bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of Tissue Thromboplastin Activity
Authors: Wijngaards G. , Hemker H.C.
Published: April 2009

The one-stage prothrombin time test for tissue thromboplastin activity was evaluated using the correction method. A strong influence of the intrinsic system was seen depending on the clotting time and the type of tubes. This test was unsuitable for quantitative comparison of thromboplastin activity of different preparations.Subsequently a two-stage assay for human thromboplastin is described using purified human factor VII, artificially prepared factor VII reagent, and a phospholipid suspension. The correction method, applicated to this two-stage assay, resulted in a rectilinear dose-response curve when the clotting times of a thromboplastin dilution series were plotted against the reciprocal of the concentrations. Using this test system it could be shown that thromboplastin from porcine tissue can activate human factor VII quite well, while that from bovine tissue cannot.
Alteration of Rat Bone Marrow Megakaryocytes Following Administration of Cytosine Arabinoside, Daunomycin and Hydroxyurea
Authors: Jerushalmy Z. , Patya M. , Pinkhas J. , de Vries A.
Published: April 2009

The in vivo effects of three cytotoxic agents – cytosine arabinoside, daunomycin and hydroxyurea on rat bone marrow megakaryocytes were studied, specifically the relative count, the type percentage and the morphology. All three agents caused thrombocytopenia, a decrease in the number of megakaryocytes relative to the nucleated bone marrow cells, and a decrease in the percentage of megakaryoblasts, associated with an increase in the percentage of granular megakaryocytes. A decreased lobulation of the megakaryocytes was observed following the administration of cytosine arabinoside and of daunomycin. Thrombosthenin, examined immunologically, remained detectable in the bone marrow smears throughout the various stages of megakaryocyte alteration.
Comparable Inhibition of Aggregation of PRP of Neonates and Adults by Aspirin
Authors: Ts’ao Chung-hsin
Published: April 2009

We have determined the minimal concentrations of 4 aggregating agents that were required to cause a 65–75 % increase in light transmission without disaggregation during the 5-min recording period in PRP samples of 14 neonates and 10 adults. We found, for example, that this degree of aggregation could be elicited in adult PRP by a mean value of 2.09 μM ADP, whereas similar aggregation of neonatal PRP required 5.16 μM ADP. Based on these figures, we observed that adult PRP was about 2.5 times more sensitive to ADP, at least 10 times to epinephrine and 2.3 times to collagen than neonatal PRP. However, neonatal PRP was approximately 20% more sensitive to ristocetin than adult PRP. By using concentrations of aggregating agents that caused comparable aggregation of neonatal and adult PRP, we noted comparable inhibition of aggregation in these samples by aspirin. While neonatal PRP was less sensitive than adult PRP to physiological aggregating agents, there was no evidence that the former was more susceptible to in vitro aspirin inhibition.
Qualitative Changes in Fibrinogen Following Exposure to Agents Used for Preparation of Fibrin Monomers
Authors: Brosstad F. , Godal H.C.
Published: April 2009

The solubility, clottability, thrombin clotting time and agarose gel chromatography pattern of human fibrinogen were studied after exposure to solvents commonly used to prepare fibrin monomers (0.0167 m acetic acid with 2.5 mM EDTA; 1 m NaBr, pH 5.2; 3.3 m urea, pH 7.4; 5 m urea, pH 7.4). When exposed to acetic acid at room temperature, fibrinogen precipitated almost immediately and quantitativley. Subsequent dialysis for 72 h against 0.3 m NaCl, pH 7.4, caused resolution of fibrinogen to a varying degree, the amount depending on the time of exposure. The redissolved fibrinogen showed reduced clottability, markedly shortened thrombin clotting time and a chromatographic profile that indicated large amounts of aggregates. Fibrinogen exposed to 1 m NaBr, pH 5.2, at room temperature for 1 h showed a slightly shortened thrombin clotting time and a broadened chromatographic profile. Exposure for 24 h to the same agent resulted in reduced solubility and clottability, a prolonged thrombin clotting time and progressive broadening of the chromatographic profile. Similar findings were obtained with fibrinogen exposed to 5 m urea, pH 7.4. Exposure to 3.3 m urea at pH 7.4 for 24 h, room temperature, led only to a moderate increase in solubility.
Factor VIII Deficient Plasma for Laboratory Tests Prepared from Normal Plasma and a Human Antibody
Authors: Exner T. , Rickard K.A. , Speers S.
Published: April 2009

Plasma from a patient with a potent factor VIII inhibitor was mixed at several low concentrations with fresh normal plasma. The factor VIII deficient plasmas obtained after a short incubation period were used as substrate plasmas in the normal one-stage factor VIII assay method. Results obtained using 0.5–1 % of the inhibitor plasma in normal plasma compared favourably with those obtained using normal haemophilia A factor VIII deficient plasma provided that tests were carried out without delay. A single plasma exchange of this patient provided enough antibody to prepare more than 500 litres of factor VIII deficient plasma by simple mixing with fresh normal plasma.
The Effect of Heparin on the Interaction of Factor VIII and Human Platelets in vitro
Authors: de Marco L. , Fabris F. , Molaro G. , Girolami A.
Published: April 2009

Heparin was found to inhibit the interaction between human factor VIII and platelets. This was noted in two different systems, namely, platelet aggregation induced by neuraminidase-treated human cryoprecipitate and platelet aggregation induced by ristocetin-human factor VIII complex. Heparin appeared to have an inhibitory effect on the above systems similar to that reported on bovine factor VIII-induced platelet aggregation.
Plasma and Platelet Phospholipids in Normal Subjects and in Patients with Chronic Arterial Obliterations
Authors: Bruhn H.D. , Jipp P. , Ravens K.G.
Published: April 2009

Normally a substantially higher percentage of phosphoethanolamine is found in thrombocytes (24 %) than in plasma (2.7 %). The thrombocytes of patients with chronic arterial occlusions contain significantly less phosphoethanolamine (16.1%) than thrombocytes of normal subjects (24.0%; p < 0.01). On the contrary, the plasma phosphoethanolamine levels of these patients are significantly elevated (4.8 %) in comparison to normal subjects (2.7 %; p < 0.05), possibly as the result of some ‘leakage’ of phosphoethanolamine from platelets. An anti-aggregating treatment did not change the phospholipid pattern of the plasma and of the thrombocytes.
Preparation of Fibrin Monomers from Human Fibrinogen in Urea, Using Soluble or Insolubilized Thrombin
Authors: Brosstad F. , Godal H.C. , Kierulf P.
Published: April 2009

Fibrin Monomers were prepared by exposing human fibrinogen to thrombin in the presence of 3.3 m urea, pH 7.4 and +20 °C. Both with thrombin covalently bound to Sepharose 4 B and with thrombin in solution (100 NIH units/ml), extensive conversion of fibrinogen to fibrin within 1 h was achieved. No spontaneous clotting occurred.The fibrin preparations consisted to a very large extent of fibrin monomers, as evidenced by N-terminal analysis, agarose gel chromatography and SDS-polyacrylamide gel electrophoresis. When the urea concentration was reduced by dilution, the fibrin monomers retained their normal clottability, polymerization speed and cross-linking ability. Identical results were obtained with radioactive fibrin monomers prepared from 125I-labelled fibrinogen. Storage for 4 weeks at +4 °C or quick freezing in liquid nitrogen and subsequent thawing caused only slightly prolonged polymerization times. Attempts to separate thrombin in solution from fibrin monomers by chromatography were unsuccessful, whereas this was easily achieved with insolubilized thrombin. Since traces of thrombin may influence the experimental results, the latter technique should be preferred.
Dissolution of Noncovalently Bonded Fibrin in the Presence of Bromide Ions
Authors: Dyr J.E. , Vorlová Z. , Vodrážka Z.
Published: April 2009

Two peaks were demonstrated after gel chromatography on Sepharose 6-B of noncovalently bonded fibrin dissolved in 1 M NaBr at pH 5.3. One was eluted at the void volume while the other one was eluted at the elution volume of fibrinogen. Both fractions were stable during rechromatography. The fraction eluted at the void volume was highly heterogeneous and consisted of noncovalently bonded aggregates of fibrin monomer units. Our experiments indicate that these high molecular weight derivatives develop during dissolution of fibrin in NaBr solution. The possible explanation of these results is discussed.
Effect of Hypovolemic Shock on the Generalized Shwartzman Reaction
Authors: Howes, jr. Edward L. , McKay Donald G.
Published: April 2009

The generalized Shwartzman reaction was produced in rabbits by two intravenous injections of endotoxin given 20–22 h apart. Prior to the second injection of endotoxin, the animals were made hypovolemic by removal of 40% blood volume or reduction of the systolic pressure to 70% of normal, whichever came first. This blood removal produced a persistent hypotensive episode. 8/9 rabbits made hypotensive in this manner had evidence of glomerular capillary thrombi compared to 8/8 in a group that did not have blood removed. It is concluded that hypovolemic shock of this magnitude does not prevent the generalized Shwartzman reaction.
Low Dose Heparin in Major Surgery: Clinical Relevance of Plasma Heparin Concentrations
Authors: Kruse-Blinkenberg H.O. , Bech-Jansen P. , Jensen J. , Schmidt A. , Gormsen Johs
Published: April 2009

56 patients undergoing elective major surgery received low dose heparin prophylaxis, 5,000 IU 2 h before surgery and every 8 h for 7 days. The patients were tested by the 125I-fibrinogen uptake test, which, if positive, was controlled by venography. The heparin concentrations were measured by the method of Denson and Bonnar and by the chromogenic method of Teien et al. in blood samples drained immediately before and after surgery and on the 1st and 5th postoperative day 2 and 4 h after the injection of heparin. The values were found higher when estimated by the method of Denson and Bonnar than by the chromogenic method. Deep vein thrombosis (DVT) developed in 11 patients, and in 10 of them within the first 48 after the operative procedure, in 1 after 4 days. The preoperative values were not lower in the patients who developed DVT, and the heparin concentrations found on the 1st and 5th postoperative day were at the same level as in those who did not develop DVT.
Photometric Assay of Platelet Factor 4 with a Chromogenic Substrate
Authors: Vinazzer H.
Published: April 2009

A photometric assay procedure for platelet factor 4 is described. The synthetic oligopeptide benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) is used as a substrate. By the action of factor Xa, p-nitroaniIine (pNA) is split form the peptide bond. The amount of pNA liberated from S-2222 per minute is in direct relation to the activity of factor Xa. This reaction permits a photometric assay. Addition of heparin to an activation system consisting of plasma, thromboplastin and calcium chloride inhibits development of Xa activity. Since platelet factor 4 neutralizes heparin, its activity can be measured in such a system when all other components are kept at a constant level. Experimental details of the reactions involved and clinical results of the assay in comparison to a clotting method are described.
Inhibition of the Fibrinolytic System in Behçet’s Disease?
Authors: Asbeck F. , Meyer-Boernecke D. , van de Loo J.
Published: April 2009

Suppression of the fibrinolytic system is a well-known phenomenon in patients with Behçet’s disease. It is generally explained by an increase in the inhibitory potential. In order to prove this theory, the fibrinolytic system was investigated in 5 male patients with severe chronic Behçet’s disease. By the venous occlusion test, a marked suppression of the activatability of the system could be demonstrated (euglobulin lysis time, various fibrin plate assays). Results of analysis of the known inhibitors could not explain the phenomenon: antithrombin III, α2-macroglobulin, fast-reacting α2-antiplasmin, C1 inactivator, and the plasmin-antiplasmin complex were normal. The only change was an elevation of α1-antitrypsin and fibrinogen in 4 patients possibly due to an acute phase reaction. In addition to the possibility of a hitherto unidentified inhibitor, therefore, a decrease in production or in release of vessel wall activators must also be considered in the pathogenesis of this disorder.
Ditazole Activity and Its Interaction with Urokinase on Experimental Thrombosis
Authors: Caprino L. , Borrelli F. , Falchetti R. , Cafiero C. , Gandolfo G.M.
Published: April 2009

The effect of ditazole, a new antiaggregant oxazole derivative as well as its possible interaction with urokinase on the formation of electrically induced thrombus, was assayed in rabbits. The activity of ditazole in reducing thrombus weight was comparable to that of aspirin. In the ditazole- or aspirin-treated animals, the microscopical examination of the thrombus showed a reduction in the fibrin component, and well-isolated platelets not undergoing a viscous metamorphosis were present. Urokinase, administered in combination with these antiaggregant drugs, did not induce a further reduction in thrombus weight. However, this additional treatment did induce clearly visible lytic areas and histological modifications as observed with the antiaggregant drugs. These data suggest that the antiplatelet drug ditazole may be an effective antithrombotic agent in man and could facilitate the penetration of urokinase into the thrombus.
A Histochemical Study of Fibrinolytic Activity and Inhibition of Plasmin in the Lungs of Some Animal Species
Authors: Smokovitis Athanasios , Astrup Tage
Published: April 2009

Determined by the histochemical fibrin slide technique, the fibrinolytic activity of lungs decreased in the following order of species: rat, mouse, pig, guinea pig, rabbit, cattle. Examined by the fibrin slide ‘sandwich’ technique, inhibition of plasmin showed the opposite mode of decrease. The possible role of these differences in the pathogenesis of various disorders of the lungs in the different species is discussed.
The Anticoagulant Effect of Heparin + Antithrombin III Using Purified Fibrinogen as Substrate
Authors: Andersen P. , Godal H.C.
Published: April 2009

The anticoagulant effect of heparin in test systems composed of purified factors [antithrombin III (AT-III), fibrinogen and thrombin] was studied. As expected, the sensitivity to heparin depended on the concentrations of AT-III and thrombin, whereas the fibrinogen level was less decisive. In addition, qualitative differences proved important. Thus, the sensitivity to heparin was greater with crude bovine thrombin than with highly purified thrombin from the same species. Further, the sensitivity to heparin increased following removal of cold-insoluble material from the fibrinogen preparation. Finally, during storage of purified AT-III at + 4 °C for more than 4 weeks, the sensitivity to heparin decreased more rapidly than expected from the amidolytic AT-III assays. Smaller amounts of heparm were required to give a prolonged thrombin clotting time than in whole plasma, indicating that components of normal platelet-poor plasma (besides AT-III and fibrinogen) interfere with the anticoagulant effect of heparin. The present test systems may prove suitable for the detection and evaluation of factors of importance for the so-called heparin tolerance.
The Antiheparin Effect of α1-Acid Glycoprotein and Platelet Material, Evaluated by the Heparin Thrombin Clotting Time
Authors: Andersen P. , Godal H.C.
Published: April 2009

The effect on the heparin thrombin clotting time (HTCT) of a so-called acute phase protein, α1-acid glycoprotein, was studied in test systems consisting of purified fibrinogen, antithrombin III, heparin and thrombin, and compared to the effect of platelet material (crude platelet factor 4). In these test systems a physiological concentration of α1-acid glycoprotein (1 g/l) inhibited heparin more effectively than did soluble platelet material from 200 × 109 platelets/1. The present observations suggest that α1-acid glycoprotein is of great significance for the so-called heparin tolerance, whereas platelet material proved less important.
Procoagulant Activity of Activated Monocytes
Authors: Hiller E. , Saal J.G. , Riethmüller G.
Published: April 2009

Endotoxin-stimulated monocytes develop a very high tissue-factor-like activity as compared to an equivalent number of monocytes in a preparation of monocyte-enriched mononuclear blood cells obtained by Ficoll®-Urovison® density gradient centrifugation which is not exposed to endotoxin. In monocytes which were obtained by plastic adherence from the monocyteenriched mononuclear blood cells, the tissue factor activity was as high as in the endotoxin-stimulated monocytes.
Elimination of Inhibition in Euglobulin Fibrinolysis by Use of Flufenamate: Involvement of C1-inactivator
Authors: Kluft C.
Published: April 2009

The fibrinolytic activity of euglobulin fractions prepared from human morning plasma and assayed on fibrin plates is strongly inhibited by the C1-inactivator present in the fractions. Flufenamate, a potent representative of the group of synthetic thrombolytic agents, eliminates this inhibition in euglobulin fractions. This elimination is an apparently irreversible reaction dependent on concentration, time and temperature. The fibrinolytic enhancing effect of flufenamate in euglobulin fractions correlated well with a similar effect of added C1s, which neutralized the C1-inactivator. The effect of flufenamate was slightly greater than that of added C1s, suggesting an additional effect of the flufenamate. The activity enhancing effect of the flufenamate at the lower molarities could be separated from an activity decreasing effect at the higher molarities. A simple technique by which inhibitory effects in euglobulin fibrinolysis are selectively eliminated is described.
Heparin-Induced Release of Plasminogen Activator
Authors: Markwardt F. , Klöcking H.-P.
Published: April 2009

The influence of heparin and heparinoids on the release of plasminogen activator was studied in the isolated perfused pig ear. Heparin enhances the release of the tissue activator of fibrinolysis in a dose-dependent manner within the range of 0.05–1.0 IU/ml perfusion fluid. The release is also caused by sulfated polyanions at concentration of 10 μg/ml.
Some Sources of Error in the One-Stage Assay of Factor VIII
Authors: Elődi Susan , Váradi Katalin , Hollán Susan R.
Published: April 2009

The error of the one-stage assay of factor VIII activity is determined by the quality of reference and substrate (factor VIII-deficient) plasmas, as well as by the accuracy of dilution and clot observation time. Storage of the fresh normal plasma used as reference and/or of the substrate plasma for 2–3 h at 0 °C may cause considerable errors, which can be eliminated by the immediate use of plasma or by the determination of an appropriate correction factor. With all precautions taken into account the average error of the assay in our hands is 20%.
Degradation of Human Fibrinogen by Plasmin
Authors: Regañon E. , Aznar J. , Vila V.
Published: April 2009

The incubation of human fibrinogen with plasmin gives rise to an early degradation product with a molecular weight of 63,000, calculated by polyacrylamide-gel electrophoresis sodium dodecyl sulfate (PAGE-SDS); this product is resistant to the action of the plasmin for short incubation times and represents 1 ± 0.2 % of the original quantity of fibrinogen. The fragment was isolated from the incubated mixture by gel filtration and has a no-identity reaction with fibrinogen fragment D against fibrinogen fragment D antiserum. The reduction gives rise to three polypeptidic chains with molecular weights of 36,000, 17,000 and 10,000 (calculated by PAGE-SDS). Study of the carbohydrate content of these polypeptidic chains from the reduced 63,000 MW fragment indicates that there is only one electrophoretic region which contains periodic acid-Schiff (PAS) positive material. As the 63,000 MW fragment has a reaction against fibrinogen fragment D antiserum and also contains PAS positive material, according to Pepper and co-workers, it can be taken to arise from the HOOC-terminal region of fibrinogen.
Experimental Defibrination with Bothropase
Authors: Kelen Eva M.A. , Rosenfeld G. , Vainzof Mariz , Machado Zafira C.
Published: April 2009

Fibrinogen was depleted in dogs injected with a single dose of bothropase even if pretreatment followed by a continuous infusion of antifibrinolytic drugs was performed during defibrination. The activation of the fibrinolytic system as a secondary effect of the defibrination syndrome induced by bothropase injection was blocked completely by aprotinin (Trasylol) but not by EACA. Plasmin activity in spite of the inhibition of plasminogen activator suggests that, either an excess of activator is released in circulation, or a plasmin-antiplasmin complex is dissociated by the circulating fibrin, according to the hypothesis of Ambrus and Markus [1], and Back et al. [4] for the mechanism of fibrinolysis in vivo. An experimental model is suggested for the study of the fibrinolytic mechanism in vivo, by the association of defibrinating agents, antivenom and antifibrinolytic drugs.
Platelet Aggregability in Microvascular Haemostasis and the Effect of Local Inflammation
Authors: Arfors K.E. , Bergqvist D.
Published: April 2009

The effects on haemostatic plug formation and individual vessel platelet aggregability of granulocytopenia, prostaglandin E1 (PGE1 and indomethacin were studied in microvessels of the rabbit mesentery. Granulocytopenia was found to have no effect. Indomethacin abolished the differences between arterioles and venules in haemostatic plug formation time and stability as well as individual vessel platelet aggregability. PGE1 significantly inhibited haemostasis and also counteracted indomethacin. It is concluded that the haemostatic differences normally seen between arterioles and venules can be explained by prostaglandin-induced inhibition of venular platelets.
Influence of Pentacyanoferrate Complexes on Platelet Aggregation
Authors: Glusa Erika , Markwardt F.
Published: April 2009

To elucidate the structural constituents of sodium nitroprusside responsible for antiaggregating effects, comparative studies were done with pentacyanoferrates possessing ligands other than nitric oxid. Among the pentacyanoferrates tested, also the nitro and thionitro compounds possess considerable antiaggregating effects. The ammine and aquo compounds are nearly ineffective. These results support the assumption that the nitrosyl cation or its secondary product, nitrous acid, is responsible for the inhibition of aggregation.
Designing of Peptide Substrates
Authors: Claeson Göran , Aurell Leif , Friberger Petter , Gustavsson Stig , Karlsson Gunilla
Published: April 2009

Chromogenic peptide substrates for serine proteases have been designed by using two approaches: (1) by using the natural substrate as a model and (2) by structure-activity correlations obtained through screening of a large number of tripeptides. Some recent examples, substrates for kallikreins and urokinase are given.
Chromogenic Substrate Assay of Plasma Prekallikrein
Authors: Stormorken Helge , Baklund Anita , Gallimore Michael , Ritland Staale
Published: April 2009

A method for assaying plasma prekallikrein has been developed applying the chromogenic substrate Chromozym PK. Different variables have been investigated, and the final assay system was found to give a reproducible and reliable assay procedure. A normal value of 99.0 ± 18% was found, the coefficient of variation being 7.9. Studies on material from patients with various liver diseases indicated that the main site of prekallikrein biosynthesis is the liver.
Changes in Plasma Levels of Prekallikrein, Kallikrein, High Molecular Weight Kininogen and Kallikrein Inhibitors during Lethal Endotoxin Shock in Dogs
Authors: Gallimore Michael J. , Aasen Ansgar O. , Amundsen Egil
Published: April 2009

Plasma levels of prekallikrein, kallikrein, high molecular weight kininogen and kallikrein inhibitors were measured in samples from dogs infused with E. coli endotoxin. Markedly reduced levels of prekallikrein and high molecular weight kininogen were seen at circulatory collapse and free kallikrein was detected. Both ‘fast-reacting’ and ‘time-dependent’ inhibitors of plasma kallikrein were significantly reduced. Fractionation of pre-infusion and collapse samples by gel filtration together with immunological procedures indicated that both α2-macroglobulin and α1-antitrypsin were the major kallikrein inhibitors in dog plasma, and that they were significantly reduced in the collapse samples.
Studies on Assays for Plasma Prekallikrein and for the Monitoring of Coumarol Therapy
Authors: Egberg Nils , Bergström Kurt
Published: April 2009

Chromogenic substrates were used to assay prekallikrein, prothrombin and factor X. Plasma prekallikrein was contact activated and allowed the splitting of HD-Pro-Phe-Arg-PNA which has a high affinity for plasma kallikrein. Prothrombin was assayed in various ways (immunologically, after activation with Ecarin or in a less specific way). Determination of factor X is considered as a possible specific method in the monitoring of coumarol therapy.
Unreliability of Chromogenic Substrates for Assay of the Clotting Activity of Thrombin
Authors: Gaffney Patrick J. , Miller-Andersson Maggie , Kirkwood Tom B.L.
Published: April 2009

The rapid loss in clotting activity of commercially available thrombins during storage is not reflected by their activity as measured by synthetic chromogenic substrates. It is suggested that conformational changes occurring during storage can affect the binding sites of thrombin for fibrinogen, whereas small synthetic substrates are incapable of monitoring these changes.
On Use of Chromogenic Substrates for Studies of Coagulation Inhibitors
Authors: Ødegård Ole Rasmus , Lie Mette
Published: April 2009

Methodological problems encountered using chromogenic substrates on thrombin and Xa are discussed: (1) influence of substrate on inhibitor; (2) influence of heparin; (3) optimal concentrations; (4) specificity; (5) denatured enzymes, and (6) natural versus chromogenic substrates.
Antithrombin III: Critical Review of Assay Methods
Authors: Ødegård Ole Rasmus , Abildgaard Ulrich
Published: April 2009

Thrombin or factor Xa added to plasma are inactivated by antithrombin III (At-III). The inactivation is accelerated by heparin, permitting assay systems which rapidly measure the At-III content of diluted plasma. Without heparin, the slow inactivation rates may be measured. Existing activity assays (fibrinogen or chromogenic substrates) and immunoassays of At-III have been reviewed. Correlation studies show a close correlation between the results of immunoassay and the results of most activity assays. In health, a narrow range of At-III has been found. The level is low in infancy. Fertile women have on the average somewhat lower levels than men. In old age, the level tends to drop. In clinical material studied with amidolytic assays, subnormal At-III levels were found in hereditary deficiency, liver disease, disseminated intravascular coagulation and in some cases with acute thrombosis. The amidolytic assays are rapid to perform, do not require experience in clotting technique and seem preferable in clinical routine work.
Fluorogenic Substrates for Sensitive and Differential Estimation of Urokinase and Tissue Plasminogen Activator
Authors: Nieuwenhuizen Willem , Wijngaards Gerrit , Groeneveld Eleonore
Published: April 2009

Two fluorogenic peptide amides have been synthesized, i.e. BOC-L-valyl-glycyl-L-arginine 2-naρhthylamide (I) and L-valyl-glycyl-L-arginine 2-naphthylamide (II). The kinetic parameters of plasmin, urokinase and human uterine tissue plasminogen activator on substrates I and II have been determined. Quite unexpectedly, the tissue activator appeared to require for its activity a blocked amino terminus on the substrate. This was further corroborated with other synthetic substrates. Plasmin and urokinase did not show this requirement.
Assessment of Plasma Fibrinolytic System with Use of Chromogenic Substrate
Authors: Latallo Z.S. , Teisseyre E. , Lopaciuk S.
Published: April 2009

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 α2-macroglobulin. All these assays could be performed within 45 min using a total of 0.4 ml of plasmin.
Rat Coagulation Factors V, VIII, XI, and XII: Vitamin K Dependent
Authors: Owen, jr. Charles A. , Bowie Walter
Published: April 2009

When rats were given single or multiple doses of warfarin, the levels of prothrombin and factors VII, IX, and X were depressed, as expected. However, modest reductions of factors V, VIII, XI, and XII, but not of fibrinogen, also occurred. The levels of all eight factors promptly returned to normal when vitamin K1was given. Warfarin-resistant rats had no depression of any of the eight factors. When vitamin K deficiency was induced by internal or external biliary fistula, factors II-VII-IX-X decreased sharply and factors V-VIII-XI-XII decreased modestly. Again, all depressions were promptly reversed by vitamin K1. Isolated livers from warfarinized rats did not generate the classic vitamin K-dependent factors during 5 h of perfusion but did generate small amounts of factors V, XI, and XII, although less than normal. The isolated rat liver apparently does not generate factor VIII.
Automated Antithrombin III Assay with a Centrifugal Analyser
Authors: Ødegård O.R. , Rosenlund B. , Ervik E.
Published: April 2009

The factor Xa inactivating function of antithrombin III is measured automatically by an amidolytic method, adapted to a centrifugal analyser. Plasma is diluted in buffer with heparin. In stage I, diluted plasma is incubated with excess factor Xa. Heparin accelerates the saturation of antithrombin with factor Xa. In stage II, remaining factor Xa is determined with the chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA. The precision of the present assay compares favourably with that of the clotting assays and immunoassay. There is a close correlation (r = 0.82) between the results obtained with this assay and the immunoassay of antithrombin III.
The Effect of Trypsin and Storage on Aggregation and Release of Human Gel-Filtered Platelets
Authors: Pechet Liberto , Tiarks Cheryl Y. , Esposito Barbara A.
Published: April 2009

Trypsin was studied as an aggregating and release-inducing agent with gel-filtered platelets (GFP) and was compared with ADP, epinephrine and collagen. GFP aggregated irreversibly with final concentrations of 0.5–4 μg/ml trypsin, 1.6–3.2 μM ADP, 2.5–5 μM epinephrine and 40 μl/ml soluble collagen. Addition of human fibrinogen to the Tyrode-suspending buffer Was required for ADP and epinephrine, but was not necessary for trypsin or collagen. Release of (14C)5HT was obtained with trypsin and collagen using the same concentrations as used in aggregation. GFP stored at room temperature for 48 h were still responsive to trypsin and collagen, whereas aggregability and (14C)5HT release induced by ADP and epinephrine were already impaired 5 h after collection of blood. CP-CPK, an ADP-removing reagent, blocked aggregation and release induced by low trypsin concentrations, suggesting that ADP plays an intermediate role in the mechanism by which trypsin activates platelets. Trypsin appears to be a valuable reagent for studying platelet physiology, particularly following storage.
Uptake by Blood Components and by Subcellular Platelet Fractions of 1-14C-Acetylsalicylic Acid
Authors: Vinazzer H. , Zolle I. , Loew D.
Published: April 2009

1-14C-Acetylsalicylic acid was used to study the distribution and the kinetics of the acetyl group in blood components. Tests were carried out in 4 patients in vivo and on four blood samples in vitro. At various intervals after tagging, whole blood, plasma, platelets and platelet fractions were examined. In vitro, relative activity was found to be higher in platelets than in all other blood components. When platelet fractions were examined, the highest specific activity was found in membranes. In vivo, rapid disappearance of activity from circulation was observed. The decrease of activity was slower in platelets than in whole blood and in plasma. The highest specific activity was again found in platelet membranes. The half-life of the 14C tag was measured in all components. It was found to be longest in platelet membranes where it corresponded to the half-life of platelets in circulation. From the results obtained, the conclusion was drawn that irreversible acetylation of the platelet membrane must be considered to be one of the main causes of platelet dysfunction induced by acetylsalicylic acid.
Ultrastructural Morphometric Investigations on Normal Human Platelets
Authors: Stahl K. , Themann H. , Dame W.R.
Published: April 2009

Platelets from normal human volunteers were investigated by ultrastructural morphometry. Preparation of blood samples was done in two different ways in order to evaluate the possible influence of the preparative method on the size and shape of platelets and their organelles. No significant differences could be demonstrated for any of the measured parameters. Relative volumes of specific granules, mitochondria and the open canalicular system were calculated according to the method of Weibel. Surface and numerical densities of some compartments were determined. Direct measurements of the long and short axes of the platelets were conducted to ascertain further information about their size and shape. Absolute data are in good agreement with the few reports in the literature.
Inhibition of PGI2 (Prostacyclin) Synthesis in the Arterial Wall Enhances the Formation of White Platelet Thrombi in vivo
Authors: Bourgain R.H.
Published: April 2009

Prostaglandins play an important role in platelet-vessel wall interactions. Cyclic endoperoxides PGG2 and PGH2 are transformed to PGI2 (prostacyclin) within the arterial wall. The inhibition of PGI2 synthetase results in a marked enhancement of white platelet thrombus formations. This finding suggests that the synthesis of PGI2 from PGG2 and PGH2 accounts for the nonthrombogenicity of the endothelium.
Effect of Temperature on Short-Term Preservation of Platelets for in vitro Tests
Authors: Rosenstein Robert , Zacharski Leo R.
Published: April 2009

We have evaluated the effect on the response of citrated platelet-rich plasma to aggregating agents of storage at 4 °C versus room temperature (21 °C) for 2 and 4 h after venipuncture. While there were small decreases in some responses to epinephrine, ADP and collagen attributable to 21 °C storage, only in the case of ristocetin-induced aggregation was a profound difference noted. Platelets stored at 4 °C for 4 h showed no significant change in response to ristocetin. In contrast, those stored at room temperature showed a marked decrease. This change is not likely to be attributable to a change of pH, although pH rose less with storage at 4 °C than at 21 °C. It is recommended that PRP for in vitro testing be stored at 4 °C.
Urokinase Inactivation Rate in the Rabbit: Effect of Circulatory Isolation of the Liver, Spleen and Kidneys
Authors: Matsuo O. , Kosugi T. , Mihara H.
Published: April 2009

After injection of urokinase (UK) in normal rabbits and in rabbits with ligated liver, spleen and kidneys, the inactivation rate constant of UK was evaluated by using the disappearance rate of fibrinolytic activity from plasma. A peripheral intravenous injection of UK in normal rabbits induced significant but transient fibrinolytic activity, whereas intraportal injection of UK induced no fibrinolytic activity. However, in rabbits with ligated liver, spleen and kidneys, the fibrinolytic activity sustained for a long period, i.e. the inactivation rate constant of UK was 6 times smaller compared with normal rabbits. The implications of these results are discussed.
Platelet Aggregation and Antithrombin III Levels in Diabetic Children
Authors: Corbella E. , Miragliotta G. , Masperi R. , Villa S. , Bini A. , de Gaetano G. , Chiumello G.
Published: April 2009

We studied platelet function and antithrombrin III levels in 30 insulin-dependent diabetic children with no clinically evident vascular complications. 9 were in-patients and 21 were out-patients. The disease had been discovered within the previous 10 years. 25 control subjects of comparable age and body weight were studied simultaneously. Template bleeding time, threshold concentrations of ADP or adrenaline required to induce irreversible platelet aggregation and plasma antiheparin activity (platelet factor 4) did not differ significantly in control and patient groups. In contrast, the immunological levels of plasma antithrombin III were significantly higher in the diabetic group. These results suggest that diabetic children, with no clinical signs of microangiopathy, show no laboratory changes suggesting increased platelet function. The unexpected increase in the antithrombin III level could reflect a very early defense mechanism against activation of the blood clotting system.
The Thrombogenicity of 6-Azauridine
Authors: Gitel Sanford N. , Grieco Anthony J. , Wessler Stanford , Snyderman Selma E.
Published: April 2009

The antimetabolite 6-azauridine blocks the de novo synthesis of pyrimidines and causes increased serum levels of several amino acids including homocystine. 6-Azauridine was withdrawn from clinical use for the treatment of psoriasis because of the occurrence of arterial and venous thromboembolic episodes in some psoriatic patients. Utilizing a standard animal model for the recognition of venous and arterial thrombosis, 6-azauridine was demonstrated in this study to cause thrombosis without producing homocystinemia when administered orally or intravenously.
Correlation of Activation and Aggregation of Platelets. Discrimination between Anti-Activating and Anti-Aggregating Agents
Authors: Patscheke Heinrich
Published: April 2009

Shape change and release reaction indicate different degrees of the complex platelet response termed activation. Aggregation is a variable consequence of activation. Aggregation shows a temperature dependency opposite to that shown by the preceding shape change. Aggregation increases at lower temperature and requires, in contrast to activation, extracellular Ca2+, stirring, and at a low degree of activation the presence of fibrinogen. Aggregation can enhance activation by triggering prostaglandin endoperoxide-thromboxane synthesis. If activation reaches a high level associated with the release reaction, activation is further amplified by prostaglandin endoperoxide-thromboxane synthesis emerging independent of aggregation. These mechanisms of amplification of activation are blocked by indomethacin, an inhibitor of prostaglandin endoperoxide-thromboxane synthesis = anti-activating agent. In contrast, anti-aggregating agents, exemplified here with n-acetyl neuraminic acid, attack the aggregation of activated platelets but neither activation nor prostaglandin endoperoxide-thromboxane synthesis. Its anti-aggregating effect, in addition, enables n-acetyl neuraminic acid to imitate the inhibitory effect of indomethacin on the feedback amplification which results from aggregation. Anti-aggregating agents as characterized here may open a new valuable concept for anti-aggregation in vivo.
Influence of a Hypolipemic Drug (Procetofene) on Platelet Functions and Lipid Composition in Type II B Hyperlipoproteinemia
Authors: Renaud S. , Morazain R. , Sauvanet J.P. , Dumont E. , Drouin P.
Published: April 2009

Platelet function was evaluated in platelet-rich plasma and in washed platelets in relation to their lipid composition, in 18 type II B hyperlipoproteinemic men treated with procetofene. The results were compared to those obtained in 15 young, healthy normolipemic controls and in 22 untreated male subjects with type II B hyperlipoproteinemia. In addition to completely normalizing platelet functions, procetofene was able to induce a 28–53% decrease in serum cholesterol and triglycerides. Solely, the aggregation to ADP (secondary), arachidonic acid and epinephrine was higher in the untreated hyperlipoproteinemic subjects. This increased susceptibility to aggregation does not appear to be related to the cholesterol or cholesterol-phospholipid content of platelets.
Warfarin Treatment during Pregnancy: a Clinical Note
Authors: Russo Rosario , Bortolotti Uberto , Schivazappa Luciano , Girolami Antonio
Published: April 2009

The case history is presented of a woman aged 38 who received warfarin treatment because of a mitral valve replacement. During the first trimester of pregnancy, her prothrombin time was 20% of normal and kept at 40% until the 38th week of gestation. Then the patient received heparin until a few days after delivery of a normal child.
Interaction between Mural Thrombi and the Underlying Artery Wall
Authors: Woolf N.
Published: April 2009

This communication describes the morphological changes which result from the induction of experimental thrombi in the artery wall of the rabbit and pig. The role of the platelet as the initiator of neo-intimal proliferation is stressed and the genesis of the cells contributing to such proliferation discussed.
Platelet-Reactivity of Isolated Constituents of the Blood Vessel Wall
Authors: Barnes M.J. , Maclntyre D.E.
Published: April 2009

Collagens I and III, in fibrillar form, bound platelets equally well; both readily induced platelet aggregation. In contrast, collagens IV and V, although pretreated as collagens I and III to induce fibrillogenesis, failed to produce aggregation. No binding of platelets was detected. Lens capsule, containing collagen type IV in situ, was also inactive. Inactivity appears due to the lack of an appropriate quaternary structure since segment-long-spacing (SLS) aggregates of collagens IV and V, as of type I, induced aggregation. Elastin and its associated microfibrillar element did not aggregate platelets; some binding of platelets to elastin only was observed.
Platelet Interactions with the Endothelium and the Subendothelium: the Role of Thrombin and Prostacyclin
Authors: Cazenave Jean-Pierre , Dejana Elisabetta , Kinlough-Rathbone Raelene , Packham Marian A. , Mustard Fraser
Published: April 2009

The adherence of 51Cr-labeled platelets to rabbit aortae everted on probes rotated in platelet-red cell suspensions has been measured. Platelet adherence to the subendothelium exposed by passage of a balloon catheter before everting the aortae was inhibited by compounds that increase platelet cyclic AMP levels (PGE1, PGI2 or dipyridamole). These agents, however, did not abolish platelet adherence to the subendothelium. Aspirin treatment of the vessel wall was used to block PGI2 production; platelet adherence to the surface of the ‘undamaged’ aorta and the subendothelium was studied following this treatment. Since aspirin treatment of the ‘undamaged’ vessel wall did not cause platelets to adhere to it, it seems unlikely that PGI2 formation by the vessel wall is the mechanism that prevents platelet adherence to normal endothelium. In addition, PGI2 formation by the vessel wall does not appear to influence platelet adherence to the subendothelium, since adherence was not increased by aspirin treatment of the damaged wall. Thrombin treatment of the ‘undamaged’ vessel wall increased platelet adherence to the surface, but the adherent platelets were seen to be adherent only to small areas where the endothelium was lost or damaged. Heparin reversed the effect of thrombin. Similar results were found when the subendothelium was exposed to thrombin or thrombin and heparin.
Injury to Cultured Endothelial Cells: the Role of Lipoproteins and Thrombo-Active Agents
Authors: Evensen Stein A.
Published: April 2009

Thrombin and low density lipoproteins (LDL) are capable of inducing damage to human endothelial cells in primary culture. These substances both induce endothelial contraction as first recognizable morphological change. However, the effect of thrombin appeared early, was reversible and prevented by hirudin, while LDL acted over hours and the injury was progressive. Addition and high density lipoprotein (HDL), together with LDL, inhibited the cellular injury induced by LDL.
Prostacyclin, Thromboxane A2 Interactions in Haemostasis and Thrombosis
Authors: Moncada S. , Amezcua J.L.
Published: April 2009

Prostacyclin and thromboxane A2 are products of arachidonic acid which play a role in the regulation of haemostatic plug and thrombus formation. Aspirin inhibits the synthesis of both compounds but is more active in blocking TXA2 formation; based on this, aspirin is suggested to have an anti-thrombotic effect. Other possible approaches to the development of anti-thrombotic drugs are discussed.
Production of Prostacyclin by Vascular Endothelial Cells
Authors: Willems Ch. , van Aken W.G.
Published: April 2009

One of the earliest events in thrombus formation is the adherence of blood platelets to the exposed subendothelium. Under normal conditions no platelet adherence to the vascular endothelium occurs. To study a possible interaction between endothelial cells and blood platelets, endothelial cells were isolated from the umbilical cord vein. These isolated cells could be identified as endothelial cells by the presence of the so-called Weibel Palade bodies and the FVIII:AG. After incubation of monolayers of endothelial cells with blood platelets an unstable substance which inhibits platelet aggregation is released from the endothelial cell. This substance could be identified as prostacyclin according to the following criteria: (1) Its formation could be blocked by pretreatment of the endothelial monolayers with indomethacin or tranylcypromin. (2) After preincubation of endothelial cells with [14C]-endoperoxide, chemically detectable amounts of [14C]-6-keto PGF1α, the stable end product of prostacyclin could be detected. (3) In incubation supernatants the presence of 6-keto PGF1α could be shown by means of gas chromatography. Monolayers of endothelial cells only synthesize prostacyclin when the cells are stimulated for example by mechanical stress or after addition of substances like arachidonic acid or thrombin. It has been suggested that apart from the aforementioned endogenous production of prostacyclin the endothelial cell may also synthesize prostacyclin using endoperoxides provided by platelets. In these studies the production of prostacyclin was measured as the inhibition of platelet aggregation. Comparison of the amount produced in the presence of buffer alone, with the amount formed in the presence of platelets, led to the suggestion that endoperoxides liberated by platelets were used by endothelial cells to synthesize prostacyclin. If, however, the retainment of the biological activity in these two systems is compared, then it appears that in the presence of plasma or platelets, prostacyclin is stabilized to a
Modulation of Platelet Function by Prostaglandins: Characterization of Platelet Receptors for Stimulatory Prostaglandins and the Role of Arachidonate Metabolites in Platelet Degranulation Responses
Authors: MacIntyre Euan
Published: April 2009

The effects of PGG2 and PGH2 on platelets are mimicked by synthetic PG analogues in which the nature and specificity of the substituents on carbons 11 and 15 (or 16) are important determinants of reactivity. Arachidonic acid and stimulatory PGs induce secretion of platelet dense granule and alpha granule constituents, but not lysomal constituents, although arachidonate metabolism is necessary for collagen-induced release of lysosomal enzymes. N0164 acts on platelets as an endoperoxide antagonist: Trimethoquinol acts as an endoperoxide and TxA2 antagonist. PGs induce platelet aggregation by combining with a specific (endoperoxide) receptor.