The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen

Agonist-generated inside-out signals enable the platelet integrin alpha(IIb )beta(3), to bind soluble ligands such as fibrinogen, We found that inhibit ing actin polymerization in unstimulated platelets with cytochalasin D or l atrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3) By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding, Cytochalasin D- and latrunculin A-induced fibrinogen wa s inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to se e cytochalasin D and latrunculin A effects. Gelsolin, which severs actin fi laments, is activated by calcium, whereas the actin disassembly factor cofi lin is inhibited by serine phosphorylation. Consistent with a role for thes e factors in regulating alpha(IIb)beta(3), function, cytochalasin D- and la trunculin A-induced fibrinogen binding was inhibited by the intracellular c alcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N' ,N' -tetra-acetic aci d acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphat ase inhibitors okadaic acid and calyculin A. Our results suggest that the a ctin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in plat elet cytosolic calcium initiate actin filament turnover. Increased actin fi lament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3) allowing it to assume the high affinity conformation required for soluble ligand binding.