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. Author manuscript; available in PMC: 2008 Sep 25.
Published in final edited form as: Biochemistry. 2007 Jun 27;46(29):8603–8610. doi: 10.1021/bi700597p

Figure 1.

Figure 1

Cleavage of PAR1-wt or mutant exodomains with α-thrombin. Reactions with purified exodomains (3-200 μM) in reaction buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) were initiated by the addition of α-thrombin (0.75 nM for A, C and D or 0.5 nM for B). Acid quenched reactions were resolved on HPLC and initial rates were determined by calculating the peak areas at early time points where the progress curves were linear and compared to a standard curve generated by completely cleaved exodomain. Curve A is α-thrombin cleavage of PAR1-wt exodomain; curve B, PAR1-D39A; curve C, PAR1-L38A; and curve D, PAR1-P40A. The data were fit to the Henri-Michaelis-Menten equation with nonlinear least squares analysis to determine the kinetic constants. Error bars represent the standard deviation and the apparent absence of error bars indicates small standard deviation. Note the change in the scaling for the y-axis in panels B and C.