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PubMed1982

The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human alpha and gamma and bovine alpha and beta-thrombins.

Human and bovine alpha-thrombins (greater than 90% alpha form) with high fibrinogen clotting activities (approximately 3,000 U.S. units/mg protein) exhibit similar Michaelis menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25 degrees C, pH 7.8 are: (Km = 4.18 +/- 0.22 and 3.61 +/- 0.15 microM; kcat = 127 +/- 8 and 100 +/- 1 s-1) for Chromozym TH and (Km = 1.33 +/- 0.07 and 1.50 +/- 0.10 microM; kcat = 91.4 +/- 1.8 and 98.0 +/- 0.5 s-1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their "non-clotting" terminal degradative forms, human gamma-thrombin (approximately 5 units/mg) and bovine beta-thrombin (approximately 200 units/mg), give increased values for these parameters (km = 14.3 +/- 2.4 and 14.4 +/- 2.2 microM; kcat = 160 +/- 9 and 124 +/- 6 s-1) for Chromozym-TH; and (Km = 2.50 +/- 0.36 and 2.99 +/- 0.33 microM; kcat = 106 +/- 3 and 106 +/- 3 s-1) for S-2238. Based on these parameters, 50% degradation of human or bovine alpha-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all alpha forms are erroneously used and assays are at 150 microM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations less than 0.1 nM or 0.01 clotting unit/ml of alpha-thrombin.

Adsorption↗
PubMed1982

Thrombin activation rate constant: one-stage chromogenic assay for the extrinsic system.

Some properties of a one-stage, chromogenic clotting assay using tissue thromboplastin as an activator are described. The chromogenic assay uses S-2238 as substrate and estimates thrombin by the rate of absorbance produced. Under assay conditions, it was observed that the log (thrombin or absorbance/min) is linear with time. The linearity appeared to extend from zero thrombin up to maximum thrombin concentration for all concentrations of each plasma type examined including factor V and factor VII deficient plasmas. Since the linear slope, b, appears to be a measure of the rate of thrombin production, it was called the Thrombin Activation Rate Constant (TARC). For dilutions of normal plasma log (b) appears to be linearly related to log (plasma concentration) and to log (PT) where PT is the standard one stage clotting time. For plasmas deficient in extrinsic clotting factors, b was linearly related to log (factor concentrations). The chromogenic assay was most sensitive to changes in prothrombin; least sensitive to changes in factor VII; and unaffected by changes in concentration of factors VIII and IX. The standard PT was least sensitive to prothrombin and more sensitive to factors X and VII. For oral anticoagulated plasmas log b and log PT were linearly correlated with a regression slope which was more negative than that from normal plasma. From theses results it was concluded that TARC is a chromogenic assay sensitive to all factors in the extrinsic pathway; that TARC might be used as both a screening assay for extrinsic deficiencies and a monitoring assay for anticoagulants; and that TARC may be more sensitive than the standard PT to the defects from oral anticoagulation.

Anticoagulants↗
PubMed1982

Measurement of the heparin enhanced-antithrombin III/thrombin reaction rate in the presence of synthetic substrate.

An assay approach, designed to allow accurate measurement of the rate of thrombin inhibition by antithrombin III in the presence of heparin, is described. In essence the approach takes advantage of the fact that synthetic substrates, e.g. N-alpha-p-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide, bind to the active site of thrombin, which slows the rate of reaction with antithrombin III. The rate of thrombin inhibition can be monitored continuously by change in absorbance at 400 nm or measured by end point determination. In either case, reproducible rate measurements can be made under a variety of experimental conditions not easily examined with conventional assay approaches.

Antithrombin III↗
PubMed1982

Perchloric acid treatment of human blood for quantitative endotoxin assay using synthetic chromogenic substrate for horseshoe crab clotting enzyme.

A new reliable human blood treatment was established for quantitative endotoxin assay using synthetic chromogenic substrate [Boc-Leu-Gly-Arg-rho-nitroanilide]. Addition of perchloric acid in a final concentration of 1.25% to platelet-rich plasma or serum in a 2:1 volume ratio completely eliminated nonspecific amidase activities as well as inhibitors. By this method, the recovery of added endotoxin was nearly 100%, and was almost independent of sample dilutions and anticoagulants.

Amidohydrolases↗
PubMed1983

Identification of a heparin activated amidolytic enzyme in carp (Cyprinus carpio) plasma.

A heparin activated amidolytic enzyme capable of cleaving synthetic thrombin sensitive chromogenic substrates was identified from the plasma of the carp (Cyprinus carpio). Activation was inhibited by KCl, protamine sulphate and human plasma. Heparin was not required for the continued action of the enzyme. Active enzyme was irreversibly inhibited by DFP. The pH and temperature optima was studied and the enzyme semi purified by Sephadex G100 superfine and Sephadex A50 chromatography. An approximate MW of 62,000 was found. Activity generated by as little as 0.002 units of heparin per ml carp plasma was detected. The trivial name carpamidase is proposed for the enzyme.

Animals↗