David A. Meh

The dimeric Aα chain composition of dysfibrinogenemic molecules with mutations at Aα 16

Abstract In the last stage of fibrinogen synthesis, two Aα-Bβ-γ half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aα16 R→H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA ★ , FPA ★ ) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aα16 R→H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R→C (four) or R→H (three) Aα16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot (‘N-DSK’) cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve ‘N-DSK’ components with two FPA ★ s (N-DSK homodimer), one FPA ★ (des A N-DSK heterodimer), or no FPAs (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aα chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aα chain are produced, both Aα chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.

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 the amino terminal end of a dimeric fibrinogen-derived substrate by thrombin or batroxobins, we studied their effects on plasmic fragment E1, a core fragment from the central domain of fibrinogen containing both A alpha chain fibrinopeptide A (FPA) sequences. Isoelectric focussing (IEF) was employed as a means of resolving des A-fragment E1, from which one FPA had been cleaved, from des AA-fragment E1 resulting from the loss of both FPAs. Using densitometric gel scanning for quantification 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 of the first fibrinopeptide A, (FPA1) or the second, (FPA2) from fragment E1, with either thrombin or batroxobin (k2:k1 ratios of 1.10 +/- 0.42, 1.34 +/- 0.26 respectively). Atroxin released FPA2 more slowly than FPA1 with a k2:k1 ratio of 0.34 +/- 0.1. Th finding that the cleavage of FPA2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombin cleavage of FPA2 may be cooperative in nature. However, the cooperativity in the cleavage sequence is insufficient to markedly suppress the evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.