Gly-Pro-Arg-Pro modifies the glutamine residues in the α- and γ-chains of fibrinogen: inhibition of transglutaminase cross-linking

Abstract

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the ϵ-amino group of lysine and the γ-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl- l-prolyl- l-arginyl- l-proline (GPRP), a terapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the α- and γ-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the γ-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polyermized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [ 3H]putrescine and [ 14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [ 3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [ 14C]putrescine incorporation into the α- and γ-chains of fibrinogen without inhibiting β-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [ 3H]putrescine into both fibrinogen and Fragment D1, without modifying [ 3H]putrescine incorporation into casein. GPRP also inhibited intermolecular α-α-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the α-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polyermization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the α- and γ-chains of fibrinogen.