Biophysical Investigation of GpIbα Binding to Thrombin Anion Binding Exosite II

Abstract

Substrates and cofactors of the serine protease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding. On the surface of platelets resides the GpIbα/β−GpIX−GpV membrane-bound receptor complex. IIa’s ABE-II is proposed to interact with an anionic portion of GpIbα which enhances IIa cleavage of PAR-1 and subsequent activation of platelets. In this work, one-dimensional (1D) and two-dimensional (2D) NMR, analytical ultracentrifugation (AUC), and hydrogen−deuterium exchange (HDX) coupled with MALDI-TOF MS were performed to further characterize the features of binding to IIa’s ABEs. The described work builds upon investigations performed in a prior study with fibrin(ogen)’s γ′ peptide and IIa [Sabo, T. M., Farrell, D. H., and Maurer, M. C. (2006) Biochemistry 45, 7434−7445]. 1D line broadening NMR (1H and 31P) and 2D trNOESY NMR studies indicate that GpIbα residues D274−E285 interact extensively with the IIa surface in an extended conformation. AUC demonstrates that both GpIbα (269−286) and γ′ (410−427) peptides interact with IIa with a 1:1 stoichiometry. When the HDX results are compared to those for the ABE-I targeting peptide hirudin (54−65), the data imply that GpIbα (269−286), GpIbα (1−290), and γ′ (410−427) are indeed directed to ABE-II. The ABE-II binding fragments reduce HDX for sites distant from the interface, suggesting long-range conformational effects. These studies illustrate that GpIbα and γ′ target ABE-II with similar consequences on IIa dynamics, albeit with differing structural features