The Effects of Hepes Buffer on Clotting Tests, Assay of Factors V and VIII and on the Hydrolysis of Esters by Thrombin and Thrombokinase

1976 ◽  
Vol 35 (01) ◽  
pp. 202-210 ◽  
Author(s):  
Phyllis S. Roberts ◽  
Haywood N. Hughes ◽  
Patricia B. Fleming
Keyword(s):  
Ionic Strength ◽  
Human Plasma ◽  
Inhibitory Effect ◽  
Clotting Factors ◽  
Bovine Thrombin ◽  
Hepes Buffer ◽  
Human Thrombin ◽  
Ethanesulfonic Acid ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

SummaryShorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiper-azine-N1-2-ethanesulfonic acid) buffer than of 50 mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HC1 buffer, pH 7.4 at 37°. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin’s enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers.Hepes may also have effects on other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl-or benzoyl-L-arginine methyl ester) at pH 7.4, 37° by purified human or bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HQ buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HQ plus NaCl for KC1). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests.Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors.The clotting times were the same in the presence of 50 mM Tris. HC1 as in Imidazole. HC1 buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HC1 buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.

The Effects of Salts and of Ionic Strength on the Hydrolysis of TAME (p-toluenesulfonyl-L-arginine methyl ester) by Thrombin and Thrombokinase

1972 ◽  
Vol 27 (03) ◽  
pp. 573-583 ◽  
Author(s):  
Phyllis S. Roberts ◽  
Patricia B. Fleming
Keyword(s):  
Ionic Strength ◽  
Alkaline Earth ◽  
Choline Chloride ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Bovine Thrombin ◽  
Human Thrombin ◽  
Alkali Chlorides ◽  
Hydrolysis Of ◽  
Alkaline Earth Chlorides

SummaryThe effects of varying concentrations of alkali chlorides (LiCl, NaCl, KC1, RbCl and CsCl), alkaline earth chlorides (BeCl2, MgCl2, CaCl2, SrCl2, and BaCl2), choline chloride and Tris,HCl (pH 8.0 at 37°) on the rates of hydrolysis of TAME by a purified preparation of human thrombin and of bovine thrombokinase were determined in 0.25 M Tris.HCl buffer, pH 8.0 at 37°. Each salt had its own individual effects on the reactions and these effects were completely different when thrombin instead of thrombokinase was used. Each salt, however, had the same qualitative effects on TAME hydrolysis by crude bovine thrombin as by purified human thrombin, but only minor effects on the hydrolysis of TAME by trypsin.With the exception of LiCl, low concentrations of alkali chlorides had inhibitory and high concentrations had acceleratory effects on both the thrombin-TAME and the thrombokinase-TAME reactions. LiCl had no accelerating effects on either reaction and it was a stronger inhibitor of thrombokinase than of thrombin. In contrast to the alkali chlorides, the alkaline earth chlorides had no acceleratory effects but had inhibitory effects on both reactions. MgCl2, however, was an exception. It weakly accelerated the thrombokinase -TAME and weakly inhibited the thrombin -TAME reaction. When comparing 0.15 M concentrations of the salts (with the exception of BeCl2 which was the strongest inhibitor of both reactions), NaCl was the strongest inhibitor of the thrombin - TAME reaction, followed by CaCl2, but BaCl2 was the strongest inhibitor of the thrombokinase -TAME reaction, followed by KC1 and LiCl.Choline chloride and Tris.HCl in concentrations up to 3 M had no significant effects on the rate of hydrolysis of TAME by either human or bovine thrombin. Increasing the ionic strength above 0.16 (lower values were not tested), therefore, may have no effect on this reaction, and all of the inhibitions and accelerations found in the presence of the alkali chlorides or the alkaline earth chlorides may be entirely due to the individual cations. On the other hand, increasing the ionic strength may produce a small inhibitory effect, as found in the presence of LiCl or MgCl2, and the lack of any effect in presence of even large concentrations of Tris.HCl or choline chloride may be due to a fortuitous balancing of the weak inhibitory effect of ionic strength by the weak acceleratory effect of the choline and Tris cations.Choline chloride and Tris.HCl had no inhibitory effects but accelerated the thrombokinase -TAME reaction. Choline chloride was the strongest accelerator tested. Rates in the presence of 0.15 and 3 M choline chloride were respectively 204 and 601 % of the controls. Although the effects of ionic strength changes on this reaction could not be established, the data indicate that increasing the ionic strength has at most only a small effect, and the inhibitions and accelerations found are due primarily to the specific cations present.

Thrombin’s Esterase Activity in the Presence of Anticoagulant and Other Salts

1969 ◽  
Vol 21 (01) ◽  
pp. 103-110 ◽  
Author(s):  
Phyllis S. Roberts ◽  
Rosalind K. Burkat ◽  
W. E Braxton
Keyword(s):  
Ionic Strength ◽  
Inhibitory Effect ◽  
Potassium Citrate ◽  
Protamine Sulfate ◽  
Small Extent ◽  
Potassium Salts ◽  
Bovine Thrombin ◽  
Ionic Strength Effect ◽  
Hydrolysis Of ◽  
Strength Effect

SummaryThe rates of hydrolysis of TAME by bovine thrombin were studied in the presence and absence of various salts at 37° in 0.25 M Tris . HCl buffer, pH 8.1, with the following results :1. The rates were inhibited to the same extent in the presence of 0.1 M sodium chloride, citrate, oxalate and sulfate and they averaged 49% of rates in the absence of added salts (controls). In contrast to this, rates in the presence of 0.1 M of the corresponding potassium salts averaged 91 % of the controls.2. 0.01 and 0.1 M LiCl, KC1, NH4C1 or CaCl2 inhibited the rates 5 and 17% respectively but the same concentrations of NaCl inhibited them 14 and 49% respectively. The inhibition by 0.1 M NaCl was not prevented or reversed by the presence of the same concentrations of any of these chlorides, except to a small extent by KCl.3. As the concentration of KC1 or NaCl was increased, the rates fell to minimum values and then increased linearly. The minimum rates occurred in the presence of from 0.15 to 0.4 M salts, and with KC1 they were 85% but with NaCl they were 52% of the controls. With 2.5 M KC1 the rates were 141 % but with 2.5 M NaCl they were 68% of the controls.4. Protamine sulfate (2 or 3 mg/ml) had no significant effects on the reaction when tested at either pH 7.4 or 8.1 (Tris buffers). Inhibition was found only when NaCl was present, whether or not protamine sulfate was also present.Since potassium citrate, oxalate, sulfate and chloride as well as lithium, ammonium and calcium chlorides inhibited the rates only slightly and all to about the same degree, it was concluded that the inhibition was probably due to non-specific ionic strength effects and not to inhibition by any of the anions or cations tested. On the other hand, the inhibitions found in the presence of the sodium salts were concluded to be due only partly to a non-specific ionic strength effect, but primarily to a specific inhibitory effect of Na+.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

Inhibition of Coagulation and Fibrinolysis by Aromatic Amidino Compounds

1971 ◽  
Vol 25 (03) ◽  
pp. 391-404 ◽  
Author(s):  
J.D Geratz
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Partial Thromboplastin Time ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Similar Degree ◽  
Contact Activation ◽  
Plasma Clot ◽  
Human Thrombin ◽  
Canine Plasma

Summary1. Aromatic diamidines which are potent inhibitors of trypsin possess a marked inhibitory effect on the clotting activity of human thrombin and on the prothrombin time and partial thromboplastin time of human plasma. They also block the contact activation phase of the coagulation process. The strongest inhibitor among the compounds tested was M & B 4596 which was followed in second place by pentamidine.2. Pentamidine was 10 times more active than ε-ACA in impeding streptokinase-induced lysis of human plasma clots. It was 100-200 times stronger than ε-ACA in inhibiting the activation of bovine plasminogen by activators formed from the interaction between streptokinase and either human plasmin(ogen) or human plasma.3. The prothrombin time and partial thromboplastin time of canine plasma were less susceptible to inhibition by pentamidine than the same tests on human plasma. Clot lysis in the canine system was inhibited by pentamidine to a similar degree as in the human system. After intravenous injection of pentamidine in the dog there occurred the expected prolongation of the partial thromboplastin time and of the clot lysis time.

Recent developments in topical thrombins

2009 ◽  
Vol 102 (07) ◽  
pp. 15-24 ◽  
Author(s):  
Thomas L. Ortel ◽  
Craig M. Kessler
Keyword(s):  
Human Plasma ◽  
Excessive Bleeding ◽  
Bovine Thrombin ◽  
Bovine Plasma ◽  
Haemostatic Agents ◽  
Coagulation Proteins ◽  
Equivalent Efficacy ◽  
Human Thrombin ◽  
Recent Developments ◽  
Surgical Bleeding

SummaryManaging blood loss is part of the surgeon’s responsibility during surgical procedures, and a variety of therapeutic strategies are available to help accomplish this. Topical haemostatic agents are among the agents used to control surgical bleeding and locally arrest blood flow. Bovine thrombin is a commonly used topical haemostatic agent; however, its use has been associated with potential risks, including well-documented cases of antibodymediated coagulopathy. This coagulopathy develops as a consequence of antibody formation directed against bovine thrombin, other bovine coagulation proteins, and their human orthologs. The fact that a coagulopathy can result in association with the use of bovine plasma-derived thrombin preparations prompted the FDA to require pharmaceutical companies to place a black-box warning in their prescribing information for products containing bovine plasma-derived thrombin. Recently, human plasma-derived thrombin and recombinant human thrombin have been approved by the FDA with the expectation that they will be less immunogenic than the bovine-derived product. In clinical studies, purified human plasma-derived thrombin and recombinant thrombin have demonstrated equivalent efficacy and safety, with improved immunogenicity profiles compared with bovinederived thrombin agents. Well-designed and adequately powered clinical trials should be conducted to indicate whether human thrombin products would improve the risk-benefit and costbenefit profiles for surgeries complicated by excessive bleeding.

Assay of the Esterase Activity of Thrombin, Plasmin and Trypsin with a Chromogenic Substrate p-Nitrobenzyl p-Toluenesulfonyl-L-Arginine

1977 ◽  
Vol 37 (02) ◽  
pp. 253-261 ◽  
Author(s):  
Tadashi Aogaichi ◽  
Gerhard W. E Plaut
Keyword(s):  
Product Formation ◽  
Enzyme Concentration ◽  
Bovine Thrombin ◽  
Absorbance Change ◽  
Bovine Trypsin ◽  
Nitrobenzyl Alcohol ◽  
Human Thrombin ◽  
Hydrolysis Of ◽  
Ester Product ◽  
Esterase Activities

Summaryp-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 μmol/ml of the ester is equivalent to an absorbance change of 4.45 cm–1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30°, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 μg/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30°, Kmis 14 μM and Vmax is 0.037 μmol/min/NIH unit/ml for bovine thrombin, Km is 78 μM and Vmax is 0.31 μmol/min/CTA unit/ml for human plasmin, and Km is 12 μM and Vmax is 138 μmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 μmol p-nitrobenzyl alcohol formed/10 min/NIH unit.

scholarly journals Comparative Hematology: Studies on Class Aves, Domestic Turkey, Meleagris Gallopavo

1977 ◽  
Author(s):  
Jessica H. Lewis ◽  
Ute Hasiba ◽  
Joel Spero
Keyword(s):  
Human Plasma ◽  
Meleagris Gallopavo ◽  
Adenosine Diphosphate ◽  
Coagulation Factors ◽  
Mammalian Brain ◽  
Large Nucleus ◽  
Extrinsic Pathway ◽  
Bovine Thrombin ◽  
Injured Area ◽  
Human Thrombin

This study compared turkey and human blood in various coagulation and cellular parameters. Turkey blood clotted slowly (± 60 minutes) in either glass or siliconized tubes and the formed clots retracted very slightly or not at all. Prothrombin times, using mammalian brain, were 12-14 seconds for human plasma and over 60 seconds for turkey plasma. On the other hand, turkey brain clotted turkey plasma in 12 to 14 seconds and human plasma in over a minute. Russell Viper Venom clotted both plasma in 17 to 22 seconds. The activated partial thromboplastin time on turkey blood was over two minutes. Bovine thrombin clotted turkey plasma in about 30 seconds compared to 15 for human. Turkey clotting times with human thrombin or reptilase were very much longer than human. Thromboplastin generation tests with all human components gave substrate clotting times of 8.6 to 9.1 seconds; with all turkey components they were greater than 40 seconds. Turkey fibrinogen averaged 450mg/dl. Assayed in systems used for human coagulation factors, turkey factor XIII was high normal, factor VIII low normal (0.48U/ml), factors II and V present in traces and the others, VII, X, IX, and XII, absent or not measurable. Turkey thrombocytes were oval, nucleated cells which aggregated during clotting but not with adenosine diphosphate, mammalian or turkey collagen or ristocetin.TEM showed thrombocytes to contain microtubules, an open canalicular system, and a few mitochondria as well as a large nucleus. Hemostasis in turkeys appears dependent upon an extrinsic pathway in which tissue factor from an injured area initiates thrombin formation, which, in turn, causes thrombocyteaggregation and fibrin formation.

The Hydrolysis of Esters Related to O-Hippuryl-2-hydroxybutanoic Acid by Carboxypeptidase A

1974 ◽  
Vol 52 (14) ◽  
pp. 2640-2647 ◽  
Author(s):  
John W. Bunting ◽  
Joe Murphy
Keyword(s):  
Ionic Strength ◽  
Substrate Inhibition ◽  
Binding Modes ◽  
Carboxypeptidase A ◽  
Acid Moiety ◽  
Hydroxybutanoic Acid ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of ◽  
Acid Esters

The hydrolysis of each of the following esters by bovine carboxypeptidase A has been studied at pH 7.5, 25°, ionic strength 0.5: O-hippuryl-, O-phenaceturyl-, O-aceturyl-, O-(N-methylhippuryl)-, and O-(N-hippurylglycyl)-2-hydroxybutanoic acids, and 2-(3-benzoylpropanoxy)-, 2-benzoxyacetoxy-, and 2-(4-phenylbutanoxy)butanoic acids. Substrate inhibition occurs with only the hippuric and phenaceturic acid esters and in the six other cases simple Michaelis–Menten kinetics are observed. The relatively minor variations in the structures of the acid moieties of these esters lead to quite large variations in Km, although kcat seems to be relatively independent of the nature of the acid moiety. Binding modes of substrate molecules at both the catalytic and inhibitory sites are discussed in the light of these observations.

Amidino-Substituted Heterocycles as Probes of the Specificity Pocket of Proteases Involved in Coagulation and Fibrinolysis

1979 ◽  
Author(s):  
R. R. Tidwell ◽  
J. D. Geratz
Keyword(s):  
Conformational Changes ◽  
Public Service ◽  
Human Factor ◽  
Potent Inhibitor ◽  
Hydrophobic Interactions ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Bovine Thrombin ◽  
Trypsin Inhibition ◽  
Human Thrombin

A series of ten newly synthesized amidine-substituted heterocycles were examined for their inhibitory effect against human and bovine thrombin, human factor Xa and plasmin, and bovine trypsin. Inhibition was competitive and reversible in all cases and the Kj values were taken to reflect binding conditions in the specificity pocket A remarkable inhibitor of the three clotting enzymes was found in 5-amidlnoindole. Compared to the standard inhibitor benzamidine, it proved to be thirty to forty times more effective against bovine and human thrombin, and factor-Xa. However b-amidinomdole proved to be the most potent inhibitor of plasmin and trypsin‘in order to explain the considerable activity of the indole derivatives, hydrophobic interactions, hydrogen bonding and charge transfer complex formation are considered Finallythe binding strength for thrombin as well as for trypsin was significantly reduced below pH 7.0. The loss in affinity may have resulted from protonation of the ammo acid triad of the catalytic site of the enzymes and consequent adverse conformational changes. This study was supported by U. S. Public Service Grants AM 10746 and HL 21540.

scholarly journals STUDIES ON THROMBIN-INDUCED PLATELET AGGLUTINATION

1961 ◽  
Vol 114 (6) ◽  
pp. 905-920 ◽  
Author(s):  
Richard W. Shermer ◽  
Reginald G. Mason ◽  
Robert H. Wagner ◽  
Kenneth M. Brinkhous
Keyword(s):  
Ionic Strength ◽  
Divalent Cations ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
High Ionic Strength ◽  
Clotting Time ◽  
Cadmium Ions ◽  
Ph Values ◽  
Human Thrombin ◽  
High Concentrations

A one-stage macroscopic test for platelet agglutination was used to study the effect of thrombin and thrombin-cation mixtures on washed platelets. Conclusions regarding platelet agglutination are as follows: (a) Canine, bovine, or human thrombin alone does not cause agglutination of canine or human platelets. (b) Thrombin with calcium or magnesium causes rapid platelet agglutination. Both calcium and magnesium are active at physiologic concentrations. Divalent manganese or cadmium ions can be substituted for calcium or magnesium. (c) The agglutination reaction is affected but little by the species of origin of thrombin or platelets, or by variations in ionic strength or pH over a broad range. (d) Temperature at which the reaction is carried out is critical; optimal temperature for the test is 28°C. (e) Agglutination is inhibited by high ionic strength, by pH values outside the range 6.4–8.6, and by temperatures outside the range 25–28°C. High concentrations of calcium have a specific inhibitory effect. (f) Platelet agglutination time is as sensitive an index of thrombin concentration as is the fibrinogen clotting time. A comparison is made between divalent cations which influence platelet agglutination induced by thrombin, TAg', and TAg. A similar comparison is made of cations influencing the action of thrombin on the "substrates," fibrinogen, TAMe, and platelets.

Sign in / Sign up

Export Citation Format

Share Document