Review of some unusual effects of calcium binding to fibrinogen

Abstract

Calcium binding curves of human and bovine fibrinogen were obtained by using a calcium sensitive electrode. The two were identical and showed 2 high, 2–3 medium and more than 15 low affinity sites. Differential scanning calorimetry at neutral pH demonstrated the presence of the D and E domains of fibrinogen; however, at pH 3.5 the D-domain was split into two. The presence of the subdomains was demonstrated also by digestion by pepsin at this pH. Combination of digestion of fibrinogen and of its fragments with different enzymes and temperatures identified up to 12 subdomains in the original molecule. Clotting of fibrinogen by thrombin at pH 7.0 was investigated also by differential scanning calorimetry. In the absence of Ca 2+ clotting elicited a 40% increase in the enthalpy of thermal denaturation of the D domain of fibrinogen, but the position of the peak increased only by 0.4 °C. However, with clotting in the presence of 10 −3 M calcium the former increased by 70–75% and the latter by 11.0 °C, while these parameters of the E-domain remained unchanged. Changes of bound calcium during clotting were also measured with the calcium sensitive electrode. These had to be corrected, because the drop in free calcium was partly compensated by release of some calcium that was already bound to fibrinogen. Log of the half time of calcium uptake plotted against log thrombin concentration indicated a first order process with respect to thrombin concentration, moreover, the rate determined corresponded to that of the conformation change measured by calorimetry. The calcium uptake was correlated with release of the fibrinopeptides. Release of fibrinopeptide B follows parallel to binding of calcium and that of fibrinopeptide A is about fourfold faster. Polymerization and formation of thick bundles of fibrin is connected with release of fibrinopeptide A. Clotting with Ancrod, an enzyme that releases only fibrinopeptide A, showed only minimal binding of calcium. The polymerization inhibiting tetrapeptide Gly-Pro-Arg-Pro also depressed binding of calcium. These data suggest that a calcium-binding site must be in the proximity of the site of release of fibrinopeptide B and of a polymerization site.