Kinin Formation Research Articles

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

Milk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropylfluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Kinins in relation to kallikrein activity, kininogen, electrolytes, aldosterone and catecholamines in urine from normal individuals.

The object of the present study was to test the hypothesis that urinary kinin excretion is an indicator of intrarenal kinin formation and to investigate urinary excretion of kinins in relation to natriuresis, kaliuresis, diuresis and urinary aldosterone and catecholamines in normal individuals on a free salt and water intake. In freshly voided urine collected from 24 normal individuals kinin concentration was directly related to kallikrein activity. Kininogen concentration was very low and neither related to kallikrein activity nor to kinin concentration. The excretion rates of kinins and kininogen were unrelated to the time interval between micturitions. In 24 hour urine collections from 50 normal individuals the excretion of kinins was positively correlated to natriuresis, kaliuresis and diuresis and also to dopamine but not to aldosterone, noradrenaline and adrenaline. Kinin excretion was inversely related to age and was lower in women than in men. On the basis of these results it is concluded that urinary kinin excretion reflects intrarenal kinin formation in normal ambulatory individuals and that urinary kinins are formed mainly in the interstitial and vascular space of the kidney. Furthermore, kinins and dopamine seem to play a physiological role in the renal handling of electrolytes and water.

Local kallikrein and trypsin responses in the rat.

1 Locally administered commercial hog pancreatic kallikrein (Depot-Glumorin) and bovine pancreatic trypsin both increased vascular permeability in the skin and paws of rats. 2 By the use of numerous antagonists and enzyme inhibitors, this vascular response was found to be the result not of kinin formation but of a direct action mostly on histamine receptors. 3 Highly purified kallikrein did not increase vascular permeability in rats, suggesting either that the effect was due to an impurity in the commercial preparation or that a structural change in the enzyme occurred on purification. 4 Soya bean trypsin inhibitor prevented the trypsin response when both were injected locally. On intraperitoneal injection, the inhibitor was effective only against local kallikrein. 5 The kallikrein inhibitor, aprotinin (Trasylol), was not effective against local kallikrein but it reduced the trypsin response when both were injected locally.

Surface activation of blood coagulation, fibrinolysis and kinin formation.

The activation of plasma prekallikrein by single-chain factor XII has been studied in the presence of high molecular weight kininogen and kaolin. The data indicate that factor XII can initiate blood coagulation, fibrinolysis or kinin generation in the presence of kaolin and does so by converting prekallikrein to kallikrein. An enzyme cascade is then generated leading to the formation of fibrin, plasmin or bradykinin in three closely related physiological events.

Purification of a high molecular weight kininogen from rat plasma.

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 degrees and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.

Activators of Inactive Renin (‘Prorenin’) in Human Plasma: Their Connection with Kinin Formation, Coagulation and Fibrinolysis

1. Human plasma was treated at 4°C with acid, trypsin, plasmin, streptokinase, urokinase, active Hageman factor fragment (β-XIIa) and β-XIIa-activated plasma prekallikrein (Fletcher factor). The conversion of inactive into active renin (activation) was studied in normal plasma (n = 10), Hageman factor-deficient plasma (n = 2), Fletcher factor-deficient plasma (n = 1) and plasminogen-free plasma (n = 4). 2. In normal plasma inactive renin was activated at pH 7·5 after treatment at pH < 4·0; at pH 3·3 the results were the same as with trypsin. This was also the case in plasminogen-free plasma. In Hageman factor-deficient plasma and in Fletcher factor-deficient plasma, however, the quantities of renin that were activated after acidification were much smaller than with trypsin. The addition of physiological amounts of active kallikrein to pH 3·3-pretreated Hageman factor-deficient plasma caused complete activation of renin. In contrast, the addition of active Hageman factor fragment to pH 3·3-pretreated Fletcher factor-deficient plasma had little or no effect. 3. Plasmin, streptokinase-activated plasminogen and urokinase-activated plasminogen activated inactive renin in pH 4·0-pretreated normal plasma as well as in pH 4·0-pretreated Hageman factor-deficient plasma and Fletcher factor-deficient plasma. 4. It is concluded that inactive renin is activated by two separate proteolytic pathways: one pathway depends on both Hageman factor and plasma prekallikrein, and the other pathway depends on plasminogen. In the Hageman factor-dependent pathway plasma kallikrein and not Hageman factor is the major activator of inactive renin. It is assumed that pH 3·3-treatment of plasma destroys the major inhibitors of kallikrein and that pH 4·0-treatment destroys the major inhibitor of plasmin.

Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.

Substrate specificities of acid kininogenases.

Two kinin forming enzymes were extracted from bovine spleen and separated from cathepsin B1 and B2 by DEAE-Cellulose chromatography. Since these catheptic kininogenases were found to release kinins from kininogens at acidic pH's, these were named acid kininogenase I and II. The presence of SH compounds was not necessary for I to have a kinin forming activity, while it was necessary for II. These have only minor difference for electrophoretic behaviors as can be seen, e.g., from a small difference in pI values, but could be separated by polyacrylamide gel electrophoresis. Their production of kinins from bovine crude bradykininogen was highly reproducible. Kininogenase I was proved to react on bovine HMW kininogen and also to release some kinin from LMW kininogen and leukokininogen. From the study of several substrates, these enzymes were revealed to have very low tryptic and little esterolytic activities and to have affinity to some hydrophobic amino acids.

Experimental disseminated intravascular coagulation (DIC)

Endotoxinemia was produced in six Japanese monkeys (7-13kg) by two suitably spaced (24hrs, Fig. 1, Experiment I) and by a continuous (8hrs, Fig. 1, Experiment II) intravascular injection with endotoxin (ET) from E. coli. The changes of concentration of ET in plasma, and faculties of coagulative, fibrinolytic, kinin forming and complement systems were investigated before and after treatment with ET. Body temperature, arterial pressure, pH, pO2, pCO2, Ht and Hb in blood were checked throughout the experiment.The main observations were as follows.[1] Two spaced injection of ET (0.3-2mg/kg, 3 monkeys)Et in plasma rose to 0.1-1μg/ml of plasma immediately after the injection of ET and thereafter decreased gradually (Fig. 1, Experiment I). Activation of coagulation system was suggested by 60 to 70% decrease in platelet count and 20 to 30% prolongation of APTT and PT except for TT. Intrinsic coagulation factors, F. XII, F. XI, F. IX, F. VIII and extrinsic factor, F. VII decreased 20 to 30% after the treatment of first ET (ET-1), and further decreased 40 to 60% after second ET (ET-2). However, fibrinogen increased about two times before the treatment. Activation of fibrinolytic and kinin forming systems was indicated by the decrease of plasminogen and prekallikrein, generation of plasmin, kallikrein and FDP after ET-2 (Fig. 2). Complement factors, C3proactivator, C3c and C4 were decreased after ET-1 and ET-2.[2] Continuous injection of ET (1-5mg/kg/hr, 3 monkeys)ET in plasma rose 1-10μg/ml of plasma immediately after the first injection of ET and maintained its concentration to the death (Fig. 1, Experiment II). Activation of coagulation system was suggested by the extreme decrease in platelet count, prolongation of APTT, PT and TT. The decreases of F. XII, F. X and F. II were observed at 3hrs after the treatment of first ET. The decreases of F. IX, F. VIII and F. V were observed at 6hrs, and F. VII and fibrinogen decreased at 9hrs (Table 1). These findings indicate that ET activates the intrinsic coagualtion system. Activation of fibrinolytic and kinin forming systems was observed markedly as compared as the experiment of two spaced injection of ET (Fig. 3). In complement system, alternative pathway was activated. In plasma inhibitors, antithrombin III decreased, but α2-macroglobulin, C1s inhibitor and α2-plasmin inhibitor slightly decreased.These results in Japanese monkeys treated with ET have led us to conclude that DIC produced with ET may have been occured initially by the activation of intrinsic coagulation factor, F. XII, accompanying by the activation of prekallikrein and plasminogen.

Differential assay method for high molecular weight and low molecular weight kininogens.

<h2>Abstract</h2> A differential assay method for high molecular weight (HMW) and low molecular weight (LMW) kininogens was developed. Plasma from human or rat was divided into three portions for the following purposes. For total kininogen, plasma was pretreated at pH 2.0 and bradykinin released by trypsin was separated and assayed on rat uterus. For HMW kininogen, plasma was incubated with glass powder in the presence of o-phenanthroline (a kininase inhibitor). The released bradykinin was separated and assayed. For LMW kininogen, plasma was incubated with glass powder in the absence of o-phenanthroline. The HMW kininogen-depleted plasma was treated in the same way as for the total kininogen assay. The HMW, LMW and total kininogen levels were as follows: 0.98 ± 0.05, 3.08 ± 0.09, 4.11 ± 0.10 for human and 0.96 ± 0.06, 0.94 ± 0.16, 1.93 ± 0.11 μg bradykinin equivalent/ml plasma for rat. From the results using heated human plasma (60°C, 1 hr), 10% of normal plasma was required to obtain full formation of kinin from HMW kininogen by 30 min incubation with this method.

EXOCRINE-GLAND FUNCTION AND THE BASIC BIOCHEMICAL DEFECT IN CYSTIC FIBROSIS

A biochemical link is proposed between secretion and reabsorption in exocrine glands, in the form of a factor (kinin E) which initiates ion reabsorption. Activation of this factor involves arginine esterases, and in cystic fibrosis these may be defective, preventing the formation of kinin E. In cystic fibrosis stimulation of exocrine glands produces abnormal reabsorption of ions, leading to the physiological abnormalities and clinical manifestations of the disease.

Fungal proteases and the mammalian kinin system. II. Kinin hydrolysis by brinolase

Brinolase, a thrombolytic fungal protease capable of forming vasoactive kinins, has been shown to hydrolyze kinins after their formation. Using synthetic bradykinin as a substrate, the kinetics and mechanism of hydrolysis have been elucidated, evidently explaining the apparently low kinin formation in vivo , Bradykinin hydrolysis proceeded rapidly in vitro with a pH optimum of 7.0–7.5, and a half-life of 5.1 min, using 250 ng/ml bradykinin and 50 μg/ml brinolase. The K m was 3.2×10 −6 M and the V max was 4.6 × 10 −8 mol/liter/min, using 5 μg/ml brinolase. Two-dimensional paper fractionation of the brinolase-bradykinin digest revealed the presence of free arginine amongst the five peptide fragment spots.

The role of kininogenases, kinin formation and kininogenase inhibition in post traumatic shock and related conditions.

The kinin system has for a long time been considered to play a role in the pathophysiology of trauma, particularly in blood pressure changes and in inflammatory effects. Recent findings necessitate a revision of this view. It is now necessary to differentiate between two kinin systems: 1. the plasma kallikrein-HMW kininogen-kinin-system, which besides forming kinin acts decisively in Hageman Factor activation, clotting and fibrinolysis; 2. the glandular and tissue kallikrein-LMW kininogen-kinin-system which is to our present day knowledge primarily involved in kinin formation. Kinins exert a variety of actions. By interfering with angiotensin II formation, kinins may contribute to blood pressure regulation. By enhancing cellular glucose uptake and/or metabolism, they regulate partly energy production. In post traumatic states death is preceded by a severe depletion of various factors of the system and an almost total loss of kinin forming capacity. Severity and time course of these phenomena suggest that early institution of direct (Trasylol) or indirect (heparins, cortocosteroids) proteinase inhibition, and if necessary a replacement of the lost factors, should be considered.

Site of formation of kinins in the dog nephron.

The site of formation of kinins in the nephron was determined by stop-flow studies in dogs. Klinin, inulin, sodium, potassium concentrations were measured in the fractions collected during the stop-flow procedures. In addition, in three of the 17 stop-flow experiments, kallikrein activity was also measured. The highest kinin concentration after correction for water reabsorption was found in the fractions that were probably trapped in the distal part of the nephron. Either one or two peak was located either in the fraction overlapping (in one instance) or in the fractions coming prior to the fractions with the highest concentration of potassium. This first peak was present in all but one of the stop-flow experiments and was greater than the second peak. The second peak of kinins was found in 13 of the 17 stop-flow exeriments and was located in the fractions with the lowest sodium concentration. Those fractions with the lowest sodium concentration. Those fractions with the lowest sodium concentration also had the highest kallikrein concentration. No evidence of kinin formation was found in the fractions representing the proximal nephron. We conclude, therefore, that kinins are formed in the distal part of the nephron, with the highest concentration found in the last part of the distal nephron and/or in the renal papilla and pelvis.

Plasma kinin activation in tranexamic acid treated patients with hereditary angioneurotic edema.

Plasma kinin formation after in vitro activation of factor XII in the coagulation system was examined in 4 patients with hereditary angioneurotic edema. Compared to controls activationcurves showed a steeper increase and higher maximum levels. Administration of tranexamic acid to the patients resulted in partial normalisation of plasma kinin activation.

Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.

Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.

Effect of kallidin, substance P, and other basic polypeptides on the production of respiratory macromolecules.

When canine tracheal explants were incubated in culture medium 199 in the presence of D-glucosamine labeled with carbon-14 for 24 hours, a significant amount of radioactivity was found in the secreted macromolecules. When kallidin was present in the culture medium, the amount of radioactivity associated with a portion of these macromolecules was increased. A met-lys-bradykinin derivative had a similar effect, but bradykinin did not. When hexadimethrine, an inhibitor of kinin formation, was present in the culture medium, the amount of radioactivity in the macro-molecular fraction was decreased. Substance P and the structurally related polypeptides, physalaemin and eledoisin, also enhanced the production of tracheal macromolecules; they were several-fold more active than kallidin. The effect of polypeptides on the activities of glycosyltransferases was also investigated. One of the enzymes present in a microsomal fraction prepared from the mucosal lining of canine trachea was uridine diphosphate (UDP)-galactose:mucin galactosyltransferase, which required a 25 mM concentration of maganese ions to be present in the assay mixture to obtain maximal enzymatic activity. When the concentration of manganese ions was decreased to 2.5 mM, there was less than one third of the maximal enzymatic activity, but full activity could be restored by the addition of kallidin. Several other basic polypeptides had a similar effect on the enzymatic activity. Kallidin had little or no effect on the activities of several other glycosyltransferases. The results suggest that basic polypeptides may be important in controlling the synthesis and/or release of respiratory glycoproteins.

Renal venous output of kinins in patients with hypertension and unilateral renal artery stenosis.

In four patients with hypertension and angiographically pronounced unilateral renal artery stenosis, kallikrein activity was estimated in each kidney separately by the determination of kinin output in the renal veins. All patients showed suppression of renin release from the kidney with a non-stenotic artery. Accordingly, plasma flow from the kidney with artery stenosis could be estimated. The ratio of venous output of kinins between the kidney with a non-stenotic artery and the one with artery stenosis was 2.6-6.5. This indicates that renal artery stenosis leads to diminished intrarenal kinin generation. Reduced kinin formation may explain the low diuresis and natriuresis found in the kidney with artery stenosis.

Possible physiological role of new peptide fragments released from bovine high molecular weight kininogen by plasma kallikrein

The possibility of a physiological role for two new peptides, Fragment 1.2 and Fragment 2 (histidine-rich peptide), released from bovine plasma high molecular weight (HMW) kininogen by the action of plasma kallikrein was examined. The former peptide, which was liberated at the same rate as bradykinin, and the latter peptide, which was derived from Fragment 1.2 on subsequent incubation with plasma kallikrein, showed negligible kinin-like activities. However, the peptides strongly inhibited kinin formation in bovine plasma exposed to glass ballotini, and prolonged the calcium clotting-time of citrated rat plasma in a manner similar to hexadimethrine bromide. Also, they inhibited the activation of Hageman factor by kaolin. The above three types of experiments showed the potency of Fragment 1.2 to be approximately three times that of Fragment 2 on a molar basis. Neither fragment showed inhibition of already activated Hageman factor, nor of the esterase activities of plasma kallikrein, plasmin and thrombin. These results suggest that liberation of these polypeptides from HMW kininogen represents a negative feedback mechanism for the contact activation of Hageman factor.

Presence of a bradykinin-like substance in pulmonary washings.

Pulmonary washings were studied for the presence of bradykinin-like substances and the capacity for their formation and degradation. Bronchopulmonary lavage with saline was performed in anesthetized, exsanguinated Syrian golden hamsters with a cell yield of 99 per cent alveolar macrophages. In the presence of inhibitors of both kinin formation (hexadimethrine) and destruction (1,10-phenanthroline), immunoreactive kinin was found in bronchopulmonary lavage fluid (12.5 ng per 5 x 10(6) cells lavaged), washings of pooled macrophages (5.62 ng per 5 x 10(6) cells washed), and lysates of alveolar macrophages (3.97 ng per 5 x 10(6) cells lysed). Higher kinin concentrations were obtained by selective inhibition of kinin-destroying enzymes with phenanthroline. Inhibition of kinin formation with hexadimethrine alone rendered nearly all concentrations below the limit of detectability. These findings demonstrated that the capacity for kinin formation and degradation exists in pulmonary washings. The precise origin of this kinin and its forming and destroying enzymes (whether from the pulmonary alveolar macrophage, other sites in the bronchopulmonary tree, or plasma) is not yet known.