The platelet cytoskeleton regulates the affinity of the integrin α(IIb)β3 for fibrinogen

Abstract

Agonist-generated inside-out signals enable the platelet integrin α(IIb)β3 to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to α(IIb)β3. By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP- induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating α(IIb)β3 function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetra-acetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains α(IIb)β3 in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on α(IIb)β3, allowing it to assume the high affinity conformation required for soluble ligand binding.