Activated coagulation factor XIII (FXIIIa) cross-links the ␥-chains of fibrin early in clot formation. Cross-linking of the ␣-chains occurs more slowly, leading to high molecular weight multimer formations that can also contain ␥-chains. To study the contribution of FXIIIa-induced ␥-chain cross-linking on fibrin structure and function, we created 2 recombinant fibrinogens (␥Q398N/Q399N/K406R and ␥K406R) that modify the ␥-chain crosslinking process. In ␥K406R, ␥-dimer crosslinks were absent, but FXIIIa produced a cross-linking pattern similar to that observed in tissue transglutaminase crosslinked fibrin(ogen) with mainly ␣-␥ crosslinks. In Q398N/Q399N/K406R, cross-links with any ␥-chain involvement were completely absent, and only ␣-chain crosslinking occurred. Upon cross-linking, recombinant normal fibrin yielded a 3.5-fold increase in stiffness, compared with a 2.5-fold increase by ␣-chain cross-linking alone (␥Q398N/Q399N/K406R). ␥K406R fibrin showed a 1.5-fold increase in stiffness after cross-linking. No major differences in clot morphology, polymerization, and lysis rates were observed, although fiber diameter was slightly lower in crosslinked normal fibrin relative to the variants. Our results show that ␥-chain crosslinking contributes significantly to clot stiffness, in particular through ␥-dimer formation; ␣-␥ hybrid cross-links had the smallest impact on clot stiffness. IntroductionAfter cross-linking by FXIIIa, fibrin becomes markedly more resistant to proteolytic and mechanical disruption. The introduction of N⑀-(␥-glutamyl)lysine isopeptide bonds between fibrin molecules within and between clot fibers has a remarkable effect on the rheologic properties of clots. [1][2][3][4] This stabilization of fibrin fibers leads to the formation of a rigid and elastic structure that is capable of stopping leakages in the circulatory system, whereas bleeding is a frequent problem without FXIIIa cross-linking. Of the 3 fibrinogen chains, only the ␣-and ␥-chains, but not the ␤-chains, become cross-linked by FXIIIa. During clot formation, at the early stages of polymerization, cross-linking occurs within emerging protofibrils between ␥K406 and ␥Q398 and/or ␥Q399, resulting in the formation of ␥-dimers. [5][6][7] Multiple cross-linking between fibrin ␣-chains results in the formation of ␣-polymers. 6 Over time, FXIIIa has been shown to generate cross-linked chain structures containing various combinations of ␣-and/or ␥-chains; mainly these are thought to be ␣ n , 8 but ␥ 3 , ␥ 4 , and hybrid ␣ p ␥ q (n, p, and q ϭ 1, 2, 3, respectively, and so forth) are also known to occur. 9,10 The effect of these different cross-linking formations on fibrin clot function has not been fully clarified. Previously, the increase of stiffness in clots has been attributed to ␣-chain cross-linking. 11 On the other hand, a recent study suggested that ␥-chain cross-linking played a major role in the determination of the viscoelastic properties of fibrin. 12 However, in these studies, chain-specific cross-linking was manipulated either by ...