The biggest effects were observed for mutations at Arg93, Arg233 and Lys236. if only a single site is usually involved, GpIb may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin’s two exosites in GpIb binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that this peptide corresponding to the C-terminal portion of GpIb and the entire extracellular domain name bind exclusively to thrombin’s exosite II. The conversation of thrombin with GpIb thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition. Abbreviations:GpIb, glycoprotein Ib; PAR, protease-activated receptor; TM, thrombomodulin; LRR, leucine-rich repeat; PPACK,d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; SPR, surface plasmon resonance; TROSY, transverse relaxation optimised spectroscopy; PEG, polyethylene glycol Keywords:crystallography, exosite, haemostasis, NMR, platelet == Graphical abstract == == Highlights == We analysed interactions of the platelet receptor GpIb with thrombin PRKACG using three complementary methods. GpIb exclusively binds to exosite II of thrombin. Exosite I remains available for binding to other ligands. GpIb recruits thrombin to the platelet membrane as a cofactor for PAR-1 cleavage. == Introduction == Thrombin is the final protease of the coagulation cascade[1],[2], and its activity is crucial for the formation of stable blood clots[3]. In addition to the cleavage of fibrinogen to fibrin, thrombin also feeds back to upregulate its own production by activating cofactors fV and fVIII, the protease fXI, and through cleavage of the protease-activated receptor (PAR)-1[4],[5]. PAR-1 is a seven-transmembrane G-protein-coupled receptor expressed on the surface of platelets. Upon cleavage Mevastatin of the N-terminal extracellular peptide at Arg41, the newly formed N-terminus inserts into the body of the receptor triggering platelet activation. The rate of platelet PAR-1 cleavage by thrombin is increased by approximately 5-fold Mevastatin in the presence of glycoprotein Ib (GpIb)[6], a member of the platelet GpIb-IX-V receptor complex[7], that has been identified as the high-affinity receptor for thrombin on the surface of platelets[8]. The crystal structure of the extracellular domain of GpIb, composed of the 300 N-terminal residues, revealed an elongated banana-like shape dominated by a leucine-rich repeat (LRR) region, followed by a flexible acidic stretch at the very C-terminus Mevastatin (Gly271Glu282) containing three sulfated tyrosine residues (276, 278 and 279)[9]. The acidic residues in this region and the posttranslational sulfation of the tyrosines are crucial for binding of GpIb to thrombin[10],[11],[12]. Thrombin has two anion binding exosites, both of which are essential for its activity and specificity (Fig. 1a and b)[13]. Exosite I is the fibrinogen recognition exosite, but it also interacts with a variety of other substrates, cofactors and inhibitors, including the N-terminal region of PAR-1, the cofactor thrombomodulin (TM; shown inFig. 1a and b) and the C-terminal peptide of the inhibitor hirudin (known as hirugen). Exosite II binds to glycosaminoglycans such as heparin (shown inFig. 1a and b), to the -chain of fibrinogen and to the second kringle domain of prothrombin, known as fragment-2 (F2). Although both exosite I and exosite II interactions are mediated by electrostatics, there is a surprising degree of exosite selectivity, with ligands often binding exclusively to one site or the other[13]. At high concentrations, however, there is evidence that some ligands, such as heparin, can interact with both sites[14],[15], although this is not thought to be of physiological relevance. It has been demonstrated that the two exosites can bind independently and simultaneously to different ligands; for example, when hirugen is bound to exosite I, GpIb or fibrinogen peptides are still able to bind to exosite II[16]. However, particularly in the absence of active-site occupation, there is a considerable degree of allosteric communication between exosites I and II, with binding at one exosite decreasing affinity of ligands for the other[17]. == Fig. 1. == Structure Mevastatin of thrombin and its exosite Mevastatin interactions with GpIb. (a) A ribbon diagram of thrombin (heavy chain only) is shown in the standard orientation coloured from its N-terminus to its C-terminus, from blue to red. In magenta are classic exosite binding ligands, TM as ribbons (exosite I) and heparin as sticks (exosite II). The anion binding exosites are indicated. (b) Thrombin is oriented as in (a) but shown in surface representation coloured according to electrostatic potential, with blue positive and red negative. The active-site cleft is the deep red pocket between the exosites. (c) The crystal structure 1OOK is shown, with thrombin in cyan and the acidic C-terminal region of GpIb as magenta sticks. The chosen asymmetric unit contained the copy of GpIb shown binding.