Cellular expression of plasma prekallikrein in human tissues.

Plasma prekallikrein (PPK) is synthesised in hepatocytes and secreted into the blood, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Recently we demonstrated by quantitative RT-PCR that the human PPK gene is transcribed not only in the liver, but also in various non-hepatic human tissues at significant levels. However, up to now no reliable information is available concerning protein synthesis in the corresponding human tissues. Here we demonstrate by immunohistochemical studies that PPK or plasma kallikrein (PK) is localised in cells of different embryologically derived human tissues. In the human nephron, single cells of the distal tubules stained intensely, while the cytoplasm of cells forming proximal tubules and collecting ducts stained uniformly. PPK/PK was localised in hepatic epithelial cells of the liver, in cells of the pancreatic islet of Langerhans, in the interstitial Leydig cells of the testes, in the follicular and thecal granulosa cells of the ovary, and in the parotid gland, oesophagus, skin, respiratory tract, prostate and breast. We conclude that the cellular localisation of PPK/PK in multiple different progenitor-derived cells indicates specific cellular functions of this enzyme, in addition to its known function in the blood.

Primary structure of guinea pig plasma prekallikrein.

A full length guinea pig plasma prekallikrein (PK) cDNA was cloned from a liver cDNA library. The nucleotide sequence with 2242 bp was analyzed and the amino acid sequence with 618 residues was deduced. Kallikrein was purified from guinea pig plasma and cleavage site in the activation was determined. The amino acid sequence around the cleavage site -368Ile-Asp-Ala-Arg-Ile-Val-Gly-375Gly- differed from that of the human PK -368Thr-Ser-Thr-Arg-Ile-Val-Gly-375Gly-. Protease substrates containing penta-peptides which mimicked the sequence of the cleavage sites from P3 to P2' of guinea pig Hageman factor (HF) and PK were synthesized, and kinetic analyses of the hydrolysis by guinea pig activated HF (HFa) and kallikrein were carried out. The combination between HFa and the PK mimicking peptide provided the best kinetics. These results in part explain why the cascade activation of PK by HFa is predominant in the guinea pig system.

Part of prekallikrein removed from human plasma together with IgG--immunoblot experiments and functional tests.

With the present study, evidence is provided that prekallikrein (PK) in human plasma might be present in two different states, one of them removed along with IgG on Protein G columns. At a plasma dilution of 1 + 2.5, small amounts of an IgG fraction were left in plasma along with all of the PK. At a dilution of 1 + 11, nearly all IgG was removed. The removal in parallel of part of the PK was shown in immunoblot experiments and confirmed in amidase assays. One monoclonal antibody against PK (13G11) and two preparations of polyclonal antibodies were used for the immunoblot experiments. Different peptide substrates (S-2302, S-2222, Bz-Pro-Phe-Arg-pNA), along with protease inhibitors (soybean trypsin inhibitor, corn trypsin inhibitor, lima bean trypsin inhibitor) were used for the amidase assays. The amidase assays indicated that factors XII and XI were reduced by Protein G columns. In all experiments with extensive removal of IgG, protein recognized by the factor XII light chain mAb C6B7 was removed at the same time. This antibody preparation did not detect purified contact factors, but it did recognize a preparation of purified beta-FXIIa, and also significant amounts of protein present in plasma deficient in factor XII and not detectable in plasma deficient in PK. This protein accordingly seems to be connected with the PK fraction removed with IgG.

Identification of a factor IX binding site on the third apple domain of activated factor XI.

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68-140% of wild type activity). The Km for activation of factor IX by factor XIa/PKA3 (12. 7 microM) is more than 30-fold higher than the Km for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 microM). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.

The shape of high molecular weight kininogen. Organization into structural domains, changes with activation, and interactions with prekallikrein, as determined by electron microscopy.

Knowledge of the organization of the kininogen gene and protein structure and function correlations has allowed the development of a model of high molecular weight kininogen. Domains 1-3 on the heavy chain are evolutionarily related to cystatin and the latter two are inhibitors of cysteine proteases. Proteolytic cleavage in domain 4 to release bradykinin causes a conformational change, exposing a surface-binding region (domain 5) on the disulfide-linked light chain. The carboxyl-terminal domain 6 contains a zymogen binding sequence for factor XI and prekallikrein which, with domain 5, accounts for its cofactor activity. To explore further the domain structure, we have determined the shapes of high molecular weight kininogen and prekallikrein by electron microscopy of rotary shadowed preparations and computer image processing. High molecular weight kininogen appears to be a linear array of three linked globular regions about 16 nm long, with the two ends also connected by another thin strand. Both prekallikrein and kallikrein have a compact globular shape, with a subdivision that is sometimes visible. Different functional domains of high molecular weight kininogen were identified by monoclonal antibodies against these regions, as well as ligand binding of prekallikrein. These studies indicate that one end globular region is the prekallikrein-binding domain, the other comprises the cysteine protease inhibitor domains and the smaller central nodule is the surface-binding domain. Cleavage of high molecular weight kininogen with plasma kallikrein to yield two-chain high molecular weight kininogen results in a striking change in conformation: the central surface-binding domain swings out so that it is still adjacent to the prekallikrein-binding domain but no longer in the middle. These structural studies provide insight into the interactions of these proteins and aspects of the mechanisms of their actions.

Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells.

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.

Effect of Neurotropin on the activation of the plasma kallikrein-kinin system.

Bradykinin (BK), an important mediator of allergic reactions and pain induction, is released by the activation of the plasma kallikrein-kinin (K-K) cascade. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus and is widely used clinically in Japan as an effective agent for these disorders. Since its mechanism of action is not clearly known, we have investigated the effects of Neurotropin on the human plasma K-K system. In dextran sulfate-activated plasma, Neurotropin inhibited the formation of BK, the cleavage of high molecular weight kininogen (HK) and the formation of kallikrein-C1 inhibitor and activated coagulation factor XII (FXIIa)-C1 inhibitor complexes. Experiments using purified enzyme of the K-K cascade indicated that Neurotropin inhibited surface-mediated activation of coagulation factor XII (FXII) and the activation of prekallikrein by FXIIa. Neurotropin also inhibited the binding of FXII and HK to the activating surface. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and consequently the formation of BK.

[Preparation of a prekallikrein concentrate and determination of its activator in blood derivatives]. / Príprava koncentrátu prekallikreinu a stanovení jeho aktivátoru v krevních derivátech.

The determination of an increased level of a fragment of the Hageman's factor (HFf) in preparations prepared from blood plasma can be an index of their potential reactivity in clinical application. The demonstration of the presence of HFf is based on the fact that the preparation containing this factor is able to activate the prekallikrein substrate. When the concentration of prekallikrein is sufficient, the amount of the developed active enzyme kallikrein is proportional to the amount of HFf. The amount of kallikrein is determined by cleaving the specific chromogenic substrate NO-Pro-Phe-Arg-pNA. The presence of salts negatively influences the rate of the activating stage of the reaction. The described preparation of the prekallikrein concentrate from the human blood plasma is a modification of the method of Levy and Sober) for the separation of the human immunoglobulin fraction. The blood plasma with an addition of hexadimethrinebromide is sorbed on DEAE Sephadex equilibrated with 0.0175 mol/l phosphate buffer pH 6.3 with hexadimethrinebromide in a vessel with an intact surface. Prekallikrein together with IgG are not sorbed on the anion exchanger under the above-mentioned conditions. The obtained supernatant can be employed as the prekallikrein substrate to determine the HFf activity in blood derivatives.

Activation of the contact system in ascites from patients with gastrointestinal cancer.

Our observations indicates that the plasma contact system is activated in ascites from patients with gastrointestinal cancer: Factor XII is activated, plasma kallikrein is present in complex with the protease inhibitor alpha 2-macroglobulin, and the plasma kallikrein substrate high molecular weight kininogen, is highly degraded. Contact activation seems to take place in spite of a high level of inhibition. Activation of the contact system generates mediators, which may play a role in the accumulation of ascites.

Computerized immunoblot analyses (CIBA) of the distribution of prekallikrein and its activation products in vivo and in vitro.

The distribution of prekallikrein and the complexes of kallikrein with C1 inhibitor (C1INH), alpha 2-macroglobulin and approximately 58-kDa protein(s) (e.g. antithrombin III), differs in normal and C1INH-deficient human plasma, activated in vitro, due to different C1INH levels. Different distribution in the deficient plasma activated in vivo or in vitro, suggests different rates of complex clearance.

Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule.

The location of 16 of the 18 disulfide bonds in human plasma prekallikrein was determined by amino acid sequence analysis of cystinyl peptides produced by chemical and enzymatic digestions. A unique structure, named the apple domain, was established for each of the four tandem repeats in the amino-terminal portion of the molecule. The apple domains (90 or 91 amino acids) contain 3 highly conserved disulfide bonds linking the first and sixth, second and fifth, and third and fourth half-cystine residues present in each repeat. The fourth tandem repeat contains an extra disulfide bond that forms a second small loop within the apple domain. The carboxyl-terminal portion of plasma prekallikrein containing the catalytic region of the molecule was found to have disulfide bonds located in positions similar to those of other serine proteases.

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.

[The use of a tripeptide chromogenic substrate for simultaneous determination of 4 indices of kallikrein-kinin system]. / Ispol'zovanie tripeptidnogo khromogennogo substrata dlia odnovremennoi otsenki chetyrekh pokazatelei aktivnosti kallikrein-kininovoi sistemy.

Evaluation of the kallikrein-kinine system activity in blood plasma is of importance due to participation of the components of this system in regulation of blood plasma proteolysis. Using D-Pro-Phe-Arg-pNA as a substrate, a procedure for simultaneous estimation of four patterns of the kallikrein-kinine system activity was developed: I. maintenance of contact phase, 2. content of prekallikrein (1.49 un/ml in normal state), 3. activity of kallikrein (0.104 un/ml), 4. antikallikrein potential (0.700 IU/ml).

Characterization of N-linked oligosaccharides of human urinary kallikrein molecules.

The micro-heterogeneity due to varied N-linked oligosaccharides of both active- and pro-types of human urinary kallikrein (HUK) in normal subjects and some patients were investigated by the methods of serial lectin affinity chromatography and crossed affino-immunoelectrophoresis. In the case of both types of normal HUK, the species carrying tri- and/or tetra-antennary oligosaccharide(s), corefucosylated bi-antennary oligosaccharide(s), and bi-antennary oligosaccharides containing outer galactose residues and an N-acetylglucosamine residue linked beta 1,4 to a beta-linked mannose residue (bisecting N-acetylglucosamine residue) amounted to approximately 36, 33 and 17% of the total of each type of HUK, respectively. On the other hand, in some diseases, i.e. essential hypertension, Bartter's syndrome and acute pancreatitis, alterations of the chromatographic and electrophoretic patters were observed and are assumed to correspond to glycosylation changes in each HUK molecule.

Purification and immunological properties of human urinary kallikrein and prokallikrein.

Human urinary prokallikrein and kallikrein have been purified from the same source of urine simultaneously. The anti-kallikrein and anti-prokallikrein antibodies were raised in rabbits using the purified preparations. With respect to solid phase enzyme immunoassay (EIA), immunoaffinity column chromatography, and single radial immunodiffusion, reactivity of each antibody with kallikrein was distinctly different from that with prokallikrein. Kallikrein could be determined by anti-kallikrein antibody-immobilized EIA below 20 ng per ml, whereas prokallikrein was undetectable. Prokallikrein became detectable at higher concentrations, although it was less reactive than kallikrein. The anti-prokallikrein antibody-immobilized EIA detected both kallikrein and prokallikrein with the same sensitivity. However, the binding capacity for kallikrein was about one-third less than that for prokallikrein. The results show that kallikrein in human urine may be determined directly and selectively. Similar difference in reactivity was observed with immunoaffinity column chromatography and single radial immunodiffusion. The presence of 3-4 antigenic sites per molecule was indicated by quantitative precipitin reaction, and it is suggested from analysis of amino acid sequence of kallikrein by the method of Hopp and Woods that four hydrophilic regions exist in kallikrein molecule.

Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants.

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.

[Contact-activated and immunoreactive prekallikrein in the plasma of patients with hypertension]. / Kontaktno-aktiviruemyi i immunoreaktivnyi prekallikrein v plazme krovi bol'nykh gipertonicheskoi bolezn'iu.

Alterations in activity of kaolin-activated (contact-activated, CA) and immunoreactive (IR) prekallikrein were studied in blood plasma of healthy persons and of patients with hypertension under conditions of rest and of dose-dependent physical loading. CA prekallikrein was dissimilarly altered depending on the type of hemodynamic structure of the pressure reaction (arterial pressure): content of CA prekallikrein was increased in hyperkinetic type and--decreased in hypokinetic type. Content of IR prekallikrein, measured by means of radial immunodiffusion using monospecific antiserum, was similar in healthy persons and in the patients at rest and after physical exercises. It was increased slightly but did not correlate with CA prekallikrein alterations. As content of CA- and IR-prekallikreins did not correlate in the disease dynamics and after physical exercises, functional impairments may cause activation of blood plasma prekallikrein in hypertension. A procedure for isolation of highly purified preparation of kallikrein, production of monospecific antiserum to prekallikrein are described. Immunochemical studies and quantitative estimation of IR-prekallikrein were carried out.