Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X

We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.     

Microplate coagulation assays.

This report describes the development of microplate-based blood coagulation assays. The assays require a kinetic microplate reader to follow changes in absorbance at 405 nm caused by the coagulating plasma. Procedures for performing prothrombin time and activated partial thromboplastin time tests are described with intra- and inter-assay variability of a few percentage points. The prothrombin time of normal plasma was 64.5 +/- 3.6 s, and the activated partial thromboplastin time was 69.8 +/- 3.2 s. Clotting times were prolonged when normal plasma was mixed with plasmas deficient in particular coagulation factors, as expected. These assays take advantage of the microplate format (small sample size and multiple simultaneous assays) and can be customized for specific purposes, such as quantifying purified factor IX or assessing protein C activity in plasma.     

Simultaneous determination of functional coagulation factors and competitive (PIVKA-) inhibitors based on enzyme kinetics

For a plasma containing the competitive (PIVKA-) inhibitors induced by anticoagulant treatment the coagulation time t is related to the concentrations of functional coagulation factors S (substrates) and competitive inhibitors I by t = tmin + el/S + gamma I/S with tmin being the minimum possible coagulation time and e and gamma the sensitivities of the test procedure towards a change in the concentration of functional coagulation factors and competitive inhibitors, respectively. The calibration of the test procedure can be achieved by performing a series of dilutions on an inhibitor-free plasma (determination of tmin and e) and, after that, on a plasma of known inhibitor content (determination of gamma) in both cases recording the parametrizing straight line which results from multiplying the respective equation by S. The content of functional coagulation factors and competitive inhibitors in the plasmas of anticoagulated patients then can be determined simultaneously by treating the patient's plasma like in the calibration for gamma. The proposed method should allow the complete metrological characterization of thromboplastin time reagents without any need for reference thromboplastins.     

Species specificity of thromboplastin. A phylogenetic study.

Having studied the influence of thromboplastin preparations derived from cold and warm-blooded species on the plasma of 12 vertebrates (carp, frog, turtle, hen, rabbit, rat, mouse, guinea-pig, ground squirrel, dog, sheep and man) we have established that among the species far from each other phylogenetically, the phenomenon of species specificity could be demonstrated in general. It was found that the extrinsic coagulation system measured by Quick times can well be activated in every examined species. Simultaneously, in plasmas of turtle and hen the intrinsic clotting system proved to be deficient.     

The calibration of rabbit tissue thromboplastins: experience of the Dutch Reference Laboratory for anticoagulant control

A number of commercial rabbit tissue thromboplastins used in oral anticoagulant control have been calibrated against the first International Reference Preparation for thromboplastins. This was done in a three-stage procedure by one laboratory, each stage representing a different level of thromboplastin comparability. The calibration model recently recommended by ICTH and ICSH was tested. This model proved to be suitable, although a statistically significant aberration was observed for some of the thromboplastins. The bias introduced by using the model in these non-ideal cases was small compared to the overall variation of the International Normalized Ratio, being the universal scale for reporting the prothrombin time during oral anticoagulant control. Batch-to-batch calibration using lyophilized pooled plasmas could be reliably performed for several commercial thromboplastins.     

Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C

Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.     

Laboratory testing for lupus anticoagulants

The diagnosis of LA requires special attention to specimen processing and careful determination of a reference interval for the screening procedures. A sensitive APTT reagent should be used as the routine screen with appropriate secondary screening procedures available for the difficult patient (e.g., KCT and dRVVT). Appropriate attention to the ratio of patient to normal plasma in mixing studies is imperative. The ratio of four parts patient to one part normal is recommended. In some instances, confirmatory tests utilizing either the PNP or other test systems may be ambiguous. In these cases it is necessary to perform factor assays for a definitive diagnosis. There is a need for reference plasmas and a common means of expressing LA titers.     

Thromboplastin (tissue factor) in plasma membranes of human monocytes.

The synthesis of thromboplastin, a potent trigger of blood coagulation, can be induced in human peripheral blood monocytes. Indirect evidence suggests that newly synthesized thromboplastin becomes in part available on the cell surface. We have attempted to study the localization and availability of thromboplastin more directly by isolating plasma membranes from isolated human peripheral blood monocytes. The specific activities of the plasma membrane markers increased 16-22-fold in these preparations with a recovery of about 15%. The contamination by mitochondria, lysosomes, nuclei and endoplasmic reticulum was low as estimated by marker enzymes and electron microscopy. In both unstimulated and stimulated monocytes thromboplastin was largely recovered in this plasma membrane fraction, providing direct evidence for its membrane localization. Phospholipase C (E.C. 3.1.4.3) is a potent inactivator of thromboplastin through its hydrolysis of the phospholipids necessary for thromboplastin activity [Otnaess, Prydz, Bjørklid & Berre (1972) Eur. J. Biochem. 27, 238-243]. About 70% of the total membrane thromboplastin activity was inactivated when whole cells were treated with phospholipase C and the membranes subsequently isolated. Following stimulation to induce thromboplastin synthesis, the plasma membranes showed a shift in their relative content of phosphatidylcholine and phosphatidylethanolamine consistent with a transmethylation process.     

Deux nouveaux anticoagulants: Dabigatran et Rivaroxaban <Emphasis Type="Bold">Leur impact sur les examens de coagulation</Emphasis>

Two new orally active anticoagulants — Dabigatran etexilate prodrug or Pradaxa® inhibitor of factor IIa and Rivaroxaban or Xarelto® inhibitor of factor Xa — have received the European and Canadian authorizations to be launched on the market for preventing venous thromboembolic accidents in major orthopedic surgery. Dabigatran is registered in North America for patients with atrial fibrillation.One of their advantages is that they do not require regular monitoring of coagulation.Nevertheless, in a small number of particular clinical settings, it might become necessary to measure their anticoagulant activity. This implies the choice of appropriate techniques and an interpretation of the results according to the time between the blood collection and the taking of the drug. Studies have used pools of normal plasmas enriched with increasing concentrations of Rivaroxaban or Dabigatran, or plasmas of treated patients.One must make a distinction between the simple tests, which are very easy to make, like prothrombin time preferred for Rivaroxaban rather than for Dabigatran, but not specific, and the tests like anti-Xa activity measurement less easily available, but specific. The activated partial thromboplastin time, and preferably the modified thrombin time (Hemoclot), and the anti-IIa activity measurement will be used for Dabigatran. In all these techniques, the use of calibrated plasmas containing known quantities of drug allow to express the results in ng or in μg/mL of plasma, in order to compare them to the values obtained in patients.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.     

Characteristics of the earliest cross-neutralizing antibody response to HIV-1

Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%-30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2-3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.     

Modeling of the composition of materials for soft X-ray sources used in research on inertial confinement fusion

A method for calculating and optimizing the composition of materials for soft X-ray sources used in research on inertial confinement fusion is described. For a target-converter, a material composition is determined with which the conversion of laser light into X radiation is highly efficient. A comparative analysis is carried out of the efficiencies of generation of soft X-ray emission in the plasmas of some composite materials of thin conductors (wires) used as loads in X-and Z-pinches. Numerical calculations of the optical plasma properties are reported whose results make it possible to judge the emissivity of plasmas of different materials. The results obtained are compared to the data from other studies.     

Coagulation and fibrinolysis in capybara (Hydrochaeris hydrochaeris), a close relative of the guinea-pig (Cavia porcellus)

Fibrinolytic and coagulation properties of capybara (Hydrochaeris hydrochaeris, LINNAEUS, 1766) plasma were analysed and the results compared to the guinea-pig (Cavia porcellus), a close relative. Capybara fibrinogen was isolated and fibrinolysis of its plasma was carried out in a homologous system and with bovine fibrin. Undiluted plasma did not have fibrinolytic activity on fibrin plates; euglobulins gave a dose-related response. Zymography of capybara and guinea-pig plasma gave the same patterns of activity as human or bovine plasma. Human urokinase (UK) and tissue plasminogen activator (t-PA) produced lysis in capybara fibrin plates. Streptokinase (SK) (500 IU/ml) did not activate capybara or guinea-pig plasma. In this system, human plasma was extensively activated. Coagulation tests for both species of rodent were prolonged. The capybara showed values for prothrombin time (PT) shorter than activated thromboplastin time (APTT). The guinea-pig, as already shown, had longer PT values. Factors X and VII were very low for capybara and guinea-pig when tested using reference curves and diagnostic kits for human plasma. It is suggested that the capybara could be a valuable laboratory animal considering its size and closeness to the guinea-pig, and this could allow for the provision of materials from one single animal when convenient or necessary.     

The protein component of human brain thromboplastin

The protein component of human brain tissue thromboplastin (factor III) has been purified by deoxycholate (DOC) extraction, ultracentrifugation, gel filtration and finally repeated preparative polyacrylamide gel electrophoresis (PGE) in the presence of sodium dodecylsulphate (SDS). The final preparations gave one band in analytical PGE. Reduced and alkylated protein appeared as a band of molecular weight about 53 000 in SDS-PGE.The protein had a low solubility in aqueous solutions in the absence of detergents. When recombined with an optimal amount of the phospholipid fraction of tissue thromboplastin (fraction B) the procoagulant thromboplastin activity was regained. Neither alone nor after recombination with phospholipid did the protein catalyze the hydrolysis of aminoacyl-β-naphthylamides or casein.     

Theoretical and experimental studies of the radiative properties of matter at high energy densities and their application to the problems of inertial confinement fusion

The paper presents the results of theoretical and experimental studies of the radiative properties of plasmas produced by heating and compression of various materials to high energy densities. The specific features of the theoretical plasma model known as the ion model, which is used to calculate the radiative characteristics of plasmas of complex chemical composition, are discussed. The theoretical approach based on this model is applied to the plasma produced during the explosion of the X-pinch wires. The theoretical estimate of the radiation efficiency is compared with the experimental data on the total energy yield from an X-pinch made of two different wires (NiCr and Alloy 188). The radiative characteristics of (C12 H16 O8) and (C8 H12 O6) plasmas are calculated for the temperature diagnostics of plasmas produced from porous targets employed in inertial confinement fusion experiments with the use of laser radiation and heavy-ion beams.     

Complex plasmas IV: Theoretical approaches to complex plasmas and their application

This paper completes a series of reviews devoted to the physics of complex plasmas, in which one of the components (dust) is in a crystalline or liquid state, while the others (electron, ions, and neutral atoms) are in a gaseous state. This review is devoted to the theoretical approaches used to describe complex plasmas so far. The main theoretical developments have been concentrated in the gaseous and weakly nonideal states of complex plasmas. Here, we describe the achievements in the new kinetic and new hydrodynamic approaches to complex plasmas. At present, only generalizations of the van der Waals approach for complex plasmas have been used to describe phase transitions and plasma condensation in complex plasmas. Here, criteria for transitions are described and compared with the existing experimental observations. Theoretical and numerical results for nonlinear structures, such as dust layers, dust voids, dust sheaths, and dust convective vortices, obtained by solving the stationary balance equations, are also discussed and compared with state-of-the-art experiments. At present, experiments in this field are progressing very fast, while theory is not advancing at the same rate of development. To further develop new theoretical models, one can use the elementary physical processes in complex plasmas described in the previous parts of the review. However, the detailed comparison of theory and experiments also needs more detailed experimental diagnostics of the phenomena observed. In the concluding part of our review, the trends in experiment and theory, as well as some existing applications, including industrial, environmental, and astrophysical ones, are described.     

Effect of feed gas composition of gas discharge plasmas on Bacillus pumilus spore mortality

AIMS: To investigate the effect of gas composition on the sensitivity of Bacillus pumilus spores to gas plasmas. METHODS AND RESULTS: Inert gas plasmas, oxygen-based plasmas and various moisturized air plasmas were used to inactivate B. pumilus spores in low gas pressure of 50 Pa. Although the treatment temperature did not exceed 55 degrees C when exciting these plasmas, spore survival varied widely depending on the composition of the gas feed. Higher spore mortality was acquired by inert gases of low molecular weight except for helium. The highest spore mortality (4.54log reduction) was obtained when air with a 0.05 molar fraction of water vapour was used as the plasma carrier gas. CONCLUSIONS: Water molecules in the plasma carrier gas play a significant role in inactivation of B. pumilus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This strong inactivation may occur through hydroxyl free radicals generated from the moisturized air plasma.     

Fatal hemorrhagic diathesis associated with mild factor IX deficiency in pl/J mice

Surgical implantation of devices into the abdomen of PL/J mice was associated with fatal hemorrhage at 9 to 11 d after surgery. Coagulation profiles were evaluated to determine the underlying cause of this effect. The mean activated partial thromboplastin time (aPTT) of untreated PL/J mice was significantly higher than that of BALB/cByJ and C57BL/6J strains. The addition of human plasmas deficient in factors VIII, XI, or XII, prekallikrein, or high molecular-weight kininogen corrected the elevated aPTT of PL/J mice, but correction did not occur when factor IX-deficient human plasma was added. When compared to an assigned factor IX activity of 100% for pooled plasma from BALB/cByJ mice, C57BL/6J and PL/J mice revealed percent activities of 67% and 16%, respectively. PL/J mice could represent a new model for the study of pathogenesis and therapy of mild factor IX deficiency that is expressed and becomes clinically apparent secondary to major surgery.     

Mechanism of heparin inactivation by thromboplastin (factor III)

Thromboplastin (a commercial one and that obtained from different tissues) is shown to inactivate heparin in proportion to the quantity of thromboplastin or to the heparin:thromboplastin ratio. A degree of inactivation of heparin changes after the modification of protein component of thromboplastin by proteases, however there is no dependence between the protein amount in the preparation and its antiheparin activity. Inactivation of heparin by thromboplastin is stipulated by the formation of associations due to electrostatic interactions between the clusters of amino acid protein residues (which dissociate under physiological conditions as bases) and heparin sulphogroups. It is suggested that factor III circulating in blood flow participates in the creation of hemostatic potential not only as a result of its ability to catalyze thrombinogenesis, but also due to the decrease of the anticoagulant blood activity.