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Biomedical subjects

Z S Latallo

Publications and source records attributed to Z S Latallo.

At least 19 recordsLinked to original sources
PubMed1986

Reaction of thrombins with human antithrombin III. I. Enzyme activity losses unrelated to the inhibitory reaction and their prevention.

Adsorption loss of thrombin may produce considerable systematic error when quantitative evaluation of its reaction with antithrombin III is based on measurements of changes in enzyme activity with time. At 25 degrees C, pH 7.8 (0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES) glass, siliconized glass, polypropylene and polystyrene surfaces adsorb thrombin in a similar manner. Its adsorption is a saturation reaction reaching an equilibrium after about three hours. At initial concentrations of enzyme convenient for assay with chromogenic substrate (10 nM or less) a large fraction of activity is lost. Binding of alpha thrombin to polypropylene is very tight. The association constants (Ka) were found to be 12.6 X 10(9) M-1 and 4.0 X 10(9) M-1 for human and bovine alpha thrombin respectively. The adsorbed enzyme has hardly any ability to hydrolyze chromogenic substrate. A small part of its activity can be recovered by displacement with PEG 6000, more when the substrate is present. The addition of non-ionic detergents and continuous stirring allows recovery of up to 30% of the enzyme activity and accelerates this process. Complete and long term (more than 20 hours at 25 degrees C) prevention of adsorption loss of human alpha and bovine alpha and beta thrombin at pH between 7.0-8.3 is achieved by the addition of 0.1% PEG 6000 to the buffer and use of tubes precoated with PEG 20,000. Alternative use of non-ionic detergents (BRIJ-35, TRITON X-100) to modify the solvent also appears effective but their possible interference with reactions studied should be borne in mind.

Adsorption↗
PubMed1986

Reaction of thrombins with human antithrombin III: II. Dependence of rate of inhibition on molecular form and origin of thrombin.

The rate of thrombin inhibition by AT III depends upon the molecular form (alpha, beta, gamma) and species origin of the enzyme. The following apparent second order rate constants (.1000/M.s) were established alpha human 11.24 +/- 0.8; alpha bovine 7.46 +/- 0.27; beta bovine 6.49 +/- 0.34 and gamma human thrombin 2.80 +/- 0.11, 25 degrees C, pH = 7.80, 0.01 M TRIS, 0.01 M HEPES buffer, 0.0025 M EDTA, 0.3 M NaCl, 1 mg/mL PEG 6000. Using these values, the concentration of active AT III in an unknown sample can be calculated from the measured apparent first order rate constant in moles/liter instead of relative units. In contrast to the reactions in the absence of heparin, in the presence of high affinity heparin, the differences between various forms of thrombin are more pronounced and the shape of the progress curves, as well as rates, are highly dependent on the ionic strength. In the presence of heparin, measurement of the rate of inhibition under pseudo first order conditions can be made only when the NaCl concentration is at least 0.3 M. The significance of the presented data for designing a functional assay of AT III is discussed.

Animals↗
PubMed1983

Cardiac output redistribution induced by noradrenaline in two murine tumor models.

The response of tumor vessels to vasoactive substances could provide useful information on experimental tumor biology. We have studied the effects of noradrenaline (20 micrograms/kg i.v.) on cardiac output (%CO) distribution in C57BL/6J mice bearing syngeneic Lewis lung carcinoma (3LL) and BALB/c mice with JW sarcoma (JWS). Mice were studied at different stages during tumor growth using microspheres labeled with 57Co (basal determination) or 58Co (after noradrenaline or saline). In control C57BL/6J mice noradrenaline induced a redistribution of CO, with an increase in the heart and brain and a decrease in the kidneys and hind limb muscle CO fractions (%CO). In 3LL-bearing mice the %CO to the tumor was not changed by noradrenaline 1 week after implantation but was significantly less after 2 and 3 weeks. %CO to the total lung tissue or to isolated metastases did not change after noradrenaline. In control BALB/c mice noradrenaline increased the %CO to the brain and decreased that to the kidneys and hind limb muscle. In JWS-bearing mice the %CO to the tumor was reduced 2 weeks after implantation, was not changed after 4 weeks and was increased after 6 weeks. These results suggest that tumor vessel reactivity to a vasoactive substance may change markedly during various phases of tumor growth and may differ in different experimental models.

Animals↗
PubMed1982

Experience with chromogenic substrates in studies of clotting and fibrinolytic systems.

Experiences in the application of chromogenic substrates for the evaluation of clotting variables are reported. The use of a special buffer system enables the determination of factor Xa by the thrombin substrate Chromozym TH. Prothrombin in plasma can be determined using the substrate S-2238 after activation with factor Xa or Ecarin. Factor VII is activated with thromboplastin and is estimated indirectly via factor Xa activity. Comparison of the amidolytic method for factor VII with prothrombin times of patients on oral anticoagulants shows good correlation. Chromogenic substrates can be also used for determining platelet factor 3 (S-2238), plasminogen and alpha 2-antiplasmin.

Blood Coagulation↗
PubMed1981

An evaluation of chromogenic substrates in the control of oral anticoagulant therapy.

A comparison has been made between the prothrombin time test using British Comparative Thromboplastin (BCT) and four modified amidolytic assays in the assessment of laboratory control of oral anticoagulant administration in short-term and long-term patients. The results were also correlated with the APTT and specific assays for factors VII and X based on established coagulation techniques. The chromogenic substrate assays did not give as close agreement with the BCT as the clotting assays for factors VII and X. A combination of the amidolytic factor II and X results did not improve the correlation. Agreement with the BCT was better in long-term patients with the substrates but there was no obvious advantage between the factor II and X amidolytic methods in this group. The development of a specific amidolytic assay for factor VII should be considered based on the evidence of this study although its reliability for oral anticoagulant control would need to be validated by clinical trial. In the interim, it may be anticipated that thromboplastins showing less sensitivity than BCT to factor VII would give a better correlation with the amidolytic assays.

Acenocoumarol↗
PubMed1981

An assessment of an amidolytic assay for factor VII in the laboratory control of oral anticoagulants.

A comparison has been made between the prothrombin time test using British Comparative Thromboplastin (BCT) and a chromogenic substrate assay for factor VII in the assessment of laboratory control of oral anticoagulants in short-term and long-term patients. Opportunity was also taken to compare the findings with parallel results obtained with the venous Thrombotest technique and a specific clotting assay for factor VII. There was good agreement between the amidolytic factor VII assay, using a method modified from Seligsohn et al (1978) with the Quick test using BCT and Thrombotest in 60 long-term patients. Tests in 53 patients within the first 3 weeks of starting oral anticoagulant administration gave less satisfactory agreement between the above amidolytic method and the conventional tests. In contrast, there was a good correlation between the two conventional tests in both groups and also between the clotting and amidolytic factor VII method. Although the results are an improvement on previous, less satisfactory correlations between the BCT prothrombin time method and amidolytic assays for factor II and X, the present study indicates the limitations of a specific clotting assay versus a broad spectrum extrinsic clotting test in oral anticoagulant control. While not warranting the routine use of the chromogenic assay for factor VII in place of the prothrombin time using BCT, the factor VII amidolytic assay offers a limited but dependable guide to dosage in long-term patients. The complexity of the technique in its present form militates against its adoption for routine anticoagulant control in hospital laboratories.

Acenocoumarol↗
PubMed1978

Assessment of plasma fibrinolytic system with use of chromogenic substrate.

A block of tests employing a chromogenic substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi AB, Stockholm) is proposed for a quantitative assessment of the plasma fibrinolytic system. It allows measurement of spontaneous plasmin activity, plasminogen content, activator activity (ability of plasma to activate plasminogen), immediate and progressive antiplasmin levels and alpha2-macroglobulin. All these assays could be performed within 45 min using a total of 0.4 ml of plasmin.

Fibrinolysin↗
PubMed1976

Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation.

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 degrees C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.

Binding Sites↗
PubMed1975

Separation of human antihemophilic factor (AHF, factor VIII) from fibrinogen by means of defibrase.

Various attempts have been made in the past to remove fibrinogen from plasma fractions rich in factor VIII. Present studies indicated the Defibrase, a preparation of a thrombin-like enzyme from the venom of Bothrops atrox, might be successfully employed for this purpose. Addition of Defibrase to plasma or low-purity factor VIII concentrates allows for the complete removal of fibrinogen without any apparent reduction in the activity. It was also shown that treatment with Defibrase neither decreases the stability of factor VIII nor apparently affects its antigens properties.

Batroxobin↗