Abstract

The product of thrombin digestion of fibrinogen, that is fibrin, binds to thrombin with high specificity. The latter interacts 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. Binding of thrombin to fibrinogen involves sequences of both Aα and Bβ chain in the fibrinogen E domain. This 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 γ chain variant, termed γ′, arising from an alternative mRNA splicing, 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 (408VRPEHPAETEYDSLYPEDDL427), rich of acidic residues. The γ′ chain, mainly heterodimerizes in the fibrinogen molecule with the more abundant γA chain, thus generating the γA/γ′ dimers (about 10% of the total γ chain content). The different expression of γ′ chain has been variably associated with both venous and arterial thrombosis. Previous genetic studies showed that the fibrinogen γ-H2 haplotype is characterized by a reduced fibrinogen γ′ levels. This haplotype is associated with a significantly increased risk for venous thrombosis. At variance with venous thromboembolism, the effect of altered expression of γ′ chain on arterial thrombosis remains largely elusive and still debated. Biochemical studies showed that γ′ chains bind to α-thrombin with high affinity and that the 408-427 region of γ′ chain binds to the anion binding exosite (ABE)-II of thrombin. We can speculate that the fibrinogen γ′ chain, through its ability to bind to thrombin, might enhance the amount of clot-bound thrombin. The latter may represent a storage pool of the enzyme, facilitating arterial thrombus formation via platelet activation. In this study, we investigated the effect of the fibrinogen γ′ and of its 20-amino acid-insertion peptide on the thrombin interaction with the platelet receptors glycoprotein (Gp) Ibα, and protease-activated receptors PAR-1 and PAR-4, involved in platelet activation. Fragment D was used as the best surrogate to selectively study the high affinity binding site for thrombin in γ chain in a conformation similar to that present in the native fibrinogen molecule. Both synthetic γ′ peptide and fragment D*, containing the elongated γ′ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC50 values of 42±3.5 μM and 0.47±0.03 μM, respectively. Solid phase binding and titration spectrofluorimetric assays analyzing the tryptophan fluorescence of thrombin showed that both fragment D* and the synthetic γ′ peptide specifically bind to ABE-II of thrombin and competitively inhibit the thrombin interaction with GpIbα with a mean Ki≈0.5 μM and ≈35 μM, respectively. HPLC assays showed that both the γ′ chain allosterically inhibited thrombin cleavage of the synthetic PAR-1 38-60 peptide, with an IC50 value of 45 μM, in good agreement with the Kd value of thrombin binding to γ′ chain derived from fluorimetric titrations. Likewise, flow cytometry studies showed that the hydrolysis of native PAR-1 molecules on intact platelets was progressively inhibited by increasing concentrations of both fragment D* and γ′ peptide, while PAR-4 cleavage was unaffected. In summary, fibrinogen γ′ chain binds with high affinity to thrombin and inhibits with combined mechanisms the thrombin-induced platelet activation. The role of different expression of γ′ chain in circulating fibrinogen may variably influence the thrombotic and hemorrhagic manifestations in different clinical settings or different phases of thrombotic diseases.

Disclosures: No relevant conflicts of interest to declare.

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