FXIIIa-mediated isopeptide gamma-gamma bonds are produced between gamma polypeptide chains of adjacent monomeric fibrin. Despite the use of the different methodological approaches there are apparently conflicting ideas regarding the orientation of gamma-gamma bonds. To identify the orientation of these bonds a novel approach has been applied. It was based on self-assembly of soluble cross-linked fibrin protofibrils ongoing in the urea solution of moderate concentrations followed by dissociation of protofibrils in the conditions of increasing urea concentration. The oligomers were composed of monomeric desA fibrin molecules created by cleavage of the fibrinopeptides A from fibrinogen molecules with thrombin-like enzyme, reptilase. The results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated an emergence of the double-stranded rod-like fibrin protofibrils. For the first time, the protofibrils are proved to exhibit an ability to dissociate under increasing urea concentration to yield singlestranded structures. Since no accumulation of alpha polymers has been found the covalent structure of soluble single-stranded fibrin oligomers is entirely brought about by gamma-gamma bonds. The results of this study provide an extra evidence to support the model of the longitudinal gamma-gamma bonds that form between the gamma chains end-to-end within the same strand of a protofibril. (C) 2015 Elsevier Inc. All rights reserved.