HL-K6-3512) as well as by grants (HL-02212 and HL-16346) from the National Institutes of Health. A Fulbright Travelling Fellowship to C. G. C. is gratefully acknowledged.

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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 *

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