Anion Binding Exosites As a Target to Understand the Activation of Thrombin

In blood coagulation, thrombin is expressed as the 72 kDa zymogen Prothrombin (ProT) that is composed of Fragment 1 (1-155), Fragment 2 (156-271), and a protease domain (272-579). ProT is physiologically activated by the Prothrombinase Complex (FXa, cofactor FVa, phospholipid, and calcium) following cleavages at R320 and R271. The resultant protease thrombin utilizes its anion binding exosites (ABE I and II) to regulate substrate specificity. Little is known about how these exosites mature as ProT is converted to thrombin. In the current project, NMR methods were used to characterize the binding of protease activator receptor (PAR) peptides to (pro)-ABE I. 1D proton line broadening and 2D transferred-NOESY studies revealed that PAR3 (43-56) and PAR1 (49-63) each bound in an extended conformation to immature pro-ABE I on ProT. Binding interactions increased in the presence of thrombin containing a mature ABE I. Unlike previous crystallography work, NMR could evaluate binding of the PAR1 acidic tail sequence (58DEEKN62) to (pro)-ABE I. 15N- HSQC NMR titration experiments were used to map the binding roles of individual PAR3 residues. With PAR3G51 (43-56), 15N-labeled F47 and 15N-labeled D54 both reported on interactions with pro-ABE I on ProT versus ABE I on thrombin. 15N-labeled D54 exhibited major line broadening during the titration with thrombin consistent with enhanced ligand affinity toward mature ABE I. By contrast, the large changes in chemical shift observed during the titration of 15N-labeled F47 with thrombin reflected a significant change in exosite environment for PAR3G51 F47 without altering the binding affinity. Results are consistent with D54 playing an electrostatic role in anchoring PAR3G51 to (pro)-ABE I followed by a supporting hydrophobic role for F47. Greater knowledge on exosite maturation will lead to new strategies for controlling procogulation and anticoagulation.