Mechanism of Surface-Mediated Activation of Bovine Factor XII and Plasma Prekallikrein

Abstract

The mechanism of kaolin-mediated activation of bovine Factor XII was studied in the presence of prekallikrein and HMW kininogen. The activated enzymes were assayed using fluorogenic peptide substrates, Boc-Glu (OBzl)-Gly-Arg-4-methyl-coumaryl-7-amide (MCA) for Factor XIIa and Z-Phe-Arg-MCA for plasma kallikrein. The rates of activation of the zymogens were separately measured by blocking either of the active enzymes with a specific inhibitor, corn inhibitor for Factor Xlla and Trasylol for plasma kallikrein. The results were as follows:
1. At the early stage of the activation reaction, kallikrein activity was first generated after a short lag phase, and then Factor XIIa activity was generated with a sigmoidal curve. In the presence of corn inhibitor, the activation of prekallikrein was observed, but in the presence of Trasylol, the activation of Factor XII was not observed. In the presence of a high concentration of Ala-Phe-Arg-CH₂Cl, which immediately inactivates both of the active enzymes, the cleavage of a single chain prekallikrein into the two chain form by Factor XII was found, as revealed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), using nonlabeled and tritiated prekallikrein.
2. The incubation of Factor XII alone in a quartz cuvette or in the presence of kaolin and HMW kininogen did not result in the activation of Factor XII. The concave upward curve due to autocatalytic activation was not observed even after the addition of Factor XIIa to the Factor XII preparation. Moreover, no limited proteolysis of Factor XII during the incubation with kaolin and HMW kininogen was shown by SDS-PAGE, using ³H-Factor XII.
3. The rates of the activation of prekallikrein by Factor XII and by Factor XIIa were approximately the same at a higher concentration of prekallikrein. However, at a lower concentration of prekallikrein, the activation curve was shown to be sigmoidal and the rate was, much slower, than that with Factor XIIa.
These results indicate that the activation of bovine Factor XII is initiated by the attack of Factor XII on prekallikrein, followed by the reciprocal activation of Factor XII by the plasma kallikrein generated. The autocatalytic activation of bovine Factor XII by Factor XIIa was not demonstrated.