Fibrin polymerizes through the interaction of sites exposed by the thrombin-mediated cleavage of fibrinopeptides in the central E region of the protein and complementary sites near the ends of the molecules, open in the D regions of both fibrinogen and fibrin. A preparation of fragment E, containing the central domain and part of the coiled-coil regions of fibrin, was used in mixtures with fibrinogen in this electron microscopy study to investigate the formation of fibrillar structures. At short times, linearly ordered oligomers of fibrinogen were observed with an additional mass of E fragments at the end-to-end junctions. At later times, long f lexible polymers made up of 30 or more fibrinogen and fragment E units, with a tendency for lateral aggregation and tangle formation, were seen. These singlestranded assemblies could be readily dissociated in dilute acetic acid into their fibrinogen and fragment E components. However, if the aggregates were treated with factor XIIIa so that all ␥ chains became ligated by N (␥-glutamyl)lysine linkages, the polymers could no longer be taken apart. Because the only ␥ chains in the preparation are present in the fibrinogen molecules interacting end-to-end, the findings show that the factor XIIIa-induced cross-linking of ␥ chains in the clotting of fibrinogen or fibrin must occur between molecules that are longitudinal (or end-to-end) rather than transverse (or half-staggered).Fibrinogen, which is made up of three pairs of polypeptide chains (A␣B␤␥) 2 , is about 47 nm long and has a molecular mass of 340 kDa (1-6). The amino-terminal ends of all three pairs of chains are joined together by disulfide bonds in the central region of the molecule. The carboxyl-terminal ends of the B␤ chains contain the proximal end regions, and the carboxyl-terminal ends of the ␥ chain contain the distal end regions (7). Each carboxyl-terminal A␣ chain consists of a globular ␣C domain that associates with the central region and a connector to the distal molecular end (8, 9). The central and end regions are connected by a three-chain ␣-helical coiledcoil. The overall shape of the molecule has been determined by coordinated x-ray crystallography and cryoelectron microscopy (10). Recently, x-ray crystallographic studies have revealed atomic resolution structures of the carboxyl-terminal ␥ chain and the end regions of the molecule (11, 12).The particular bonds of fibrin(ogen) cleaved by plasmin and the products of such proteolysis have been characterized (13,14). The carboxyl-terminal segment of the A␣ chain and the amino-terminal segment of the B␤ chain are removed first, followed by splitting of all three chains in the middle of the coiled-coil to yield fragment D, which includes the end domains and distal coiled-coil, plus fragment Y, which contains the remainder of the molecule. Fragment Y can then be split to yield another fragment D and fragment E, which contains the central domain and both proximal portions of coiled-coil (Fig. 1A).Because these different fragments contain distinctly d...