Autoactivation of human plasma prekallikrein.
Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity toward the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase that was followed by a relatively rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, lima bean, and ovomucoid trypsin inhibitors did not. The Ki of the reversible inhibitor benzamidine for autoactivation (240 microM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors. This indicates that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis of the time course of activation showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated by kallikrein. The apparent second-order rate constant was 2.7 X 10(4) M-1 s-1 (pH 7.2, 50 microM sulfatides, ionic strength I = 0.06, at 37 degrees C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the second-order rate constant of the reaction at high salt concentrations. In support of the autoactivation mechanism it was found that increasing the amount of kallikrein initially present in the reaction mixture resulted in a significant reduction of the lag period and a rapid completion of the reaction while the second-order rate constant was not influenced. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein.
D016264:Dextran Sulfate; D003911:Dextrans / Q000494:pharmacology; D010450:Endopeptidases; D004789:Enzyme Activation; D006801:Humans; D007610:Kallikreins / Q000502:physiology*; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011288:Prekallikrein / Q000502:physiology*; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D012965:Sodium Chloride / Q000494:pharmacology; D013433:Sulfoglycosphingolipids / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D003911:Dextrans; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D013433:Sulfoglycosphingolipids; D014361:Trypsin Inhibitors; D012965:Sodium Chloride; C026148:prolyl-phenylalanyl-arginine-4-nitroanilide; D016264:Dextran Sulfate; D011288:Prekallikrein; D010450:Endopeptidases; D007610:Kallikreins; D012697:Serine Endopeptidases