New Hypothesis for the Molecular Mechanism of Surface-Dependent Activation of Hageman Factor (Factor XII)

The surface-dependent mechanism of activation of highly purified human Hageman factor (HF) was studied using ³H-DFP uptake as a quantitative active site titrant. HF was treated in various ways and the reaction mixture was exposed to 3 mM ³H-DFP for 5 min at 37°. Following addition of SDS and removal of free DFP by dialysis, the reaction products were analyzed on SDS gels. In solution, the HF zymogen at 80,000 MW took up 0.015 mol DFP per mol HF. HF bound to kaolin, celite, or ellagic acid with or without high MW kininogen took up the same 0.015 mol DFP per mol HF. However, HF bound to celite or kaolin with high MW kininogen and kallikrein took up 0.9 mol DFP per mol HF into a 28,000 MW fragment of HF. In approximately half of these activated HF molecules, this 28,000 MW fragment was linked by disulfide bonds to a 52,000 MW fragment in a surface-bound 80,000 MW form of activated HF. In clotting assays, DFP did not inhibit kaolin-bound HF unless the surface-bound HF first had been proteolytically activated by kallikrein.

Kinetic studies of the cleavage of ¹²⁵I-HF by kallikrein or by plasmin in the presence of high MW kininogen showed that kaolin-bound HF was cleaved more than 20 times faster than HF in solution.

These results suggest that binding to kaolin or celite or ellagic acid, classically known as “activating surfaces”, does not convert a detectable fraction (< 1%) of the bound HF molecules to active enzymes. Rather, surface-binding makes HF molecules much more susceptible to proteolytic activation in the presence of high MW kininogen, and the reciprocal proteolytic activations of HF and prekallikrein are thus greatly stimulated by “activating surfaces”.