Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

The following peptide was synthesized by classical methods in solution: Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-8). The Michaelis-Menten parameters for the hydrolysis of the Arg-Gly bond in F-8 by thrombin were determined to be Kcat = 31 X 10(-11) M [(NIH unit/L) s]-1 and KM = 310 X 10(-6) M. Comparison of these values with those determined previously for native fibrinogen and for a series of similar synthetic peptides, together with information about the amino acid sequences of this portion of the A alpha chain of abnormal fibrinogens, suggests an important role for Asp at position P10. Differences in the Michaelis-Menten parameters between F-8 and the 51-residue N-terminal CNBr fragment of the A alpha chain of fibrinogen correspond to only 1-2 kcal/mol in binding affinity.