Confirmation of Mendelian properties of heterodimeric fibrinogen moleculesin 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 heter ozygous dysfibrinogenemias with this mutation suggested that incorporation of the mutant chains into the molecules was ordered in a manner yielding on ly (1) homodimeric normal (RFPARFPA) atroxin-coagulable molecules and (2) h omodimeric abnormal (HFPAHFPA) atroxin-incoagulable molecules in equal quan tities. Although heterodimeric molecules (RFPAHFPA) could not be found in s tudies on the intact protein, Meh et al. demonstrated their existence by sh owing 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 bet ween the NDSKs of fibrinogen and cr-fibrin. An opportunity to confirm and b etter quantify the heterodimers arose with the recent development of a meth od (GPRphoresis) for identifying molecules lacking only one FPA, which is a pplied here in study of a newly presenting case of an A alpha 16R-->H dysfi brinogenemia, "fibrinogen Amarillo." GPRphoresis uses electrophoretic shift s, staged with GPRP-NH2 to separate the self-aggregating fibrin monomers la cking 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, RF PAHFPA, and HFPAHFPA molecules in nearly perfect Mendelian 1:2:1 proportion s. In turn, the high levels of the semifibrin in the terminal atroxin diges ts provide genetic phenotypic evidence supporting fidelity of the GPRphores is method. (C) 2001 Elsevier Science Ltd. All rights reserved.