A potential role for α-actinin in inside-out αIIbβ3 signaling

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for alpha-actinin in inside-out integrin activation. In resting human platelets, alpha-actinin was associated with alpha IIb beta 3, whereas inside-out signaling (alpha IIb beta 3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated alpha-actinin from alpha IIb beta 3. We evaluated the time-dependent changes of the alpha IIb beta 3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient alpha IIb beta 3 activation, whereas PAR4-activating peptide induced long-lasting alpha IIb beta 3 activation. When alpha IIb beta 3 activation signaling dwindled, alpha-actinin became rephosphorylated and reassociated with alpha IIb beta 3. Compared with control platelets, the dissociation of alpha-actinin from alpha IIb beta 3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained alpha IIb beta 3 activation was markedly impaired. Overexpression of wild-type alpha-actinin, but not the mutant Y12F alpha-actinin, increased its binding to alpha IIb beta 3 and inhibited PAR1-induced initial alpha IIb beta 3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of alpha-actinin augmented PAR-induced alpha IIb beta 3 activation in CMK. These observations suggest that alpha-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating alpha IIb beta 3 activation by inside-out signaling. (Blood. 2011;117(1):250-258)