Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

Abstract

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 ...

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

“…By contrast, GpIb␣ binding can promote hydrolysis of PAR1 on platelets (44). Similar effects were not observed with a comparable PAR4 peptide substrate sequence (11). The Fbg ␥Ј peptide may be propagating an effect over to the active site region that has greater influence on the PAR1 LDPR sequence than the PAR4 PAPR sequence.…”

“…Examining the Influences of Dual Occupancy-Our studies provide the first examples of probing ligand binding to both anion-binding exosites using HDX. In complementary fluorescence-based approaches, dual occupancies with Fbg ␥Ј peptide or GpIb␣ at ABE II and then Hirudin (54 -65) or DNA aptamer at ABE I have been observed (10,11). The HDX studies provide the advantage of demonstrating that Hirudin(54 -65) focuses its effects on ABE I and then allowing Fbg ␥Ј or GpIb␣ peptides to target specific ABE II segments and influence other more distant regions (Fig.…”

“…Studies in this research area have often focused on fluorescence and/or surface plasmon resonance effects. Some reports indicate that thrombin can readily accommodate ligands at both exosites (7,10,11). With these examples, the conversion from a binary (enzyme-ligand) to a ternary (ligand 1-enzyme-ligand 2) complex does not lead to major changes in the K D value of either ligand.…”

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…[43][44][45][46] Recently, Lancellotti et al reported that the ␥Ј chain reduces thrombin-induced platelet aggregation through a combined mechanism. 47 They showed that the ␥Ј chain, either in fragment D or the synthetic ␥Ј peptide, hinders both thrombin binding to GpIb␣ and thrombin cleavage of PAR1 on platelets. Lovely et al showed similar results in a study using synthetic ␥Ј peptides.…”

Fibrinogen γ′ increases the sensitivity to activated protein C in normal and factor V Leiden plasma

“…12 Although this anticoagulant activity of fibrin(ogen), known as "antithrombin I," is still poorly understood, it has been recently shown that the interaction of the C-terminus of the fibrinogen g9 chain with thrombin exosite II inhibits thrombin-mediated activation of FVIII 13 and FV, 14 as well as platelets. 15,16 The physiological relevance of this anticoagulant mechanism is underscored by the two-to threefold increased risk of venous thrombosis associated with the common FGG H2 haplotype, which is associated with reduced levels of fibrinogen g9 without affecting total fibrinogen levels. There is an Inside Blood Commentary on this article in this issue.…”