Paxillin binding to the PH domain of kindlin‐3 in platelets is required to support integrin αIIbβ3 outside‐in signaling

Abstract

BackgroundKindlin‐3 is essential for supporting the bidirectional signaling of integrin αIIbβ3 in platelets by bridging the crosstalk between integrin αIIbβ3 and the cytoplasmic signaling adaptors. ObjectiveIn this study, we identified a previously unrecognized paxillin binding site in the pleckstrin homology (PH) domain of kindlin‐3 and verified its functional significance. MethodsStructure‐based approaches were employed to identify the paxillin binding site in the PH domain of kindlin‐3. In addition, the bidirectional signaling of integrin αIIbβ3 were evaluated in both human and mouse platelets. ResultsIn brief, we found that a β1‐β2 loop in the PH domain of kindlin‐3, an important part of the canonical membrane phospholipid binding pocket, was also involved in mediating paxillin interaction. Interestingly, the binding sites of paxillin and membrane phospholipids in the PH domain of kindlin‐3 were mutually exclusive. Specific disruption of paxillin binding to the PH domain by point mutations inhibited platelet spreading on immobilized fibrinogen while having no inhibition on soluble fibrinogen binding to stimulated platelets. In addition, a membrane‐permeable peptide derived from the β1‐β2 loop in the PH domain of kindlin‐3 was capable of inhibiting platelet spreading and clot retraction, but it had no effect on soluble fibrinogen binding to platelets and platelet aggregation. Treatment with this peptide significantly reduced thrombus formation in mice. ConclusionTaken together, these findings suggest that interaction between paxillin and the PH domain of kindlin‐3 plays an important role in supporting integrin αIIbβ3 outside‐in signaling in platelets, thus providing a novel antithrombotic target.