Identification of peptide antagonists to glycoprotein Ibα that selectively inhibit von Willebrand factor dependent platelet aggregation

GPIb alpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIb alpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIb alpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIb alpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIb alpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-I and OS-2, determined their equilibrium dissociation GP1b alpha binding constants (K(D)s) of 64, 0.74, and 3.1 nM, respectively. Isothermal calorimetry corroborated the K-D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIb alpha-F-C in a concentration-dependent manner. All three peptides inhibited GPIb alpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIb alpha-vWF-A1 interaction.