Fig. 3

Activation of latent antithrombin activity in fusion proteins. Purified albumin fusion protein preparations were first reacted with specific purified proteases, then diluted into the presence of thrombin and its chromogenic substrate S2238, and the velocity of the thrombin-catalysed amidolysis reaction was measured and expressed as a percentage of the uninhibited control reaction. Panel A: EPR-HV3HSA was reacted with FXIa for zero (open squares) or 60 min (closed triangles) prior to dilution into the S2238 reaction and measurement of the rate of coloured product formation by thrombin over time. Buffer controls introduced phosphate buffer PPNE (see Materials and Methods) and FXIa (open circles) but no fusion protein into the S2238 reaction. Data represents the mean ± SEM, n = 7. Panel b: As in Panel a, EPR-HV3HSA or HSAEPR-HV3 proteins were incubated for zero (0′, white bars) or 60 min (60′, black bars) prior to dilution into S2238 reactions and determination of the rate of thrombin-mediated amidolysis. EPR-HV3HSA reactions are the slope of the same progress curves shown in Panel a as a percentage of the Buffer control; HSAEPR-HV3 reactions were determined analogously. Panel c: The mean (n = 6 ± SEM) of thrombin reaction velocities supplemented with activation reactions of the three fusion proteins labelled to the right of the progress curves with FXIa is shown, at six time points, in PPNE buffer as in Panels a and b. Panel d: As in Panel a and b, except that HBS / 5 mM CaCl₂ was used as the buffer instead of PPNE and except that EPR-HV3HSA was combined with the proteases identified on the x axis prior to determination of second stage thrombin-mediated reaction velocity (white bars, FVIIa, FXa, FXIIa, or Plasmin; black bars FXIa). Control reaction “FXIa + No EPR-HV3HSA” contained FXIa but no EPR-HV3HSA in the activation reaction. Buffer reaction contained only HBS / 5 mM CaCl₂ Data represents the mean ± SEM, n = 7