Fibrinogen Hershey IV: A Novel Dysfibrinogen with a γ V411I Mutation in the Integrin αIibβ3 Binding Site.

The C-terminal segment of the fibrinogen γ chain plays a crucial role in platelet aggregation via its interaction with the platelet receptor αIIbβ3. It is well established that the last four amino acids of the γ chain (408 to 411) are critical for this function, as mutations or deletions of this region abrogate fibrinogen’s ability to bind αIIbβ3. We describe here the first naturally occurring fibrinogen mutation affecting the C-terminal region of the γ chain and investigate its effects on platelet interactions. The proband, a 49-year-old woman, was diagnosed with dysfibrinogenemia based on a prolonged thrombin time and low fibrinogen activity (55 mg/dL). Her bleeding history was significant for menorrhagia and one episode of post-operative hemorrhage. DNA sequencing of the fibrinogen genes demonstrated heterozygosity for two mutations, γ R275C and γ V411I. The latter γ V411I mutation represents a novel mutation affecting the C-terminal amino acid of the γ chain. We hypothesized that this mutation would decrease fibrinogen’s affinity for the platelet receptor αIIbβ3. In order to isolate the effects of this mutation on fibrinogen-platelet binding, γ 400-411 dodecapeptides were synthesized to mimic the C-terminal γ chain sequence. One peptide contained the wild-type sequence ending in valine (γ 400-V411), and the second peptide incorporated the isoleucine mutation (γ 400-I411). Previous studies have demonstrated that the wild type γ 400-V411 dodecapeptide inhibits platelet aggregation by competing for fibrinogen binding to αIIbβ3. We performed platelet aggregation studies comparing inhibition of aggregation with the wild-type γ 400-V411 and the mutant γ 400-I411 peptides. Washed platelets were obtained from a normal donor, and platelet aggregation monitored using the agonist ADP. The IC₅₀ for the initial rate of aggregation with the γ 400-I411 peptide was 214 μM, compared to 133 μM with the wild-type peptide. We then examined the extent of aggregation in the presence of either wild-type or mutant peptide. Consistent with the previous results, total aggregation was lower with the wild-type peptide compared to the mutant peptide. The IC₅₀ for the γ 400-I411 peptide was 450 μM compared to 250 μM with the γ 400-V411 peptide. Overall, these findings suggest that the γ I411 mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available amount of wild-type fibrinogen may be sufficient to support platelet aggregation. The bleeding diathesis observed for the proband could therefore reflect other factors, especially the γ R275C mutation.