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PubMed1992

Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.

Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1978

In vitro prothrombin synthesis from a purified precursor protein. II. Partial purification of bovine carboxylase.

In this paper, we describe the isolation and partial purification of an enzyme system that converts bovine decarboxyfactor II (PIVKA-II) into prothrombin (factor II). It is shown that the increase in factor II activity occurs in parallel with 14CO2 incorporation into BaSO4 adsorbable proteins. The system is not strictly vitamin K-dependent because it is obtained from the livers of normal healthy cows. By preincubating the enzyme(s) with an excess of warfarin, an absolute vitamin K1-dependence can be obtained. The reaction is inhibited by its own product, factor II.

Animals↗
PubMed1981

Tryptophan exposure in various conformational isomers of bovine prothrombin fragment 1. An acrylamide quenching study.

In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity.

Acrylamides↗
PubMed1979

An automated amidolytic assay of thrombin generation: an alternative for the prothrombin-time test.

An automated method is presented for the determination of thrombin generation in plasma during activation by thromboplastin. The thrombin generated is allowed to split the chromogenic substrate Tol-Gly-Pro-Arg-pNA yielding p-nitroaniline. The increase in absorbance at 410 nm is recorded. This assay may serve as a specific, precise and fast alternative for the conventional clotting tests: "Prothrombin time" and "Thrombotest".

Amides↗
PubMed1980

Prothrombin time standardization and temperature problems.

The reaction temperature is crucial for enzymatic reactions. Difficulties in controlling the temperature in the tilt test tube technique for the prothrombin time test are illustrated. An improved detailed description of the technique is suggested for standardization purposes. Mixing of the specimen with the reagent should be done in the water bath. The test tubes should be immersed deeply into the bath. Recording of the temperature in the reaction mixture is recommended. When using plastic test tubes the temperature problems may be greater than with glass test tubes. Some heat denaturation of coagulation enzymes may take place at 37 degrees C. Lowering the temperature of the reaction to 30 degrees C and perhaps photometric recording of the coagulation should be included in a suggested future optimized reference method.

Evaluation Studies as Topic↗
PubMed1987

Structure and order of the protein and carbohydrate domains of prothrombin fragment 1.

The three-dimensional structure of prothrombin fragment 1 has been determined by X-ray crystallography at 3.8 A resolution. The fragment is composed of a number of structural units, some of which are ordered while others are disordered. The ordered part of the structure includes a compact kringle unit, a helical domain and a carbohydrate chain. The kringle structure is organized around a close pair of buried disulfide bridges. One of its carbohydrate chains, that attached to Asn 101, is fully ordered, but the carbohydrate chain attached to Asn 77 appears to be disordered. The calcium binding unit is composed of a disordered part containing all ten gamma-carboxyglutamic acid residues and an ordered part forming the helical domain. The highly conserved residues Phe 41, Trp 42 and Tyr 45, which form a hydrophobic cluster on the first helix, interact around a crystallographic two-fold axis with the equivalent residues in another molecule to form a dimer in the crystal.

Amino Acid Sequence↗
PubMed1988

Structure of prothrombin fragment 1 refined at 2.8 A resolution.

The structure of prothrombin fragment 1, solved at 2.8 A resolution (1 A = 0.1 nm) by a combination of multiple and single isomorphous replacement methods utilizing solvent flattening, has been refined by restrained least-squares methods (R = 0.24), solvent not included, using fairly stringent restraints on the molecular geometry and individual thermal parameters. The inner kringle loop possesses significantly lower B-values than the outer loops even though the former also constitutes a surface of the folded kringle structure. This surface forms the Lys sub-site of the fibrin binding site of other kringles. The hydrogen bonding network and ion pair interactions of fragment 1 appear to maintain a compact folded structure among the various loops of the kringle structure. On the other hand, since there is only one hydrogen bond between the kringle and its preceding 30 residues, considerable flexibility is suggested for the Gla-domain consistent with its disorder in crystals. A chitobiose has been located at the Asn77 glycosylation site, but only a single N-acetyl-glucosamine is ordered at Asn101. The lysine binding site region of other kringles is not properly developed in fragment 1, accounting for its lack of Lys/fibrin affinity. Most of the conserved sequence among 11 different kringles is associated with either: (1) protecting the inner loop disulfides Cys87-127, Cys115-139 upon which the folding is based; or (2) a requirement of the lysine binding site. The remainder of the conservation is generally associated with the ten reverse turns of the folding; of these 40 residues, or about half the sequence, 14 are conserved among eight different turns. The intermolecular packing consists of infinite helical columns of fragment 1 molecules related by a crystallographic 4(3) screw axis, which are held together by van der Waals' interactions of aromatic clusters from different molecules related by a crystallographic 2-fold rotation axis.

Amino Acid Sequence↗
PubMed1991

Structure of bovine prothrombin fragment 1 refined at 2.25 A resolution.

The structure of bovine prothrombin fragment 1 has been refined at 2.25 A resolution using high resolution measurements made with the synchrotron beam at CHESS. The synchrotron data were collected photographically by oscillation methods (R-merge = 0.08). These were combined with lower order diffractometer data for refinement purposes. The structure was refined using restrained least-squares methods with the program PROLSQ to a crystallographic R-value of 0.175. The structure includes 105 water molecules with occupancies of greater than 0.6. The first 35 residues (Ala1-Leu35) of the N-terminal gamma-carboxy glutamic acid-domain (Ala1-Cys48) of fragment 1 are disordered as are two carbohydrate chains of Mr approximately 5000; the latter two combine to render 40% of the structure disordered. The folding of the kringle of fragment 1 is related to the close intramolecular contact between the inner loop disulfide groups. Half of the conserved sequence of the kringle forms an inner core surrounding these disulfide groups. The remainder of the sequence conservation is associated with the many turns of the main chain. The Pro95 residue of the kringle has a cis conformation and Tyr74 is ordered in fragment 1, although nuclear magnetic resonance studies indicate that the comparable residue of plasminogen kringle 4 has two positions. Surface accessibility calculations indicate that none of the disulfide groups of fragment 1 is accessible to solvent.

Amino Acid Sequence↗
PubMed1986

Chromogenic assay for the prothrombin activator ecarin from the venom of the saw-scaled viper (Echis carinatus).

Ecarin, by limited proteolysis and subsequent autocatalytic reactions, causes the conversion of prothrombin into three products with amidolytic activity: meizothrombin, meizothrombin 1 and lpha-thrombin. Ecarin action may be abolished by ethylenediaminetetraacetic acid and the activity of alpha-thrombin can, with a high degree of selectivity, be inhibited by heparin. Thus, ecarin potency may be assayed by measuring the meizothrombin activity generated by ecarin action on human plasma in the presence of heparin. The chromogenic substrate Tosyl-glycyl-L-prolyl-L-arginine-p-nitroanilide (Chromozym TH) is used in this assay.

Animals↗
PubMed1982

Activated prothrombin complex concentrates: approaches to their preparation.

Locally produced prothrombin complex concentrate was activated by different methods and the resultant inhibitor bypassing activity compared with commercially produced activated concentrates using an assay based on the shortening of the clotting time of haemophilic plasma containing inhibitor to factor VIII. Analytical chromatography suggested that for most of these products the inhibitor bypassing activity could be explained in terms of classical activated coagulation factors. In addition, a preparation of Bridge Anticoagulant Neutralising Activity was examined. The results indicate this product merits in vivo investigation as a possible treatment for inhibitor patients.

Blood Coagulation Factors↗
PubMed1983

The interaction of salicylate and vitamin K in synthesis of Factor II (prothrombin).

The effects of sodium salicylate on synthesis of a vitamin K-dependent plasma coagulation protein, Factor II (prothrombin) was studied using the isolated rat liver perfused for 10 hours in vitro. Cumulative synthesis of Factor II was measured by a standard coagulation assay, by activation with E. carinatus venom, and by rocket immunoelectrophoresis. When sodium salicylate 5 mg or 25 mg was added to the liver perfusate (volume 100 ml) at the outset of the perfusion, cumulative synthesis of both coagulation activity and immunoreactive protein was significantly less than that seen in control perfusions containing no salicylate. The inhibitory effect of salicylate was prevented by pretreatment of rat liver donors with supplemental vitamin K injected 24 hours before sacrifice. Although some interaction between salicylate and vitamin K was apparent from these experiments, the results from vitamin K-deficient rat liver donors were quite different from those containing salicylate. There was no assayable Factor II coagulation activity produced in 10 hours of perfusion of vitamin K-deficient rat livers, but cumulative synthesis of immunoreactive Factor II was quite comparable to that seen in control perfusions.

Animals↗
PubMed1985

Novel rhodamine tripeptide substrate for manual and automated colorimetric prothrombin time test.

A chromogenic prothrombin time test is described using the tripeptide substrate, (Sar-Pro-Arg)2-Rhodamine 110. The method is both precise and sensitive to the factors of the extrinsic pathway. The accuracy of the method was demonstrated by comparison of patient sample values to standard clotting test results. The manual substrate test was adapted to two different automated chemistry analyzers.

Autoanalysis↗
PubMed1990

The effect of fluorescent humic substances existing in the well water of Blackfoot disease endemic areas in Taiwan on prothrombin time and activated partial thromboplastin time in vitro.

Fluorescent humic substances (FHS) in well water of Blackfoot disease endemic areas were purified and fractionated by Sephadex G-25 column chromatography. Four fractions of the purified FHS were isolated. The purified FHS and their fractions were then added to normal human pool plasma in vitro separately to detect the abilities of the effects on prothrombin time (PT) and activated thromboplastin time (APTT). Results showed that all of the four fractions of the purified FHS prolonged both PT and APTT in the higher concentration ranges (10 mg/ml - 20 mg/ml), but shortened both PT and APTT in the lower concentration ranges (0.5 mg/ml - 5 mg/ml). Among the four fractions of the FHS, the fraction 1, the humic substance with the highest molecular weight among all the four FHS, showed the most obvious effects. Owing to the effects of the FHS on PT and APTT values, we supposed that there is a close relationship between the FHS and the cause of Blackfoot disease.

Arsenic↗
PubMed1990

A prothrombin time-based functional assay of protein S.

Protein S activity was measured as the degree of prolongation of a prothrombin time-based clotting assay in which diluted test sample, protein S-depleted plasma previously incubated with Protac to fully activate protein C, bovine thromboplastin and calcium ions are mixed. Assay specificity was first demonstrated by observing that the prolongation of the clotting time was dependent on protein S and was subsequently confirmed by testing plasma samples from patients with conditions known to affect protein S activity. High sensitivity, reproducibility (interassay coefficient of variation lower than 5%) and easy handling of samples and reagents make this assay suitable for screening of congenital and acquired protein S deficiency.

Anticoagulants↗
PubMed1990

Thrombin generation in prothrombin-complex-preparations; its effect on a chromogenic substrate, fibrinogen and platelets.

The kinetics of thrombin generation in various prothrombin-complex-preparations (PCC) were investigated using a chromogenic substrate assay. The rate of thrombin formation and the total activity of thrombin generated varied considerably among different preparations. The thrombin generation velocity influenced the rate of fibrinogen to fibrin conversion, measured as plasma viscosity alteration per unit of time. It also influenced thrombin induced platelet aggregation with regard to maximum aggregation and aggregation velocity. The kinetic parameters measured photometrically and coagulometrically showed reduced generation velocities and less thrombin effects with thrombin formed in PCCs than with thrombin generated in plasma. The quantitative differences between thrombin effects in various PCCs on platelet aggregation did not correlate with the data of the amidolytic and fibrinogenolytic measurements. The significance of the results is discussed with regard to the question of whether, or to what extent, PCCs could cause or accelerate a disseminated intravascular coagulation.

Blood Coagulation Factors↗
PubMed1991

Influence of anticoagulants used for blood collection on plasma prothrombin fragment F1 + 2 measurements.

The levels of prothrombin fragment F1 + 2 were measured by a double antibody radioimmunoassay in blood samples collected into different anticoagulant solutions. We evaluated healthy males between the ages of 42 and 77, asymptomatic patients with hereditary deficiencies of protein C or protein S, and persons receiving tumor necrosis factor infusions. The results in specimens collected in an anticoagulant containing ACD, EDTA, adenosine, and 25 U/ml of heparin (a) were highly correlated with those collected in an anticoagulant containing a synthetic thrombin inhibitor, EDTA, and aprotinin (b). However, in asymptomatic patients with congenital antithrombin III deficiency, we found that the plasma levels of F1 + 2 in blood collected in anticoagulant (a) were usually substantially higher than those collected in anticoagulant (b). We determined that this phenomenon was not attributable to the venipuncture procedure itself, but rather appears to be due to the action of low concentrations of heparin in the presence of reduced blood levels of antithrombin III. Our data show that the previously documented elevations in plasma F1 + 2 levels in patients with congenital antithrombin III deficiency appear to be caused by the above in vitro anticoagulant effect, and that this population does not exhibit evidence of a prethrombotic state as defined by the F1 + 2 assay.

Adult↗
PubMed1991

The influence of time, temperature and packed cell on activated partial thromboplastin time and prothrombin time.

Activated partial thromboplastin time (APTT) and prothrombin time (PT) were performed in four groups of studies in order to evaluate the influences of time, temperature, and different forms of plasma storage to the result. Different designs for storage of the plasmas were studied, including the plasmas stored either with or without packed cells, the plasmas stored in the cuvette with exact volume for performing the test or in the test tube. The temperatures for store of the plasma were at room temperature, at 4 degrees C and at -70 degrees C. The time for store of the plasma was from 1 hour up to 7 hours. The plasmas included normal pooled plasmas and diseased plasmas. From this study, it is found that the PT test was not easily affected by the temperature, storage time and the form of storage in comparison with the APTT test which was much easily affected by the above conditions. APTT should be done within 2 hours after sampling and the plasma should be stored with the packed cells at 4 degrees C in order to obtain a reliable result. PT could be done within 7 hours without influence to the result if the plasma was stored with the packed cells at 4 degrees C. No significant cold-induced shortening of PT could be noted when the plasma was incubated at 4 degrees C up to 7 hours. In either PT or APTT, the most suitable condition for storing the plasma should be with the packed cells at 4 degrees C.

Blood Preservation↗