FIBRINOGEN KAISERSLAUTERN (GAMMA 380 LYS TO ASN) - A NEW GLYCOSYLATEDFIBRINOGEN VARIANT WITH DELAYED POLYMERIZATION

An adult woman diagnosed with cerebral thrombosis following a caesarea n section was found to have severely prolonged thrombin and reptilase times, Five other family members also had prolonged, but variable, thr ombin and reptilase times. Analysis of purified fibrinogen on reducing SDS-PAGE revealed an additional band, in all family members, which mi grated immediately below the normal B beta band, Western blotting indi cated that this band was a gamma chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide side chai n. Partial acid hydrolysis localized the new oligosaccharide to the C- terminus of the gamma chain, Amplification of this region by PCR and s ubsequent DNA sequencing demonstrated a single base substitution alter ing the normal 380 Lys (AAG) codon to Asn (AAT), producing a new Asn-L ys-Thr glycosylation site, The propositus and one other family member were homozygous for this mutation but the remaining four family member s were heterozygous. The polymerization of purified fibrin monomers fr om the propositus was grossly abnormal; however, the polymerization cu rve was almost normalized by the removal of terminal sialic acid resid ues. This suggests that the polymerization defect was primarily caused by additional negatively charged sialic acid residues present on the new oligosaccharide. Further analysis of the D domain of purified fibr inogen established that calcium binding to the high affinity site rema ined unaffected by the bulky carbohydrate side chain or negatively cha rged sialic acid residues.