Functional role of Bβ‐chain N‐terminal fragment in the fibrin polymerization process

Abstract

Fibrinogen is a central protein of the blood coagulation system. The human fibrinogen molecule (molecular mass 344 kDa) consists of two identical subunits connected by disulfide bonds [1]. Each monomer subunit is formed by three nonidentical polypeptide chains, Aa, Bb and c. The N-terminal ends of all six polypeptide chains are situated in the fibrinogen central region, which is known as the E-domain. Two 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 Bb15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bb26-36. 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 Fabfragment) 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' (Bb15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbSARGHRPLDKKREEA(12-26), , , and , which imitate the various sequences in the N-terminal region of the fibrin Bb-chain, have been investigated. Peptides Bb12-26 and Bb26-46, but not Bb40-46, Bb19-26, and Bb26-36, proved to be specific inhibitors of fibrin polymerization. The IC 50 values for Bb12-26 and Bb26-46 were 2.03 · 10 )4 and 2.19 · 10 )4 m, respectively. Turbidity and electron microscopy data showed that peptides Bb12-26 and Bb26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bb12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aa17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bb12-46 of one fibrin desAA molecule and the D-domain of another has been constructed. ; NaCl ⁄ P i , 0.02 M potassium phosphate buffer (pH 7.4) with 0.13 M NaCl; t-NDSK, thrombin-treated N-terminal disulfide knot of fibrin; TPBS, NaCl ⁄ P i with 0.05% Tween-20.

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