SUBSTITUTION OF TYROSINE FOR PHENYLALANINE IN FIBRINOPEPTIDE-A RESULTS IN PREFERENTIAL THROMBIN CLEAVAGE OF FIBRINOPEPTIDE-B FROM FIBRINOGEN

Phenylalanine at residue 8 in the A alpha chain of fibrinogen is a hig hly conserved amino acid that is believed to be critical for binding a nd catalysis by the serine protease thrombin. We have examined the req uirement for Phe at this position by constructing a variant recombinan t fibrinogen with a conservative substitution of tyrosine for phenylal anine, A alpha F8Y fibrinogen. We found that the variant fibrinopeptid e A (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cle avage of an A alpha 1-23 peptide and an A alpha 1-50 fusion protein wi th the same substitution. This result indicates that fibrinogen residu es other than A alpha 1-50 participate in thrombin binding and fibrino gen proteolysis. We found, for the first time, that thrombin-catalyzed lysis of the fibrinogen B beta chain preceded lysis of the A alpha ch ain, such that fibrinopeptide B (FpB) was released prior to F8Y 1-16. Kinetic analysis demonstrated that F8Y 1-16 was a very poor substrate for thrombin, with a specificity constant 280-fold lower than normal f ibrinopeptide A. FPB was also a poor substrate, but the specificity co nstant for FpB was only 4-fold lower than normal. Consequently, FpB wa s preferentially released from A alpha F8Y fibrinogen. This ''role rev ersal'' had a dramatic effect on polymerization, such that the rate of A alpha F8Y fibrinogen polymerization was 13% of the rate of normal r ecombinant fibrinogen. These results confirm the importance of phenyla lanine at A alpha chain residue 8 for efficient thrombin-catalyzed pro teolysis of fibrinogen, and further demonstrate that sequential fibrin opeptide release has an important role in normal polymerization.