Inhibition of Platelet Integrin α IIb β 3 by Peptides That Interfere With Protein Kinases and the β 3 Tail

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Abstract
—α-Thrombin stimulation of human platelets initiates inside-out signaling to integrin αIIbβ3 (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of αIIbβ3 via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the β3 subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and αIIbβ3 ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited α-thrombin–induced ligand binding to αIIbβ3, but a reversed peptide with Y→F substitutions (KFSGFIF) had no effect. Ligand binding to αIIbβ3 was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and β2-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the β3 subunit. The LLITIHDR peptide, matching the membrane-proximal domain of β3 (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of β3, reduced α-thrombin–induced ligand binding by 60±4%, 97±1%, and 97±2% (n=3) at 500 μmol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin αIIbβ3 and identify target domains in β3 that contribute to the regulation of this integrin.