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

Stefano Lancellotti,Sergio Rutella,Vincenzo De Filippis,Nicola Pozzi,Bianca Rocca,Raimondo De Cristofaro

doi:10.1074/jbc.m803659200

Abstract

The expression of the elongated fibrinogen gamma chain, termed gamma', 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 gamma' chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibalpha (GpIbalpha), protease-activated receptor (PAR) 1, and PAR4. Both synthetic gamma' peptide and fibrinogen fragment D*, containing the elongated gamma' chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC(50) values of 42+/-3.5 and 0.47+/-0.03 microm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic gamma' peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbalpha with a mean K(i) approximately 0.5 and approximately 35 microm, respectively. Both these gamma' 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 gamma' 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.

Highlights

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
This study investigated whether and how ␥؅ chain binding to anion-binding exosite (ABE)-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ib␣ (GpIb␣), protease-activated receptor (PAR) 1, and PAR4
This study showed for the first time that the fibrinogen sequence 408 – 427 in the elongated ␥Ј chain inhibits the thrombin-induced aggregation of platelets through a combined mechanism, impairing both GpIb␣ and PAR1 interactions

Summary

Introduction

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. 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. Fragment D was used as the best surrogate to selectively study the high affinity binding site for thrombin in ␥ chain in a conformation similar to that present in the native fibrinogen molecule and suitable for thrombin binding studies This experimental approach was aimed at assessing whether ␥Ј chain can affect platelet activation by inhibiting competitively the interaction between the enzyme and GpIb␣ and by acting as an allosteric effector on PAR hydrolysis by thrombin. The obtained results may shed light on the possible role of fibrinogen ␥Ј chain on the thrombin-induced platelet activation and on possible implications on both anti-thrombotic and pro-thrombotic properties of fibrinogen in arterial circulation, where platelets play a central role in thrombo-hemorrhagic syndromes

Methods

Findings

Conclusion