bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization Of A Crosslink-Containing Fragment Derived From The A Polymer Of Human Fibrin And Its Application In Immunologic Studies Using Monoclonal
Antibodies
Authors: Sobel J H , Birken S , Ehrlich P , Friedman R , Moustafa Z , Canfield R E 
Published: July 2018

A high molecular weight crosslink-containing fragment derived from cyanogen bromide (CNBr) digests of the a polymer component of human fibrin has been isolated and characterized. The material has been used to generate monoclonal antibodies toward the goals of (1) producing fibrin-specific probes for use in the early detection of thrombosis and (2) generating monoclonal lines to single determinants in the COOH-terminal region of the Aα chain for use in structural studies of fibrinogen and fibrin.Biochemical and immunologic characterization data indicate the fragment is comprised, predominantly, of equimolar quantities of the CNBr peptides spanning residues #241-476 (CNBr 8) and #518-584 (CNBr 10) in the original Aα chain. The acceptor and donor units are crosslinked via an average of 2.8-3.2 ε-(γ-glutamyl) lysine bonds per mole of CNBr 8 + CNBr 10 producing heterogeneously sized fragments in the range of 80,000-200,000 daltons.Two types of monoclonal lines have been obtained. The first react with regions of primary structure and in one instance immunoreactivity could be localized to the Aa tryptic peptide #253-268. The second type appear to recognize conformational determinants as shown by one antibody that reacts well with the CNBr crosslinked fragment but poorly with its constituent peptides.
Activation of Bovine Factor IX By Bovine Factor XIa . Active Site Titration Of Factor IXa and Development of A spectro-Photometric assay for Factor IX
Authors: Tans G , Janssen-Claessen T , Dieijen G v , Rosing J , Hemker H C 
Published: July 2018

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate.Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.
Prostacyclin Prevents Endothelial & Platelet Damage During Haemodialysis
Authors: Turney J H , Dodd N , Weston M J 
Published: July 2018

We have previously demonstrated that prostacyclin (PGI) enhances the biocompatibility of extracorporeal circuits. Dialysis with & without PGI were studied in 17 patients. We measured platelet count, (β-thromboglobulin, factor VIII related antigen, & Antithrombin III (Bick method). Results are presented as percentage change + SEM of initial values.The change in all values during dialysis with heparin alone was significant (p<0.005). Additional PGI prevented any change (p<0.0001 compared with heparin at 300 minutes). We conclude that platelet activation & consumption persists throughout dialysis with heparin alone. The rise in factor VIII-RA & AT III reflects vascular endothelial damage induced by the reinfusion of activated blood components. Thus PGI not only protects platelets but also prevents dialysis-induced vascular endothelial damage. Longterm use of PGI should tend to reverse the prothrombotic state in dialysed uraemic patients and may therefore reduce their risk of atherosclerotic cardiovascular disease.
Low Affinity Platelet Factor 4/β-Thromboglobulin (LA-PF4/βTG) Antigen And Circulating Platelet Aggregates During Prostacyclin (PGI2) Therapy
Authors: Musiat J , Szczeklik A , Nizankowski R 
Published: July 2018

We have found that continuous intraarterial infusion of PGI2 alleviates rest pain and promotes healing of ischemic ulcers in patients with advanced arteriosclerosis obliterans. Since these effects could be related to the anti-platelet actions of prostacyclin, we studied serum levels of the platelet specific protein, a possible marker of platelet activation in vivo, named LA-PF4/βTG, and compared it with those of serum circulating platelet aggregates. The former was assessed by radioimmunoassay, the latter by method of Wu and Hoak. Determinations were carried out in 16 patients with arteriosclerosis obliterans, initially and during the second day of intraarterial infusion of PGI2 in a mean dose of 6 n9/kg/min. This therapy led to an almost uniform fall in circulating platelet aggregates. In contrast, levels of LA-PF4/β-TG antigen at the beginning of second day or the infusion /mean = 19.19 ng/ml, ± S.D. a 8.97/ did not differ significantly from the initial values /mean = 23.59 ng/ml, ± S.D. = 13.12/.In conclusion: 1/ In our patients vascular disease did not produce any apparent increase in serum LA-PF4/β-TG antigen levels; 2/ Infusions of PGI2 did not affect basal levels of this platelet specific protein, while it markedly depressed the percent of circulating platelet aggregates.
The Plasmin Inhibitor From Human Platelets Inhibits The Binding Of Modified Plasminogen To Fibrin
Authors: Sandbjerg Hansen M 
Published: July 2018

Modified plasminogen (Lys-plg) is known to bind to fibrin and this binding is competitively inhibited by omega-aminocarboxylic acids. An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human platelets. This material contained none of the known plasma proteinase-inhibitors when studied by crossed immunoelectrophoresis and electroimmuno assay.Human fibrinogen was clotted by thrombin in the presence of radiolabelled Lys-plg and the fraction of radioactivity associated with the washed fibrin clot was determined. The presence of the inhibitor preparation in the clotting mixture caused a significant decrease in the fraction of radiolabelled Lys-plg adsorbed onto fibrin. The fibrin-adsorbed fraction decreased as a function of an increasing inhibitor concentration. The incorporation of purified cold insoluble globulin (fibro- nectin) into the fibrin clot did not influence the adsorption of Lys-plg neither in the presence nor the absence of the inhibitor. The presence of calciumions or albumin in various concentrations did not influence the reactions. The addition of 6-aminohexanoic acid to the clotting mixture strongly enhanced the effect of the inhibitor preparation. It has previously been demonstrated that the inhibitor binds to immobilized plasminogen. These results may indicate that lysine-binding sites are important for the inhibitor-enzyme interaction.
A Kinetic Approach To The Definition Of Pathophysiology Of Low Antithrombin III (AT) Levels In Patients
Authors: Reeve E B , Leonard B D , Bies R D , Ambruso D R , Hathaway W E 
Published: July 2018

Patients with 50 percent of normal levels of AT are at serious risk of deep venous thrombosis. Possible causes of 50 percent levels are (1) a halving of synthetic rate, or (2) a doubling of fractional catabolic rate. In health fractional catabolic rate is 50 percent of the plasma AT per day. Doubling of fractional catabolic rate-might be due to (a) the presence of a structurally defective AT molecule or (b) might be directly related to a great excess of AT consumption by complex formation with released clotting proteases. These possible causes of low AT levels can be distinguished by turnover studies with 131I-iabelled AT from the patient and from a healthy donor.We have developed simple, rapid methods of preparing highly purified AT from 30 to 60 ml of citrated blood and of labelling it with 131I without denaturation at about 0.5 iodine atom per AT molecule. A turnover study requires intravenous injection of 4-6 µC of 131I.Studies in a female patient with hereditary AT deficiency (40-50 percent AT level) compared to normal subjects indicates our approach. Fractional breakdown rate of her own 131l-AT was 50 percent per day indicating no obvious structural abnormality. Fractional breakdown rate of 131I_labelled donor AT was also 50 percent per day further indicating no difference in the behavior of her own and of normal AT and no excess consumption by clotting proteases. The patient's ability to compensate for increased AT-III consumption during an episode of thrombosis was impaired. Thus the patient suffered from a synthetic defect resulting in the ability to synthesize only 50 percent of her AT requirements per day.AT turnover studies provide an approach to the definition of the etiology of depressed AT levels in various disease states.
Comparison Of Mean Platelet Survival Time In A Non-Radioisotope Method Calculatend With New Model With Mean Survival Time In CR-51 Methods
Authors: Tsukada T , Tango T 
Published: July 2018

Platelet survival time using non-radioisotope method measuring malondialdehyde(MDA) generation stimulated by arachidonate prior to and after intake of aspirin(ASA) and Cr-51 method was measured simultaneously in 18 cases with ITP. In cases with normal platelet survival time MDA generation curve showed linear or the letter-S pattern. On the other hand, all cases with shorter survival time had MDA generation curve with letter-S pattern. To describe the S pattern of MDA generation curve following model was devised on the assumption : 1) ASA inhibits MDA generation in megakaryocytes so that newly produced platelets show impaired MDA generation, 2) ability of MDA generation in platelets exposed to ASA recovers during circulating.where a is the disappearance time of inhibiting effect of ASA on megakaryocyte MDA generation and b is the time necessary for recovery of MDA generation in platelets exposed to ASA. Mean survival time(MST) is calculated by l/λ.The difference of MST between non-radioisotope method and Cr-51 method( gamma model) in 11 cases with MST of less than 4 days in Cr-51 method were 2.9 ± 0.4 days in platelet survival time (PST) at which MDA generation attained to pre-ASA levels, 2.7 ± 0.4 days in MST calculated with linear regression of MDA generation curve and 1.8 ± 0.4(SEM) days in MST calculated by the above model. On the other hand, in cases with MST of more than 4 days in Cr-51 method the difference of MST in both methods were 1.4 ± 0.3(SEM) days in PST, 0.6 ± 0.2 days in linear regression and 0.6 ± 0.2 days when MST was calculated with the above formula.These results suggest that the model reported is so far the better fitting model for the MDA generation curve in cases with normal and shortened platelet survival.
Release Of Slow-Reacting Substance (SRS) From Rabbit Platelets
Authors: Mencia-Huerta J M , Hadji L , Benveniste J 
Published: July 2018

In the guinea-pig, platelet aggregation by several agonists results in a bronchoconstriction. Thus we speculated that SRS could be the mediator for this phenomenon, eventhough it has not been yet described as originating from glatelets.Washed rabbit platelets (2.5 × 108) stimulated by plateletactivating factor (PAF-acether) (0.72 nM), thrombin (0.15 unit/ml) or arachidonic acid (AA) (10 uM) released 2.7 ± 0.9, 2.5 ± 0.2 and 1.4 ± 0.8 units of SRS, respectively (5 exp., 1 unit = guinea-pig ileum contraction induced by 5 ng histamine). By contrast, ADP (10-100 uM) aggregated platelets but did not induce the release of SRS. Preincubation of platelets with aspirin (0.1 mM : 10 min), or the presence of indomethacin (5 uM) in the incubating media, did not modify the yield of SRS, thus excluding prostaglandins as the contracting substance. SRS release was not correlated with the aggregation since aspirin-treated platelets failed to aggregate in the presence of AA but still released SRS, whereas untreated platelets ADP-stimulated, did aggregate without releasing SRS.L-cysteine (20 mM) increased 10 fold the yield of SRS and eicosatetraynoic acid (ETYA) (10-100 uM) caused a dose-dependent inhibition of SRS release.Platelet SRS was resistant to base but not to acid hydrolysis and its action on guinea-pig ileum was reversed by FPL 55712 (1 ug/ml). It was further purified on reverse phase high pressure liquid chromatography (RP-HPLC) using a discontinuous gradient of methanol in water. Platelet-SRS was eluted as 3 peaks of activity (retention times : 39 min, 46 min, 54 min) as did SRS from ionophore-stimulated rat peritoneal cells. Synthetic leukotrienes C and D exhibited retention times of 46 min and 54. min respectively. SRS obtained from platelets preincubated with 14C- AA was also analyzed on RP-HPLC. Only one peak of activity (54 min) coincided with radioactivity suggesting the incorporation of AA in this SRS molecule.These data bring evidence for the release from stimulated rabbit platelets of a SRS closely related to leukotrienes.
Kinetics Of Antithrombin III During Severe Consumption Coagulopathy In An Infant Measured Without Radioactive Tracer Proteins
Authors: Schmidt B , Wais U , Witt I , Pringsheim W , Künz W 
Published: July 2018

The precise diagnosis of disseminated intravascular coagulation remains difficult to achieve: Decreased production of coagulation factors and/or thrombocytes can mimic the laboratory pattern of disseminated intravascular coagulation. The measurement of increased turnover rates for coagulation proteins would provide more convincing criteria.During the course of severe coagulopathy in an infant suffering from septicaemia and shock, antithrombin levels were determined repeatedly before and during treatment with Antithrombin concentrate: Activities and concentrations were measured, using chromogenic substrates and immunodiffusion plates, respectively. By mathematical analysis of these data, using a biexponential function, the plasma elimination half-life of the antithrombin III was estimated to be 7.5 to 10.5 hours. Compared with known plasma half-lives of radioactively labelled antithrombin III in adults, the increase was five- to ten-fold. This indicates an accelerated consumption of antithrombin III in this case of severe coagulopathy.
Quantitative Endotoxin Determination In Blood With A Chromogenic Substrate
Authors: Thomas L L M , Sturk A , Schaap M C , ten Cate H , ten Cate J W 
Published: July 2018

A method, its optimalization and a solution to specific problems is described for the quantitative determination of endotoxins in blood. The method is based upon the endotoxin-dependent activation of a proenzyme, present in limulus ame- bocyte lysate. This activated enzyme is measured with the chromogenic substrate S2222. Possible inhibitors and activated coagulationfactors interfering in the assay are removed by dilution and boiling. The method is shown to be fast (2½ hr), sensitive and reproducible with a detection limit of 10 pg/ml.The endotoxin assay was found to have a good correlation with results of blood cultures.The detection threshold of the endotoxin assay compared with the number of circulating gramnegative organisms and CRD-measurements will also be shown.
Simple Chromogenic Peptide Substrate Assays For Determining Components Of The Plasma Kallikrein System
Authors: Gallimore M J , Friberger P 
Published: July 2018

Over the past few years the introduction of chromogenic substrates has facilitated studies on components of the plasma defence systems. During this period it has become apparent that there is a need for simple, easy to perform, assays for measuring components of these systems. We have therefore developed simple assays for determining prekallikrein (PKK), kallikrein inhibition (KKI) and kailikrein-1ike activity (KK). These assays have a plasma dilution step, followed by repeat single volume pipettings of reagents. Both “rate” and “end-point” assays have been developed. For the PKK assay a new activator has been developed which contains Hageman factor and high mol. wt. kininogen. This overcomes the problems found by several groups with other activators when studying patients with low plasma levels of Hageman factor, PKK and high mol. wt. kininogen. For the KKI assay a stable preparation of human plasma KK has been produced. This assay detects mainly Cl'- esterase inhibitor. The assay for KK-like activity is required where activation of PKK has occured and spontaneous activity towards the substrate is found. Plasma samples from 25 normal donors were tested for PKK, KKI and KK. The PKK and KKI values were calculated from standard curves made with a standard plasma. When compared with PKK and C1INH antigen levels correlations of 0.84 and 0.85 were obtained. All of the plasmas had KK activities lower than 5% of the total PKK levels.These assays should prove valuable in the rapid evaluation of the plasma KK system in patients.
Comparison Of The Thrombogenic Potential Of Two Membranes During Haemodialysis
Authors: Rajah S M , Crow M J , Donaldson D R , Sreeharan N , Davison A M 
Published: July 2018

The thrombogenicity of two dialysis membranes was compared by observing the changes in platelet function, heparin sparing effect and scanning electron microscopy (SEM) findings following haemodialysis. Ten patients undergoing regular dialysis treatment using a Meltec Multipoint Kiil board dialyser of 1.5 sq.m, surface area, have been investigated. The two membranes studied were Cuprophane (C) and Polycarbonate (P). All patients entering the study had not received any antiplatelet therapy in the 14 day period prior and the heparin schedule remained unchanged for both membranes. The thrombogenic potential was assessed by chaig- es in platelet count, platelet adhesion, platelet aggregation with collagen, plasma B-Thromboglobulin (B-Tg) during dialysis, changes in heparin concentration post-dialysis and detailed examination of the membranes by SEM at the end of dialysis.There was a significant fall in platelet count (p< 0.001) and platelet aggregation with collagen (p<0.05) with the C membrane, whereas the comparable values for the P membrane were not significant. Plasma B-Tg was very significantly raised with the C membrane (p<p.001) but to a lesser degree with the P membrane (p<0.05). Plasma heparin concentrations were significantly higher at the end of dialysis (p<0.05) with the P membrane compared to the C membrane, indicating a heparin sparing effect. SEM demonstrated consistently less platelet aggregates and fibrin deposition with the P membrane.The results of this study indicate that the polycarbonate membrane has less thrombogenic potential (hence greater overall efficiency) than the Cuprophane membrane.
Need For Large (But Simple) Trials
Authors: Peto R , Yusuf S 
Published: July 2018

Every year at least a million people are admitted to hospital with a diagnosis of suspected acute MI. Within a year about 15% are dead, some in hospital and seme after discharge. Reduction of 15% to (say) 12% would save same 30,000 lives per year, perhaps 10,000 of whom would be people in middle, rather than old, age. Such a reduction might well be conferred by certain of the treatments currently envisaged (e.g. limitation of infarct size by intravenous beta-blockade or by fibrinolytic treatment, or maintenance for several months following discharge on antiplatelet, anti-coagulant, beta-blocking or various other drugs), though it is much less plausible that large risk reductions (e.g. from 15% dead to 5% or 10% dead) could be conferred. Most trials are far too small to detect a reduction from 15% dead to 12% or 13% dead; for example, the mean size of the 36 randomised trials of beta-blockers or of fibrinolytic agents in the acute phase of (suspected) MI is 250 patients per trial. Such trials cannot reliably assess the sort of moderate reductions in mortality which it is medically realistic to hope for. This ideally requires not 250 per trial but more like 10,000 per trial. Some treatments (e.g. fibrinolytic agents, acute-phase i.v. beta-blockers, and post-discharge anti-platelet agents) which have failed to gain wide acceptance do seem to prevent death when all of the relevant trials are viewed together.
If such an overview of many trials is not accepted as sufficient evidence, the need for a few really large trials will be underlined. There is no need for expensive investigation of each patient in, large trials whose chief aim is to study mortality, and there may be more prospect of success in such trials if they are made unusually simple.The point is not that all trials should be replaced by large, simple trials, but rather that unless a few large, simple trials are undertaken we shall not know which treatments prevent death.
The Use Of Peptide Fluorogenic Substrates In Cytochemistry
Authors: Smith R E 
Published: July 2018

Cytochemical demonstration of proteases by fluorescent techniques is only a recent achievement. Localization of cellular proteases through the use of synthetic peptide substrates (as opposed to use of antigen-antibody in immuno-cytochemistry) depends upon the enzyme-substrate complex theory which assumes combination of enzyme with substrate in phase I and liberation of the enzyme and products in phase II. Determination of intracellular protease activity depends on liberation of a fluorophore attached to the peptide by an arylamide or ester bond. To localize the site of enzymatic hydrolysis of the substrate, the product must remain immobilized. Overcoming diffusion of reaction product is not a new problem in chromometric detection of enzymes, but it is enhanced with fluorometric detecting systems. Fluorescence microscopy allows for more precision in localizing reaction product, therefore diffusion is more noticeable. In flow cytometry, monodispersed cells are in a liquid transport media; thus diffusion of reaction product from a cell is unacceptable for it may be entrapped by another cell.Although peptide derivatives of naphthylamines have been used in cytochemistry as chromogenic substrates for over 20 years, their fluorogenic potential was not useful until development of the concept of wavelength shifting. Naphthylamines fluoresce at short UV wavelengths, so the reaction product is difficult to quantitate above background interference. Furthermore, the product is soluble and easily washed out of cells or tissue. Coupling of 4-methoxy-2-naphthylamine to 5-nitrosalicylaldehyde provides a method of reaction product immobilization as well as extending the emission wavelength into the yellow-orange region of the spectrum. Nevertheless the technique has certain limitations, unavoidable in a two-step reaction product capture method. Recently, we synthesized peptide derivatives of several types of coumarins; some are proving to be useful fluorogens in the cellular localization of proteases.