ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

• Published in: Blood 2014-05, Vol.123 (20), p.3156-3165
• Main Authors: Kasirer-Friede, Ana, Kang, Jian, Kahner, Bryan, Ye, Feng, Ginsberg, Mark H., Shattil, Sanford J.
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Animals; Blood Platelets - cytology; Blood Platelets - metabolism; CHO Cells; Cricetinae; Cricetulus; Cytoskeletal Proteins - metabolism; Fibrinogen - metabolism; Humans; Membrane Proteins - metabolism; Mice; Neoplasm Proteins - metabolism; Platelet Glycoprotein GPIIb-IIIa Complex - metabolism; Platelets and Thrombopoiesis; Protein Binding; Protein Interaction Mapping; Talin - metabolism
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2013-08-520627

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. •ADAP interacts with talin and kindlin-3 in platelets.•ADAP is a hematopoietic component of the molecular machinery that promotes activation of and stable fibrinogen binding to αIIbβ3.