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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1651.)
© 2000 American Heart Association, Inc.

Thrombosis

Inhibition of Platelet Integrin _IIbß₃ by Peptides That Interfere With Protein Kinases and the ß₃ Tail

Ingeborg Hers; José Donath; Pieter E. M. H. Litjens; Gijsbert van Willigen; Jan-Willem N. Akkerman

From the Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, Utrecht, the Netherlands. Dr Hers is now at the Department of Biochemistry, School of Medical Sciences, Bristol, UK.

Correspondence to Prof Dr J.W.N. Akkerman, Department of Haematology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail j.w.n.akkerman{at}laboratory.azu.nl

Abstract—-Thrombin stimulation of human platelets initiates inside-out signaling to integrin _IIbß₃ (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of _IIbß₃ 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 ß₃ subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and _IIbß₃ ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited -thrombin–induced ligand binding to _IIbß₃, but a reversed peptide with YF substitutions (KFSGFIF) had no effect. Ligand binding to _IIbß₃ 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 ß₂-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 ß₃ subunit. The LLITIHDR peptide, matching the membrane-proximal domain of ß₃ (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 ß₃, reduced -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 _IIbß₃ and identify target domains in ß₃ that contribute to the regulation of this integrin.

Key Words: integrin _IIbß₃ • glycoprotein IIb/IIIa • protein kinase C • protein tyrosine kinase • platelets

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