Titration of the Acceptor Cross‐linking Sites in Fibrin*

Abstract

In terms of physiological function, the cross-linking sites of fibrin constitute the active sites of this large protein molecule. They provide the y-glutamyl-clysine peptide bridges between the fibrin units of a fully developed, elastic clot structure.' According to the nucleophilic displacement model shown in FIGURE 1, they can be further divided into amine (electron donor or donor, for short) and carbonyl (or acceptor) subsites. Proper reactivity of these sites may literally mean the difference between life and death. We have recently completed studies on a patient in whose blood the clotting time as well as the activity of the fibrin stabilizing factor system was normal; yet, severe bleeding occurred because an antibody selectively directed against fibrin prevented enzymatic cross-linking. Evaluation of the reactivity, number and submolecular localization of crosslinking sites in fibrin is a topic of great interest. Enzymatic substitution in the amine-acceptor groups forms the subject of this paper. Acceptor Site LabelingThe idea of covalently introducing specific tracers into the acceptor sites of fibrin arose from the discovery 2. I . that certain synthetic amines inhibited the enzymatic cross-linking of fibrin. If amines-inhibitory in rnillimolar (or lesser) concentrations-can be shown to become rapidly incorporated into fibrin, the conclusion would be justified that specific blocking in the acceptor cross-linking sites took place (FIGURE 1 ) . Indeed, such specific amine incorporation would constitute one of the best arguments in favor of the enzymatic nature of the cross-linking process. It would also be proof of an acylenzyme intermediate formed between fibrin and fibrinoligase on the cross-linking pathway (FIGURE 2 ) .A variety of the amine inhibitors was shown to become rapidly incorporated into fibrin. The finding that the relative ordering of the reciprocal apparent Michaelis constants for amine incorporation was the same as that of the inhibitory potencies measured by the clot solubility bioassay was further confirmation that specific labeling in the acceptor sites was achieved. Thus, these amines really act as substitute or pseudodonors in the enzymatic reaction with fibrin. Hydroxylamine and hydrazine could be used as chemical markers.'; (It will be recalled that the fibrin hydroxamate was employed in the experiments providing the first proof that y-glutamyl residues served as acceptors in fibrin.;) Glycine ethyl ester 'i.were used as C" comand isonicotinic acid hydrazide *

Studies on the structural abnormality of fibrinogen Paris I.

“…Only one glutaminyl residue on the 'y-chain participates in cross-linking (36), although several are present in the COOH-terminal region. Despite the fact that 8-chains of fibrin do not participate in cross-linking (37,38,48) and fail to incorporate monodansylcadaverine in the presence of Factor XIIIa (27), S-sulfo-,8-chains can undergo considerable cross-linking (49).…”

“…The final concentration of the components of the cross-linking mixture were: thrombin 0.4 U. S. U/ml, Factor XIII, 36 Loewy (25) Flutorescent amine incorporation into fibrin. Factor XIIIacatalyzed incorporation of the fluorescent amine, dansylcadaverine (dansylcadaverine sulfate, a gift from Dr. Rolf Huseby), into acceptor sites on fibrin (27) was carried out under the general conditions for cross-linking of fibrin described above. The final concentration of Factor XIII was 4 Loewy (25) U/ml; monodansylcadaverine, 9 mM.…”

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines

“…With an apparent Michaelis constant of about 1 x 10-4M, this compound has already been used successfully for such diverse biological investigations as the quantitative study of hereditary deficiency of the enzyme precursor in plasma (Lorand etal., 1970), titration of the order and extent of reactivity of the cross-linking sites in the y and a chains of fibrin (Lorand et al, 1972b), and activity staining of the enzyme on disc-gel electrophoretograms (Lorand et al, 1974).…”

A gamma methionine-310 to threonine substitution and consequent N-glycosylation at gamma asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi.

“…They include proteolytic cleavage of fibrinopeptides A and B by thrombin, polymerization of fibrin monomers to form fibrin gels, and intermolecular cross-linking mediated by activated blood coagulation factor XIII (Factor XIIIa) (4,5). Although polymerization and gelation of fibrin have been extensively studied (1)(2)(3), little is known about the mechanisms involved in the reactions.…”

“…The carboxy-terminal region of the fibrinogen y chain has been shown to participate in various functions of fibrinogen including fibrin polymerization (1)(2)(3)(4)(5)(6)(7).…”