Exposure of ligand-binding sites on platelet integrin αIIB/β3 by phosphorylation of the β3 subunit

Abstract

The exposure of ligand-binding sites for adhesive proteins on platelet integrin αIIB/β3 (glycoprotein IIB/IIIA) by platelet-activating factor (PAF) is transient, whereas sites exposed by α-thrombin remain accessible. The same difference is seen in the phosphorylation of the β3 subunit. Inhibition of protein kinases (1 μM staurosporine) and protein kinase C (10 μM bisindolylmaleimide) closes binding sites exposed by both agonists and induces dephosphorylation of β3. Inhibition of Tyr-kinases (20 μM Herbimycin A) has only a slight effect. Inhibition of Ser/Thr-phosphatases (1 μM okadaic acid, 30 s preincubation) changes the transient exposure and β3 phosphorylation by PAF into the ‘permanent’ patterns induced by α-thrombin. Inhibition of Tyr-phosphatases (100 μM vanadate) has little effect. Preincubation with okadaic acid makes exposed binding sites and phosphorylated β3 insensitive to staurosporine, resulting in exposed αIIB/β3 independent of concurrent phosphorylation/dephosphorylation. The stoichiometry of β3 phosphorylation by α-thrombin is 0.80±0.10. Thus, one of the mechanisms that regulates exposure and closure of ligand-binding sites on the αIIB/β3 is phosphorylation/dephosphorylation of a Ser/Thr-residue in the β3 subunit.