B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.
BACKGROUND
Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous.


OBJECTIVE
To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val.


METHODS
We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis.


RESULTS
Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants.


CONCLUSIONS
These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.
Okumura
N
N
Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, Japan. nobuoku@gipac.shinshu-u.ac.jp
Terasawa
F
F
Haneishi
A
A
Fujihara
N
N
Hirota-Kawadobora
M
M
Yamauchi
K
K
Ota
H
H
Lord
S T
ST
D012103:Batroxobin / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D026122:Factor XIIIa / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000737:chemistry / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D009154:Mutation; D009391:Nephelometry and Turbidimetry; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000378:metabolism*
D009842:Oligopeptides; C027539:fibrinopeptides gamma; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C048770:glycyl-histidyl-arginyl-proline; C034585:glycyl-prolyl-arginyl-proline; D005340:Fibrinogen; D026122:Factor XIIIa; D012103:Batroxobin; D013917:Thrombin