Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process.
Four mAbs of the IgG(1) class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bbeta15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bbeta26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fab-fragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bbeta15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbetaSARGHRPLDKKREEA(12-26), BbetaLDKKREEA(19-26), BbetaAPSLRPAPPPI(26-36), BbetaAPSLRPAPPPISGGGYRARPA(26-46) and BbetaGYRARPA(40-46), which imitate the various sequences in the N-terminal region of the fibrin Bbeta-chain, have been investigated. Peptides Bbeta12-26 and Bbeta26-46, but not Bbeta40-46, Bbeta19-26, and Bbeta26-36, proved to be specific inhibitors of fibrin polymerization. The IC(50) values for Bbeta12-26 and Bbeta26-46 were 2.03 x 10(-4) and 2.19 x 10(-4) m, respectively. Turbidity and electron microscopy data showed that peptides Bbeta12-26 and Bbeta26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bbeta12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aalpha17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bbeta12-46 of one fibrin desAA molecule and the D-domain of another has been constructed.
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine. lougovskoy@yahoo.com
E V
EV
Lugovskoy
P G
PG
Gritsenko
L G
LG
Kapustianenko
I N
IN
Kolesnikova
V I
VI
Chernishov
S V
SV
Komisarenko
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001704:Biopolymers; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005337:Fibrin / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D001704:Biopolymers; D000939:Epitopes; D005337:Fibrin