Fibrinogen Hillsborough: a novel γGly309Asp dysfibrinogen with impaired clotting

Abstract

We present a novel γ-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed a γ-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal γ chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)→GAC (Asp) at codon 309 of the γ chain gene. This Gly→ Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue γ309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. γGly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting γD domains of condensing fibrin monomers during polymerization.