Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release.
During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen.
Mullin
J L
JL
Departments of Chemistry and Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill 27599-7525, USA.
Gorkun
O V
OV
Binnie
C G
CG
Lord
S T
ST
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors
D010455:Peptides; D011994:Recombinant Proteins; D005345:Fibrinopeptide B; D005340:Fibrinogen; D013917:Thrombin