Synthesis and radioimmunoassay of canine fibrinopeptide A

Canine fibrinopeptide A, the 16 residue NH₂-terminal segment of the Aα chain of canine fibrinogen released by thrombin proteolysis and its NH₂-tyrosyl analogue, were synthesized by stepwise solid-phase syntheses. Following cleavage, the synthetic peptides were purified by ion-exchange chromatography, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis and Edman degradation analysis. Rabbits immunized with carbodiimide-coupled synthetic canine fibrinopeptide A-bovine albumin conjugates produced antibodies which bound up to 70% of available counts using ¹²⁵I-N-tyrosyl canine fibrinopeptide A as tracer. Quantitative displacement of bound tracer could be effected by both synthetic canine fibrinopeptide A and clot supernates prepared from purified canine fibrinogen. Fifty percent displacement of radiolabelled tracer could be achieved with 1.1 picomole of peptide antigen. Antiserum to canine fibrinopeptide A was about 50-fold less sensitive on a molar basis to canine fibrinogen as compared to the free peptide in solution. This suggests that antigenic determinants on the canine fibrinopeptide are altered by attachment of the peptide to its parent molecule. The molar reactivity of human fibrinopeptide A was about 25% in comparison with canine fibrinopeptide A, indicating that both the human and canine peptides share some antigenic determinants. Using this assay, fibrinopeptide A immunoreactivity was measured in canine plasma following removal of fibrinogen by ethanol precipitation and dialysis. The mean fibrinopeptide A level 4 normal dogs (18 determinations) was found to be 1.5±0.98 pmols/ml anticoagulated plasma.