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 inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin 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 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 alpha(IIb)beta(3) 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'-tetraacetic 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 alpha(IIb)beta(3) 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 alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.
Bennett
J S
JS
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. bennetts@mail.med.upenn.edu
Zigmond
S
S
Vilaire
G
G
Cunningham
M E
ME
Bednar
B
B
D000199:Actins / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*
D000199:Actins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen