Free radical-induced fibrinogen coagulation: modulation of neofibe formation by concentration, pH and temperature

The reaction of fibrinogen with Cu(II) (10-100 microM) and ascorbate (0.1-2.0 mM) leads to the formation of an insoluble clot-like material, "neofibe", which is dependent on experimental conditions. The reaction is observed with human and bovine fibrinogen, in the presence of 0.1-0.3 N NaCl, is optimal at the pH range of 7.4-7.7, and has characteristics typical of a site-specific Fenton reaction. Thus, it is inhibited by EDTA and catalase. Inhibition by mannitol is observed only at relatively high concentrations of this scavenger (greater than 100 mM). Concomitantly, the rate of oxygen utilization increases linearly with the concentration of reagents. The energy of activation of oxygen utilization by ascorbate and Cu(II) in the absence or presence of fibrinogen is Ea = 6.1 and 7.9 Kcal/mol, respectively. These values suggest that the rate-limiting step is dictated by a reaction of oxygen with "free" or protein-bound copper cations. The temperature dependency of the extent of transformation of human and bovine fibrinogen into neofibe is unusual in that it is biphasic-increasing from 5 to 25 degrees C, and decreasing thereafter, to 43 degrees C. This is not due to the temperature stability of fibrinogen. The essential requirement for copper, ascorbate and oxygen or hydrogen peroxide, as well as the low efficiency of mannitol as a scavenger, are in accord with the most likely interpretation of these data that fibrinogen undergoes a site-specific Fenton reaction. This modifies the protein and results in the formation of insoluble, polymeric, neofibe aggregates.