Integrin αIIb-mediated PI3K/Akt activation in platelets

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 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.

Figure 1. SFK(s) and PI3K propagate αIIb-initiated signaling that elicits platelet activation, TxA2 production and granule secretion.

Normal human platelets and β3Δ724 platelets were stimulated by the LIBS-specific monoclonal antibody D3 (30 µg/ml) in the presence of Fg (250 µg/ml) with or without 10 µg/ml of human αIIbβ3 specific monoclonal antibody 7E3, DMSO, 10 U/ml of apyrase, 75 µM of indomethacin, 10 µM of Src family kinase inhibitor PP2, 100 nM of PI3K inhibitor wortmannin, or 10 µM of MAPK inhibitor U0126. (A) The agglutination and/or aggregation of normal human platelets (NP) and β3Δ724 human platelets, respectively induced by D3 plus Fg with or without inhibitors treatment. The absence of shape change indicates agglutination, rather than aggregation. (B) The TxB2 production and granule secretion of normal human platelets and β3Δ724 platelets induced by D3 plus Fg with or without inhibitor treatment. Each bar represents the mean of quadruplicate determinations. The error bars correspond to the standard deviations of the data.

Figure 2. Akt phosphorylation in β3Δ724 platelets elicited by D3 plus Fg is blocked by peptide p-RKEFAKFEEER.

Normal and β3Δ724 platelets, respectively were treated with the LIBS-specific monoclonal antibody D3 (30 µg/ml) in the presence of Fg (250 µg/ml), with or without 10 µM of PP2, 100 nM of wortmannin, 10 µM of a p-control peptide, 10 µM of p-RKEFAKFEEER peptide, 75 µM of indomethacin or 10 U/ml of apyrase. Immunoblots of the platelet lysates were treated with anti-phospho-Akt (Ser473) and anti-actin antibodies. (A) The Akt Ser473 phosphorylation by normal human platelets and β3Δ724 platelets, respectively induced by D3 plus Fg with or without inhibitors treatment. (B) The peptide p-RKEFAKFEEER inhibits the aggregation of β3Δ724 platelets induced by D3 plus Fg. (C) The peptide p-RKEFAKFEEER inhibits Akt Ser473 phosphorylation in β3Δ724 platelets aggregating in response to D3 plus Fg. The experiments were repeated for 3 times.

Figure 3. Akt phosphorylation in response to PAR4 stimulation in mouse platelets is secretion- and αIIbβ3-dependent.

(A) Aggregation of normal and Tp deficient mouse washed platelets induced by 160 µM of the PAR4 agonist peptide AYPGKF. Aggregation of Tp deficient mouse platelets induced by 160 µM of the PAR4 agonist peptide AYPGKF with and without 10 U/ml of apyrase, ± 250 µg/ml of Fg, ± 10 µg/ml of mAb 1B5. (B) Akt Ser473 phosphorylation of normal and Tp deficient mouse platelets treated with and without 10 U/ml of apyrase, ± 250 µg/ml of Fg, ± 10 µg/ml of mAb 1B5. (C) Aggregation of normal and β3 deficient mouse washed platelets induced by 160 µM of peptide AYPGKF. (D) Akt Ser473 phosphorylation of normal and β3 deficient mouse washed platelets induced by 160 µM of peptide AYPGKF. The experiments were repeated for three times.

Figure 4. Akt phosphorylation in response to PAR4 stimulation in mouse platelets is SFK and PI3K-dependent, and inhibited by p-RKEFAKFEEER.

(A) Aggregation of normal mouse washed platelets induced by 160 µM of PAR4 agonist peptide with or without DMSO, 10 µM PP2, or 100 nM of wortmannin. (B) Akt Ser473 phosphorylation of normal mouse washed platelets induced by 160 µM of PAR4 agonist peptide with or without DMSO, 10 µM of PP2, or 100 nM of wortmannin. (C) Aggregation of normal mouse washed platelets induced by 160 µM of PAR4 agonist peptide with or without 10 µM of p-RKEFAKFEEER or the scrambled p-control peptide. (D) Akt Ser473 phosphorylation of normal mouse washed platelets induced by 160 µM of PAR4 agonist peptide with or without 10 µM of p-RKEFAKFEEER or the scrambled p-control peptide. The experiments were repeated for three times.

Figure 5. Outside-in signaling mediated by αIIb in CHO cells expressing αIIbβ3.

(A) Spreading of CHO cells expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724, respectively on immobilized Fg for 90minutes. (B) Quantification of area (pixel number) in 4 random fields (mean±SE) at all 6 time points. At the 90 minute time point, the size of CHO cells, CHO cells expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724, was 24303.43 ± 4851.921 pixels, 97055.33 ± 21284.32 pixels, 133012.3 ± 71539.87 pixels, 24124 ± 11043.93 pixels, respectively. Statistical analysis performed using Student t test. (C) Akt Ser473 phosphorylation by CHO cells in suspension expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724 induced by 5 µg/ml D3 and 100 µg/ml Fg. These experiments were repeated for three times.