Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates

Biochemistry ◽  
1981 ◽  
Vol 20 (25) ◽  
pp. 7196-7206 ◽  
Author(s):  
Brian J. McRae ◽  
Kotoku Kurachi ◽  
Ronald L. Heimark ◽  
Kazuo Fujikawa ◽  
Earl W. Davie ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Sites ◽  
Factor Xa ◽  
Plasma Kallikrein ◽  
Bovine Thrombin ◽  
Factor Ixa ◽  
Factor Xiia ◽  
Factor Xia ◽  
Peptide Thioesters

scholarly journals The synthesis of arginylfluoroalkanes, their inhibition of trypsin and blood-coagulation serine proteinases and their anticoagulant activity

1990 ◽  
Vol 265 (2) ◽  
pp. 539-545 ◽  
Author(s):  
T Ueda ◽  
C M Kam ◽  
J C Powers
Keyword(s):  
Blood Coagulation ◽  
Human Factor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Activated Partial Thromboplastin Time ◽  
Serine Proteinases ◽  
Bovine Thrombin ◽  
Bovine Trypsin ◽  
Factor Xiia ◽  
Factor Xia

Seven arginylfluoroalkanes (‘arginine fluoroalkyl ketones’) were synthesized by using a modified Dakin-West procedure. The structure of benzoyl-Arg-CF2CF3 was analysed by 19F-n.m.r. spectroscopy and m.s. and the compound was shown to exist primarily as a hydrate or cyclic carbinolamine. Arginylfluoroalkanes are good inhibitors of blood-coagulation serine proteinases and were found to be slow-binding inhibitors for bovine trypsin with Ki values of 0.2-56 microM. Benzoyl-Arg-CF2CF3 was the best inhibitor for bovine thrombin and human Factor XIa, and inhibited thrombin and Factor XIa competitively with Ki values of 13 microM and 62 microM respectively. The best inhibitor for pig pancreatic kallikrein was p-toluoyl-Arg-CF3, with a Ki value of 35 microM. Benzoyl-Arg-CF3 and benzoyl-Arg-CF2CF3 inhibited human plasma kallikrein competitively, with Ki values of 50 microM. None of the seven arginylfluoroalkanes was a good inhibitor of human factor Xa or of Factor XIIa. The arginylfluoroalkanes were tested in the prothrombin time (PT) and activated partial thromboplastin time (APTT) coagulant assays. Two fluoroketones, benzoyl-Arg-CF2CF3 and 1-naphthoyl-Arg-CF3, had significant anticoagulant activity. Benzoyl-Arg-CF2CF3 was found to prolong the PT 1.8-fold at 120 microM and to prolong the APTT 2.4-fold at 90 microM, whereas 1-naphthoyl-Arg-CF3 only prolonged the APTT 1.7-fold at 100 microM.

scholarly journals The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin

1981 ◽  
Vol 193 (2) ◽  
pp. 395-400 ◽  
Author(s):  
E Holmer ◽  
K Kurachi ◽  
G Söderström
Keyword(s):  
Molecular Weight ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Molecular Weight Dependence ◽  
Factor Xiia ◽  
Factor Xia ◽  
Heparin Fractions ◽  
Inhibition Of Thrombin

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.

scholarly journals Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes

1983 ◽  
Vol 215 (2) ◽  
pp. 287-294 ◽  
Author(s):  
R R Cook ◽  
J C Powers
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Activated Protein C ◽  
Factor Xa ◽  
Bovine Thrombin ◽  
Rapid Formation ◽  
Factor Xiia ◽  
Chymotrypsin A ◽  
Ellman's Reagent

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4′-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4′-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

Fluorogenic Peptide Substrates For Proteases In Blood Coagulation, Kallikrein-kinin And Fibrinolysis Systems: Substrate For Plasmin And Factor XIa

1979 ◽  
Author(s):  
S Iwanaga ◽  
H Kato ◽  
I Maruyama ◽  
N Adachi ◽  
Y Ohno ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Molar Ratio ◽  
Assay Method ◽  
Plasma Kallikrein ◽  
Urinary Kallikrein ◽  
Fluorogenic Substrates ◽  
Peptide Substrates ◽  
Factor Xia ◽  
Fluorogenic Peptide

Fluorogenic peptide substrates, peptidyl-4-methylcoumarin amides (MCA), are now commercially available and proved to be useful for the assay of α-thrombin, Factor Xa, urokinase, Limulus clotting enzyme, plasma and urinary kallikreins. We have newly synthesized the fluorogenic substrates for plasmin and Factor XIa and tested for their possible use as specific substrates for these enzymes. Among six peptidyl-Lys-MCA, Boc-Glu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA were good substrates for bovine and human plasmins, which were activated by urokinase. Streptokinase-activated plasmin complex (molar ratio with 1 to 1) hydrolyzed the former but not the latter. Using Boc-Phe-Ser-Arg-MCA and Boc-Leu-Thr-Arg-MCA, a specific assay method for Factor XIa activated with Factor XHa in the presence of HMW kininogen and kaolin, was developed. The kinetic parameters of various proteinases towards the specific substrates were as follows: α-Thrombin (Boc-Val-Pro-Arg-MCA, Km=2.1x10-5M), Factor Xa (Boc-Ile-Glu-Gly-Arg-MCA, 1.6x10-4), Plasma kallikrein (Z-Phe-Arg-MCA, 2.4xl0-4M), Pancreatic kallikrein (Pro-Phe-Arg-MCA, 1.6x10-4M), Urinary kallikrein (Pro-Phe-Arg-MCA, 2.2x10-4M), Urokinase (Glutaryl-Gly-Arg-MCA, 4.4xl0-5M), Limulus clotting enzyme (Boc-Leu-Gly-Arg-MCA, 2.7X10-5M), Plasmin (Boc-Glu-Lys-Lys-MCA, 6.7xl0-4M and Boc-Val-Leu-Lys-MCA, 2.5x10-4M).

scholarly journals Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1431-1436
Author(s):  
CF Scott ◽  
HR Wenzel ◽  
HR Tschesche ◽  
RW Colman
Keyword(s):  
Human Plasma ◽  
Serine Proteases ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Disease States ◽  
Reactive Center ◽  
Experimental Drug ◽  
Factor Xiia ◽  
Factor Xia ◽  
Altered Form

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine- directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15- aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

Crotalase and Thrombin: Cohomology of Amino Acid Sequences as Evidence for a Common Ancestral Gene

1979 ◽  
Author(s):  
Hubert Pirkle ◽  
Francis S. Markland ◽  
Ida Theodor ◽  
Richard Baumgartner
Keyword(s):  
Amino Acid ◽  
Mathematical Formulation ◽  
Evolutionary Relationship ◽  
Factor Xa ◽  
Amino Acid Sequences ◽  
Terminal Sequence ◽  
Ancestral Gene ◽  
Heavy Chains ◽  
Factor Ixa

Crotalase is a coagulant eniyme from the venom of C. adamanteus which releases specifically fibrinopeptide A from fibrinogen and cleaves the single thrombin-vulnerable bond of prothrombin. Edman degradation of crotalase yielded a six-step N-terminal sequence which contained only one residue in common with the N-terminal sequence of the B-chain of thrombin (Magnussen; Thompson et al.). When the two sequences were compared with the N-terminal sequences of the heavy chains of factor IXa (Enfield) and factor Xa (Titani), extensive identity was noted in five of the six positions, indicating evolutionary relationship among the four proteins and the suitability of the crotalase molecule for structure-function studies of thrombin-like actions. Our method for demonstrating that crotalase and thrombin are evolutionarily related is novel and is termed cohomology. We regard two amino acid sequences to be cohomologous when they each exhibit appreciable similarity to one or more di’ferent sequences but not to each other. We have found that this approach to the determination of evolutionary relationship, while employed intuitively here, is amenable to rigorous mathematical formulation (to be published).

scholarly journals Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1431-1436 ◽  
Author(s):  
CF Scott ◽  
HR Wenzel ◽  
HR Tschesche ◽  
RW Colman
Keyword(s):  
Human Plasma ◽  
Serine Proteases ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Disease States ◽  
Reactive Center ◽  
Experimental Drug ◽  
Factor Xiia ◽  
Factor Xia ◽  
Altered Form

Abstract Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine- directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15- aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

scholarly journals Lignosulfonic Acid Sodium Is a Noncompetitive Inhibitor of Human Factor XIa

2021 ◽  
Vol 14 (9) ◽  
pp. 886
Author(s):  
Srabani Kar ◽  
Page Bankston ◽  
Daniel K. Afosah ◽  
Rami A. Al-Horani
Keyword(s):  
Human Plasma ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Factor Xiiia ◽  
Inhibition Mechanism ◽  
Factor Xi ◽  
Fibrin Polymerization ◽  
Factor Xiia ◽  
Factor Xia ◽  
Lignosulfonic Acid

The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis–Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa–antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure–activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.

A comparision of the abilities of plasma kallikrein, β-factor XIIa, factor XIa and urokinase to activate plasminogen

1983 ◽  
Vol 29 (4) ◽  
pp. 407-417 ◽  
Author(s):  
Lindsey A. Miles ◽  
Judith S. Greengard ◽  
John H. Griffin
Keyword(s):  
Plasma Kallikrein ◽  
Factor Xiia ◽  
Factor Xia
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