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 <40 nm. In human platelets spread on fibrinogen, a 16-fold increase in the number of proximity ligation signals per platelet was detected when antibodies to ADAP and talin were employed in the assay compared to when control antibodies were used (P < 0.01). In addition, ADAP partially co-localized with talin and kindlin-3 in platelets as determined by fluorescence deconvolution microscopy. In order to exclude a role for SKAP2 in these ADAP interactions, we turned to CHO cells that stably express αIIbβ3, a model cell system that normally lacks SKAP-2 and ADAP but can be used to reconstitute inside-out αIIbβ3 signaling by expression of relevant proteins. Following transient transfection of ADAP into αIIbβ3-CHO cells, ADAP was found to specifically co-immunoprecipitate with endogenous talin and ectopically-expressed kindlin-3. When these cells were further engineered to conditionally express the platelet PAR1 thrombin receptor and increased levels of talin, the presence of ADAP significantly enhanced PAR-1-mediated αIIbβ3 activation, as detected with antibody PAC-1 (P < 0.05). Although overexpression of kindlin-3 in these cells failed to enhance PAR1-mediated, talin-dependent PAC-1 binding, αIIbβ3 activation was moderately enhanced when kindlin-3 was co-expressed with ADAP (P < 0.05). Altogether, these studies establish that ADAP functions in close proximity to talin and kindlin-3 to promote agonist-dependent αIIbβ3 activation in platelets in a manner independent of SKAP2. Therefore, the mechanism whereby ADAP modulates integrin activation in platelets is subtly, if not fundamentally, different than in lymphocytes. This raises the potential of selectively targeting ADAP's integrin-activating function in platelets or lymphocytes in thrombotic or immune disorders, respectively, while leaving integrin function in the other cell type unaffected.