Molecular basis of platelet activation by an αIIbβ3-CHAMPS peptide

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 alpha IIb- and alpha v-integrin subunits, which induce selective activation of integrins alpha IIb beta 3 and alpha v beta 3, respectively [1]. Objectives: In the present study, we have investigated the ability of an alpha IIb-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 gamma-chain, but only weak phosphorylation of PLC gamma 2. 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 A(2) (TxA(2)) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA(2). Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin alpha IIb beta 3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. Conclusions: The present study demonstrates that the alpha IIb-CHAMPS peptide induces platelet activation through integrin alpha IIb beta 3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alpha IIb-CHAMPS peptide to study integrin alpha IIb beta 3 function is compromised by non-integrin-mediated effects.