A mechanism of platelet integrin aIIbb3 outside-in signaling through a novel integrin aIIb subunit-filamin-actin linkage

Abstract

The communication of talin-activated integrin aIIbb3 with cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin cross-linker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive aIIbb3, is displaced from aIIbb3 by talin to promote the integrin activation (inside-out signaling) and how filamin further functions remains unresolved. Here we show that while associating with the inactive aIIbb3, filamin also associates with the talin-bound active aIIbb3 to mediate platelet spreading. FRET-based analysis reveals that while associating with both aIIb and b3 cytoplasmic tails (CTs) to maintain the inactive aIIbb3, filamin is spatiotemporally re-arranged to associate with aIIb CT alone on activated aIIbb3. Consistently, confocal cell imaging indicates that integrin a CT-linked filamin gradually delocalizes from b CT-linked focal adhesion marker - vinculin likely due to the separation of integrin a/b CTs occurring during integrin activation. High-resolution crystal and NMR structure determinations unravel that the activated integrin aIIb CT binds to filamin via a striking a-helix→b-strand transition with strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin aIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of aIIbb3, phosphorylation of FAK/Src kinases, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.