Figure 7

Expression and activation of prothrombin variants and resulting activity of thrombin upon activation. (A) Measurement of prothrombin in the cell media by ELISA demonstrated that expression of prothrombin variants in HEK293T cells resulted in reduced secretion levels that were indistinguishable from control and (B) corresponding measurement of prothrombin in the cell lysate demonstrated decreased biosynthesis. (C) Rates of prothrombin activation by prothrombinase measured using DAPA. Once prothrombin variants were fully activated by prothrombinase, their ability to cleave synthetic substrate S-2238 (D) or fibrinogen (E) were monitored, with significant differences in the latter, but not the former. For both S-2238 and fibrinogen, the rate was calculated during the initial reaction phase when the substrate is non-limiting and the conditions are presumed to be steady-state. Rate was then normalized to enzyme concentration. (F) Clotting profile between wild-type and C138A/Δ15 demonstrated delayed clot initiation and altered turbidity. (G) The rates of clot formation were determined from the clotting profile and reduced in the mutant.