A re-examination of the cleavage of fibrinogen and fibrin by plasmin

• Published in: The Journal of biological chemistry Vol. 250; no. 18; pp. 7210 - 7218
• Main Authors: Ferguson, E W, Fretto, L J, McKee, P A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-09-1975
• Subjects: Amino Acids - analysis; Cadaverine; Dansyl Compounds; Fibrin; Fibrinogen; Fibrinogens, Abnormal; Fibrinolysin; Humans; Macromolecular Substances; Molecular Weight; Peptide Fragments - analysis; Protein Binding; Urokinase-Type Plasminogen Activator
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)40930-7

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights
of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000
to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three
disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of
COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from
its gamma' subunit of 38,000 molecular weight; however, the beta'' subunit of 42,000 molecular weight and the alpha'' subunit
of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin
had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The
molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation
equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine
into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species
of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated
during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains
and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen
molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three
pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link
acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled
peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled
fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent
plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both
alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.