Molecular basis of platelet activation by an αIIbβ3-CHAMPS peptide

Abstract

Background: A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind αIIb- and αv-integrin subunits, which induce selective activation of integrins αIIbβ3 and αvβ3, respectively [1]. Objectives: In the present study, we have investigated the ability of an αIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. Methods: The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. Results: IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR γ-chain, but only weak phosphorylation of PLCγ2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A2 (TxA2) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA2. Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin αIIbβ3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. Conclusions: The present study demonstrates that the αIIb-CHAMPS peptide induces platelet activation through integrin αIIbβ3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR γ-chain and Syk. The use of the αIIb-CHAMPS peptide to study integrin αIIbβ3 function is compromised by non-integrin-mediated effects. © 2009 International Society on Thrombosis and Haemostasis.