Sequential Plasmin Proteolysis Of The Nh₂-Terminus Of The Bβ Chain Of Human Fibrinogen

Recent clinical data have shown that early plasmin proteolysis of human fibrinogen results in the release of fibrinopeptide B (FPB) immunoreactivity into plasma. Upon thrombin treatment this activity increases indicating that the immunoreactivity arises from larger FPB-containing peptides. The presence of degradation products from the Bβ chain may be an index of fibrinogen proteolysis not only in normal hemostasis but also in pathological disorders such as thrombosis and disseminated intravascular coagulation. Therefore the capability of detecting events leading to progressive plasmin degradation at the Bβ chain could be of major diagnostic and therapeutic significance. Toward this goal, we have isolated the various peptides released from human fibrinogen by plasmin proteolysis using HPLC on a reverse phase C₁₈ column. These peptides were identified using amino acid analysis and radioimmunoassays for FPB. Bβ 1-42 was the earliest fragment released during limited plasmin proteolysis. These levels reached a maximum and then began to fall during the course of the digestion. In addition, increasing levels of Bβ 1-21 and of FPB were shown to lag behind the production of Bβ 1-42. Using purified Bβ 1-42, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components, Bβ 1-14 and Bβ 22-42 and a minor component composed of Bβ 15-42. The rate of this reaction is not affected by the addition of hirudin indicating that it is not caused by trace amounts of thrombin. Taken together these data indicate a sequence of events in which Bβ 1-42 is initially cleaved from fibrinogen during plasmin digestion. Free from its parent molecule, Bβ 1-42 can undergo further plasmin attack, preferentially at the 21-22 bond to yield Bβ 1-21 and B8 22-42. Plasmin then attacks Bβ 1-21 at the 14-15 bond to release FPB.