Direct Analysis of Plasma Fibrinogen-Derived Fibrinopeptides by High Performance Liquid Chromatography: Investigation of Aα-Chain N-Terminal Heterogeneity

Abstract

SummaryFibrinopeptide A (FPA), released from the fibrinogen Aa-chain by thrombin, can be resolved by high-performance liquid chromatography (HPLC) into three forms, the intact peptide (A), a modified peptide phosphorylated at the serine in position 3 (AP), and an N-terminally degraded form (AY). A new method has been developed, using HPLC, that allows direct measurement of the proportions of AP, A, and AY released by thrombin from fibrinogen in plasma samples of 200 ul or less. The method was used to examine variations in the proportions of AP and AY expressed as a % of total FPA in a number of patient and control groups. The mean percentages of AP and AY of plasma fibrinogen were found to be 21.7 and 14.2%, respectively, in normal laboratory controls. In older, apparently normal, individuals these figures were 27.0 and 15.5%, respectively. Cord plasma exhibited very high AP and slightly reduced AY levels (41.6 and 12.4%, respectively) compared with normal adults. Patients with liver failure had low AP levels and high AY levels (11.6 and 21.1%, respectively). Patients, recovering from major surgery or acute thrombotic stroke showed an acute-phase rise in fibrinogen level that was accompanied by an increase in AP and variable reduction in AY. Incubation of heparinized whole blood for 8 days in vitro demonstrated a gradual decrease in the proportion of AP and increase in AY of plasma fibrinogen. These results provide some support for the idea that an increased “aging” of fibrinogen in the circulation may result in a decrease in the AP content of fibrinogen accompanied by a more variable increase in AY.

Hydrodynamic characterization of recombinant human fibrinogen species

“…No densitometric analyses were conducted on the hrα251-FG sample, as a single Aα chain band was evident. Note that the computed values for the hrHMW-FG and hpHMW-FG Aα chain species differ slightly because, as will be shown next, the hrHMW-FG apparently completely lacks the phosphorylation on the FpA which is normally present in ~20% of the chains [37]. Note also that the Aα chain C-terminally degraded species are likely to be overestimated due to non-linearity effects in the Western blots staining, and for the confounding effect of baseline noise on the smaller peaks.…”

“…Instead, the degraded (desArgB lacking the C-terminal arginine residue, FpBY2 lacking the N-terminal pyroglutamic acid and glycine residues) and deamidated (N→D) forms of FpB [32] appears to be present in similar amounts in all species. While the absence of FpAP in hrHMW-FG could be due to the particular expression system used [27], the lack of FpAY in both recombinant species is likely due to in vivo processes of potential physiological significance [37]. …”

Should neurologists measure fibrinogen concentrations?

“…Thrombin cleaves the Aa and Bh chains to release fibrinopeptides A and B, respectively, from the amino-terminal ends. After the fibrinopeptides are released, the resulting fibrin monomers undergo polymerization to form an insoluble fibrin clot [3]. Fibrinogen also shows heterogeneity due to cleavage in the circulation, at the carboxyl termini of the polypeptide chains, giving rise to a series of molecules of varying size [4].…”

Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples

“…Intact fibrinogen is present only in plasma. In the conversion of plasma to serum, fibrinogen is proteolytically cleaved by thrombin to form fibrin and fibrinopeptide A [10]. In theory, it should be possible to distinguish between the intact and processed fibrinogen using a sandwich ELISA.…”