ADAPtation of Platelet Integrin αIIbβ3 to Inside-Out Activation Signals

Abstract 188 Platelets respond to excitatory agonists by binding adhesive ligands, such as fibrinogen, and by aggregating during hemostasis and thrombosis. These processes require the activation of αIIbβ3 from a low- to a high-affinity state. Activation of αIIbβ3 is mediated by inside-out signaling and ultimately by interactions of the molecular adaptors, talin and kindlin-3, with the β3 cytoplasmic domain. While certain key elements in the circuitry connecting agonist receptors and αIIbβ3 have been identified, some critical aspects of inside-out signaling remain undefined. The role of the hematopoietic adapter protein, ADAP, is a case in point. Platelets deficient in ADAP exhibit decreased αIIbβ3 activation in response to several agonists and reduced thrombus formation in vivo. How ADAP interfaces with the signaling machinery of platelets to regulate αIIbβ3 is unknown. Promotion of integrin adhesive function in lymphocytes by ADAP requires SKAP1, an ADAP-binding partner that is not expressed in platelets. While platelets express the SKAP1 homologue, SKAP2, the latter is dispensable for αIIbβ3 activation since SKAP2-null platelets are normal. We hypothesized that ADAP modulates αIIbβ3 function through interactions with talin and/or kindlin-3. Immunoprecipitation of ADAP from human or mouse platelets showed that ADAP was associated with a pool of talin and kindlin-3. To evaluate the proximity of this association in intact platelets, a proximity ligation assay was employed whereby antibody and DNA amplification techniques are combined to identify protein-protein interactions at the single cell level that occur at distances Disclosures: No relevant conflicts of interest to declare.