NORMAL BINDING OF CALCIUM TO 5 FIBRINOGEN VARIANTS WITH MUTATIONS IN THE CARBOXY-TERMINAL PART OF THE GAMMA-CHAIN

Calcium ions are known to accelerate polymerization of fibrin monomers . Each of the two carboxy terminal domains of normal fibrinogen contai ns one high-affinity calcium binding site that seems to be situated cl ose to the polymerization site in the gamma-chain. Most hitherto descr ibed functionally defective fibrinogen variants showed impaired clot f ormation. Since the tightly bound calcium ions may influence the confo rmation of the polymerization site, the question arises whether the ab normal clotting of a dysfibrinogen might be due to defective calcium b inding. We investigated binding of calcium to fibrinogen and the effec t of calcium on the clotting properties of five heterozygous fibrinoge n variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinoge ns has one single amino acid substitution in the carboxyterminal part of the gamma-chain: fibrinogen Claro (gamma 275 Arg --> His), Milano V (gamma 275 Arg --> Cys), Milano I (gamma 330 Asp --> Val), Bern I (ga mma 337 Asn --> Lys), and Milano VII (gamma 358 Ser --> Cys). The shor test thrombin clotting time and the earliest onset of turbidity increa se were observed in fibrinogen gamma 358 Ser --> Cys; both parameters were little affected by calcium concentration. In the variant gamma 33 7 Asn --> Lys, the thrombin time was abnormally prolonged at 0.01 mM C a2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen gamma 330 Asp --> Val was barely i mproved at increasing calcium concentrations. Both variants with the s ubstitution of gamma 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and th e lowest turbidity of fibrin clots at any Ca2+ concentration used. Hig h affinity calcium binding was found to be normal in all five fibrinog en variants studied, suggesting that their abnormal clotting was not d ue to defective binding of calcium. The gamma-chain in the fragment D- 1 derived from the variant gamma 337 Asn --> Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas frag ments D-1 from the other four gamma-chain variants as well as from nor mal fibrinogen were protected against plasmic degradation in the prese nce of 1 mM Ca2+.