SEQUENCE OF RELEASE OF FIBRINOPEPTIDE-A FROM FIBRINOGEN MOLECULES BY THROMBIN OR ATROXIN

During the conversion of fibrinogen to fibrin, two amino-terminal fibr inopeptides A (FPAs) are cleaved by thrombin from each molecule. Durin g early phases of conversion, fibrin intermediates lacking one of two FPAs (des A fibrin) are produced, the level of which depends on whethe r the FPA cleavage sequence from each molecule is random or concerted. Random cleavage of FPA would produce higher levels of des A fibrin at any thrombin concentration than would concerted cleavage, and the lev el of this intermediate product would have an important effect on the ultimate structure of the fibrin clot. Because evidence bearing on thi s subject is conflicting, we carried out experiments to assess the FPA release sequence from fibrinogen by thrombin or by an FPA-cleaving sn ake venom enzyme, Atroxin. At timed intervals the enzymatic reaction w ets terminated by precipitation with trichloroacetic acid, and the pre cipitate was then treated with cyanogen bromide to produce a dimeric a mino-terminal fragment, These disulfide-linked amino-terminal fragment s of fibrinogen, containing both, one, or neither FPA, were then separ ated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their distribution quantified by densitometry, The rates of cleavage o f the first FPA, k(1), and of the second FPA, k(2), were computed by f itting the data to equations for a consecutive chemical reaction. This analysis indicated that cleavage by either enzyme resulted in substan tial amounts of des A fibrin intermediates. The ratio of the cleavage rates (k(2)/k(1)) was higher for thrombin (1.2 +/- 0.3) than it was fo r Atroxin (0.7 +/- 0.2) but indicates in both cases that the release r ate of the second FPA is nearly the same as that of the first FPA. The se findings lead us to conclude that the process of FPA release from f ibrinogen by thrombin or Atroxin is random rather than concerted.