Low Molecular Weight Kininogen Research Articles

Acute phase plasma proteins in kininogen-deficient Brown Norway rats

We examined whether Brown Norway rat plasma (BN/May Pfd f) contains alpha 1-cysteine proteinase inhibitor (alpha 1-CPI), also called major acute phase alpha 1-protein or T-kininogen. T-kininogen is a low molecular weight kininogen from which kinin can be released by trypsin but not by kallikreins. The BN plasma reacted with rabbit anti-alpha 1-CPI gamma globulins. Purified alpha 1-CPI released a kinin-like activity with trypsin and with homogenate of salivary glands, as Brown Norway rat plasma did. High concentration of added rat urine induced a small release (10%) of kinin from alpha 1-CPI. Preincubation of Brown Norway rat plasma with rabbit anti-rat alpha 1-CPI gamma-globulins nearly suppressed the kinin-forming substrate of trypsin in this plasma. These results indicated that plasma of our Brown Norway rats contains only alpha 1-CPI as kinin-forming substrate. This plasma contains low amount of alpha 2-macroglobulin, while its content in orosomucoid and haptoglobin was a little larger than that of Wistar rat plasma.

Hemodynamics of the isolated perfused rat kidney in the absence and presence of kallikrein substrate.

Rat kidneys were isolated and perfused for 120 minutes at 80 mm Hg with Krebs-Henseleit buffer containing 7 gm% bovine albumin fraction V. Analysis of the bovine albumin revealed the presence of a contaminating kallikrein substrate which exhibited a MW of 55,000 upon gel-filtration on Sephadex G-200, similar to the reported MW of bovine low molecular weight kininogen. The mean values for renal perfusate flow rate (RPF) and glomerular filtration rate (GFR) in kidneys perfused with this bovine albumin depleted of the kallikrein substrate were significantly lower when compared to standard perfusions. In two other groups of isolated kidneys perfused with kallikrein substrate depleted bovine albumin, purified bovine low molecular weight kininogen (bLMWK, 8.5 micrograms protein in 50 microliters per minute) or synthetic bradykinin (Bk, 1 ng in 10 microliters per minute) were infused after the first 30 minute clearance period into the renal artery. The induced hemodynamic changes were similar in that RPF and GFR significantly increased. These studies show that depletion of bovine albumin from kallikrein substrate results in a concomitant decrease of RPF and GFR, while overall renal function is unaffected. Infusion of both bLMWK or BK to kallikrein substrate depleted bovine albumin has similar effects resulting in a simultaneous increase in RPF and GFR. This suggests that endogenous kallikrein from the perfused kidney releases kinin from infused bLMWK and indicates that kinin primarily affects the afferent arterioles.

Urinary Kallikrein Quantity and Activity in Patients with Pregnancy Induced Hypertension in Third Trimester

In this study, urinary kallikrein quantity (UKQ) and activity (UKA) were measured by the kallikrein direct RIA and kininogenase activity with human low molecular weight kininogen in 32 non pregnant women, 20 normal pregnant women and 18 patients with pregnancy induced hypertension (PIH) in third trimester. There was a significant difference (p<0.001) in UKQ between PIH patients (n=18, 22.5±3.3μg/day, mean±SE) and non pregnant women (n=32, 64.0±6.3μg/day). Also, there was a significant difference (p<0.001) between PIH patients and normal pregnant women (n=20, 68.1±10.1μg/day). UKA of PIH patients (n=18, 82.6± 13.6μg/min/day) was significantly (p<0.0001) lower than non pregnant women (n=32, 496.2+57.2μg/min/day), and significantly (p<0.001) lower than normal pregnant women (n=20, 319.5±48.1μg/min/day). UKA of normal pregnant women was significantly (p<0.05) lower than non pregnant women.These data suggest that there may be renal damage and renal kallikrein-kinin system may be damaged in PIH patients.

The relationship between rat major acute phase protein and the kininogens.

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.

Single-step purification of "kallikrein-resistant" kininogen from rat plasma using monoclonal-antibody immunoaffinity chromatography.

Monoclonal antibody to rat plasma kininogen, obtained after immunization of mice with the kininogen prepared by conventional methods, was purified from ascites fluid and coupled to CNBr-activated Sepharose-4B. Monoclonal-antibody affinity adsorbant thus prepared provided a rapid single-step method of purifying to homogeneity plasma kininogen. Purified rat plasma kininogen showed identical molecular weight and immunological cross-reactivity to rat plasma low molecular weight (LMW) kininogen purified by conventional procedures. Rat plasma kininogen differed from LMW kininogen from other species by virtue of its resistance to cleavage by either plasma or glandular kallikreins.

Immunoglobulin E-mediated release of a kininogenase from purified human lung mast cells.

Certain manifestations of the allergic response, such as vasodilation and edema, may be attributed to the involvement of kinins, but, as yet, little data exist to show how these peptides could be produced during IgE-mediated events. We now report that purified human lung mast cells contain a kininogenase that is released in a dose-dependent fashion by anti-IgE. Release of kinin-generating activity parallels that of histamine and, like histamine release, kininogenase release is temperature-dependent. Kininogenase release also parallels increasing histamine release when mast cells of increasing size are stimulated with an optimal level of anti-IgE. Finally, when mast cells of greater than 99% purity were optimally challenged, kininogenase activity again paralleled histamine levels in the release supernatant and residual cell pellet. The mast cell kininogenase appears to be a preformed mediator in that it occurs in lysates of unstimulated mast cells of greater than 99% purity. Optimal generation of kinin from highly purified human low molecular weight kininogen occurred at pH 5.5 and in 8 preparations was 380 +/- 237 ng kinin/h/10(6) mast cells (means +/- SD). Thus, human lung mast cells contain a kininogenase that is released during, and may participate in, IgE-mediated inflammatory disorders in humans.

T-kininogen - the major plasma kininogen in rat adjuvant arthritis

Total kininogen in plasma of Freund's adjuvant treated rats increased 20-fold 7 days following the injection. Analysis of the kininogens demonstrated that increases in T-kininogen was the major reason for the rise in kininogen. High molecular weight and low molecular weight kininogens showed little or no change. The increase in T-kininogen paralleled the inflammatory condition. Anti-inflammatory agents which reduced paw swelling also reduced plasma T-kininogen levels. Unidentified peaks on HPLC of kinin following plasma treatment by trypsin were shown to be oligopeptides containing T-kinin (Ile-ser-bradykinin). The relationship of T-kininogen to the inflammatory response is discussed.

Experimental arteriosclerosis and the plasma kininogen and low molecular weight kininogen antigen concentration in rabbits

A decrease in trypsin-releasable kinin and in low molecular weight kininogen antigen concentrations was observed in plasma of rabbits with cholesterol-induced arteriosclerosis. In rabbits receiving normal diet for 30 days after 30 days of cholesterol almost complete normalization of the morphological status was observed, which was accompanied by normalization of plasma kinin concentration and by higher than normal concentration of kininogen antigen measured. These changes were not observed in animals receiving normal diet for 30 days after 60 days of cholesterol administration. No data were obtained suggesting a massive kinin release during induction of arteriosclerosis by experimental hypercholesteremia. The possibility of intense kinin formation during a period of repair of the morphological changes was discussed.

Changes in the kallikrein kinin system during acute pancreatitis in man

Changes in the kallikrein-kinin system were analysed in 19 attacks of acute pancreatitis in man and correlated to the severity and clinical course of the disease. Prekallikrein, kininogen and kallikrein inhibition were significantly lower in blood in severe attacks than in moderate or mild attacks. These changes were even more pronounced in peritoneal fluid, where kallikrein activity was above normal, while kininogen and kallikrein inhibition were nil in severe attacks. Both high and low molecular weight kininogen were decreased, denoting an activation also by kininogenases other than plasma kallikrein. These changes indicate an activation of the kallikreinkinin system in acute severe pancreatitis in man, especially in the abdominal cavity. Although there is a great deal of evidence for activation of the kinin system in experimental shock slates in animals (1–4), there are to date rather few studies showing that such an activation takes place in human acute pancreatitis. This lack of clinical studies is mainly explained by the difficulty in measuring activated components of the system, especially since these components are rapidly inactivated in different ways in vivo (5).

Identification of a kallikrein-like latent serine protease in human erythrocyte membranes

We have discovered and characterized a kallikrein-like latent serine protease in intact human erythrocytes and ghosts. The enzyme is activatable by trypsin. The solubilized enzyme has esterolytic activity with a pH optimum of 9; but the membrane-associated activity increases almost linearly up to pH 10. The activated enzyme releases kinin from bovine low molecular weight kininogen. Enzyme activity is inhibited by TosLysCH 2Cl, phenylmethylsulfonyl fluoride, aprotinin and amiloride, and weakly by soybean or lima bean trypsin inhibitor. It is inhibited by Co 2+, Zn 2+ and Mn 2+ but is stimulated by Fe 2+, deoxycholate and phospholipase A 2. An erythrocyte membrane protein (M r = 88,000) with an active site serine residue was identified with [ 14C]-diisopropylphosphorofluoridate labeling. Consistent with the finding of tryptic activation of the latent erythrocyte serine protease, trypsin treatment reduced the density of labeling of this protein and revealed a lower molecular weight form (M r = 64,000). Possible relationships between the activity of this newly identified serine protease and events such as erythrocyte membrane ion fluxes might be of interest.

Kininogenase activity of tonin.

Tonin, known for its specific and direct generation of angiotensin II, was highly purified from rat submaxillary gland and investigated for kininogenase activity. For the substrate, heat-treated plasma from ox blood, and highly purified low-molecular-weight (LMW) and high-molecular-weight (HMW) kininogens, were used. The reaction product formed at pH 8.0 well satisfied the characteristics of kinin, i.e., depressor and oxytocic activities and reactivity with antibradykinin antiserum. Kinin formed by tonin from purified LMW kininogen was identified with bradykinin in high performance liquid chromatography and radioimmunoassay. The results revealed tonin's new capability of forming kinin in addition to the hitherto known pressor angiotensin II, indicating tonin, too, is a member of the "kinin- tensin enzyme system."

Radioimmunoassays of human high and low molecular weight kininogens in plasmas and platelets.

Radioimmunoassays of human high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) were developed using antibodies directed against the light and the heavy chains of kallikrein-cleaved HMWK. With the anti-light chain antibodies, the radioimmunoassay was specific for HMWK with a detection limit of 0.4 ng. The anti-heavy chain antibodies were used to quantify the concentration of total kininogen antigens. In four different plasmas with a congenital deficiency in HMWK procoagulant activity, there was no detectable antigen in two cases and trace amounts, less than 1 micrograms/ml in the other plasmas (normal concentration: 72 +/- 6 micrograms/ml). In the absence of HMWK, the radioimmunoassay performed with the anti-heavy chain antibodies was specific for LMWK. The amount of LMWK was different in each of these patients' plasmas, ranging from no detectable antigen, i.e. less than 0.15 micrograms/ml, to a normal content. Antigens immunologically indistinguishable from plasma kininogens were detected in lysates of five times washed platelets. HMWK antigen concentration was 3.17 +/- 0.87 micrograms per 10(11) platelets (mean value in 11 donors). LMWK was also present in platelet lysates and the relative concentration versus HMWK was the same as in plasma.

Purification and characterization of rat low molecular weight kininogen.

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.

The effect of high molecular weight kininogen on surface-adsorbed fibrinogen

High molecular weight kininogen (HMWK) plays an important role in altering the association of plasma fibrinogen with surfaces. Plasma initially deposits fibrinogen onto most materials, but on hydrophilic surfaces within 10 min adsorbed plasma fibrinogen cannot be detected on the surface by antifibrinogen antisera. However, using HMWK-deficient plasma, fibrinogen remains immunologically identifiable. The inter-relationship of adsorbed plasma fibrinogen with kininogen on hydrophilic surfaces is studied further using glass slides stained for protein with Coomassie Blue, and oxidized silicon crystal slices in an automated ellipsometer. On glass slides when plasma that is deficient in both low molecular weight kininogen (LMWK) and HMWK, is reconstituted with HMWK (0.40 Units/ml), fibrinogen is no longer detected on the surface. This finding is specific for HMWK, since, when the same plasma is reconstituted with LMWK (220 μg/ml), the amount of fibrinogen detected on the surface is unchanged. The alteration of surface-adsorbed fibrinogen by HMWK is not due to plasmin-induced fibrinolysis, since it occurs in plasminogen-free plasma. In the ellipsometer, surface adsorption of normal plasma is associated with a significantly less (p < 0.0005) thick protein layer (1.99 ± 0.08 degree change in azimuth) than plasmas deficient in HMWK (2.32 ± 0.11). Using ellipsometry, HMWK in plasma is shown to shorten the time in which immunologically detectable surface-adsorbed fibrinogen was removed or altered. These studies in a whole plasma system present further evidence that HMWK specifically modifies the association of plasma fibrinogen with hydrophilic surfaces.

A radioimmunoassay method for measurement of urinary kinins.

A sensitive and specific radioimmunoassay for measuring urinary kinins was developed. Antibodies against bradykinin were induced in rabbits by injecting bradykinin coupled to bovine albumin. One of the antisera generated was used at a final dilution of 1:18,000 to obtain a 30% total binding of bradykinin-(8-tyrosine)-[125I]-triacetate. Synthetic bradykinin (5-1,000 pg) was used as standard in the curves. The sensitivity of the assay was 5 pg. The recovery of bradykinin added to urinary samples was 86.85 +/- 6%. The intraassay and interassay coefficients of variation were 3.3% (n = 12) and 4.4% (n = 5), respectively. The antiserum showed no cross-reactivity with oxytocin or low molecular weight kininogen and cross-reacted with kallidin (lys-bradykinin), met-kallidin, and angiotensin I, but cross-reaction with angiotensin I (2.5%) was low enough to be disregarded. The mean urinary levels of total kinins in 12 normal subjects were 23.2 +/- (SEM) 2.2 micrograms/day.

The influence of the kallikrein-kinin system in the development of the pancreatic shock.

Kallikrein and other kininogenases like trypsin and plasmin release kinins by limited proteolysis from high (HMWK) — and low molecular weight kininogen (LMWK). The kinins are vasoactive peptides with fairly unknown physiology.

Identification of tissue kallikrein in brain and in the cell-free translation product encoded by brain mRNA.

A monoclonal antibody against purified rat urinary kallikrein was coupled to agarose and used to isolate kallikrein from rat brain. The purified enzyme has N alpha-tosyl-L-arginine methyl esterase activity with a pH optimum at 9.0, kinin-releasing activity from a purified low molecular weight kininogen, and a parallelism with standard curves of rat urinary kallikrein in a direct radioimmunoassay. Brain kallikrein is inhibited by a series of tissue kallikrein inhibitors with IC50 values similar to those for urinary kallikrein. The purified brain enzyme was labeled with [14C]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. Electrophoretic mobility of the enzyme was closely similar to that of urinary kallikrein with estimated Mr of approximately 38,000. With Western blot analyses using a rabbit anti-kallikrein antibody, both brain and urinary kallikrein were visualized at identical positions by immunoperoxidase staining and by autoradiography with 125I-protein A binding. Brain mRNA was used to direct cell-free protein synthesis in wheat germ and rabbit reticulocyte lysate systems. [35S]Methionine-labeled kallikrein was identified by fluorography of sodium dodecyl sulfate-polyacrylamide gels after the translation products were subject to immunoprecipitation with affinity-purified kallikrein antibody. Collectively, the data show that tissue kallikrein exists in brain and can be synthesized by brain mRNA.

Malignant hypertension: a syndrome accompanied by plasmatic diminution of low and high molecular weight kininogens.

Total kininogen (Kgn), kallikrein, and prekallikrein were measured in patients with malignant hypertension (MH), essential hypertension (EH), normotensive control (NC), and hypertension and chronic renal failure (HRF). These components of the kallikrein-kinin system were related to the levels of creatinine and fibrinogen. High molecular weight Kgn and low molecular weight Kgn were also measured in blood samples from a peripheral vein, arterial blood, and suprahepatic vein in NC, EH, and MH. Results showed that total Kgn levels were diminished in MH and this diminution could not be ascribed to decreases in renal function, hematocrit, or fibrinogen levels. Appropriate antihypertensive treatment for over 1 year did not normalize Kgn levels in 10 of 11 patients. High molecular weight Kgn and low molecular weight Kgn were both diminished in MH (0.26 +/- 0.04 nmol bradykinin/ml and 0.93 +/- 0.12 nmol lysyl-bradykinin/ml, respectively) as compared to NC (0.39 +/- 0.07 and 1.92 +/- 0.16) and EH (0.51 +/- 0.07 and 1.65 +/- 0.13). Higher concentrations of high molecular weight Kgn were demonstrated in the suprahepatic vein as compared to arterial blood, demonstrating its synthesis by the liver. However, patients with MH had a diminished capacity to synthetize high molecular weight Kgn. A decrease in synthesis of high molecular weight Kgn may be a partial explanation for low levels of total Kgn. It is suggested that a lack of Kgn may play a role in the pathogenesis of MH.

A single gene for bovine high molecular weight and low molecular weight kininogens.

A potent vasoactive peptide, bradykinin, is liberated from two distinct kininogens designated low molecular weight (LMW) and high molecular weight (HMW) kininogens. We have recently cloned and sequenced cDNAs for bovine LMW prekininogens and indicated that LMW prekininogens are encoded by two very similar but distinct mRNAs. This study concerns the structural basis for the relationship between HMW and LMW prekininogen mRNAs by examination of cDNA clones for bovine HMW prekininogens and a genomic clone. Two types of cDNAs for HMW prekininogens have been identified. The deduced sequences of these two mRNAs are identical with those of two LMW prekininogen mRNAs up to the regions preceding the sequences specifying the divergent C-terminal regions of HMW and LMW kininogens, with one-to-one correspondence. In genomic DNA, the sequence precisely corresponding to the divergent 3'-terminal region of LMW prekininogen mRNA is located after 87 nucleotides downstream from the sequence specifying the 3'-untranslated region of HMW prekininogen mRNA. Based on these findings, we conclude that HMW and LMW prekininogen mRNAs are transcribed from the same gene.

Purification of single-chain human low-molecular-weight kininogen and demonstration of its cleavage by human urinary kallikrein

Human low-molecular-weight kininogen (LMWK) was purified to apparent physical and functional homogeneity by a six-step procedure consisting of ion-exchange chromatography, reverse ammonium sulfate gradient solubilization, hydrophobic chromatography on phenyl-Sepharose, gel filtration, and removal of contaminating proteins by their affinity for Affi-Gel blue and zinc. The recovery averaged 15.6% ( n = 4). Purified LMWK presented as a single stained band on alkaline polyacrylamide gel electrophoresis which corresponded to the region of function in eluates from a duplicate gel. The apparent homogeneity was also observed in sodium dodecyl sulfate (SDS)-gel electrophoresis, where the protein presented as a single band of M r = 65,000 without reduction and 68,000 with reduction. A mole of substrate released 0.8 mol of kinin in 5 min when cleaved by human urinary kallikrein (HUK), and 0.9 mol after 30 min. Cleavage of the single-chain LMWK released kinin from within a disulfide loop as indicated by the SDS-gel electrophoresis of reduced and unreduced kinin-free LMWK. The heavy chain exhibited an M r = 62,000, which is similar to the M r of the amino-terminal chain of human HMWK and is consistent with their antigenic relatedness. In contrast to the M r = 64,000 procoagulant chain of human HMWK, the small (<10,000) carboxy-terminal chain of LMWK has no procoagulant activity and may serve only to protect the kinin moiety in the intact substrate.