Evaluating How Binding Interactions for PARs Change as Prothrombin is Converted to Thrombin

Abstract

Thrombin is generated during the final stages of blood coagulation from its inactive precursor prothrombin (ProT). The resultant serine protease plays important roles in procoagulation, anticoagulation, and platelet activation. Thrombin's specificity is regulated by its active site, surface loops, and regulatory Anion Binding Exosites I and II (ABE I and ABE II). Binding of physiological ligands to ABE I can allosterically control thrombin conformation and activity. Protease Activated Receptors (PARs) function as tethered ligands that elicit transmembrane signaling for platelet activation and aggregation. Both PAR1 (49-62) and PAR3 (44-56) have been hypothesized to bind thrombin ABE I with the help of a hirudin-like motif (56FEEI59). Unexpectedly, some ligands can already bind to pro-ABE I of ProT. Ongoing NMR studies are comparing the binding environments encountered as PAR1 (49-62) interacts with immature pro-ABE I and mature ABE I relative to that of PAR3 (44-56). 1D proton line broadening confirmed that PAR1 and weaker binding version PAR1G already exhibited line broadening with ProT and such broadening increased when bound to thrombin. 2D tr-NOESY revealed PAR1 (49-62) bound to pro-ABE I in an extended conformation with the proline adopting a trans conformation. 1H-15N HSQC titrations provide quantitative estimates of binding interactions for selectively 15N-labeled PAR aminoacids interacting with ProT vs Thrombin. Similar to PAR3, PAR1 peptides revealed the hydrophobic pocket (F34, L65, and I82) of pro-ABE I is partially available for peptide binding. When bound to thrombin, the acidic C-terminal tail of PAR1 (49-62) remains unresolved by crystallography. NMR titrations are making it possible, for the first time, to monitor binding of PAR1 (58DEEKN62) to pro-ABE I and ABE I. Knowledge gained from this project may help develop drug candidates that target or avoid specific hots spots on (pro)-ABE I.