The IGF receptor inhibitor NVP-AEW541 and the neutralising antibody αIR3 block IGF-1–mediated PKB phosphorylation and reduce PAR-1–mediated aggregation. (i) Washed platelets were incubated for 10 minutes with vehicle (DMSO) or the indicated concentration of NVP-AEW541 (A) or platelets were incubated for 3 minutes with saturating concentration of the F(ab′) fragment IV.3 to block the IgG receptor FcγRIIa, followed by incubation for 3 minutes with the indicated concentration of the IGF receptor neutralization antibody IR3 (B). Platelets were subsequently stimulated with 100 nM IGF-1 for 5 minutes, followed by extraction. IGF receptor immunoprecipitates or whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with antiphosphotyrosine (PTyr) and anti-P–PKBSer473, respectively. Membranes were stripped and reprobed with the appropriate antibodies to confirm equal loading. (ii) Alternatively, washed platelets were incubated with vehicle (control), 0.5 μM NVP-AEW541 for 10 minutes (A), or incubated for 3 minutes with saturating concentration of the F(ab′) fragment IV.3, followed by incubation for 3 minutes with 5 μg/mL αIR3 (B). Platelets were subsequently stimulated with 0.5 μM SFLLRN or 0.7 μM SFLLRN and aggregation was recorded for a total of 5 minutes. Results are representative of 3 experiments.