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.

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.

Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor

Biochemistry ◽  
1988 ◽  
Vol 27 (12) ◽  
pp. 4231-4237 ◽  
Author(s):  
Joost C. M. Meijers ◽  
Deon H. A. J. Kanters ◽  
Riek A. A. Vlooswijk ◽  
Helmy E. Van Erp ◽  
Martin Hessing ◽  
...  
Keyword(s):  
Human Plasma ◽  
Protein C ◽  
Plasma Kallikrein ◽  
Protein C Inhibitor ◽  
Factor Xia

S1′ and S2′ subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

2008 ◽  
Vol 389 (12) ◽  
Author(s):  
Aurelio Resende Lima ◽  
Fabiana M. Alves ◽  
Pedro Francisco Ângelo ◽  
Douglas Andrade ◽  
Sachiko I. Blaber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Receptor Agonist ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Charged Amino Acids ◽  
B1 Receptor ◽  
Lower Specificity ◽  
Natural Amino Acids ◽  
Hydrolysis Of

AbstractThe S1′ and S2′ subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO3H2)]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P1′ and P2′ positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS390S391RI-NH2was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S390and S391phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO3H2)391RI-NH2was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg9)-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S1′ subsite, with lower specificity for the S2′ subsite. Abz-MISLMKRPPGFSPFRSSRI-NH2was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO3H2) were poorly hydrolyzed. In conclusion, S1′ and S2′ subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.

Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds

1999 ◽  
Vol 45 (1-3) ◽  
pp. 145-149 ◽  
Author(s):  
Maria Luiza V Oliva ◽  
Sonia Andrade ◽  
Isabel F.C Batista ◽  
Misako U Sampaio ◽  
Maria Juliano ◽  
...  
Keyword(s):  
Human Plasma ◽  
Factor Xa ◽  
Plasma Kallikrein ◽  
Factor Xa Inhibitor ◽  
Tissue Kallikrein ◽  
Reactive Site

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

scholarly journals Specificity of S'1 and S'2 subsites of human tissue kallikrein using the reactive-centre loop of kallistatin: the importance of P'1 and P'2 positions in design of inhibitors

2003 ◽  
Vol 371 (3) ◽  
pp. 1021-1025 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Sandro E. FOGAÇA ◽  
Robson L. MELO ◽  
Luiz JULIANO ◽  
Maria A. JULIANO
Keyword(s):  
Human Tissue ◽  
Serine Proteases ◽  
Potent Inhibitor ◽  
Competitive Inhibition ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Loop Sequence ◽  
Reactive Centre Loop ◽  
Hydrolysis Of ◽  
Peptide Analogues

We have demonstrated that the S´1 and S´2 subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P´1 and arginine at P´2 resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P´1 and P´2 in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (Ki). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 inhibited hK1 in the range 20–30 nM (letters in italics denote the d-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 was a weak inhibitor for other serine proteases, as indicated by the higher Ki values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a Ki value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH2 can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.

Ultrasensitive Fluorogenic Substrates for Serine Proteases

1997 ◽  
Vol 78 (04) ◽  
pp. 1193-1201 ◽  
Author(s):  
Saulius Butenas ◽  
Maria E DiLorenzo ◽  
Kenneth G Mann
Keyword(s):  
Serine Proteases ◽  
Factor Viia ◽  
Activated Protein C ◽  
Factor Xa ◽  
High Specificity ◽  
High Rate ◽  
Fluorogenic Substrates ◽  
Type Plasminogen Activator ◽  
Factor Xia ◽  
Coagulation And Fibrinolysis

SummarySelective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a kcat value of 170 s-1 and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by > 10,000-fold and > 100-fold for activated protein C (APC). Seven of these substrates have a over 100 s-1 and three of them have a KM below 1 μM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor Vila/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.

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