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A talin mutant that impairs talin-integrin binding in platelets decelerates ?IIb?3 activation without pathological bleeding.

ABSTRACT: Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin ? cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the ?3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of ?IIb?3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to ?3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of ?IIb?3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-?3 integrin binding affinity results in decelerated ?IIb?3 integrin activation and protection from arterial thrombosis without pathological bleeding.

SUBMITTER: Stefanini L

PROVIDER: S-EPMC3999757 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbβ3 activation without pathological bleeding.

Stefanini Lucia L Ye Feng F Snider Adam K AK Sarabakhsh Kasra K Piatt Raymond R Paul David S DS Bergmeier Wolfgang W Petrich Brian G BG

Blood 20140228 17

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin β cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing t ...[more]

PMID: 24585775

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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

A talin mutant that impairs talin-integrin binding in platelets decelerates ?IIb?3 activation without pathological bleeding.

Publications

A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbβ3 activation without pathological bleeding.

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