Monitoring Conformational Changes that Occur to Blood Coagulant Prothrombin as it is Activated to Thrombin

Abstract

The blood coagulation protein thrombin is originally expressed as the zymogen prothrombin (ProT). Prothrombin contains the F1.2 (Gla + Kringle 1 + Kringle 2) domain and the inactive serine protease prethrombin-2 (PT2). Kringle 2 of F1.2 protects an immature anion binding exosite on ProT called pro-ABE II. The prothrombinase complex converts ProT to thrombin. Cleavage of ProT after R271 releases F1.2 from PT2. Cleavage of PT2 at R320 helps to generate the active, two-chain species thrombin that contains an A and B chain linked by a disulfide. Hydrogen-Deuterium Exchange coupled with MALDI-TOF mass spectrometry was used to monitor the conformational dynamics of ProT, F1.2 + PT2, PT2, and thrombin. During the activation process, the A-chain and the autolysis loop undergo changes in solvent accessibility that respond to the individual R271 and R320 cleavages. The resultant conformational properties aid in creating a protease that can accept substrates at the active site. Importantly, this coagulation protein system also uses anion binding exosites to control its functions. F1.2 protects pro-ABE II on both ProT and PT2 from solvent exposure. Moreover, this binding event exhibits a long-range effect over to pro-ABE I suggesting an influence on development of inter-exosite communication. As F1.2 is removed and PT2 is activated to thrombin, pro-ABE I and II become more solvent exposed and mature into the functional exosites ABE I and II. HSQC NMR titrations with GpIbalpha (269-286, 15N-labeled L275, 15N-labeled D277) demonstrate that ligand binding affinity at pro-ABE II / ABE II increases as ProT is converted to PT2 and then thrombin. The final thrombin enzyme effectively accommodates substrates at its serine protease active site and utilizes its mature exosites to regulate several coagulation related activities.