bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human and Bovine Plasminogen-Free Thrombin, Purified by Means of Gel Filtration and Ion Exchange Chromatography
Authors: Berg W , Korsan-Bengtsen K , Ygge J 
Published: July 2018

SummaryA simple method for preparation of plasminogen-free human and bovine thrombin is described.Crude thrombin was prepared in the usual manner from oxalated plasma by means of adsorption on BaSO4, elution with trisodium citrate and activating the eluate from BaSO4 with tissue thromboplastin.This crude thrombin was purified by means of gel-filtration and chromatography on CM-Sephadex A-50.The gel-filtration was performed on three types of Sephadex, G-75, G-50, and G--25. By means of Sephadex G-75 the thrombin was well separated from the main part of inert protein and this type of Sephadex was used for the purification in large-scale. Separation of thrombin from protein of higher molecular weight was also obtained with Sephadex G-50 but not with Sephadex G-25 indicating a molecular weight of thrombin between 4000 and 10,000.The importance of using an elution buffer of sufficient high ionic strength for gel-filtration is shown. A great deal of the thrombin was adsorbed to the Sephadex if the gel-filtration was performed at a too low ionic strength.The final preparation contained 30,000 NIH units of thrombin per mg tyrosin and no detectable plasminogen.The commercial preparation “Topostasine” was also purified in the same manner, but the plasminogen content in “Topostasine” was high and could not be completely separated from thrombin.
Preparation of Human Plasminogen Suitable as Substrate for Quantitative Determination of Plasminogen Activators
Authors: Berg W , Korsan-Bengtsen K , Ygge J 
Published: July 2018

SummaryA simple method for preparation of plasminogen with low spontaneous activity and soluble at a neutral pH and at physiological ionic strength is described. Euglobulin made from fresh, oxalated, BaSO4-adsorbed, human plasma was first purified by means of gel filtration on Sephadex G-200. After gel filtration, further purification and concentration was done on DEAE-sephadex A-50. The activity was 100-130 casein units per mg tyrosine.
Über den Nachweis der Plasminogenverarmung in retrahierten Vollblutgerinnseln und über deren Bedeutung für die Lysierbarkeit mit Streptokinase
Authors: Gottlob R , Blümel G 
Published: July 2018

Summary1. Blood clots are lysed by SK before the beginning of retraction. Retracted clots are only very little lysed.2. Recalcified citrated blood is easily lysed since the excess of added calcium ions inhibits the retraction.3. The determination of the water content of the clots gives information on their retraction state.4. The determination of water content made it possible to prove that thrombus retraction takes place in vivo. Already one day after thrombosis the water content is clearly lower than in fresh clots. Later the water content increases again slightly.5. Clots formed in vitro and in vivo are lysed by SK after retraction if they come into contact with fibrin or fibrinogen containing plasminogen.6. Heated fibrin plates or heated fibrin tubes both with SK, casein degradation and an immunological method allowed to prove that whole blood clots loose their activable plasminogen during retraction. One assumes that plasminogen is expressed during retraction.7. The possibility of a therapeutical fibrinolysis with SK is due to the fact that SK can penetrate into the clot and the SK containing thrombus continuously comes in contact with circulating blood rich in plasminogen. The importance of these results for thrombolysis with SK is discussed.
Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists
Authors: Cook Nigel S , Zerwes Hans-Günter , Tapparelli Carlo , Powling Max , Singh Jagjit , Metternich Rainer , Hagenbach Alex 
Published: July 2018

SummaryPlatelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation.Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.These results highlight important species differences in the response of platelets to activation by two different agonists and also in their inhibition by GP-IIb/IIIa antagonists. In particular, platelets from the rat and hamster were insensitive to agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded with an affinity similar to human platelets. Since these studies were performed in whole blood, the results should be representative of those expected in animal experiments. These recently developed methods for studying platelet responses in small aliquots of whole blood are simple to perform and provide important information concerning the optimal choice of species for subsequent in vivo studies with these compounds.
A Mutation in the Protein S Pseudogene Is Linked to Protein S Deficiency in a Thrombophilic Family
Authors: Ploos van Amstel Hans K , Reitsma Pieter H , Hamulyák Karly , Die-Smulders Christine E M de , Mannucci Pier M , Bertina Rogier M 
Published: July 2018

SummaryProbands from 15 unrelated families with hereditary protein S deficiency type I, that is having a plasma total protein S concentration fifty percent of normal, were screened for abnormalities in their protein S genes by Southern analysis. Two probands were found to have a deviating DNA pattern with the restriction enzyme Mspl. In the two patients the alteration concerned the disappearance of a Mspl restriction site, CCGG, giving rise to an additional hybridizing Mspl fragment.Analysis of relatives of both probands showed that in one family the mutation does not co-segregate with the phenotype of reduced plasma protein S. In the family of the other proband, however, complete linkage between the mutated gene pattern and the reduced total protein S concentration was found: 12 heterozygous relatives showed the additional Mspl fragment but none of the investigated 26 normal members of the family. The mutation is shown to reside in the PSβ gene, the inactive protein S gene. The cause of type I protein S deficiency, a defect PSα gene has escaped detection by Southern analysis. No recombination has occurred between the PSα gene and the PSβ gene in 23 informative meioses. This suggests that the two protein S genes, located near the centromere of chromosome 3, are within 4 centiMorgan of each other.
α2-Adrenoreceptor Status of Human Platelet Subpopulations Separated by Continuous Flow Electrophoresis
Authors: Crook M , Crawford N 
Published: July 2018

SummaryHuman platelets were separated into subpopulations using continuous flow electrophoresis, which uses electrophoretic mobility as the separative parameter. The platelets with the greatest electrophoretic mobility showed higher amounts of total sialic acid than the less electrophoretically mobile subpopulation (74.3 ± 18.2 nmol/109 platelets vs. 49.2 ± 20.1 nmol/109 platelets, p <0.05 paired Student’s t-test). Furthermore, neuraminidaselabile sialic acid was also elevated in the more electrophoretically mobile platelet subpopulation (29.1 ± 6.0 nmol/109 platelets vs. 21.8 ± 10.4 nmol/109 platelets, p <0.05 paired Student’s t-test). We also found that the sialic acid enriched platelet subpopulation had more α2-adrenoreceptors than the less electrophoretically mobile platelets (457 ± 104 vs. 302 ± 164 receptors per cell, p <0.05 paired Student’s t-test).
Pathogenic Role Of Thrombus Formation In The Microcirculation Of Mucosa In The Gastrointestinal Defence Mechanism
Authors: Kondo M , Yoshikawa T , Furukawa Y , Takemura S , Fukumoto K 
Published: July 2018

There is no doubt that impaired blood supply to the gastrointestinal (GI) tract causes various pathological changes in the digestive mucosa by disrupting the defence mechanism. Ischemic change of the intestinal mucosa is classified into (1) occlusive mesenteric ischemia and (2) non-occlusive mesenteric ischemia, and intravascular clot formation has been investigated mainly in the former category.However, GI-bleeding of unknown origin is often experienced during the management of patients in disseminated intravascular coagulation (DIC), while to which neither the mechanism nor the specific treatment has been indicated. In the present study, the role of the microcirculation in the GI- defence mechanism is investigated from the standpoint of DIC.Single injection of E. coli endotoxin to dogs resulted in hemorrhagic necrosis of the GI mucosa within 8 hrs, and this was prominent in the small intestine where hemorrhagic change started at the top of villi. Microscopic examination showed tortuous thrombus formation in the microcirculation of the mucosa.When the jejunal mucosa was treated directly by protease inhibitors such as tranexamic acid or aprotinin prior to endotoxin, hemorrhagic necrosis of the area was successfully prevented, suggesting that proteolytic enzymes either in the GI-cavity or in the tissue digested the ischemic mucosa.Systemic pretreatment of rats with heparin was effective in preventing the hemorrhagic necrosis, strongly indicated that blood supply to the microcirculation of the GI-mucosa played a role in the GI-defence mechanism.It is concluded that in the managing patients with DIC, one should be careful to protect GI-bleeding which often appear in the form of hemorrhagic necrosis. Besides the use of systemic anticoagulants, direct contact of protease inhibitors with GI-mucosa might be of help in the protection or the treatment of GI-bleeding in DIC.
Effect Of Neuraminidase On Plasmin Formation By Urokinase And The Vascular Plasminogen Activator
Authors: Geiger M , Binder B R 
Published: July 2018

It was the aim of the present study to determine whether sialic acid residues in the activator or substrate molecule play a role in the activation of plaminogen to plasmin by the vascular plasminogen activator (VPA) and urokinase (UK).Using a purified test system with UK (Mr=31.000, human urine), VPA (Mr=70.000, cadaver vessel eluates) as activators, Glu-plasminogen as substrate, and pretreatment with neuraminidase (C.perfringens,1.51U/mg) it could be shown by quantification of the formed plasmin with S-2251 as substrate that neuraminidase treatment enhances plasmin formation by UK as well as by VPA. Using Sepharose-bound neuraminidase (C.perfringens, 20-30U/g Sepharose) it could be shown that neuraminidase affects the substrate in a dose and time dependent fashion, while the activity of the activators seems to remain unchanged. Treatment of plasminogen with neuraminidase in a concentration of 2.5 mU per μmole plasminogen for 90 min. at 37°C increased the plasmin formation by VPA by about 50% which was the same as for UK.Kinetic analysis of plasmin formation from neuraminidase treated plasminogen by UK and VPA suggests increased binding of the substrate to the activators (unchanged Vmax and decreased KM). The enhancing effect of fibrin on plasmin formation by VPA seems to remain unaltered by neuraminidase treatment of the plasminogen.These data suggest that, as in the case of other protease substrates, removal of sialic acid residues results in increased cleavage of the substrate, i.e. enhanced plasminogen activation, regardless of the type of activator.
Plasminogen Activator (PA) Activity Of Urokinase (UK) , The Vascular Plasminogen Activator (VPA) , Plasma Kallikrein (PK) , And Hageman Factor (HF) In The Presence And Absence Of Fibrin
Authors: Binder B R , Beckmann R , Jörg M 
Published: July 2018

It could be shown previously by us and others that the PA activity of purified VPA is increased in the presence of fibrin, while the activity of UK remains unchanged. However, there had been some discussion on the mechanisms involved and the relative potency of the different PAs in man. In the present study kinetic analysis of plasminogen activation with different PAs was performed in the presence and absence of fibrin.Human UK (urine, Mr=56.000) , VPA (cadaver vessel eluates) , PK (HF-fragments activated) were purified to apparent homogeneity, and active sites were determined by 3H-DFP incorporation. Plasminogen activation was evaluated by incubation of different amounts of PAs with Glu-plasminogen at different substrate concentrations with and without fibrin (fibrinogen- Sepharose treated with thrombin) at 37°C for 3 to 120 minutes; the plasmin generated was measured by a synthetic para- nitroanilide substrate.Under these conditions VPA exhibited an apparent KM≃0.5 μM and a kcat≃0.15 s-1. In the presence of fibrin KM decreased significantely (<<0.01 μM) while kcat remained unchanged; untreated fibrinogen-Sepharose had no effect. UK, on the other hand, showed an apparent KM≃1 μM and a kcat≃0.5 s-1 which were both unaffected by fibrin. PK cleaved plasminogen with an apparent KM≃0.2 μM and a kcat≃0.004 s-1 regardless of the presence of fibrin. Preliminary data using intact HF together with kaolin and phospholipids revealed an apparent KM for plasminogen activation of that mixture of 3.5 μM and a kcat≃0.07 s-1 which were again unaffected by fibrin.From these data it can be concluded that in the absence of fibrin UK is a more effective PA than the others, while in the presence of fibrin VPA is by fare the most effective one. From kinetic analysis PK and HF should play only a minor role in plasminogen activation.
On The Fibrinolytic Properties Of Single-Chain And Two- Chain Human Tissue Plasminogen Activator
Authors: Rijken D C , Hoylaerts M , Collen D 
Published: July 2018

Tissue plasminogen activator from pig hearts may be isolated as a single-chain or as a two-chain molecule (Wallén et al., 1980). The present report deals with the two molecular forms of a plasminogen activator (tissue plasminogen activator-like) secreted by human melanoma cells in culture. A single-chain form was prepared by addition of aprotinin to the culture media and during the purification procedure while a two-chain form or a mixture of both was obtained in the absence of aprotinin.The fibrinolytic activities of the two forms were comparable on fibrin plates and in a clot lysis time system. Analysis of the molecular structure of 125I-labeled plasminogen activator by dodecyl-sulfate-gel electrophoresis, revealed that the single-chain form was converted into a two- chain form during the lysis of fibrin. The plasminogen activating properties of the two molecular forms were therefore measured in the presence of aprotinin, which prevents the conversion. Aprotinin (1,000 KIU/ml) was incorporated in a fibrin clot (1 mg/ml) containing plasminogen activator (20 ng/ml), 125I-labeled Glu-plasminogen and varying amounts of unlabeled Glu-plasminogen. The amount of plasmin formed after 30 min was quantitated by measuring the radioactivity migrating in the position of the plasmin B-chain on dodecyl-sulfate-gel electrophoresis under reducing conditions. The rate of plasmin formation obeyed Michaelis-Menten kinetics with Km = 2.5 μM and kcat = 0.6 s-1 for the single-chain form and Km = 1.1 μM and kcat = 0.3 s-1 for the two-chain form.Although the conversion of the single-chain tissue plasminogen activator into a two-chain form during fibrinolysis might have a regulatory function, these kinetic parameters of the plasminogen activation do not support this hypothesis.
Binding To Phospholipid Protects Factor VIII From Inhibition By Human Antibodies
Authors: Barrowcliffe T W , Gray E , Kemball-Cook G 
Published: July 2018

Previous studies with activated Factor IX concentrates have suggested that they may contain a form of Factor VIII clotting activity (VIII:C) which is partly protected from inactivation by antibodies. A possible mechanism for such protection is binding to phospholipid. The interaction between Factor VIII, phospholipid and human antibodies to Factor VIII was studied by a two-stage clotting assay, and by a fluid-phase immunoradiometric assay for Factor VIII clotting antigen (VIII C:Ag).In the two-stage thrombin generation assay, Factor VIII:C was rapidly destroyed by human antibodies, even in the presence of optimal phospholipid. However, preincubation of Factor VIII with phospholipid before addition of antibody protected the Factor VIII from inactivation, resulting in the production of much more thrombin.In assays of VIII C:Ag, pre-incubation of Factor VIII with phospholipid before addition of labelled antibody reduced the amount of detectable antigen. The reduction was greater with increasing phospholipid concentration, up to 60% of the original antigen being ‘lost’ at a total phospholipid concentration of around 250 μg/i.u.These results suggest that human antibodies to Factor VIII are directed largely at its phospholipid binding site. The protection of Factor VIII from inactivation by complexing with phospholipid could have important clinical applications in treatment of haemophiliacs with inhibitors.
A New Clinical Preparation Of High-Purity Porcine Factor VIII:C Concentrate
Authors: Costello W P , Middleton S M , Malia R G 
Published: July 2018

A polyelectrolyte-fractionated porcine Factor VIII concentrate (Hyate:C, Speywood Laboratories, average specific activity: 10 U Factor VIII:C/mg, platelet aggregating factor (PAF): 0.25 U/ml) has been recently available. Clinical use of this product for the treatment of patients with antibody to Factor VIII (13 haemophilic and 1 non- haemophilic patients for a total of 26 courses of treatment) has shown that: 1/ multiple courses of therapy can be given with both good clinical tolerance and good clinical efficacy, 2/ with one exception, no patient developed thrombo- cytopaenia and 3/ with the exception of one case, in which the patient has been treated with human Factor VIII prior to the infusion of porcine Factor VIII. the expected anamnestic response did not occur. These clinical findings prompted us to further purify the porcine Factor VIII concentrate for clinical use.Cryoprecipitate, prepared from frozen porcine plasma, is dissolved in low ionic strength Tris buffer at pH 6.8 and adsorbed with aluminium hydroxide gel. The ionic strength is then adjusted to 0.3 M NaCl before loading on a column of charged and equilibrated polyelectrolyte E-5. After washing the column with 4 bed volumes of buffer, the proteins were eluted with 1 M NaCl at pH 6.5 and precipitated by 20 % polyethylene glycol 4000. The redissolved precipitate is then chromatographed on a preparative column of Sepharose CL-4B. The void volume is sterile-filtered and lyophilised. The new preparation has a specific activity of approximately 100 U Factor VIII:C/mg with a PAF level of 3.3 U/ml. This represents an improvement of 10 fold in specific activity over the existing concentrate.It is expected that the new concentrate will also show low immunogenicity which can only be revealed by clinical results. A clinical trial is envisaged within the next two months.
Postoperative Platelet Accumulation In The Lungs: Can It Be Measured In Surgical Patients And Does It Matter?
Authors: McCollum C N , Hawker R J , Norcott H C , Hail C , Drolc Z , Miller M R 
Published: July 2018

Pulmonary microembolisation as a result of trauma, sepsis or major surgery may be an aetiology for shock lung. The postoperative kinetics of 111-Indium labeled autologous platelets have been studied to determine whether they accumulate in the lungs, and whether this relates to subsequent pulmonary function.One day prior to major surgery platelets from 10 patients were labelled with 111-Indium-oxine and reinjected. Isotope emissions were counted over the lungs and aortic arch preoperatively, 2.5 hours following surgery and daily for 7 days. Screen filtration pressure (SFP) measurements of aggregates in femoral vein blood was estimated 30 minutes and 3 hours postoperation. Arterial PO2 was measured preoperation and on days 1 and 7 following surgery.The ratio of isotope emissions lung/aortic arch increased following surgery indicating pulmonary accumulation of platelets. The preoperative ratio (mean ± SEM) of 0.38 ± 0.017 rose to 0.47 ± 0.039 (p<0.05) 2.5 hours following surgery and to 0.56 ± 0.052 (p<0.01) the following day. This ratio had returned to preoperative levels by day 4. Immediate postoperative platelet accumulation in the lung (the rise in lung/ aortic arch ratio) correlated closely with SFP measurements (r 0.89, p<0.01) and the fall in arterial PO2 one week following surgery (r 0.69, p<0.05).These results indicate that circulating platelets aggregate during surgical shock and then accumulate in the lungs. This process is associated with postoperative hypoxia.
Pulmonary Embolism: Trends In Edinburgh Surgical Units Over Twenty Years
Authors: Ruckley C V 
Published: July 2018

The annual statistical records (audit) of ten general surgical units for the years 1959, 1964, 1969, 1974 and 1979 have been analysed to find the trends in incidence of pulmonary embolism in patients who died - with particular reference to patients normally expected to have a good prognosis.The operations for the 5 years totalled 59,880. There were 2,593 deaths and 1,181 autopsies (46%). The total annual operations rose from 9,757 in 1959 to 13,830 in 1979 while the death rate fell from 6% to 4%.Pulmonary embolism proven at autopsy (150) plus clinical diagnosis when autopsy was not performed (41) was reported in 191. There was a fall in incidence from 0.4% of operations in 1959 to 0.2% in 1979; but the validity of this was obscured by a fall in autopsy rate from 59% to 34% over the same period. However in patients below the age of 80 who should have had a good prognosis (i.e. did not have metastatic malignancy or end-stage cardiorespiratory disease) the autopsy rate remained relatively constant at a mean of 76% throughout the period. In this important group the incidence of pulmonary embolism fell progressively from 0.27% in 1959 to 0.06% in 1979.In general surgical patients who would normally be expected to have a good prognosis the risk of fatal pulmonary embolism has steadily diminished over the past twenty years, although prophylactic methods have only been in general use for the past 5 years.