No abstract

SummaryHuman plasma was adsorbed to celite. The celite eluates were chromatographed on QAE-Sephadex A-50, followed by gel filtration, isoelectric focusing and preparative polyacrylamide electrophoresis.By anion-exchange chromatography, factor XI a, kallikrein and y-globulin were isolated from the excluded protein peak. Factor XIa (MW ∼170,000) corrected the deficiency in plasmas deficient in factors XII or XI, but had no effect on the kininsystem. Its isoelectric point was about 8.0-8.5. Kallikrein had a molecular weight of ∼ 90,000 and a pi of 7.8-8.0 The two substances could be isolated also by polyacrylamide electrophoresis.Highly anionic fractions contained a fragment of factor XII a (prekallikrein activator), which activated prekallikrein and retained some of the clot-promoting property of the intact factor XIIa molecule. Its molecular weight was about 37,000, the pi about 4.4 and it migrated as a prealbumin by disc electrophoresis.It is concluded that, by massive contact exposure, most of factor XII a converts to the highly anionic, low-molecular-weight prekallikrein activator. Since the prekallikrein activator is a fragment of factor XII a, we recommend that it be referred to as “XII f”.

Immune aggregates of bovine serum albumin (BSA) and rabbit anti-BSA presumably adsorb Hageman factor (factor XII) and induce its conversion t o prekallikrein activator (PKA), which can be eluted from the precipitates and purified by chromatography. N o clotting studies could be done with the material desorbed from the immune aggregates incubated with serum, because thrombin was present in t h e preparations.When Al(OH)3-adsorbed plasma (free of prothrombin) was incubated with celite, large quantities of PKA were recovered in the celite eluates, together with activated factor XI1 (XIIa), activated factor XI (XIa) and kallikrein. These components could be separated by anion exchange chromatography and gel filtration. PKA had a molecular weight of approximately 31 000, 33 000 and 3 6 000 as determined by sucrose density gradient ultracentrifugation amino acid analysis and gel filtration, respectively; its sedimentation coefficient was 2.7 -2.9; in fully activated form it migrated as a sharp prealbumin band by disc gel electrophoresis. The isoelectric point of PKA was approximately 4.4.The moderately anionic factor XIIa had an estimated molecular weight of 120 000. The cationic factor XIa and kallikrein were separated by gel filtration, the clot-promoting substance having a molecular weight of 170 000 --180 000, while kallikrein was found t o be about 9 6 000. A kinin-forming, BAEe-hydrolysing fraction with an elution volume greater than that of egg albumin was recovered and it is assumed that it represents a fragment of kallikrein.It is concluded that during massive surface adsorption, most of the adsorbed factor XIIa becomes fragmented. This fragment acquires prekallikrein-activating activity. The fragment retains some of the property of the parent molecule by being able to partially correct the clotting defect of factor XIIdeficient plasma. These findings are in good agreement with those obtained under similar conditions with human plasma.

The activation of human factor VIII by thrombin has been demonstrated by a new experimental approach. This method permitted investigation of the interaction of thrombin and factor VIII in the absence of most other clotting factors. The activation effect of thrombin is susceptible to inhibition by diisopropylfluorophosphate. Trypsin cannot replace thrombin in the activation reaction, and it destroys factor VIII activity rapidly.