Substitution of tyrosine for phenylalanine in fibrinopeptide A results in preferential thrombin cleavage of fibrinopeptide B from fibrinogen.
Phenylalanine at residue 8 in the Aalpha chain of fibrinogen is a highly conserved amino acid that is believed to be critical for binding and catalysis by the serine protease thrombin. We have examined the requirement for Phe at this position by constructing a variant recombinant fibrinogen with a conservative substitution of tyrosine for phenylalanine, Aalpha F8Y fibrinogen. We found that the variant fibrinopeptide A (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cleavage of an Aalpha 1-23 peptide and an Aalpha 1-50 fusion protein with the same substitution. This result indicates that fibrinogen residues other than Aalpha 1-50 participate in thrombin binding and fibrinogen proteolysis. We found, for the first time, that thrombin-catalyzed lysis of the fibrinogen Bbeta chain preceded lysis of the Aalpha chain, 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 fibrinopeptide A. FpB was also a poor substrate, but the specificity constant for FpB was only 4-fold lower than normal. Consequently, FpB was preferentially released from Aalpha F8Y fibrinogen. This "role reversal" had a dramatic effect on polymerization, such that the rate of Aalpha F8Y fibrinogen polymerization was 13% of the rate of normal recombinant fibrinogen. These results confirm the importance of phenylalanine at Aalpha chain residue 8 for efficient thrombin-catalyzed proteolysis of fibrinogen, and further demonstrate that sequential fibrinopeptide release has an important role in normal polymerization.
Rooney
M M
MM
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill 27599, USA.
Mullin
J L
JL
Lord
S T
ST
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D005340:Fibrinogen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D010446:Peptide Fragments; D011108:Polymers; D011994:Recombinant Proteins; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D014443:Tyrosine; D010649:Phenylalanine; D005340:Fibrinogen; D013917:Thrombin