Formation of an association between factor XII and kallikrein in human plasma--significance of storage of plasma and the functional state of plasma kallikrein.

In a previous study evidence was provided that zymogen FXII might associate with part of the kallikrein generated by acetone treatment of human plasma in the presence of benzamidine (Thromb. Res. 61, 123-133, 1991). Some results also suggested an increase in such a complex formation upon storage of plasma, and two questions were raised in the present study: Does kallikrein activated by acetone-treatment of plasma exist in modifications with different abilities to associate with FXII? And will -70 degrees storage of plasma increase the liability to complex formation? S-2302 amidase assays carried out in mixtures of normal plasma and plasma genetically deficient in prekallikrein (PK) suggested an inhomogeneity of the kallikrein generated. A minor and unstable part of it could be blocked by corn trypsin inhibitor, thus indicating the presence of an association with FXII. In fractions from gel filtration of acetone-activated plasma, kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassay, and FXII, PK and HK were studied in PAGE immunoblot experiments. When freshly collected plasma was used, an amidase double peak (mol. wts. 400 and 300 kD) indicated an inhomogeneity of the kallikrein present, HK being observed in both peaks. FXII eluted separately over a gel. mol. wt. range of 90-55 kD. When plasma was stored at -70 degrees for 10 months before use, the more low-molecular part of the kallikrein double-peak had disappeared and was recovered, in a highly unstable state, adsorbed to the column material together with HK and FXII. Accordingly both functional assays and the results of immunoblot experiments indicated an inhomogeneity of the kallikrein present, and also a tendency of the minor part of it to associate with FXII, a tendency increased upon storage of plasma at -70 degrees.

Assay of factor XII in human plasma using prekallikrein or the chromogenic peptide S-2222 as substrates--significance of the functional state of plasma kallikrein.

Factor XII was assayed in acetone-treated and kaolin-activated human citrated plasma (total plasma dilution 1.0 + 0.3 v/v during activation with kaolin, 1.8 mg/ml incubate). Measurements were performed with the tetrapeptide Bz-Ile-Glu-Gly-Arg-pNa (S-2222) and with prekallikrein as substrates. The correlation of both methods to another S-2222 based method recently described was good, r = 0.90 and 0.85 for the two methods respectively. Under the assay conditions used, FXIIa was present as a S-2222 amidase that could be blocked by corn inhibitor, whereas plasma kallikrein was found to be present partly as an amidase blocked by a low concentration of soybean trypsin inhibitor, and partly in a functional state not inhibited and adding to the measured level of FXII. The presence of benzamidine 0.7 to 2.1 mM during acetone treatment increased the measured level of FXII assayed both as prekallikrein activator and as S-2222 amidase.

Contact activation factors in plasma from pregnant women--increased level of an association between factor XII and kallikrein.

The plasma levels of FXII, prekallikrein (PK), and high- and low molecular weight kininogens (HK and LK) were studied in pregnant women in the last trimester and in non-pregnant controls. FXIIa and plasma kallikrein were assayed in acetone-treated citrated plasma (CPLa) with the tetrapeptide S-2222 as substrate, using soybean trypsin inhibitor and corn inhibitor to exclude kallikrein and FXIIa respectively. No difference in PK-level could be registered for the two kinds of plasma, but the level of FXII had increased to about 150% in the pregnancy plasma. No difference in HK-level was observed, whereas the LK-level was significantly higher in pregnancy plasma, about 250% and 160% in rocket immunoassay and bioassay respectively. In fractions from gel filtration of plasma acetone-activated in the presence of benzamidine (BPLa), kallikrein was assayed as S-2302 amidase, HK and LK were measured in rocket immunoassay, and HK and FXII were studied in PAGE immunoblot experiments. In contrast to previous results obtained upon gel filtration of CPLa, not only kallikrein and HK, but in addition also FXII now appeared together in the same fractions and as two separate peaks. One peak eluting in early fractions (gel mol. wt. 300-400 KD), and one late eluting peak of proteins adsorbed to the gel material. The first peak was notably marked in pregnancy plasma. The results provide support for the assumption of an association in plasma between the three contact activation factors studied.

Amidolytic assay of factor XI in human plasma--significance of kallikrein for the activity measured.

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper (Thromb. Res. 74, 477-485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibitor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at -70 degrees C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

Contact activation factors in plasma from women on estrogen replacement therapy after ovariohysterectomy.

The plasma levels of factor XII, prekallikrein, factor XI, and high molecular weight kininogen were studied in women with bilateral oophorectomy and hysterectomy who received hormone replacement therapy with a 2 mg daily dose of estradiol valerate. Also plasminogen activator activity was investigated. The observations made provide support for the assumption that the low doses of estrogen used in hormone replacement therapy do not significantly affect the levels of contact activation or fibrinolytic factors in plasma. Plasma obtained from young, healthy women was used as a standard reference material. Significantly higher levels of factor XII and prekallikrein were registered in functional tests in the ectomized women than in the reference material, an increase not observed in the immunological assays. These observations are discussed in light of recently published data from our laboratory on an increase in the measured level of factor XII obtained upon the removal of IgG before assay. Also a marked increase in urokinase activity was registered in the ectomized women. The high levels of factor XII, prekallikrein, and urokinase, as compared with the reference material, seemed to be age dependent, being also observed in a group of naturally postmenopausal women.

Functional correlation between kallikrein and factor XII activated in human plasma.

Plasma kallikrein and FXIIa were assayed in acetone-treated human citrated plasma (CPLa) with the chromogenic peptide Bz-Ile-Glu-Gly-Arg-pNA (S-2222) as substrate. In end point assays with short incubation periods (1-10 min.) nearly all kallikrein present could be blocked by a low concentration of soybean trypsin inhibitor (STI). In 30 min. assays the main part of the kallikrein was recovered in a functional state not inhibited by STI, and at the same time the level of FXIIa (as amidase activity blocked by corn inhibitor, C.I.) was reduced to about 2/3 of the initial value. The formation of an association between FXIIa and kallikrein is suggested. In fractions from gel filtration of CPLa kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassays, and HK and FXII were studied in PAGE immunoblot experiments. Kallikrein appeared as one peak together with HK (gel mol. wt. 300 KD), about 40% of HK was free (220 KD), and no FXII was observed in the kallikrein or HK peaks, but in two areas corresponding to 78-79 KD and 39-42 KD. When experiments, however, were carried out with plasma acetone-activated and gel filtered in the presence of benzamidine (5 mM), part of the amidase activity present in kallikrein peak fractions was blocked by C.I. This observation supports the above suggestion of an association between FXIIa and kallikrein.

Contact factors in plasma from women on oral contraception--significance of factor XI for the measured activity of factor XII.

The plasma levels of contact activation factors were measured in women using a low estrogen dose oral contraceptive (OC). Basic values for factor XII (FXII), factor XI (FXI), prekallikrein (PK), and high molecular weight kininogen (HK) were obtained in immunoassays by comparing with control plasma. The plasma levels of FXII and PK were significantly increased in OC plasma, to 147% and 146% respectively, whereas no significant increase could be registered for FXI (106%) or for HK (107%). Functional assays carried out with different peptide substrates (S-2222 for FXIIa, and S-2222, S-2302 and Bz-Pro-Phe-Arg-pNA for kallikrein) showed increases in OC plasma to about 150% for both proteases, in accordance with results obtained in radial immunodiffusion (RID). However, when FXIIa was measured with the high molecular weight substrate PK, no significant increase could be registered. Further experiments suggested this result to be due to the low level of FXI present in OC plasma, as compared to the levels of FXII and PK. Assays were carried out in mixtures of test plasma (OC or control plasma) and plasma deficient in FXI or FXII. The results obtained suggested that FXIa was present in some kind of association with part of FXIIa and part of kallikrein present. At low concentrations of FXI the functional activity of FXIIa was reduced, and the assay data indicated that an appropriate level of FXI was required to obtain maximum rate of hydrolysis of prekallikrein by FXIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact activation factors in plasma from women using oral contraceptives--increased levels of factor XII, kinin-free high molecular weight kininogen and acetone-activated kallikrein.

The plasma levels of FXII, prekallikrein (PK) and high- and low molecular weight kininogens (HK and LK) were measured in women on low estrogen dose (30-40 micrograms ethinylestradiol) oral contraceptives (OC) and in controls. FXIIa was assayed in acetone-treated citrated plasma (CPL) with PK and the tetrapeptide S-2222 as substrates, and in acetone-treated citrated plasma with benzamidine (BPL) with PK as substrate. The level of FXII was found to be about 20% higher in OC-plasma than in control plasma. Kallikrein was assayed in CPL with S-2222 as substrate and in BPL with the tripeptide S-2302 as substrate. No difference in PK-level was observed in the CPL-based method, whereas an increase in kallikrein activity of about 30% was registered in BPL. The levels of HK and LK were estimated both in rocket immunoassay and in bioassay of released kinin. No difference in HK-level could be registered in immunoassay, whereas the bioassay revealed a HK-level in OC-plasma of 40% of the control level. Immunoblot studies showed that a substantial part of HK in OC-plasma was present as kinin-free protein (mol. wt. 103 KD), assumed to possess a higher cofactor potency than that of native HK. Both in bioassay and immunoassay the level of LK was found to be 60% higher in OC-plasma than in control plasma. Considered together the observations on contact factors made in this study provide support for the assumption of an increased readiness for contact activation in plasma from women using OC.

A new case of total kininogen deficiency.

Six families with total kininogen deficiency have been described in the literature. We report herein an additional case in a Pakistanese woman. The defect was discovered accidentally due to lack of normal clot formation in a preoperative routine blood sample. She had a borderline prolonged bleeding time, and reported occasional hematuria, but was otherwise without symptoms. Absence of both kininogen species was proven by functional and immunological methods, and by lack of kinin formation both by plasma kallikrein and hog pancreas kallikrein. Prekallikrein was reduced, probably because the main part circulates complexed to high molecular kininogen. Activation of intrinsic fibrinolysis was grossly hampered, and cold activation of coagulation absent with epsilonaminocaproic acid and greatly retarded by dextran sulfate, kaolin and ellagic acid. Together with other evidence the findings indicate the following order of importance for contact activation in plasma--F.XII, high molecular weight kininogen, prekallikrein.

Increased level of cAMP in the rat intestinal mucosa caused by sodium lauryl sulphate.

The level of cyclic AMP in the jejunal mucosa from tied loops of anaesthetized rats was found to be significantly increased (27-50%) when sodium lauryl sulphate (SLS) was added to the loop fluid (2-27 mM). Imidazole (25 mM) did not significantly alter the resting level of cyclic AMP, but reduced the increase caused by SLS (17 mM). Theophylline (25 mM) significantly increased the intestinal level of cyclic AMP, and potentiated the increase caused by SLS. Ouabain (2.5 mM) did not alter the level of cyclic AMP in the presence or in the absence of SLS. The results of previous experiments on the increases in intestinal absorption caused by SLS or by dibutyryl cyclic AMP (Briseid et al., 1974, 1976) are discussed in light of the present data. It is concluded that the SLS-effect on absorption can only partly by ascribed to its effect on the intestinal level of cyclic AMP.

Increased intestinal absorption in the rat caused by sodium lauryl sulphate, and its possible relation to the cAMP system.

The increases in the absorption of ouabain, phenolsulphonphthalein and pralidoxime caused by 17 mM sodium lauryl sulphate (SLS) from jejunal loops of anaesthetized rats were significantly reduced if sodium and chloride (Briseid et al., 1974) or chloride and bicarbonate were replaced by other ions in the loop fluid. Separate substitutions of sodium, chloride of bicarbonate did not significantly alter the SLS-caused absorption, except that the substitution of choline for sodium reduced the absorption of pralidoxime, both in the presence and in the absence of SLS. The increases in the absorption of phenolsulphonphthalein and pralidoxime caused by SLS were potentiated by theophylline (25 mM) and reduced by imidazole (25 mM). The addition of dibutyryl cyclic AMP (2.5 mM) to the loop fluid increased this absorption of the test substances. This effect was reduced by imidazole, but under the experimental conditions it was not potentiated by theophylline. Determinations of cyclic AMP in the rat intestinal mucosa showed that the level of this substance was significantly higher in the presence than in the absence of SLS. The experimental conditions were as described for the absorption experiments. It is concluded that the data obtained support the idea of an increased level of cyclic AMP as the main basis for the effect of SLS on the absorption.

Dextran-induced lowering of parameters of the kallikrein-kinin system in rat plasma.

Pretreatment of rats with tranexamic acid inhibited the rapid lowering of the plasma levels of acetone/kaolin-activated prekallikrein proactivator and prekallikrein caused by intravenous injection of dextran, but did not inhibit the reduction in the level of plasminogen, and potentiated the lowering of high molecular weight kininogen. By acetone/kaolin activation of normal rat plasma a mixture of surface-bound factor XIIa and unbound XIIf was obtained, and a BAEe-esterase (MW about 47,000) possessing weak kininogenase activity was present in addition to kallikrein. In activated plasma from dextran-treated rats the cleavage of XIIa was strongly reduced, and the second esterase was almost absent. It is suggested that dextran induces the loss of a plasma factor which is important for the cleavage of factor XIIa in the adopted procedure. This factor was not high molecular weight kininogen, and the lowering of plasminogen was too small to account for the reduction in PKA-activity.

Reduced or unchanged cofactor function of human high molecular weight kininogen induced by human plasma kallikrein.

Plasma kallikrein activated spontaneously during the purification of prekallikrein (I) and acetone-activated plasma kallikrein (II) were at pH 7.4 both capable of reducing the capacity of purified human high molecular weight kininogen (HMrK) to function as cofactor in the contact phase activation of factor XII in a crude plasma preparation. At pH 6.8 only I had such an effect. SDS polyacrylamide gel electrophoresis with reduction indicated that both I and II contained kallikrein as a cleaved 'three-chain molecule. I contained in addition a Mr 49,000 fraction reflecting possibly uncleaved heavy chain. The registration of reduced cofactor function of HMrK induced by plasma kallikrein is discussed in view of the assay procedure used.

Rocket immunoassay of high and low molecular weight kininogens in human plasma.

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) in human plasma could be rapidly (36 min.) separated on a DEAE-Sepharose Fast Flow column (1.0 x 5 cm and 0.50 ml plasma) by applying a NaCl step gradient. Quantification was then carried out by the Laurell rocket method with an antiserum raised against HMWK. Standard preparations for the assays were (I) crude HMWK and (II) crude LMWK prepared by the one-step procedure mentioned. In disc PAGE (8% with 0.1% SDS) immunoblot showed two main bands in I, migrating to apparent mol.wts. of 180,000 and 120,000. The 180,000 band predominated in native plasma. Purified HMWK (spec.act. 14 micrograms bradykinin/A 280) yielded in addition a band corresponding to a mol.wt. of 100,000. Immunoblot of II showed one broad zone over the mol.wt. range 65-70,000. The average assay values obtained in human plasma specimens from 10 males were 85 micrograms/ml for HMWK (range 65-130) and 174 micrograms/ml for LMWK (range 164-183). HMWK occasionally lost immunoreactivity during purification without a corresponding loss of kinin. Such a loss of immunoreactivity seemed to run parallel with a reduced release of kinin induced by hog pancreas kallikrein.

Assay of prekallikrein as plasminogen proactivator in plasma specimens from reactors to dextran or to contrast media.

In a previous study of plasma specimens from reactors to clinical dextran, it was concluded that a factor activated by acetone converted the high-molecular-weight kininogen (HMrK) into a non-functional state. We recently identified the HMrK-destroying factor as a plasma kallikrein modification with high plasminogen activator (PGA) activity. The aim of the present work was to investigate whether the level of plasma kallikrein, assayed as PGA on fibrin plates, was higher than normal in plasma from patients reacting to dextran or radiographic contrast media. Plasma specimens from 10 reactors and 16 controls were examined. No difference in PGA level could be detected between reactors and controls in citrated plasma stabilized with benzamidine 9 mM, whereas a significant loss of PGA activity took place in citrated reactor plasma during the acetone activation procedure. The loss of PGA activity could not be due to known inhibitors of plasma kallikrein. It was only partial, and did not influence the amidase effect against the tripeptide substrate H-D-Pro-Phe-Arg-pNA. The results might indicate the presence in plasma of different amounts of an unknown constituent important for the obtainable level of plasma kallikrein with high PGA activity. The significant loss of PGA activity in reactor plasma might indicate an abnormally high level of the unknown plasma factor.

Assay of kallikrein inhibitors and levels of acetone-activated kallikrein in plasma specimens from reactors to dextran or to contrast media.

The average level of kallikrein assayed as acetone-activated plasminogen activator (PGA) in plasma specimens from 10 reactors to clinical dextran or to radiographic contrast media did not differ from the level in 16 controls. This result was obtained in plasminogen-free citrated plasma stabilized with benzamidine. In the absence of benzamidine a significant reduction of PGA activity was registered in the reactor plasma, but not of activity against the tripeptide substrate S-2302 or the ester substrate BAEe. After storage of the plasma specimens for 12 to 18 months at -70 degrees, the PGA activity obtainable in reactor plasma had increased to the level registered in control plasma. Known inhibitors of plasma kallikrein and of histidine-rich glycoprotein (HRG) were assayed by radial immunodiffusion. The levels of alpha 2-macroglobulin, antithrombin-III and HRG were within the normal range in plasma from reactors, the level of C1-esterase inhibitor was slightly increased (116%), and the level of alpha 1-antitrypsin was higher than normal (124%). Evidence is provided that the loss of PGA activity taking place during acetone activation of fresh reactor plasma could not be due to a transition of native alpha-kallikrein to 3-chain beta-kallikrein. It is concluded that a plasma constituent unstable during storage is responsible for the selective and partial loss of PGA activity registered in reactor plasma.

Activation of factor XII in rat plasma: protection by benzamidine of the cofactor function of high molecular weight kininogen.

Factor XII has been assayed as kaolin-activated prekallikrein activator in rat citrated plasma pretreated with acetone (Briseid et al. 1978 & 1979; Briseid & Berstad 1979). In the present work benzamidine added during blood collection increased the extent of activation by a factor of 6. Rat high molecular weight kininogen (HMWK) added to acetone-treated citrated plasma likewise increased the activation, providing evidence of the protection by benzamidine of the cofactor function of HMWK. All cofactor capacity was retained after the removal of the kinin part of HMWK. Experiments carried out with plasminogen-free plasma showed that plasmin could hardly be the the factor responsible for the destruction of HMWK. The stoichiometric factor XII concentration-effect curve obtained by diluting acetone-treated rat plasma with acetone-treated human factor XII deficient plasma showed that factor XII is present in functional excess, the concentration of HMWK deciding the extent of activation. By diluting acetone-treated rat plasma with buffer, HMWK concentration-effect curves were obtained which were approximately linear over a range of 0.03-0.40 microgram (bradykinin equivalents) per ml kaolin incubate. No further activation of factor XII was obtained at 0.80 microgram/ml.

Inhibition by low molecular weight dextran of the blood pressure fall and the lowering of plasminogen proactivator induced by clinical dextran in the rat.

The intravenous injection into rats of dextran with a molecular weight of 1,000 (LMr dextran) in doses greater than or equal to 200 mg/kg induced rapid, but transient falls in blood pressure. Pretreatment of the rats with LMr dextran 200 mg/kg caused a partial inhibition of the profound blood pressure fall induced by the injection of clinical dextran with a molecular weight of 70,000 (HMr dextran), 40 mg/kg. In accordance with previous works (Briseid & Berstad 1981; Berstad & Briseid 1982; Berstad 1982) it was found that the intravenous injection of HMr dextran lowered the plasma levels of plasminogen proactivator (pro-PGA) and functionally active high molecular weight kininogen (HMrK). Also LMr dextran, 200 mg/kg, induced significant reductions in the mentioned parameters, but less extensive than those obtained by HMr dextran, 40 mg/kg, and pretreatment of the rats with LMr dextran inhibited the subsequent effects of HMr dextran. It is suggested that a dextran-activated plasminogen activator might be an early link in the mechanism underlying the dextran-induced state of shock in the rat.

Dextran-induced lowering of plasminogen proactivator and functionally active high molecular weight kininogen in the rat.

Incubation of plasminogen-free rat citrated plasma with acetone (23% v/v) yielded enzyme preparations with high levels of plasminogen activator (PGA) and kininogenase (kallikrein), but with a low concentration of high molecular weight kininogen (HMWK) active as cofactor for kaolin-induced activation of factor XII. When benzamidine (4.0 mM) was present during acetone activation, a high yield of functionally active HMWK was obtained. Gel chromatography separated PGA into one high molecular weight fraction (HMW-PGA) without kininogenase and BAEe esterase activity, and one fraction (LMW-PGA) eluting together with plasma kallikrein. Injection of dextran (100 mg/kg intravenously) reduced the amount of LMW-PGA to 40%, without altering the concentration of HMW-PGA, and with only a small reduction of the kininogenase activity.