Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbβ3 on platelet activation and aggregation

alpha(IIb)beta(3), the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of alpha(IIb)beta(3) changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence (1000)LEEDDEEGE(1008) of alpha(IIb) (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of alpha(IIb), is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of alpha(IIb) and beta(3). The aim of the present study was to investigate the synergistic effect of the intra- and extracellular-peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin alpha(IIb)beta(3) activation.