Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering

Karen P Fong,Hua Zhu,Lisa M Span,David T Moore,Kyungchul Yoon,Ryo Tamura,Hang Yin,William F Degrado,Joel S Bennett

doi:10.1074/jbc.m116.716613

Abstract

αIIbβ3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the β3 cytosolic tail. Exogenous peptides designed to interact with the αIIb transmembrane (TM) domain activate single αIIbβ3 molecules in platelets by binding to the αIIb TM domain and causing separation of the αIIbβ3 TM domain heterodimer. Here we asked whether directly activating single αIIbβ3 molecules in platelets using the designed peptide anti-αIIb TM also initiates αIIbβ3-mediated outside-in signaling by causing activation of β3-associated c-Src. Anti-αIIb TM caused activation of β3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to αIIbβ3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual αIIbβ3 molecules can initiate αIIbβ3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-αIIb TM resulted in the redistribution of αIIbβ3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered αIIbβ3. Thus, these studies indicate that not only is resting αIIbβ3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.

Highlights

␣IIb␤3 activation in platelets is followed by ␣IIb␤3 clustering [1] and ␣IIb␤3-mediated “outside-in” signal transduction [2] that is initiated by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail (CT)3 [3,4,5]
As noted previously by Liu et al [17], when platelets were stimulated by ␥-thrombin, c-Src activation appears to potentiate the effects of weaker platelet stimuli such as anti-␣IIb TM and 0.1 units/ml thrombin, but increasing the strength of the platelet stimulus, in this case increasing the thrombin concentration to 5 units/ml, largely bypasses the c-Src requirement
Activation of the c-Src associated with the ␤3 CT in platelets triggers a protein phosphorylation cascade that results in outside-in platelet signaling [4]

Summary

Introduction

␣IIb␤3 activation in platelets is followed by ␣IIb␤3 clustering [1] and ␣IIb␤3-mediated “outside-in” signal transduction [2] that is initiated by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail (CT)3 [3,4,5]. To test this conclusion and to address whether anti-␣IIb TM-induced ␣IIb␤3 activation requires c-Src kinase activity, we measured anti-␣IIb TM-induced fibrinogen binding to platelet ␣IIb␤3 in the absence or presence of PP2. These results indicate that anti-␣IIb TM initiates platelet activation by binding and altering the conformation of ␣IIb␤3 and that this effect on ␣IIb␤3 is independent of platelet secretion and c-Src activity.

Results

Conclusion