COMPARISON OF THE SEQUENCE OF FIBRINOPEPTIDE-A CLEAVAGE FROM FIBRINOGEN FRAGMENT-E BY THROMBIN, ATROXIN, OR BATROXOBIN

In order to investigate the sequence of fibrinopeptide release from th e amino terminal end of a dimeric fibrinogen-derived substrate by thro mbin or batroxobins, we studied their effects on plasmic fragment E1, a core fragment from the central domain of fibrinogen containing both Aalpha chain fibrinopeptide A (FPA) sequences. Isoelectric focussing ( IEF) was employed as a means of resolving des A-fragment E1, from whic h one FPA had been cleaved, from des AA-fragment E1 resulting from the loss of both FPA's. Using densitometric gel scanning for quantificati on of the levels of intact fragment E1, des A-fragment E1, and des AA- fragment E1, in mixtures incubated with enzyme for various periods of time, we found similar catalytic rate constants (k1, k2) for release o f the first fibrinopeptide A, (FPA1) or the second, (FPA2) from fragme nt E1, with either thrombin or batroxobin (k2 : k1 ratios of 1.10 +/- 0.42, 1.34 +/- 0.26 respectively). Atroxin released FPA2 more slowly t han FPA1 with a k2 : k1 ratio of 0.34 +/- 0.1. Th finding that the cle avage of FPA2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombi n cleavage of FPA2 may be cooperative in nature. However, the cooperat ivity in the cleavage sequence is insufficient to markedly suppress th e evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.