RESUMO
Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin ß 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 ß3 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 αIIbß3 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 ß3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbß3 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-ß3 integrin binding affinity results in decelerated αIIbß3 integrin activation and protection from arterial thrombosis without pathological bleeding.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/genética , Talina/metabolismo , Trombose/metabolismo , Animais , Cálcio/metabolismo , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica , Hemostasia , Camundongos , Camundongos Transgênicos , Mutação , Ligação Proteica , Resistência ao Cisalhamento , Estresse Mecânico , Ressonância de Plasmônio de SuperfícieRESUMO
Abstract Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases.