Failure of thrombin-mediated proteolytic release of the mutant fibrinopeptide A of fibrinogen isolated from Fib^AEK mice. (A) Reaction mixtures of purified WT fibrinogen (left) and fibrinogen^AEK (right) were incubated with thrombin and Ca²⁺ for various times, and the fibrinogen chains analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The positions of the Aα, cleaved α, Bβ, cleaved β, and γ chains are indicated. Note that cleaved α chain was never observed in reaction mixtures containing fibrinogen^AEK regardless of incubation time. (B) HPLC chromatograms of reaction mixtures containing either WT fibrinogen (left) or fibrinogen^AEK (right) following incubation with thrombin and Ca²⁺ for various times. Indicated are the positions of peaks corresponding to fibrinopeptide A (FpA), fibrinopeptide B (FpB), and mutant fibrinopeptide A (FpAEK). The identification of peak positions was independently established by resolving synthetic peptides corresponding to the predicted fibrinopeptide sequence. Note that FpAEK is never observed in chromatograms derived from fibrinogen^AEK reactions regardless of incubation time with thrombin and Ca²⁺. (C) Fibrinopeptide release curves were prepared by plotting the percent of fibrinopeptide released versus time. FpA data were fitted with a simple first-order equation, and the FpB data from normal fibrinogen were fitted to a standard equation describing 2 consecutive first-order processes. (D) Table of specificity constants (k_cat/K_M), which were determined by dividing first-order rate constants by the thrombin concentration.