The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa

The HHLGGAKQAGDV (H-12) sequence at the carboxyl termini of the gamma chain s and the RGD sequences in the A alpha chains of human fibrinogen are poten tial recognition sites for the binding of soluble fibrinogen to glycoprotei n IIb-IIla (GPIIb-IIIa) on activated human platelets. Thus, addition of eit her H-12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS h ad relatively little inhibitory effect on these functions of rabbit platele ts. In the present study, we found that H-12 inhibited ADP- and thrombin-in duced aggregation of rabbit platelets in a dose-dependent manner. Specifici ty was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to i nhibit ADP-induced aggregation.;Furthermore, flow cytometric analyses demon strated that H-12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interac tion of H-12 with rabbit GPIIb-IIIa, we performed affinity chromatography b y applying an octylglucoside extract of rabbit platelet proteins onto an af finity matrix containing the fibrinogen gamma chain sequence. Proteins of s imilar to 135 kDa and similar to 95 kDa were specifically eluted by soluble H-12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoc lonal antibody against human GPIIIa. Tn control samples, no detectable prot ein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H-12 inhibits aggregati on of and fibrinogen binding to rabbit platelets by directly interacting wi th rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would se rve as a suitable thrombosis model for testing the efficacy of peptide mime tics derived from H-12.