Fibrinogen coagulation without thrombin: reaction with vitamin C and copper(II).

We describe a novel method for inducing fibrinogen derived clots. The addition of vitamin C (0.1-1 mM) to a solution of fibrinogen (1 mg/ml) and Cu(II) (20-150 microM) results in protein coming out of solution. This phenomenon can be "read" by fibrometers as "clotting time". The reaction requires Cu(II) and can be prevented by a chelating agent, such as citrate, as well by a hydroxyl radical scavenger, such as mannitol. The insoluble protein, called "neofibe", is soluble in 4 M urea and 2% SDS. Isoelectric focusing and SDS-electrophoretic comparison of native fibrinogen with neofibe reveal molecular modifications of the starting protein. This reaction is an interesting example of a free radical mediated transformation of soluble protein into insoluble material. Some findings on the connection between hemostasis, vitamin C and Cu(II) are discussed.