UC San Diego

Thrombin is a tightly regulated serine protease that acts at the terminus of the blood coagulation cascade, performing proteolytic activation of platelets and converting soluble fibrinogen into insoluble fibrin. In complex with thrombomodulin, thrombin acts as an anticoagulant by activating protein C. The strict regulation of thrombin is essential to organism survival, as under-regulation of clot formation results in amplified blood loss and over-regulation leads to pathological clot formation. In human health, hemophilia, pulmonary emboli, venous thrombi, myocardial infarctions and ischemic strokes are all tied to the misregulation of thrombin. Structurally, thrombin consists of a double [Beta]-barrel core that is conserved among members of the trypsin-like protease family with extended active site loops that are not conserved. Recent evidence has suggested that the regulation of thrombin is tied much more to dynamics than previously thought and NMR is the ideal method of study. Chapter II outlines the process of isotopic labeling, expression, refolding, activation and purification of recombinant thrombin from E. coli. Once this barrier was overcome, the next hurdle in the study of thrombin by NMR was resonance assignments, discussed in Chapter III. Results from resonance assignment of thrombin with PPACK occupying its active site vs. the S195M mutant representing apo-thrombin showed major chemical shift perturbations between the forms and a large degree of resonance line broadening that abrogated the signal, an effect that was mostly abolished upon active site ligation by PPACK. In Chapter IV, using a combined NMR and computational approach (performed by P. Gasper and P. Markwick of the McCammon lab) the dynamics of PPACK- thrombin are characterized and the solution ensemble is modeled. The results indicate that even with the active site occupied by PPACK, thrombin undergoes a large degree of structural fluctuations on timescales ranging from picoseconds all the way to milliseconds. The equivalent study was undertaken on S195M-thrombin as discussed in Chapter V. As compared to PPACK-thrombin, the apo-like S195M-thrombin, apo-thrombin displays a remarkable degree of dynamics in the [mu]s-ms timescale. These results reinforce the paradigm shift towards an ensemble view of thrombin activity states and substrate recognition