Abstract 527: Design of Activation-specific GPIIb/IIIa Inhibiting Peptides
Backround: The clinically used GPIIb/IIIa blockers are accompanied with adverse effects (e.g. bleeding, thrombocytopenia). In previous studies we established an activation specific GPIIb/IIIa blocking single chain antibody (SA2), that is accompanied with less bleeding. Small inhibitory peptides offer additional advantages, like potential oral application. We hypothesized that small peptide inhibitors can be generated on the basis of the heavy chain CDR3 region of this antibody.
Methods and Results: The CDR3 region of the antibody SA2 was synthesized as linear peptide (SA2lin: ATYTSRSDVPDQTS). The inhibitory potency of this peptide was evaluated in flow cytometry by measuring fibrinogen binding to ADP (20 μM) activated platelets. SA2lin demonstrates an IC50 of 166 μM, reflecting a significant lower affinity compared to the complete antibody (IC50 = 1.5 μM) and to the non-activationspecific GRGDSP peptide (IC50 = 8 μM). Thus, we aimed to improve the affinity by various strategies:
Shortening of the peptide did not cause any improvement of affinity.
A cyclic, SH-bonded peptide (constructed by the addition of a cystein residue on both sides) resulted in a significant reduction of the IC50 (54 μM).
The exchange of the serine in the RXD-pattern against a glycine (SA2cyc (S-G):CATYTSRGDVPDQTSC ), as it is found in the original fibrinogen molecule, caused a major increase in affinity (IC50 = 7 μM).
Activation-specificity was proven by measuring the GPIIb/IIIa occupancy in flow cytometry: Complete occupancy was induced by 1mM GRGDSP as well as by 1mM SA2cyc(S-G) on ADP-activated platelets. In contrast to that, occupancy was only induced by GRGDSP peptide and not by SA2cyc(S-G) on resting platelets . Finally we were able to demonstrate a differential inhibitory effect on adhesion of GPIIb/IIIa expressing CHO-cells on fibrinogen under arterial flow. The non activation specific inhibitor eptifibatide inhibited activated as well as non activated cells, whereas SA2cyc(S-G) still allowed adhesion mediated by non activated GPIIb/IIIa.
Conclusion: In conclusion we were able to generate function-dependent, conformation-specific small molecular GPIIb/IIIa inhibitors as a potential basis for new therapeutic agents.