Figure 7

Activation of CnPC by Protac, followed by detection of amidolytic activities with Spectrozyme. All measurements were performed in duplicate, and the average of the two measurements was used as one true experimental replicate. Activity levels were expressed in optical density units measured at wavelength =405 nm. Each data point shown is the mean (±SEM) of three experiments using plasma from three different dogs. (A) Amidolytic activities detected at ten time points following addition of Spectrozyme. These results show that purified CnPC was activatable and retained its amidolytic activities. (B) Linear regression analysis depicting the relationship between the amount of amidolytic activities detected (expressed by optical density measured at wavelenghth =405 nm) and concentration of CnPC present at various time points. These results show that the linear range of 0–10 μg/mL in this assay was valid if the samples were analyzed within 5 minutes following addition of Spectrozyme. Each point is the mean (±SEM) of three experiments using plasma from three different dogs. The dotted lines delineate the 95% confidence intervals for the curves. (C) Activation of protein C in canine plasma and a plasma fraction obtained following barium chloride and ammonium sulphate precipitation. Final total protein concentration was adjusted to 100 ng/mL. Protein C was activated by Protac, a specific activator of protein C, followed by detection with the chromogenic substrate Spectrozyme. Measurements were taken at ten time points following addition of Spectrozyme in the system.