Investigation of a number of dysfibrinogenaemias and their impact on fibrinogen function

Abstract

An understanding of fibrinogen activation and polymer formation has been derived from the study of natural fibrinogen variants (dysfibrinogenaemias). This thesis details the investigation of a number of dysfibrinogenaemias and how they impact on fibrinogen function.

Fibrinogen Lincoln results from translation of an Aα chain gene containing a 13 bp deletion (nt 4758-4770), which causes a frameshift and translates as four new amino acids before termination at a stop codon. Turbidity curves of fibrin monomers revealed that polymerisation was delayed and the final turbidity was half that of the control. Decreased final turbidity suggested that the fibrin fibres formed were of a thinner diameter than normal. Scanning electron micrographs supported these findings revealing a clot network composed of thinner fibres and smaller pores. The decreased pore size of the fibrin network was substantiated by permeation data, which revealed that clots were 25 fold less permeable.

Fibrinogen Otago was found in a woman diagnosed with afibrinogenaemia. She was later found to have a functional fibrinogen concentration of 0.06 mg/ml. Plasma protein electrophoresis showed an absence of normal fibrinogen and a novel 270 kDa component, which was confirmed as fibrinogen by immunofixation. Reducing SDS-PAGE showed that the expected 67 kDa Aα chain was missing and replaced by a 30 kDa band. This aberrant chain was not detected by the monoclonal antibody F-103. Cycle sequencing of the DNA encoding the F-103 epitope revealed the homozygous insertion of cytosine within a block of four cytosines at position 4133 bp of the Aα gene sequence. The new sequence translates with three new amino acids before termination at a stop codon. Turbidity studies of fibrin monomers revealed delayed polymerisation, with a final turbidity higher than the control. Although this suggested that the final fibre diameter was larger than normal, scanning electron micrographs revealed clots composed of thinner fibres and smaller pores.

Fibrinogen Kaiserslautern was found in an adult woman with prolonged thrombin and reptilase times. Analysis of five other family members also revealed prolonged, but variable, thrombin and reptilase times. Reducing SDS-PAGE revealed an additional protein band, in all family members, which migrated immediately below the normal Bβ band. Western blotting confirmed that this band was a γ chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide. Partial acid digestion defined the location of the new oligosaccharide to the C-terminus of the γ chain. Cycle sequencing of this region revealed a single base substitution altering the normal Lys (AAG) codon to Asn (AAT), producing a new Asn-X-Thr glycosylation site. The patient and one other family member were homozygous for this mutation while the remaining four family members were heterozygous. The polymerisation of fibrin monomers from the patient was abnormal, however, it was normalised by the removal of sialic acid residues with neuraminidase. This suggested that the polymerisation defect was primarily caused by negatively charged sialic acid residues present on the oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site was unaffected by the oligosaccharide side chain or negatively charged sialic acid residues. Factor XIIIa catalysed crosslinking of the fibrinogen was delayed whereas the crosslinking of fibrin was normal. Scanning electron micrographs of plasma from the patient revealed clots which were similar to the control.

A number of mutations were detected in the N-terminal of the Aα chain. The Aα 16 Arg to His mutation was characterised using PCR and restriction digestion. The mutation in Fibrinogen Oslo IV (Aα 19 Arg→Gly) was found by DNA sequencing of exon II of the Aα chain gene and confirmed by N-terminal protein sequencing. This is the third reported case of this mutation. Protein sequencing of fibrin from Otago II detected two amino acid sequences. The first sequence was the normal Gly¹⁷-Pro-Arg-Val-Val... and the second was Asp²⁰-Val-Glu-Arg ... This suggested that the patient was heterozygous for a point mutation, which alters Aα 20 valine to aspartate. This mutation creates a new proprotein processing site. These three variants illustrate different mechanisms by which point mutations can affect protein structure and function.