The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its ␣ chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIII a -catalyzed end-to-end ligation of the ␥ chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIII a reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of ␣ chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.The clotting of fibrinogen in human plasma is regulated by a remarkable series of biochemical controls (for a recent summary, see ref. 1). The key for unlocking the self-assembly potential of fibrinogen in its conversion to fibrin is the limited proteolytic attack by thrombin that, through removal of the N-terminal fibrinopeptide moieties (2, 3), generates a new set of Gly end groups (4, 5). The short sequence of amino acids including and following the newly exposed N-terminal Gly residues of the two ␣ chains of fibrin (6, 7) seems to be the necessary ligand for causing the noncovalent, reversible aggregation of fibrin into a clot (5,8,9). Gly-Pro-Arg-Pro, which resembles the natural Gly-Pro-Arg-Val sequences (knobs) in the fibrinopeptide A-denuded central E domain of fibrin, can prevent clot formation (10). The tetrapeptide blocks the polymerization pockets or holes located in the ␥ chains at the two D end domains of the protein (11-16). It is important to bear in mind that these polymerization pockets are present not only in fibrin but also in the parent fibrinogen molecule, i.e., thrombin action is not required for opening them for Gly-ProArg-Pro to bind. Thus, the two N-terminal knobs in the central E domain of the protein unmasked by thrombin seem to act as a bifunctional ligand for the end-to-end, noncovalent assembly of fibrin units into the well known, half-staggered, double array of protofibrils seen in the electron microscope (17, 18). This brings together each E domain with the D domains of two other molecules.The assembly process, as organized by the thrombinmodified E domains, imparts a major advantage for the next enzymatic phase of the coagulation cascade, the factor XIII acatalyzed covalent stabilization of fibrin filaments by N (␥-glutamyl)lysine crosslinks. Fibrinogen in solution is about an order of magnitude weaker substrate for factor XIII a than clotted fibrin (19,20), which, from the physiological point of view, is obviously an important feed-forward regulatory con...