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Peptide LSARLAF induces integrin ?3 dependent outside-in signaling in platelets.

ABSTRACT: Peptide LSARLAF (LSA) can bind and activate integrin ?IIb?3 in the absence of 'inside-out' signal. The active ?IIb?3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than ?IIb?3, and determine the roles of Src, PLC?2, FcR?-chain, and SLP-76 in LSA-induced platelet activation.Ligand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing ?IIb?3 or ?V?3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed ?3, Src, FcR?-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA.Ligand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to ?IIb?3 or ?V?3. LSA also enhanced CHO cells with ?IIb?3 or ?V?3 expression spreading on immobilized fibrinogen. ?3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLC?2 and Syk was also ?3 dependent. Src, FcR?-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA.LSA-induced platelet activation is ?3 dependent, and signaling molecules Src, FcR?-chain, SLP-76 and LAT play crucial roles in LSA-induced ?3 mediated signaling.

SUBMITTER: Niu H

PROVIDER: S-EPMC3393848 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Peptide LSARLAF induces integrin β3 dependent outside-in signaling in platelets.

Niu Haixia H Xu Zhenlu Z Li Ding D Zhang Lin L Wang Kemin K Taylor Donald B DB Liu Junling J Gartner T Kent TK

Thrombosis research 20120405 2

<h4>Introduction</h4>Peptide LSARLAF (LSA) can bind and activate integrin αIIbβ3 in the absence of 'inside-out' signal. The active αIIbβ3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbβ3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation.<h4>Method</h4>Ligand-receptor binding assay was p ...[more]

PMID: 22482832

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Project description:BackgroundBidirectional integrin ?IIb?3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function.ResultsAn antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin ?IIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin ?IIb?3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling.ConclusionsThese findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin ?IIb?3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

Dataset Information

Peptide LSARLAF induces integrin ?3 dependent outside-in signaling in platelets.

Publications

Peptide LSARLAF induces integrin β3 dependent outside-in signaling in platelets.

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