The Mechanism of Kindlin-Mediated Activation of Integrin αIIbβ3

Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis [1-5]. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering [6] and depends on integrin-talin interactions [5]. Loss of kindlins results in reduced activation of integrins [7-13]. Kindlins might promote talin binding to integrins through a cooperative mechanism [5, 14-16]; however, kindlins do not increase talin association with integrins [17]. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric alpha IIb beta 3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular alpha IIb beta 3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces alpha IIb beta 3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied alpha IIb beta 3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin alpha IIb beta 3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.