Limited proteolysis of fibrinogen by fibrinogenase from Echis multisquamatis venom.

Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase's hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26-42 and Aα 20-78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin.