The expression of the elongated fibrinogen ␥ chain, termed ␥, derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how ␥ chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ib␣ (GpIb␣), protease-activated receptor (PAR) 1, and PAR4. Both synthetic ␥ peptide and fibrinogen fragment D*, containing the elongated ␥ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC 50 values of 42 ؎ 3.5 and 0.47 ؎ 0.03 M, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic ␥ peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIb␣ with a mean K i ≈ 0.5 and ≈35 M, respectively. Both these ␥ chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen ␥ chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.Fibrinogen is a key molecule in both primary and secondary hemostasis, because of its role in forming the platelet plug by connecting activated platelets and in forming plasma fibrin clot upon thrombin cleavage. Fibrinogen consists of two symmetric half-molecules, each containing a set of three different polypeptide chains termed A␣, B, and ␥. The latter contains several sites that interact with different ligands such as other fibrin(ogen) molecules, coagulation enzymes, growth factors, and integrins (1). The product of thrombin digestion of fibrinogen, i.e. fibrin, binds with a considerable specificity thrombin, so that in the early studies fibrin was termed antithrombin I (2). Thrombin has a divalent interaction with two classes of binding sites on fibrin, one of low affinity in the E domain and the other of high affinity in the D domain of fibrin(ogen) molecules (3). Binding of thrombin to fibrinogen involves sequences of both A␣ and B chain, which contain recognition sites in the fibrinogen E domain. These recognition sites are still able to interact with thrombin after cleavage of fibrinopeptide A and B and form the low affinity binding site for the enzyme. The D domains contain a ␥ chain variant, termed ␥Ј, arising from an alternative mRNA splicing (4), resulting in an elongated chain composed of 427 instead of 411 residues. The inserted region at the C terminus is composed of 20 amino acids ( 408 VRPEH-PAETEYDSLYPEDDL 427 ), rich of acidic side chains and two sulfate anions linked to Tyr 418 and Tyr 422 (5). The elongated ␥ chain, termed ␥Ј, mainly heterodimerizes in the fibrinogen molecule with the more abundant ␥A chain, thus generating ...