Confirmation of Mendelian properties of heterodimeric fibrinogen molecules in a heterozygotic dysfibrinogenemia, "fibrinogen amarillo," using GPRphoresis to differentiate SemiFibrin molecules from fibrinogen and fibrin

The fibrinogen molecule consists of two sets of A alpha, B beta, and gamma chains assembled into a bilateral disulfide linked (A alpha, B beta gamma)(2) structure. Cleavage of the two A-fibrinopeptides (FPA, A alpha1-16) from normal A alpha chains with arginine at position 16 (R-FPA) by thrombin or the venom enzyme atroxin transforms fibrinogen into self-aggregating fibrin monomers (alpha, B beta, gamma)(2). Mutant A alpha 16R-->H fibrinopeptide (H-FPA) cannot be cleaved from fibrinogen by atroxin. Many studies on heterozygous dysfibrinogenemias with this mutation suggested that incorporation of the mutant chains into the molecules was ordered in a manner yielding only (1) homodimeric normal (RFPARFPA) atroxin-coagulable molecules and (2) homodimeric abnormal (HFPAHFPA) atroxin-incoagulable molecules in equal quantities. Although heterodimeric molecules (RFPAHFPA) could not be found in studies on the intact protein, Meh et al. demonstrated their existence by showing that CNBr digests of fibrinogens from atroxin-treated A alpha 16R-->H heterozygotic dysfibrinogenemias consistently yielded N-terminal fragments (NDSKs) with partially resolved electrophoretic bands predominantly in between the NDSKs of fibrinogen and cr-fibrin. An opportunity to confirm and better quantify the heterodimers arose with the recent development of a method (GPRphoresis) for identifying molecules lacking only one FPA, which is applied here in study of a newly presenting case of an A alpha 16R-->H dysfibrinogenemia, "fibrinogen Amarillo." GPRphoresis uses electrophoretic shifts, staged with GPRP-NH2 to separate the self-aggregating fibrin monomers lacking both FPAs from weakly aggregating "semifibrin" molecules lacking one FPA An antifibrin alpha 17-23 antibody is used to measure and differentiate the semifibrin from fibrinogen with FPA fury intact. Applying GPRphoresis to atroxin digests of fibrinogen Amarillo clearly demonstrated RFPARFPA, RFPAHFPA, and HFPAHFPA molecules in nearly perfect Mendelian 1:2:1 proportions. In turn, the high levels of the semifibrin in the terminal atroxin digests provide genetic phenotypic evidence supporting fidelity of the GPRphoresis method. (C) 2001 Elsevier Science Ltd. All rights reserved.