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Low amount of sulfatides initiated intrinsic coagulation and the appearance of kallikrein activity in normal human plasma. The initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of Factorr XII, high molecular weight kininogen and prekallikrein. Because the activated partial thromboplastin clotting times in prekallikrein deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon was studied and found to be even more pronounced in the presence of sulfatides. Shortening of the clotting time was essentially completed in 5 min in the presence of sulfatides whereas a pre-incubation of 15 to 20 min was required in the presence of kaolin. The limited proteolysis of 125I-Factor XII in plasma during incubation was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. Factor XII cleavage in prekallikrein deficient plasma was completed in less than 5 min in the presence of sulfatides and in less than 15 min in the presence of kaolin. Thus, the appearance of Factor XII-dependent coagulant activity correlates with the limited proteolysis of Facto XII when normal or prekallikrein deficient plasma is activated by sulfatides or by kaolin. These observations are consistent with the hypothesis that Factor XII is cleaved in plasma to generate maximal Factor XIIa activity.

Plasma prekallikrein (kallikreinogen) and kallikrein inhibitor, assayed with the kaolin activable esterase method, have been evaluated in 20 patients with hepatic cirrhosis, in 12 cases with jaundice from acute viral hepatitis, and in 9 normal. A significant reduction of the plasma prekallikrein in cirrhosis has been found. A lowering of plasma prekallikrein has also been observed in viral hepatitis; in this condition, however, the modifications were less important than those obtained in cirrhosis. In three cases of hepatitis, the behaviour of the plasma prekallikrein and kallikrein inhibitor have been controlled during the period of the disease and compared with the behaviour of some conventional parameters, such as serum transaminases and bilirubin. An important increase of the prekallikrein level has been observed during the improvement of hepatitis. These data confirm the implication of the prekallikrein-kallikrein system in severe liver diseases, and indirectly points out the role of the liver in maintaining the physiological balance of the kallikrein system.

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 peptides, Boc-Glu (OBzl)-Gly-Arg-4-methylcoumaryl-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 specific inhibitors, corn inhibitor for Factor XIIa (Ki = 6.7 nM) and Trasylol for plasma kallikrein (Ki = 3.9 nM). The result was as follows: (1) At the early stage of the activation reaction, kallikrein activity was first generated after short lag time, 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 high concentration of Ala-Phe-Arg-Ch2Cl, which inactivates immediately both of the active enzymes, the cleavage of a single chain prekallikrein into the two chain form by Factor XII was shown by SDS-PAGE, using nonlabelled 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 an autocatalytic activation was not observed even after the addition of Factor XIIa to Factor XII preparation. Moreover, no structural change of Factor XII during the incubation with kaolin and HMW kininogen was shown by SDS-PAGE, using 3H-Factor XII. (3) The rates of activation of prekallikrein by Factor XII and by Factor XIIa were approximately the same at higher concentration of prekallikrein. However, at lower concentration of prekallikrein the rate of activation of prekallikrein by Factor XII was shown to be a sigmoidal curve and slower than that by 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 plasma kallikrein generated. The autocatalytic activation of bovine Factor XII by Factor XIIa was not demonstrated.