Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors

1979 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Amundsen ◽  
M. Larsbraaten ◽  
K. Lyngaas ◽  
E. Fareid
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Plasma Kallikrein ◽  
Peptide Substrate ◽  
Total Inhibition ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plus Fraction ◽  
Time Dependent Inhibition ◽  
Esterase Inhibitor

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.

scholarly journals Studies on Human Serum Inhibitors of Plasmin, Urokinase and Plasma Kallikrein Using Chromogenic Substrates

1977 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Fareid
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Time Dependent ◽  
Low Molecular Weight ◽  
Plasma Kallikrein ◽  
Serum Samples ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plasmin Inhibitors ◽  
Time Dependent Inhibition

Human serum inhibitors of plasmin, urokinase and kallikrein were studied using chromogenic substrate assays,(Plasmin substrate, S-22 51, Kabi, Sweden, Urokinase substrate, Chromozyme -UK and Plasma Kallikrein substrate, Chromozyme -PK, Pentapharm, Basle, Switzerland). In whole serum both “immediate” and “time dependent” inhibition of plasmin and kallikrein was observed, whilst only very weak inhibition of urokinase was detected. When serum samples were fractionated by Sephadex G-200 gel-filtration the “immediate” plasmin inhibitors were identified as α2-macroglobulin and low molecular weight antiplasmin whilst α2-macroglobulin and C1-esterase inhibitorwere immediate inhibitors of kallikrein.“Time-dependent” inhibition of both enzymes was observed in the ai-antitrypsin containing fractions.

scholarly journals Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

1974 ◽  
Vol 143 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Sten Müllertz
Keyword(s):  
Human Plasma ◽  
Protease Inhibitors ◽  
Gel Electrophoresis ◽  
Protease Activity ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Crossed Immunoelectrophoresis ◽  
Low Concentrations ◽  
Α2 Macroglobulin ◽  
Esterase Inhibitor

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a KD value of 0.18 by gel filtration and post β1 mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a KD value of 0 and α2 mobility appeared, which was probably plasmin in a complex with α2 macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same KD value by gel filtration as plasminogen (0.35), but β1 and γ mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with γ mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin–α2 macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin–α2 macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, α1 antitrypsin, inter-α-inhibitor, antithrombin III, and C1-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The β1 and post β1 components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.

scholarly journals Changes in Levels of Plasma Pre-Kallikrein, Kallikrein and Kallikrein Inhibitors During Endotoxin Shock in Dogs

1977 ◽  
Author(s):  
M. J. Gallimore ◽  
A. O. Aasen ◽  
E. Amundsen
Keyword(s):  
Late Phase ◽  
Endotoxin Shock ◽  
Time Dependent ◽  
Plasma Kallikrein ◽  
Kinin System ◽  
E Coli ◽  
Dependent Inhibition ◽  
Kallikrein Kinin System ◽  
Time Dependent Inhibition ◽  
Kallikrein Inhibitors

Plasma protease activity is known to be increased during endotoxin shock and recent studies have indicated that the plasma kallikrein-kinin system becomes activated by circulating endotoxin. Plasma levels of pre-kallikrein kallikrein and kallikrein inhibitors were therefore determined in samples from dogs infused with E. coli endotoxin, using assays with a chromogenic substrate for plasma kallikrein(Chromozyme -PK, Pentapharm, Basle, Switzerland). “Fast-reacting” and “time-dependent” inhibitors of kallikrein were studied using purified human plasma kallikrein. Considerably reduced levels of plasma pre-kallikreiri and increased levels of kallikrein were detected in the late phase of shock and significant reductions in “fast-reacting” and “time-dependent” inhibition of kallikrein was observed. These results show that during endotoxin shock plasma pre-kallikrein becomes activated to kallikrein and indicate that kallikrein inhibitors play an important mediatory role in the pathophysiology of endotoxin shock.

Studies on Components of the plasma kallikrein-Kinin System in Normal Subjects and Patients with Septicemia

1979 ◽  
Author(s):  
A.O. Aasen ◽  
M.J. Gallimore ◽  
K. Lyngaas ◽  
M. Larsbraaten ◽  
E. Amundsen ◽  
...  
Keyword(s):  
Enzyme System ◽  
Normal Subjects ◽  
Plasma Kallikrein ◽  
Plasma Samples ◽  
Kinin System ◽  
The Past ◽  
Α2 Macroglobulin ◽  
Kallikrein Kinin System ◽  
Fatal Sepsis ◽  
Esterase Inhibitor

Over the past decade evidence that the plasma kallikrein-kinin system is activated during septicemia has accumulated. We have used several tests, including newly developed chromogenic peptide substrate assays, to study components of this proteolytic enzyme system in plasma samples from normal subjects and patients with septicemia. The parameters studied were Hageman factor(HF), plasma kallikrein (KK), plasma prekallikrein (PKK), high molecular weight kininogen(HMwK), functional kallikrein inhibition (KKI), c1-esterase inhibitor (CIINH) , α2-macroglobulin (α2 M)and α1-antitrypsin (α1AT ). In samples from patients with fatal sepsis, levels of HF, PKK, HMwK, α2M and KKI were all markedly reduced and spontaneous KK activity was detected. CIINH and α1AT levels were much higher than normal. In plasma samples from three patients with septicemia who subsequently recovered mean plasma levels of PKK, KKI, HMwK and CIINH were higher than in the samples from the patients who died. Our results emphasize that the plasma kallikrein-kinin system becomes activated during septicemia and suggest that functional kallikrein inhibition contributes to survival.

scholarly journals HUMAN PLASMA ALPHA 2-MACROGLOBULIN

1970 ◽  
Vol 132 (2) ◽  
pp. 329-352 ◽  
Author(s):  
Peter C. Harpel
Keyword(s):  
Trypsin Inhibitor ◽  
Esterase Activity ◽  
Gel Filtration ◽  
Basic Amino Acid ◽  
Soybean Trypsin Inhibitor ◽  
Plasma Kallikrein ◽  
Angioneurotic Edema ◽  
Α2 Macroglobulin ◽  
Hereditary Angioneurotic Edema ◽  
Esterolytic Activity

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α2-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α2-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α2-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α2-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α2-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α2-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α2-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α2-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α2-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.

scholarly journals Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis

1996 ◽  
Vol 313 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Christopher C. LAWRENCE ◽  
Wendy J. SOBEY ◽  
Robert A. FIELD ◽  
Jack E. BALDWIN ◽  
Christopher J. SCHOFIELD
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Ferrous Ion ◽  
Typical Member ◽  
Apparent Homogeneity ◽  
Initial Characterization ◽  
Dependent Inhibition ◽  
Proline Hydroxylation ◽  
Time Dependent Inhibition

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (Mr approx. 38000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Use Of Chromogenic Peptide Substrate Assays In The Evaluation Of Surgical Treatment Of Intraabdominal Sepsis

1981 ◽  
Author(s):  
N Smith-Erichsen ◽  
A O Aasen ◽  
E Amundsen
Keyword(s):  
Surgical Treatment ◽  
Antithrombin Iii ◽  
Normal Values ◽  
Peptide Substrate ◽  
Α2 Macroglobulin ◽  
Septic Focus ◽  
At Iii ◽  
Coagulation Assay ◽  
Intraabdominal Sepsis ◽  
Esterase Inhibitor

Chromogenic peptide substrate assays were included in the evaluation program of 14 patients having surgical treatment for intraabdominal sepsis.Six of the patients survived, whereas eight died. The six survivors had a total of 19 operations (1-5 operations /patient). The eight fatal cases had a total of 14 operations (1-2 operations/patient). Plasma prekallikrein (PKK), functional antikallikrein (KKI), plasminogen (Pig), fast antiplasmin (AP) and functional antithrombin III (AT- III) values were determined using chromogenic peptide substrate assays. Hageman factor levels (HF) were determined using a coagulation assay with a HF deficient plasma. α2-Antiplasmin (α2AP), CI-esterase inhibitor (CIINH), α2- Macroglobulin (α2M) and AT III were determined immunochemically.In both the fatal cases and the survivors PKK, HF, Plg and AT III values were found markedly reduced during sepsis. The reductions of PKK and AT III were significantly more pronounced in the fatal cases than in the survivors. During sepsis the ratio of functional activity versus immunochemical values decreased when compared with normals for α2AP, CIINH and AT III. Whereas the parameters remained reduced in the fatal cases, gradual increases towards normal values for PKK, HF, Plg and AT III were found within two weeks in the survivors. Clinically and by autopsy all the fatal cases were evaluated to have a persistant intraabdominal septic focus.It is concluded that chromogenic peptide substrate assays appear to be of great value in evaluating surgical treatment of intraabdominal sepsis.

scholarly journals The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1974 ◽  
Vol 139 (2) ◽  
pp. 359-368 ◽  
Author(s):  
Zena Werb ◽  
Mary C. Burleigh ◽  
Alan J. Barrett ◽  
Phyllis M. Starkey
Keyword(s):  
Sodium Dodecyl ◽  
Synovial Cell ◽  
Polymorphonuclear Leucocyte ◽  
Collagenolytic Activity ◽  
Gel Chromatography ◽  
Subsequent Reaction ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Time Dependent Inhibition ◽  
Metal Proteinase

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by α2-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to α2-macroglobulin; α1-trypsin inhibitor was not inhibitory. α2-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of α2-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by α2-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with α2-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by α2-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by α2-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by α2-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by α2-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced α2-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of α2-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with α2-macroglobulin are the same as those of other proteinases.

Simple Chromogenic Peptide Substrate Assays For Determining Components Of The Plasma Kallikrein System

1981 ◽  
Author(s):  
M J Gallimore ◽  
P Friberger
Keyword(s):  
Plasma Levels ◽  
Rapid Evaluation ◽  
Plasma Kallikrein ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Chromogenic Substrates ◽  
Hageman Factor ◽  
The Past ◽  
Dilution Step ◽  
Esterase Inhibitor

Over the past few years the introduction of chromogenic substrates has facilitated studies on components of the plasma defence systems. During this period it has become apparent that there is a need for simple, easy to perform, assays for measuring components of these systems. We have therefore developed simple assays for determining prekallikrein (PKK), kallikrein inhibition (KKI) and kailikrein-1ike activity (KK). These assays have a plasma dilution step, followed by repeat single volume pipettings of reagents. Both “rate” and “end-point” assays have been developed. For the PKK assay a new activator has been developed which contains Hageman factor and high mol. wt. kininogen. This overcomes the problems found by several groups with other activators when studying patients with low plasma levels of Hageman factor, PKK and high mol. wt. kininogen. For the KKI assay a stable preparation of human plasma KK has been produced. This assay detects mainly Cl'- esterase inhibitor. The assay for KK-like activity is required where activation of PKK has occured and spontaneous activity towards the substrate is found. Plasma samples from 25 normal donors were tested for PKK, KKI and KK. The PKK and KKI values were calculated from standard curves made with a standard plasma. When compared with PKK and C1INH antigen levels correlations of 0.84 and 0.85 were obtained. All of the plasmas had KK activities lower than 5% of the total PKK levels.These assays should prove valuable in the rapid evaluation of the plasma KK system in patients.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

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