Evidence for the promoting role of the intra-uterine kinin release in the development of late hypertonic saline-induced abortion.

Blood plasma kininogen (K), kininases (KS), kallikrein (KK), prekallikrein (PKK), and PGF2a were estimated in the common circulation of pregnant women during late saline-induced abortion and also in retroplacental blood after foetus delivery. The results provide evidence for intra-uterine kinin release from circulating blood K by locally activated KK from the very beginning of abortion. The greatest kinin release coincided with the strongest KS activity decrease at the time of foetus delivery. The pre-abortive KS levels correlated directly with abortus duration. Uterine PG biosynthesis was activated, but appeared to be a secondary process.

PIP: Although human kininogen involvement in hormonal homeostasis during pregnancy and activation of full-term delivery has been documented, the role of intrauterine kinin release in saline-induced late abortion has not been explored. Thus, kinin-related components in the circulation of 53 women with second-trimester (16-27 weeks) hypertonic saline-induced abortions and 20 nonpregnant controls were investigated. Observed were increases in both inactive precursors such as kininogen (K) and prekallikrein (PKK) and the active enzymes kallikrein (KK) and kinases (KS). The prostaglandin (PG) F-2 alpha-KK level in abortion patients did not differ from that in controls. The period from saline instillation to delivery of the fetus (26.5 +or- 2.4 hours) was accompanied only by K and KS changes; these levels decreased simultaneously, reaching their lowest value at the time of fetal expulsion. A 6-fold increase of KK-like activity was detected at fetal expulsion. Thus, the greatest contractile activity of the uterus coincided with the highest concentrations of free kinins and their longest life-time in the systemic circulation. The duration of abortion was directly associated with KS activity level, suggesting that kinins can be used to estimate abortion duration. Tissue KK synthesized in human myometrium and endometrium is considered responsible for local intrauterine kinin release after saline instillation. Finally, a sharp PGF-2 alpha increase (3.6-fold in the systemic circulation and 33-fold in retroplacental blood) was observed at the time of fetal expulsion. The high myometrial contractile activity at the final stage of saline-induced abortion appears to be achieved through the combined effects of locally released kinins and kinin-stimulated prostaglandins.

Kininase of the Latrodectus tredecimguttatus venom: a study of its enzyme substrate specificity.

The specificity of the peptide hydrolyzing action of a highly purified preparation of kininase from Latrodectus Tredecimguttatus venom was studied by the method of TLC on silica gel with the use of various synthetic peptides as substrates. It was shown that the enzyme cleaves the -Pro(7)-Phe(8)-bonds in BK and AI molecules liberating, correspondingly, the C-terminal dipeptide and tripeptide. Exopeptidase specificity was not revealed in the enzyme activity with the use of a number of free and N-substituted tri- and pentapeptides. The results obtained characterize the spider venom kininase as a thiol endopeptidase, which cleaves internal peptide bonds at the proline carboxyl end.

[The intravascular formation of kinins in the brain studied as a new approach to the problem of the re-circulatory complications in the surgical treatment of cerebral ischemia]. / Izuchenie vnutrisosudistogo obrazovaniia kininov v golovnom mozge kak novyi podkhod k probleme retsirkuliatornykh oslozhnenii pri khirurgicheskom lechenii tserebral'noi ischemii.

The improvement of blood supply in the ischemic brain (post-ischemic cerebral reperfusion), which results from surgical treatment, has been shown to improve the neurological status only in the patients who had no lower baseline levels of kininogens (the production of free kinins) in cerebral circulation. Poor surgical outcomes were noted in the cases wherein there was preoperative activated kinin production when blood passed through the brain. Postischemic brain reperfusion was concurrent with kinin production in the cerebral vascular bed in all examinees, but in patients with postoperative complications this process is more active and involves the two kinin-forming systems tissue and plasma ones. It is hypothesized that in the latter case kinins convert from the compensatory to the pathogenetic factor of secondary circulatory disorders. The correlations found may be used to substantiate the use of antikinin therapy and to develop a biochemical test that predicts possible complications after surgical treatment of cerebral circulatory ischemia.

Kininogen consumption in cerebral circulation of humans during brain ischemia and postischemic reperfusion.

1. Total kininogen, high molecular weight kininogen and low molecular weight kininogen were quantitated as bradykinin equivalents in the blood flowing to and from the brain in patients with stenotic and occlusive carotid damage in the course of neurosurgical treatment. Although considerable improvement in blood supply of ischemic brain areas was established after surgery in all patients, improvement in postoperative neurological status was seen only in four patients (group I), while in six cases there were no or negative neurological changes (group II). 2. The biochemical study confirmed the principal difference between these two groups: 1) prior to surgery in the patients of group II, but not of group I, total kininogen in blood flowing from the brain was markedly lowered compared to its arterial level, the latter being close to normal; the decrease was due only to low molecular weight kininogen. 2) After surgery, cerebral venous total kininogen levels were significantly lowered in patients of both groups; however, for patients of group II, these changes were more pronounced and they showed a decrease in both high and low molecular weight kininogen. 3. The major involvement of low molecular weight kininogen implicates tissue kallikrein in this process. The reduction of kininogen indicates that kinin formation occurred in the cerebral intravascular space during brain ischemia and following brain reperfusion and was most likely associated with the well-known actions of kinin on cerebral vessels, i.e., vasodilatation and brain edema.

Kinetics of Lys-bradykinin release by porcine pancreatic kallikrein from rabbit low molecular weight kininogen.

The initial rates of Lys-bradykinin release by porcine pancreatic kallikrein from rabbit low molecular weight kininogen are found to follow the Michaelis-Menten kinetics with kc = 0.62 sec-1 and Km = 1.93 microM at substrate concentrations 0.3-1.3 microM, but at higher ones the Michaelis dependence is broken. Inhibition of the reaction by its product(s) with Kp < Km is revealed with integral analysis methods in a range of 4.5-270 microM kininogen.

[Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin]. / Kininaza iada pauka Latrodectus tredecimguttatus. Kharakteristika fermenta kak tiolovoi éndopeptidazy, rasshchepliaiushchei sviaz' -Pro7-Phe8- v bradikinine.

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.

[Effect of covalent binding of aprotinin with a polysaccharide carrier on its inhibition of kallikrein from human plasma, porcine pancreatic kallikrein and trypsin]. / Vliianie kovalentnogo sviazyvaniia aprotinina s polisakharidnym nositelem na ingibirovanie im kallikreina plazmy krovi cheloveka, pankreaticheskogo kallikreina svin'i i tripsina.

The inhibition of trypsin, human blood plasma kallikrein and porcine pancreatic kallikrein by aprotinin (native and immobilized on carboxymethyl ester of dextran) was investigated. The experimental values of Ki of native and immobilized aprotinin--enzyme complexes are equal to 0.037 and 0.045 nM for trypsin, 0.38 and 112.3 nM for pancreatic kallikrein and 34.4 and 454.5 nM for plasma kallikrein with N alpha-benzoyl-L-arginine ethyl ester as substrate, and to 82.6 and 231.7 nM for plasma kallikrein with a natural substrate--kininogen. These data suggest that covalent binding of aprotinin to the water-soluble polysaccharide carrier does not interfere with its interaction with trypsin, whereas the inhibition of kallikreins decreases, especially that of pancreatic kallikrein. The experimental results indicate the marked differences in the structure of the binding site of the active center (or its environment) of plasma and pancreatic kallikreins, on one hand, and trypsin, on the other, as well as the differences between the plasma and pancreatic kallikreins. A high requirement of kallikreins to the maintenance of the native conformation of aprotinin during immobilization is postulated.

[Single-chain, low molecular weight kininogen in the blood plasma of rabbits: isolation and fragmentation by porcine pancreatic kallikrein]. / Odnotsepochechnyi nizkomolekuliarnyi kininogen plazmy krovi krolika: vydelenie i kharakter fragmentatsii pankreaticheskim kallikreinom svin'i.

A highly purified preparation of low molecular weight kininogen (LMrK) was isolated from the plasminogen-free rabbit blood plasma, using chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA 34 and Sephadex G-100 as well as gradient chromatography on a hydroxylapatite column. The yield of the 320-fold purified LMrK was 16%. Trypsin released 13-14 micrograms-eq. of bradykinin (BK) from 1 mg of LMrK or 0.85-0,95 mol of BK per mol of kininogen. Rabbit LMrK consists of one polypeptide chain of Mr 69 000 and pI 4.63. Porcine pancreatic kallikrein splits off kinin from the LMrK polypeptide chain by disrupting two peptide bonds resulting in the formation of S-S-bound two chain molecule. After reduction of the S-S bonds by dithioerithritol the latter is separated into a heavy (Mr 61 000) and light (Mr 6 800) chains. A biologically active peptide was isolated from the products of CNBr cleavage of LMrK. This peptide consists of Lys-BK elongated from the C-terminal with several amino acid residues. Rabbit LMrK closely resembles human LMrK in terms of Mr, pI and location of the kinin fragment in the protein molecule.

[Identification of kinins releasing by pig pancreatic kallikrein from rabbit low molecular weight kininogen]. / Identifikatsiia kininov, osvobozhdaemykh pankreaticheskim kallikreinom svin'i iz nizkomolekuliarnogo kininogena krolika.

Pig pancreatic kallikrein is found to release kinins from LMrK with the rate of 800 mkg. eq. of bradykinin per min, per 1 mg of the enzyme. This kininogenase reaction is not accompanied by a ddep fragmentation of the kininogen molecule, because only a high molecular weight fragment which Mr is practically equal to that of intact LMrK (55-60 000), and a peptide identified as bradykinin, are found in hydrolysate. In the same conditions human salivary kallikrein is found to form kallidin from LMrK.

[Amino acid makeup, structural characteristics and substrate specificity of rabbit plasma kininogen]. / Aminokislotnyi sostav, osobennosti stroeniia i substratnaia spetsifichnost' kininogena plazmy krolika

Highly purified kininogen preparation with the activity of 16-18 int. units per mg was isolated from rabbit blood serum. Its molecular weight was estimated to be 54 000 by gel filtration through Sephadex G-200. Leucine was identified as N-terminal amino acid by the dansylation method. Rabbit kininogen consists of 394 amino acid residues (except tryptophane). Amino acid composition of kininogen is characterized by a high content of dicarbonic amino acids, proline and by a low content of methionine. Kininogen molecule does not contain SH-groups. 13.1-13.5 SH-groups were found in kininogen after the reduction of S-S bonds with beta-mercaptoethanol in the presence of 8 M urea, thus indicating the presence of 6-7 S-S bonds in kininogen molecule. Kininogen group does not occupy C-terminal position in the molecule, because the treatment of the protein with carboxypeptidase B does not change the content of bradykinine in it. Purified kininogen preparation is a substrate for kallikrein from rabbit blood plasma, human saliva and trypsin. Unlike trypsin, kallikreines from human blood plasma and saliva release kinines from kininogen with reduced S-S bonds. Under spontaneous reoxidation of reduced S-S bonds up to 90%, substate properties of kininogen for tripsin recover only by 50%. Rabbit kininogen is similar to beef kininogen II in its molecular weight, amino acid composition and the number of S-S bonds.