Glycoprotein (GP) Ibα, a member of the GP Ib-IX-V receptor complex, is one of the most abundant platelet membrane receptors. Besides mediating the interaction with von Willebrand factor, which is necessary to initiate the tethering of circulating platelets to the injured vessel wall, it mediates the binding of α-thrombin. Sulfation of three tyrosine residues (Y276, Y278, Y279) is thought to be important for binding α-thrombin. With the goal of understanding the mechanism of thrombin binding to platelet GP Ib, we first generated an amino terminal (residues 1–290) GP Ibα fragment, called GPIbαN, which bears all the determinants for α-thrombin binding. In a previously published crystal structure of the GPIbαN/thrombin complex, the negatively charged sulfate groups on Y276 and Y279 were found to mediate interactions with two positively charged clusters of α-thrombin residues, namely exosites I and II.To evaluate tyrosine sulfation levels, we digested GPIbαN with Abalone sulfatase and the resulting species were analyzed by ion exchange chromatography. Digestion of the fully sulfated form (peak 4) progressively generated 3 additional species, called peak 3, peak 2 and peak 1, which were assumed to contain 2, 1 and no sulfated tyrosine residues, respectively. With time, all peak 4 could be converted to peak 1. The GPIbαN peak 4, mixed in a 3:1 ratio with α-thrombin, formed a stable complex that could be separated by gel permeation chromatography and incorporated all the α-thrombin added into the mixture. Peak 3 (the species that was previously crystallized in complex with thrombin and found to lack sulfation on Y278) formed a comparable amount of complex relative to peak 4, while peak 2 bound only 39% and peak 1 12% of the added thrombin. A degradation product of α-thrombin that lacks exosite I, γ-thrombin, formed the same amount of complex with GPIbαN peak 3 as α-thrombin, while meizothrombin, that lacks a fully functional exosite II, was unable to generate a stable complex. Moreover heparin and oligonucleotide HD22, specific inhibitors of exosite II, completely blocked complex formation, while Hirugen and oligonucleotide HD1, exosite I inhibitors, had no effect. The mutants Y279F (containing sulfotyrosine 276 and 278) and Y278F (containing sulfotyrosine 276 and 279) generated 87% and 92% of the complex as compared to WT, while the mutant Y276F (containing sulfotyrosine 278 and 279) generated only 10% of the complex. These results indicate that sulfotyrosine 276 in GPIbαN is essential for forming a complex with thrombin, a conclusion consistent with the crystal structure in which the sulfate group on Y276 mediates close contacts with exosite II. Altogether, these findings support the concept that exosite II is necessary and sufficient for the formation of a stable complex with a soluble GP Ibα fragment. To study thrombin binding to GP Ibα on platelets and the role of tyrosine sulfation in the process, we generated mice that express human GP Ibα either wild type (TKK) or bearing the mutation Y279F. TKK mouse platelets bound α-thrombin with an affinity similar to human platelets. Neither meizothrombin (lacking a full exosite II) nor γ-thrombin (lacking exosite I) bound to TKK platelets or human platelets. Accordingly, the exosite-specific inhibitors described above completely and independently suppressed α-thrombin binding to platelets. Mouse platelets bearing the mutation Y279F exhibited no significant thrombin binding. Since sulfated Y279 makes contacts mainly with thrombin exosite I, this can be seen as a further evidence that exosite I is involved in α-thrombin binding to platelets. Altogether, these data suggest that α-thrombin binds to platelets through a process that involves concurrently both exosites. This process is not properly reflected by the use of soluble GP Ibα fragments that can interact with thrombin solely engaging exosite II.
Disclosures: No relevant conflicts of interest to declare.