Functional role of Bβ‐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 Bβ15–26, and those for two other mAbs, I‐5G and I‐3B, were in fragment Bβ26–36. 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’ (Bβ15–17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BβSARGHRPLDKKREEA(12–26), BβLDKKREEA(19–26), BβAPSLRPAPPPI(26–36), BβAPSLRPAPPPISGGGYRARPA(26–46) and BβGYRARPA(40–46), which imitate the various sequences in the N‐terminal region of the fibrin Bβ‐chain, have been investigated. Peptides Bβ12–26 and Bβ26–46, but not Bβ40–46, Bβ19–26, and Bβ26–36, proved to be specific inhibitors of fibrin polymerization. The IC 50 values for Bβ12–26 and Bβ26–46 were 2.03 × 10 −4 and 2.19 × 10 −4   m , respectively. Turbidity and electron microscopy data showed that peptides Bβ12–26 and Bβ26–46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bβ12–46 took part in fibrin protofibril formation simultaneously with site ‘A’ (Aα17–19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bβ12–46 of one fibrin desAA molecule and the D‐domain of another has been constructed.