Separation of Plasma Kallikrein and a Kallikrein‐like Plasminogen Activator Generated by Acetone in Rat Plasma

Abstract

Plasminogen activator (PGA), kininogenase (Kase) and benzoyl arginine ethyl ester (BAEe) activities generated in plasminogen‐free rat plasma by incubation with acetone (23% v/v) at 22° were purified. The activities passed unadsorbed through columns of DEAE‐Sephadex A‐50 (pH 7.8) and arginine methylester‐Sepharose 4B (pH 8.5). Part of the activities (rat plasma kallikrein) was adsorbed onto a soybean trypsin inhibitor (SBTI)‐ Sepharose 4B column at pH 8.5. At pH 7.0 a fraction with higher ratios PGA/BAEe esterase and Kase/BAEe esterase was also adsorbed. Both fractions could be eluted with 5 mM sodium hydroxide. The fraction not adsorbed at pH 8.5, but adsorbed at pH 7.0 was designated low molecular weight plasminogen activator (LMr‐PGA), a plasminogen activator fraction with higher molecular weight, but without esterase activity being also present (Berstad & Briseid 1982). LMr‐PGA was strongly inhibited by tranexamic acid (AMCA) 0.10 mM, whereas the fraction designated rat plasma kallikrein was not. By polyacrylamide gel electrophoresis Mr‐values in the range 120,000 to 130,000 were established for native samples of both rat plasma kallikrein and LMr‐PGA, whereas Mr‐values of 78,000 to 80,000 were established after treatment with SDS.

Activation of Factor XII in Human Plasma: Protection by Benzamidine of the Cofactor Function of High Molecular Weight Kininogen

“…In accordance with the above-mentioned statements in relevant literature on the stability of the kinin-free protein against further destruction by plasma kallikrein, we found no loss of cofactor capacity when purified human HM,K was incubated with purified human plasma kallikrein before being tested in cofactor assays (table 4). The results accordingly seem to indicate the presence in acetone-treated human plasma of a serine-protease capable of lowering the cofactor function of kinin-free HM,K, as has previously been registered in rat plasma (Briseid & Berstad 1981;Berstad & Briseid 1982;Briseid & Briseid 1983).…”

“…A further purified kallikrein was used to assess the lack of effect of the enzyme on the cofactor capacity of HM,K. Plasma kallikrein was adsorbed onto a column of soybean trypsin inhibitor (SBT1)-Sepharose at p H 8.5 and eluted with 5 mM sodium hydroxide, as described for rat plasma kallikrein by Johansen & Briseid (1983). Deierminaiion of factor XII (PKA) as a measure of the amount present offunctionally aciive HMK.…”

“…In experiments with purified factors from human plasma Meier et al (1977) also registered an inhibitory effect of HM,K on the PKA-activity generated when high concentrations of HM,K were used. In assays with plasminogen-free fibrin plates (Berstad & Briseid 1982;Johansen & Briseid 1983) very weak fibrinolytic activities were regularly detected in acetone-activated BPL-P. To investigate if these activities, if due to trace amounts of plasmin, might contribute to the rapid and extensive loss of cofactoractive HMrK seen upon removal of benzamidine, HM,K was incubated with plasmin and then tested for cofactor activity. Table 4 shows that incubation for 30 min.…”

“…When the incubation was carried out in the presence ofbenzamidine 7 mM, the cofactor capacity of high molecular weight kininogen (HM,K) was protected against destruction by a serine protease which was not plasma kallikrein. By analogy with studies in rat plasma this protease might be a plasminogen activator (Berstad & Briseid 1982;Johansen & Briseid 1983). Factor XI1 in the plasma preparation was activated to unfragmented factor XII.…”

“…Studies of rat plasma, however, indicated that a plasminogen activator might be responsible for the loss of cofactor capacity of HM,K in rat plasma in the absence of benzamidine (Briseid & Berstad 1981;Berstad & Briseid 1982). This activator has been partially purified (Johansen & Briseid 1983) and was found to cause an extensive lowering of the functional activity of human HM,K too (unpublished experiments).…”