Isolation of prekallikrein activator from guinea pig serum or plasma adsorbed to immune precipitates or celite: Possible relationship to Hageman factor

Abstract

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.

Conversion of Hageman factor (Factor XII) of the guinea pig to prekallikrein activator and inhibition of the formed kallikrein by a natural plasma inhibitor

“…The physicochemical and biological properties of Factor XI1 obtained from guinea pig plasma are very similar t o the clotting substance of man. A slight discrepancy was found between the molecular weight of activated Factor XI1 obtained from celite eluates [7] and the partially activated Factor XI1 obtained from whole plasma. Whether this is due to methodological differences or represents a difference due to degree of activation, remains to be ascertained.…”

“…As described in the preceding paper [7], the activator of guinea pig prekallikrein [8] probably derives from Factor XII.…”

“…As described in the preceding publication [7], upon incubation of serum or plasma with immune precipitates or celite, respectively, prekallikrein activator (PKA) is adsorbed and can be desorbed from these "surface active" substances. It was postulated that the PKA is a fragment (XIlf) of activated Hageman factor (XIIa [23].…”

“…Plasma ( 3 0 ml in the first, and 35 ml in the second run) was collected as described in the preceding paper, and chromatography was done essentially in the same way [7].…”

“…Gel filtration was carried out on a 2.5 x 90 cm ( K 25/100, Pharmacia, Montreal, Quebec) column packed with Sephadex Polyacrylamide electrophoresis, isoelectric focusing, esterase assays, determination of prekallikrein, kinin assays, clotting tests, and tests for enhanced vascular permeability were done as described in the preceding paper [7] and in another publication [5].…”