Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.

Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.

Mechanism of activation of inactive renin in human plasma by puff adder venom.

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.

Purification of guinea-pig plasma prekallikrein. Activation by prekallikrein activator derived from guinea-pig skin.

Prekallikrein was purified from guinea-pig plasma. The prekallikrein appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 82 000 by SDS-polyacrylamide gel electrophoresis, 99 000 by gel filtration on a Sephadex G-150 column and 84 500 (protein part) by amino acid analysis. The isoelectric point was approx. 9.0. The purification method yielded 3.8 mg (A280 3.800) of prekallikrein from 500 ml of plasma. Kallikrein was generated from the prekallikrein by limited proteolytic action of a prekallikrein activator which was derived from guinea-pig skin. From analysis using SDS-polyacrylamide gel electrophoresis, the kallikrein has two fragments with apparent molecular weights of 52 000 and 40 000 which are linked by disulfide bond(s). The 40 000 molecular weight fragment was shown to incorporate [3H]diisopropylfluorophosphate. The kallikrein hydrolyzed the synthetic substrates containing the Phe-Arg sequence at the COOH-terminal, and it cleaved carbobenzyloxy-Phe-Arg-4-methylcoumaryl-7-amide more readily than Pro-Phe-Arg-methylcoumaryl-7-amide. The Km for the kallikrein with carbobenzyloxy-Phe-Arg-methylcoumaryl amide was 2 times 104 M. Also, the kallikrein showed negligible activities on peptide-methylcoumaryl amide-substrate for alpha-thrombin, Factor Xa or plasmin.

Purification of prekallikrein from porcine plasma and its conversion to active kallikrein.

Plasma prekallikrein, a precursor protein of kallikrein [EC 3.4.21.8], was highly purified from porcine plasma by chromatography on a DEAE-Sephadex A-50 column, followed by rechromatography on a DEAE-Sephadex A-50 column, chromatography on a CM-Sephadex C-50 column and affinity chromatography on a p-aminobenzamidine-epsilon-aminocaproic acid-Sepharose 4B column. By this procedure, 3.3 mg of purified material was obtained from 1.6 liters of porcine plasma and about 240-fold purification was achieved from the first DEAE-Sephadex A-50 chromatography. The purified protein was found to give a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel disc electrophoresis. This preparation did not contain kallikrein, Factor XII (Hageman factor) of the blood coagulation system, high molecular weight (HMW) kininogen or plasma kininase. Thus, the material is presumed to be functionally pure. The molecular weight of prekallikrein was estimated to be about 88,000 by SDS-polyacrylamide gel electrophoresis, and prekallikrein consists of a single polypeptide chain. Activation of prekallikrein by trypsin [EC 3.4.21.4] was found to involve the cleavage of a single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. This trypsin-activated kallikrein released kinin rapidly from bovine HMW kininogen. However, liberation of kinin was extremely slow from bovine low molecular weight (LMW) kininogen. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI) and Trasylol, but not by Polybrene or egg-white trypsin inhibitor (EWTI).

Contact phase of blood coagulation in cardiogenic pulmonary oedema (CPO) and adult respiratory distress syndrome (ARDS).

In order to assess the role of the kallikreinkinin (k-k) system in the pathogenesis of pulmonary oedema, we studied the contact phase factors of blood coagulation, as well as the haemodynamics and blood gas changes in 34 patients with pulmonary oedema, 23 of them with Adult Respiratory Distress Syndrome (ARDS) and 11 with cardiogenic pulmonary oedema (CPO). We have verified significant differences in the haemodynamic pattern and blood gases between the two groups of patients, which corroborate the previously established differences between both types of pulmonary oedema. Our results reveal k-k system activation in ARDS patients, with a significant fall in factor XII (p less than 0.05), prekallikrein (p less than 0.01), alpha-2-macroglobulin (p less than 0.01) and high molecular - weight kininogens (p less than 0.005), with a rise in C1-esterase inhibitor (p less than 0.001) in comparison with patients with CPO. All of the CPO patients had normal prekallikrein levels, whereas 15 out 23 ARDS cases (65%) had decreased prekallikrein values. Our results suggest that the k-k system activation could play a role in the pathogenesis of ARDS. Estimation of prekallikrein levels may be helpful in the differential diagnosis of ARDS.

Effects of an oral antidiabetic drug on the fibrinolytic system of blood in insulin-treated diabetic patients.

Selected variables of the fibrinolytic system were assessed in 23 men with insulin-treated diabetes with no measurable pancreatic beta-cell function. Gliclazide, a second-generation sulphonylurea drug, was administered to the patients over a period of 6 months in daily doses of 160 mg or 240 mg, and blood samples were obtained before, during, and after treatment. Determined by global assays, the drug did not significantly change plasminogen activator activities in euglobulins. Measurements of specific components of the system of fibrinolysis showed a marginal increase during administration of gliclazide of tissue-type plasminogen-activator antigen and prekallikrein activity in plasma, whereas the activities in euglobulins of the intrinsic plasminogen proactivators remained nearly the same during the study. Levels in plasma and euglobulin of C1-inactivator antigen and in plasma of factor XII antigen and t-PA inhibition capacity remained constant throughout the study. There were no changes of the increase in concentration of t-PA activity and t-PA antigen following venous occlusion. The metabolic state remained the same during the whole study. It is concluded that gliclazide induces small, but significant, non-insulin-dependent extrametabolic effects on the extrinsic (t-PA) and intrinsic (prekallikrein) system of fibrinolysis. Whether these changes are of physiological importance remains to be demonstrated.

Effect of bromelain on kaolin-induced inflammation in rats.

The effects of stem bromelain on the plasma kallikrein system, bradykinin levels and plasma exudation at the inflammatory site were examined in rats with a kaolin-induced inflammation of an air pouch. Bromelain (5, 7.5 mg/kg) caused a dose-dependent decrease of bradykinin levels at the inflammatory site and a parallel decrease of the prekallikrein levels in sera. Plasma exudation was also reduced dose dependently. Bradykinin-degrading activity in sera was elevated after treatment with bromelain, although it was unchanged in the pouch fluid. These data indicate that bromelain inhibits plasma exudation through inhibiting the generation of bradykinin at the inflammatory site via depletion of the plasma kallikrein system.

Plasma prokallikrein and kininogens in burned patients.

Using specific immunological (1) and enzymatic (2) methods, we have measured prokallikrein, total, high, and low molecular weight kininogens in 36 severely burned patients. At admission to the intensive care unit, all constituents were significantly decreased when compared to previously defined reference intervals. The values remained low during the three first days after burn. The changes affecting total and low molecular weight kininogens were significantly correlated (p less than 0.05) with the severity of the burn area. Prokallikrein and kininogens levels were also closely related to the concentrations of C3c and C4 complement factors.

Clotting activities and antigen concentrations of contact factors in kidney disease.

Factor XII, prekallikrein (PK) and high molecular weight kininogen (HMWK) clotting activities and antigen concentrations in kidney disease patients were studied. Chronic hemodialysis led to a reduction of F XII, PK and HMWK clotting activities. F XII and PK antigen levels were also low. In contrast, the HMWK antigen was normal with respect to concentration as well as electrophoretic migration behaviour on 2 D-immunelectrophoresis. In kidney transplant recipients more than three month after the transplantation, we found an increased of F XII and PK clotting activities, which appeared to depend on the time elapsed since the operation. However, the antigen levels remained normal. The group of chronic kidney disease patients not requiring hemodialysis exhibited normal mean values of all three clotting activities as well as normal F XII and HMWK antigen levels. The PK antigen was reduced. However, a separate evaluation of F XII clotting activities (F XII:C) in patients with glomerular nephritis showed elevated F XII:C which correlated with the BUN values. During acute processes of the kidney disease we observed very high PK clotting activities, which normalized with stabilization of the patients. Our results show that the contact phase coagulation factors behave abnormally in certain kidney disease patients.

Murine monoclonal antibodies to human factor XI.

Murine monoclonal antibodies to human factor XI (F.XI) are described. The monoclonal antibodies (2-1, 4-1, 7-1 and 10-1) consisted of IgG1. 4-1 inhibited the activation of F.XI completely in the presence of high molecular weight kininogen and kaolin and the others did so partially, whereas these antibodies had no effect on the activation of F.XI with activated factor XII (beta-XIIa). Four antibodies had no effect directly on the amidolytic activity of activated F.XI (F.XIa). 10-1 inhibited the activation of factor IX in coagulant assay for F.XIa by Mannhalter. And 4-1 and 7-1 did so partially, whereas 2-1 did not. In immunoblotting analysis, all antibodies bound to F.XI, its reduced form and F.XIa. All were directed against the heavy chain of F.XI. All antibodies recognized F.XIa-alpha 1 antitrypsin complex.

Decrease of plasma prekallikrein and kallikrein inhibitors during intravenous phlebography.

Complex contact activation systems may have major involvement in side effects of i.v. contrast media. To investigate this, quantitative measurements of several factors (plasma prekallikrein, kallikrein inhibitory activity, haematocrit, alpha-2-macroglobulin, antithrombin III, alpha-1-antitrypsin and beta-thromboglobulin) were made before and after i.v. contrast phlebography in two groups of patients (each containing 21 patients) with no thrombosis, using a high- (meglumine iodamide) and a low-osmolality (ioxaglate) contrast medium. A statistically significant decrease in plasma prekallikrein was observed after the high-osmolality contrast medium, which is a sign of the activation of the kallikrein-kinin system and an indicator of the activation of the intrinsic coagulation. These events may play an important role in the adverse effects of contrast media.

Plasmatic prekallikrein and kallikrein inhibitor in pregnancy, labor and in newborn.

In order to reveal the possible physiological role of the kallikrein during the stress of birth, the prekallikrein and kallikrein inhibitor were evaluated in 15 pregnant women and in 15 newborns from terminated pregnancies. The results show a very low level of prekallikrein in the funiculus blood at the moment of birth and a still lower level in venous blood 24 hours after birth. No important changes resulted in the mother's blood either in pregnancy, nor at the end of the labour.

Interactions among Hageman factor (HG, Factor XII), plasma thromboplastin antecedent (PTA, Factor XI), plasma prekallikrein (PK, Fletcher factor) and high molecular weight kininogen (HMW-K, Fitzgerald factor) in blood coagulation.

Studies of plasmas from individuals with Hageman trait (factor XII deficiency), plasma thromboplastin antecedent (PTA, factor XI) deficiency, Fletcher trait (plasma prekallikrein deficiency) and Fitzgerald trait (high molecular weight-kininogen deficiency) have revealed the importance of these proteins in blood coagulation. The interactions among them, however, are not fully elucidated. We have studied these reactions by two different approaches. (1) In a purified system, high molecular weight kininogen was absolutely required for activation of PTA by HF and ellagic acid (EA). The yield of activated PTA was proportional to the amount of HF, HMW-K, and PTA in the mixtures, suggesting that these three proteins may form a complex in the presence of EA. (2) In experiments with whole plasma, we took advantage of the adsorption of EA to Sephadex gels. When normal plasma or plasma deficient in HF, PK, HMW-K or PTA was exposed to Sephadex-EA and was separated by centrifugation, each supernatant plasma except that deficient in HF shortened the prolonged partial thromboplastin time (PTT) of HF-deficient plasma. Plasma simultaneously depleted of HMW-K, PK and PTA also shortened the PTT of HF-deficient plasma and of plasma depleted of HF and PK, but had virtually no procoagulant effect upon the PTT of plasma depleted of HF and MHW-K. Thus, exposure of HF in plasma to Sephadex-EA appeared to generate a clot-promoting form of HF in the absence of other clotting factors, but its expression required the presence of HMW-K.

Fujiwara trait: the first case of kininogen deficiency in Japan.

Asymptomatic identical twins were found to show the prolonged activated partial thromboplastin time, which was corrected by addition of normal, Hageman factor deficient or Fletcher trait plasma but not corrected by Fitzgerald or Williams plasma. The prolonged activated partial thromboplastin time was also corrected by addition of highly purified bovine high molecular weight kininogen but not by low molecular weight kininogen. When total kininogen was measured as the amount of bradykinin released by trypsin on acid treated plasma, only trace amount was detected in Fujiwara and Williams plasmas, although Fitzgerald plasma showed approximately 50% of the total kininogen of normal plasma level. Acetone-kaolin activated amidase activity of plasma kallikrein was not generated by Fujiwara plasma. Substitution with normal plasma in various ratios showed plasma kallikrein activity proportionally to the normal plasma contents. Extrapolation with the values at 120 min after activation gave the prekallikrein content of Fujiwara plasma as 30% of the normal value.

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.

Independent consumption of high and low molecular weight kininogens in vivo.

The levels of high molecular weight (HMW) kininogen and pre-kallikrein in rat plasma were markedly reduced after single injection of bromelain (10 mg/kg, i.v.) and gradually recovered over a 72 hour period. The level of low molecular weight (LMW) kininogen, however, was not changed during this period. Rat pleurisy was induced by intrapleural injection of lambda-carrageenin. The levels of HMW kininogen and prekallikrein, but not of LMW kininogen, in the exudate were markedly decreased, when compared with those in plasma of the same animals. After pretreatment with disulfiram, oral administration of ethanol (2 g/kg) or intravenous injection of acetaldehyde (10 mg/kg) to rats caused significant decrease in the plasma level of LMW kininogen with no significant effect on the plasma HMW kininogen and prekallikrein levels. These results suggest that HMW and LMW kininogens may be consumed separately in vivo and play different roles.

Hageman factor dependent pathway in local vascular permeability enhancement.

The possibility of an involvement of the kallikrein-kinin system in increasing vascular permeability induced by intradermal injection of the guinea pig activated Hageman factor (beta HFa) was examined. In vitro system, the kinin generation was observed in guinea pig plasma when the plasma was incubated with beta HFa. This kinin generation was dependent upon the dose of beta HFa added and upon the presence of plasma prekallikrein, since 97 percent of the kinin release was diminished by removing the prekallikrein in plasma by treatment with anti-prekallikrein antibody. These results suggested that the Hageman factor-kallikrein-kinin cascade in guinea pig was similar to those in human and bovine plasma. In the permeability experiment in vivo, a simultaneous injection of soybean trypsin inhibitor (10(-6) M), which is the inhibitor of guinea pig plasma kallikrein, inhibited the permeability response to beta HFa by more than 90 percent. The permeability response to beta HFa was attenuated 5 fold in animals depleted of the circulating plasma prekallikrein by intraarterial antibody administration. These results indicated the participation of plasma prekallikrein in the permeability reaction to beta HFa. A simultaneous injection of a kinin destructive enzyme, carboxypeptidase B, diminished the permeability reaction to beta HFa, without any inhibition of the amidolytic activity of beta HFa or plasma kallikrein. A simultaneous injection of an inhibitor of a kinin destructive enzyme (kininase II), SQ 20,881(Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), augmented the permeability reaction to beta HFa 10 fold, without any effect on the amidolytic activity of beta HFa or plasma kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)

Prekallikein and kallikrein inhibitor in liver cirrhosis and hepatitis.

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.

Histamine and kallikrein system in man.

Histamine infusion modifies the kallikrein system, studied by kaolin contact method, in man. The main modifications are the increase of the spontaneous esterase activity and the prekallikrein lowering. Apparently the histamine administration activates the kallikrein, and consequently a release of kinin can take place.