Inhibition of contact activation by a kininogen peptide (HKH20) derived from domain 5.

Contact activation can be initiated by interaction of Factor XII, prekallikrein (PK) and high molecular weight kininogen (HK) with inorganic negatively charged biologic macromolecules, or upon cell surfaces, or interaction with membrane protein derivatives such as aggregated beta amyloid. The latter two examples are zinc-dependent. The interaction with cells is dependent on peptides derived from HK domains 3 and 5 termed LDC27 and HKH20, respectively. We have tested the ability of each of these peptides to inhibit HK-dependent contact activation. HKH20 inhibited activation of prekallikrein when a mixture containing HK, prekallikrein and Factor XII was incubated with dextran sulfate, gC1qR, amyloid beta or endothelial cells. Comparable quantities of LDC27 had no effect. The binding of biotinylated HK or biotinylated Factor XII was inhibited in a dose response fashion by increasing concentrations of HKH20 while LDC27, again had no effect. The N-terminal region of HKH20 (amino acids 475-485) is of particular importance for binding and histidine 485 prominently enhances the reaction as assessed employing overlapping and deleted peptides. Since there is a role for HK heavy chain in binding to endothelial cells and LDC27 can be employed as an affinity ligand to isolate the binding proteins, we increased the LDC27 concentration from 10-fold to 250-fold to determine whether it is functional. Inhibition of endothelial cell-dependent prekallikrein activation required 100-fold greater concentration of LDC27 when compared to HKH20 to achieve significant inhibition. We conclude that the interactions of the light chain of HK via HKH20 is of particular importance for activation of the bradykinin forming cascade in zinc-dependent or independent reactions and is true for all "surface" initiators tested thus far.