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.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

Abstract This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

scholarly journals Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 126-126
Author(s):  
Daniël Verhoef ◽  
Mark Schreuder ◽  
Ka Lei Cheung ◽  
Pieter H. Reitsma ◽  
Mettine H.A. Bos
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Factor Xa ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Bleeding Events ◽  
Surgical Interventions ◽  
Anionic Phospholipids ◽  
Active Site Pocket

Abstract The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG. Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography. Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket. We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma. Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. Disclosures Bos: Bayer Hemophilia Awards: Research Funding.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

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 Pharmacokinetics of activated protein C in guinea pigs

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2174-2184 ◽  
Author(s):  
H Jr Berger ◽  
CG Kirstein ◽  
CL Orthner
Keyword(s):  
Protease Inhibitors ◽  
Active Site ◽  
Guinea Pigs ◽  
Protein C ◽  
Dose Range ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S

Abstract Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D- phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. The other mechanism involves the direct catabolism of APC by the liver via a pathway that is nonsaturable over a substantial dose range and independent of the active site. This pattern of elimination is distinctly different from that observed with the homologous coagulation enzymes thrombin, factor IXa, and factor Xa.

scholarly journals Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

1973 ◽  
Vol 131 (1) ◽  
pp. 107-117 ◽  
Author(s):  
G. W. Jameson ◽  
D. V. Roberts ◽  
R. W. Adams ◽  
W. S. A. Kyle ◽  
D. T. Elmore
Keyword(s):  
Factor Xa ◽  
Spectrophotometric Titration ◽  
Chymotrypsin A

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine α-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for α- and β-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for α-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02–3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p′-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N2-acetyl-N1-benzylcarbazate respectively. p-Nitrophenyl N2-acetyl-N1-(9-anthrylmethyl)carbazate has been synthesized; it did not react with α-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.

scholarly journals Pharmacokinetics of activated protein C in guinea pigs

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2174-2184
Author(s):  
H Jr Berger ◽  
CG Kirstein ◽  
CL Orthner
Keyword(s):  
Protease Inhibitors ◽  
Active Site ◽  
Guinea Pigs ◽  
Protein C ◽  
Dose Range ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S

Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D- phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. The other mechanism involves the direct catabolism of APC by the liver via a pathway that is nonsaturable over a substantial dose range and independent of the active site. This pattern of elimination is distinctly different from that observed with the homologous coagulation enzymes thrombin, factor IXa, and factor Xa.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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