Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering

Abstract

␣IIb␤3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail. Exogenous peptides designed to interact with the ␣IIb transmembrane (TM) domain activate single ␣IIb␤3 molecules in platelets by binding to the ␣IIb TM domain and causing separation of the ␣IIb␤3 TM domain heterodimer. Here we asked whether directly activating single ␣IIb␤3 molecules in platelets using the designed peptide anti-␣IIb TM also initiates ␣IIb␤3-mediated outside-in signaling by causing activation of ␤3-associated c-Src. Anti-␣IIb TM caused activation of ␤3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to ␣IIb␤3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual ␣IIb␤3 molecules can initiate ␣IIb␤3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-␣IIb TM resulted in the redistribution of ␣IIb␤3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered ␣IIb␤3. Thus, these studies indicate that not only is resting ␣IIb␤3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.␣IIb␤3 activation in platelets is followed by ␣IIb␤3 clustering (1) and ␣IIb␤3-mediated "outside-in" signal transduction (2) that is initiated by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail (CT) 3 (3-5). Activated c-Src then initiates an intracellular signaling cascade culminating in the reorganization of the platelet cytoskeleton, platelet spreading, and fibrin clot retraction, events important for efficient hemostasis in the hemodynamic environment of flowing blood (6).Recently, we reported studies of the interaction of c-Src with the ␤3 CT (5). We detected little to no interaction in unstimulated platelets, but following platelet stimulation with thrombin, c-Src associated with ␤3 in a time-dependent manner and underwent transient activation. We also found that the ␤3 CT binds to the c-Src SH3 domain at a site that is occupied by the linker connecting the c-Src SH2 and kinase domains in inactive c-Src. Following platelet activation, c-Src is "unlatched," allowing ␤3 CT binding to the SH3 domain when the linker binding site is vacated. However, the full catalytic activity of c-Src requires phosphorylation of Tyr 419 (numbered according to the UniProtKB/Swiss-Prot entry P12931 for human c-Src), located in a loop between the two lobes of the c-Src kinase domain (7). In platelets, this occurs by trans-autophosphorylation when ␣IIb␤3 clustering brings ␤3 CT-bound c-Src into proximity (3). Thus, ␣IIb␤3 clustering is an essential step in the outside-in signaling that follows ␣IIb␤3 activation.Transmembrane (TM) helix-helix interactions play an important role in maintaining ␣I...

RGS10 Negatively Regulates Platelet Activation and Thrombogenesis

“…Platelet aggregation occurs in part through a conformational change in the integrin αIIbβ3 on the platelet membrane, leading to integrin activation [18–20]. We measured cell surface expression of activated αIIbβ3 by flow cytometry.…”

Increased Cardiovascular Risk Associated with Chemical Sensitivity to Perfluoro–Octanoic Acid: Role of Impaired Platelet Aggregation

“…The altered permeability to ions has been claimed as one of the major consequence of increased membrane fluidity in different cell models [36,37]. In particular, increases in intracellular calcium concentration are early upstream events in platelets' aggregation, resulting in fibrinogen receptor phosphorylation and, in turn, in full platelet activation and aggregate stabilization [38][39][40][41]. Accordingly, the effect of PFOA exposure on calcium trafficking in platelets activated with thrombin receptor activator peptide 6 (TRAP-6) was evaluated by the use of calcium fluorophore Fluo4M, a calcium indicator that increases the fluorescence emission upon calcium binding.…”

Multiple protein disulfide isomerases support thrombosis

“…Alternatively, a subpopulation of αIIbβ3 that controls platelet aggregation could be targeted. For example, a subpopulation of αIIbβ3 that interacts with the platelet cytoskeleton is required for activation of αIIbβ3 [40], and activation of individual αIIbβ3 molecules rapidly initiates αIIbβ3 clustering of intracellular signal-generating complexes [41]. Thus, a limited number of disulfide isomerase-catalyzed reactions targeting a subpopulation of αIIbβ3 molecules could amplify platelet responses required for aggregation.…”