New Chromogenic Peptide Substrates for Factor X&lt;sub&gt;a&lt;/sub&gt;

Aurell, L.; Simonsson, Roger; Arielly, S; Karlsson, Gunilla B.; Friberger, P.; Claeson, Göran

doi:10.1159/000214241

Cited by 6 publications

(2 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The structure of synthetic substrates and their efficiency for various blood derived serine proteases such as plasmin, factor Xa, thrombin, kallikreins and urokinase, factors VIIa, IXa, XIa, and XIIa, and activated protein C were analyzed in several previous publications (Sherry et al, 1966;Morita et al, 1977;Aurell et al, 1978;Claeson et al, 1978; Lonsdale -Eccles et al, 1980;McRae et al, 1981;Lottenberg et al, 1983Lottenberg et al, , 1986Castillo et al, 1983;Cho et al, 1984;Harnois-Pontoni et al, 1991;Butenas et al, 1992Butenas et al, , 1993Butenas et al, , 1995. The data concerning the influence of the substrate P n -P 1 -P n ′ structure on its efficiency for a given enzyme are controversial (Morita et al, 1977;McRae et al, 1981;Lottenberg et al, 1983;Cho et al, 1984) and do not provide clear answers about the most favorable amino acids at the various P positions (Harnois-Pontoni et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

1997

View full text Add to dashboard Cite

A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.

show abstract

Section: Discussionmentioning

confidence: 99%

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

1997

View full text Add to dashboard Cite

show abstract

“…With our series of substrates, the effect seems to be totally in kat. Factor Xa has been widely studied with synthetic substrates (Iwanaga et al, 1978;Lottenberg et al, 1981;Lonsdale-Eccles et al, 1980;Svendsen et al, 1972), and some subsite mapping has been carried out at P3 (Aurell et al, 1976(Aurell et al, , 1977(Aurell et al, ,1978. Many of these studies reported only relative reactivities without kinetic constants and did not examine a wide variety of P3 amino acid residues.…”

Section: Discussionmentioning

confidence: 99%

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates

Cho¹,

Tanaka²,

Cook³

et al. 1984

Biochemistry

View full text Add to dashboard Cite

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Synthetic Peptide Substrate Assays for Hemostasis Testing

Zolton¹

1986

Advances in Clinical Chemistry

View full text Add to dashboard Cite

New Chromogenic Peptide Substrates for Factor X<sub>a</sub>

Abstract: The factor-X_a-sensitive substrate Bz-Ile-Glu-Gly-Arg-p-nitroanilide has been made more sensitive by making ester and amide derivatives of the γ-carboxyl group of the glutamyl residue. The morpholinyl and piperidyl amides react 2.5 times more rapidly with factor X_a.

Cited by 6 publications

References 2 publications

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates

Synthetic Peptide Substrate Assays for Hemostasis Testing

Contact Info

Product

Resources

About

New Chromogenic Peptide Substrates for Factor X<sub>a</sub>

Abstract: The factor-Xa-sensitive substrate Bz-Ile-Glu-Gly-Arg-p-nitroanilide has been made more sensitive by making ester and amide derivatives of the γ-carboxyl group of the glutamyl residue. The morpholinyl and piperidyl amides react 2.5 times more rapidly with factor Xa.

Cited by 6 publications

References 2 publications

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates

Synthetic Peptide Substrate Assays for Hemostasis Testing

Contact Info

Product

Resources

About

Abstract: The factor-X_a-sensitive substrate Bz-Ile-Glu-Gly-Arg-p-nitroanilide has been made more sensitive by making ester and amide derivatives of the γ-carboxyl group of the glutamyl residue. The morpholinyl and piperidyl amides react 2.5 times more rapidly with factor X_a.