets treated with D3+Fg ± apyrase and indomethacin, meaning that activation of Akt, and p38 appears to be directly coupled to αIIb-mediated signaling, and not coupled to TxA2 and ADP-driven signaling. Phosphorylation of p38 and Akt, but not ERK, was totally blocked by 10mM PP2. Wortmannin (100nM) inhibited phosphorylation of Akt, but not P38 and ERK. Therefore, SFK-PI3K/Akt-PKC play an important role in αIIb-mediated signaling. Akt phosphorylation in VTGβ3Δ724 platelets stimulated by D3+Fg was also specifically inhibited by a palmitoylated β3R724-R734 peptide but not by a palmitoylated scrambled control peptide, which further proves that SFK-PI3K signaling pathway is mediated specifically by αIIb. The role of αIIb-mediated SFK-dependent PI3K/Akt signaling was investigated in normal washed platelets by stimulating those platelets with a level of the PAR4 peptide that caused secretion-dependent aggregation. The aggregation initiated αIIbβ3-mediated outside-in signaling in both mouse and human washed platelets. The PAR4 peptide-induced Akt phosphorylation, TxA2 production and granule secretion were αIIbβ3, aggregation, and PI3K-dependent, in the absence of ADP and TxA2 initiated signaling. The palmitoylated peptide β3R724-R734 (10mM), but not a control peptide blocked ATP secretion, TxA2 production and Akt phosphorylation in normal platelets stimulated with the PAR4 peptide. Therefore, the αIIb-initiated SFK(s), PI3K/Akt, PKC signaling pathway is responsible for TxA2 production and granule secretion, in both VTGβ3Δ724 and normal platelets. SFK-dependent p38 activation is also triggered by αIIb-mediated signaling, but it is not Akt-dependent and the function of p38 in αIIb-mediated signaling events is unknown. Finally, ERK activation is αIIbβ3-dependent, but not αIIbβ3-mediated; therefore ERK activation probably is an effect of non-αIIb-mediated contact-dependent signaling. The work presented here provides a new understanding of αIIbb3- outside-in signaling.