Human Fibrinogen Heterogeneities

Abstract

The heterogeneity of human plasma fibrinogen manifested as differences in solubility reflects the presence of early catabolic intermediates, which are more soluble, have a longer thrombin-clotting time, and are of a lower molecular weight than the parent material from which they are formed. In this study the S-sulfo subunits from fibrinogen fractions of low (I-4) and high (I-8 and I-9) solubility were compared. Separation of Aα, Bβ, and γ chains was achieved by gradient elution chromatography on CM-cellulose. The tryptic peptide maps were characteristic for each type of chain. The only observable differences were in the maps of Aα chains; in those of I-8 and I-9 a considerable number of spots were absent or reduced relative to I-4. NH2-terminal analyses of isolated chains before and after thrombin treatment showed the characteristic residues of the Aα, Bβ, and γ chains, respectively. Thus the NH2-terminal portions of all chains of I-4, I-8, and I-9 were intact. Molecular weight estimation of subunit chains was made by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under conditions in which differences in mobility due to the presence or absence of A or B peptides were distinguishable. Electrophoresis in this medium before and after reptilase or thrombin treatment served for identification of the chains. The γ chains moved as a single band in this system (molecular weight, 50,700) and were indistinguishable from one another; the same was true of Bβ chains (molecular weight, 60,400). However, electrophoresis of Aα chains revealed the presence of 13 more-or-less distinct bands (numbered in order of increasing anodal mobility), of which all but two (Band 3, a Bβ contaminant and Band 5, a γ chain contaminant) were shown to be intact Aα chain or COOH-terminally degraded Aα remnants. The molecular weights ranged from that of intact Aα chain (70,900) to 15,400 (Band 13). The highest proportion of intact Aα chains was in I-4; the lowest was in I-9, which had virtually no detectable intact Aα chain. Conversely, I-9 contained the highest proportion of Aα remnant chains; I-4, the lowest. These data suggested that cleavage at any or all of at least 10 sites along the Aα chain resulted in the formation of early catabolic intermediates. Comparisons with highly clottable derivative fractions I-8D and I-9D produced by plasmin treatment of I-4 in vitro showed that they were quite similar to their respective plasma counterparts (I-8 and I-9) in that the γ and Bβ chains appeared to be intact, but few or no intact Aα chains were present. These observations thus strengthen the notion that it is plasmin which catalyzes the formation of early intermediates in vivo. Examination of Fragment ‘X', another fibrinogen derivative produced by plasmin digestion, revealed considerable depletion of intact Bβ as well as of Aα chains, indicating that Fragment ‘X' was more extensively degraded than I-8D or I-9D.