Platelet Glycoprotein Ibα Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin

Abstract

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb␣-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the aminoterminal fragment of GPIb␣ reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb␣ caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate D-Phe-Pro-Arg-pNA. The K d for the glycocalicin-thrombin interaction was 1 M at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 M at 100 mM NaCl (⌫ salt ‫؍‬ ؊4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb␣-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb␣. Both the allosteric inhibition of fibrinogen turnover caused by GPIb␣ binding to these mutants, and the K d values for their interactions with GPIb␣ were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K d values for the interactions of these thrombin mutants with GPIb␣ by 10-and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb␣. These data demonstrate that GPIb␣ binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate D-Phe-Pro-Arg-pNA.

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

“…Our findings confirm the role of exosite II (29)(30)(31)(32)(33)(34) and define that of exosite I (35), proving its necessary involvement in binding to immobilized but not soluble receptor. Although hirugen and HD1, exosite I ligands, lack inhibitory effect on FIIa interaction with GPIbα fragments linked to agarose (31) or plastic (30), we found that both inhibit binding to GPIbα fragments linked to immobilized antibodies and to membraneanchored platelet GPIb, the biologically relevant receptor.…”

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…This confirms a role for electrostatic effects in ABE-II binding to GPIb␣, first suggested by its salt dependence (42). Whether binding to GPIb␣ allosterically modifies the structure of thrombin is still in question because conflicting results have been published indicating no effect on PAR1 peptide hydrolysis (42), but inhibitory effects on fibrinopeptide A release and D-Phe-ProArg-p-nitroanilide hydrolysis (43) and, more recently, FVIII activation (44).…”

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

“…Our current understanding of thrombin-mediated platelet activation is that its initial high-affinity binding to platelet surface GPIb-alpha is via exosite 2, which facilitates subsequent binding of a second thrombin molecule via exosite 1 (Li et al 2001;Celikel et al 2003;Dumas et al 2003). The interaction with GPIb-alpha also promotes thrombin binding to its proteolytically activated receptor-1 (PAR-1) via exosite 1, which leads to receptor cleavage .…”