Comparison of human plasma fibrinogen subfractions and early plasmic fibrinogen derivatives.

Abstract Human plasma fibrinogen Subfractions I-8 and I-9 have previously been shown to consist of catabolic intermediates of fibrinogen characterized by the lack of COOH-terminal portions of Aα chains. The present study was undertaken to determine the structural relationships between these catabolites and core derivatives formed in vitro by the action of plasmin. First, these fractions were compared with plasma Subfraction I-4, which is representative of "native" fibrinogen, by polyacrylamide gel electrophoresis of unreduced samples in sodium dodecyl sulfate. Fraction I-4 resolved into two major bands, designated I and II (the higher the number the greater the anodal migration and the smaller the molecular size), and a trace component (Band IIIa). Fractions I-8 and I-9 exhibited four bands (IIIa, IV, V, and VI) of which Band IIIa was the major component and VI, the least abundant. Fractions I-8 and I-9 were also compared with subfractions isolated from stage 1 plasmic digests of Fraction I-4 (termed I-9Dn, where n = clottability of the digest from which the subfraction was obtained). An early plasmic subfraction, I-9D88, contained Bands IIIa (major component), V, and VI plus a minor amount of Band VII; a subfraction from a more advanced stage 1 digest (I-9D50) contained a faster migrating Band III (IIIb) and relatively increased amounts of Bands VI and VIII. Despite the fact that Fraction I-9D88 closely resembled its plasma counterpart (I-9) in clottability (96%), electrophoretic, chromatographic, and solubility behavior, several differences were apparent. Band IV was detected only in the plasma fractions; Band VII was present only in the plasmic derivatives. Each of the 10 core Aα chain remnants (viz., Aα/2, Aα/4, Aα/6 to Aα/13) identifiable by gel electrophoresis of reduced plasma fibrinogen subfractions can apparently be generated in vitro by plasmin digestion. However, whereas Aα/8 and Aα/9 were the most abundant of the small Aα/ remnants (mol wt g40,000) in Fractions I-8 and I-9, Aα/11 predominated in I-9D88. Moreover, Bβ chain degradation was somewhat more advanced in I-9D88 than in its plasma counterpart. The failure to demonstrate complete identity between I-9 and I-9D88 underscores the present uncertainty as to whether plasmin alone catalyzes the formation of circulating catabolites in vivo. Experiments involving gel slicing, reduction of the eluted protein, and re-examination by gel electrophoresis, plus related electrophoretic procedures and chromatographic analyses, yielded the following results. Formation of species migrating within Bands II, IIIa, IV, and V could be accounted for by the loss of COOH-terminal portions of the Aα chains; species migrating in Band IIIa or more anodally exhibited no intact Aα chains. Formation of Band IIIb (from species like those in Band IIIa) occurs by the loss of the NH2-terminal region of one Bβ chain; material within Bands VI and VII is associated not only with the removal of this region from the other Bβ chain but also with internal cleavages such as those eventually leading to the separation of Fragments D and E.