bluuebunny/crossref_metadata_2025_split huggingface dataset:
Binding of Various Thrombin Fractions to Fibrin and the Influence of AT-III on Their Adsorption
Authors: Nilsen D W T , Brosstad F , Kierulf P , Godal H C 
Published: July 2018

SummaryHuman thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent a-thrombin, 28 per cent β-thrombin and only 2 per cent γ-thrombin. Although a-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved.High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin.o avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/1. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration.Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/1. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin.Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/1). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.In its monomeric form fibrin-adsorbed thrombin was inactivated by AT-III and could then be extruded from the clot upon syneresis and retraction, but a small fraction remained attached to polymerized fibrin, exhibiting minor amidolytic activity.
Increased Plasma Noradrenaline During Severe Sodium Restriction Does Not Stimulate Platelet Release in Essential Hypertension
Authors: Kjeldsen S E , Lande K , Gjesdal K , Jackson M B , Westheim A , Frederichsen P , Leren P , Eide I 
Published: July 2018

SummarySeventeen 50-year old hypertensive men, previously untreated with blood pressure 157 ± 4/110 ± 2 mmHg (means ± SE) were given a low sodium diet for 2 weeks. During the second week, the diet was supplemented with potassium. The urinary Na+/K+ excretion ratio changed from 2:1 to 1:4 and 1:11, respectively.Sympathetic noradrenergic tone increased considerably during the first week. Thus, venous plasma noradrenaline increased from 254 ± 22 to 347 ± 28 pg/ml (p <0.001) and arterial concentration from 253 ± 36 to 317 ± 42 pg/ml (n = 10, p <0.001). No significant change was observed in sympathetic adrenal tone as reflected by normal plasma adrenaline in venous (42 ± 5 vs 43 ± 6 pg/ml, ns) or arterial blood (71 ± 10 vs 82 ± 15 pg/ml, n = 10, ns) or in venous plasma concentration of the blood platelet release product beta-thromboglobulin (BTG) (50 ± 8 vs 43 ± 5 ng/ml, ns). During the second week sympathetic noradrenergic tone remained highly significantly elevated compared to baseline but still no change in plasma adrenaline or plasma BTG was found.Thus, whereas sodium depletion did increase plasma noradrenaline concentration markedly in these hypertensive men, no change in adrenaline concentration was observed, and blood platelet release reaction was unchanged. Plasma noradrenaline within the physiological concentration range does not seem to serve as a regulator of in vivo platelet function.
An Enzyme-Linked Immunosorbent Assay (ELISA) for the Measurement of Plasmin-α2-Antiplasmin Complex in Human Plasma - Application to the Detection of In Vivo Activation of the Fibrinolytic System
Authors: Holvoet Paul , Boer Auke de , Verstreken Maria , Collen Désiré 
Published: July 2018

SummaryAn enzyme-linked immunosorbent assay (ELISA) is developed for the measurement of plasmin-α2-antiplasmin complex in human plasma. Microtiter plates were coated with a mixture of two murine monoclonal antibodies directed against human α2-antiplasmin and bound plasmin-α2-antiplasmin complex was quantitated with a peroxidase-conjugated monoclonal antibody directed against human plasminogen. The lower limit of sensitivity of the assay was 0.01 nM of plasmin-α2-antiplasmin complex in 100-fold diluted human plasma, allowing detection of 1 nM in undiluted plasma samples. After 100-fold dilution of the plasma samples, the assay was no longer influenced by the presence of the precursors plasminogen and α2-antiplasmin.At a concentration of 2.0 nM of plasmin-α2-antiplasmin complex in plasma, intra- and interassay variation coefficients were 4.2 and 5.5 percent respectively. In plasma samples of 25 control subjects the levels of plasmin-α2-antiplasmin complex were below 1 nM. Extensive in vivo activation of the fibrinolytic system during thrombolytic therapy with streptokinase resulted in the generation of elevated levels of plasmin-α2-antiplasrnin complex up to 690 ± 150 nM.No measurable levels of plasmin-α2-antiplasmin complex were found in the plasma of 32 patients with acute deep vein thrombosis nor in the plasma of 11 patients with recurrent deep vein thrombosis.These findings indicate that plasmin-α2-antiplasmin complex is generated during in vivo activation of the fibrinolytic system and that its assay may be useful to monitor thrombolytic therapy but not for the diagnosis of venous thrombosis.
Aggregation of Washed Platelets from Non-Anticoagulated Human Blood Is Not Reversible
Authors: Bowry S K , Müller-Berghaus G 
Published: July 2018

SummaryMost of the knowledge acquired on platelet function and biochemistry has been obtained from platelets prepared from blood anticoagulated with sodium citrate. Using washed platelets from human blood (PNB) to which no anticoagulants were added, we report on responses not observed with platelets prepared from citrate-anticoagulated blood. Native blood was passed rapidly (within 5 min of venepuncture) through a Sephadex G-25/G-50 column to remove divalent ions and thus prevent coagulation. Platelets were separated from the gel-filtered blood by differential centrifugation. Responses of PNB to thrombin, collagen, calcium ionophore, ristocetin, release of 14C-5hydroxytryptamine and p-thromboglobulin, and generation of thromboxane A2 were similar to those observed for citrated platelets. Comparison of PNB with thrombin-treated platelets, which demonstrate an increase of platelet factor 3 activity, a reduction of the adenylate energy charge and an impairment of clot retraction, indicated the absence of platelet activation. Unlike citrated platelets, however, aggregation of PNB in response to ADP was irreversible in the presence of Ca2+ and fibrinogen, even at concentrations as low as 0.2 μM ADP, with aggregation taking up to 25 times longer to reach the same extent of aggregation as for citrated platelets. PNB did not aggregate to epinephrine even in the presence of Ca2+ and fibrinogen. Sodium citrate impaired ADP-induced aggregation and clot retraction of PNB. Thus citrate affects platelet function and may cause changes resulting in the unphy-siological behaviour and responses of platelets.
Urinary Beta-Thromboglobulin Correlates with Impairment of Renal Function in Patients with Diabetic Nephropathy
Authors: Hopper A H , Tindall H , Davies J A 
Published: July 2018

SummaryTBeta-thromboglobulin (βTG) is a platelet-specific protein and since its concentration in plasma rises when platelets are activated, it has been used as an indicator of platelet involvement in vascular disease. Since platelets might be involved in the pathogenesis of diabetic microvascular disease we measured urinary βTG in 20 insulin-dependent diabetics with nephropathy and compared the results with those from 20 normal subjects. Measurement of βTG in urine was undertaken to avoid errors induced by blood sampling and to gain information over a prolonged period using a single assay. Measurements were made of βTG, β2-microglobulin and total protein in urine collected for 24 h and creatinine and β2 microglobulin in plasma. Survival of indium-111-labelled platelets was measured in nine patients. Urinary PTG was significantly (p <0.02) increased in the 20 patients compared with 20 normal volunteers (median value 1.3 vs 0.8 μg/24 h). There was a strong correlation between urinary βTG excretion and plasma creatinine concentration (r = 0.8, p <0.0001) and plasma β2-microglobulin concentration (r = 0.9, p <0.0001). Urinary βTG concentration did not correlate with platelet survival. The results indicate that although urinary βTG is significantly increased in patients with diabetic nephropathy its concentration in urine correlates with indicators of glomerular filtration rather than with a test of platelet activation.
An Enzyme Immunoassay (ELISA) for the Quantitation of Human Factor VII
Authors: Boyer C , Wolf M , Rothschild C , Migaud M , Amiral J , Mannucci P M , Meyer D , Larrieu M J 
Published: July 2018

SummaryA new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab′)2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21 to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VIIC was observed. In “compensated” DIC (n = 5), levels of F VII Ag varied from 60 to 186%, with significantly higher F VII C levels (from 143 to 189%). In contrast, in “decompensated” DIC (n = 7), low F VII Ag and F VII C levels were observed (from 7 to 27%). In patients with deep-vein thrombosis (n = 25), high levels of F VII Ag (from 102 to 136%) and F VII C (from 110 to 150%) were demonstrated. In surgical patients, no significant difference was observed before and one day after intervention.
Effect of Pentosan Polysulphate Administration in Man on the Formation of Covalent Complexes Between Heparin Cofactor II and Thrombin Generated in Plasma by Contact Activation
Authors: Dol F , Sié P , Dupouy D , Boneu B 
Published: July 2018

SummaryAn assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified 125I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 μg/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man.The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 μg/ ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.
Haemophilia B Leyden in Greece
Authors: Mandalaki T , Louizou C , Dimitriadou C , Briët E 
Published: July 2018

SummaryIn this paper, a five generation Greek family is described with haemophilia B. The disease is characterized by a normal ox-brain prothrombin time, normal levels of the vitamin-K dependent clotting factors VII and X and a proportional reduction of factor IX acttivity and antigen levels all of which is consistent with the cross-reacting material negative form of haemophilia B. However, in this family the factor IX levels in the three patients of generation V are around 1 U/dl while the three older patients in generation III have factor IX levels ranging from 28 to 44 U/dl. In the oldest patient of generation V we observed a rise of the factor IX level from 1 U/dl up to the age of 13 to 10 U/dl at age 14. In addition, the older patients have very mild bleeding symptoms or none at all, while the young ones have occasional spontaneous haemorrhages in muscles and joints, compatible with severe or moderately severe haemophilia. The disease appears to be similar to haemophilia B Leyden which has been described in a Dutch family.
Evaluation of p-Amidinophenyl Esters as Potential Antithrombotic Agents
Authors: Pizzo Salvatore V , Turner Denise , Porter Ned A , Gonias Steven L 
Published: July 2018

SummaryThree p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4])+. In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 μM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better α-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of α-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 αl of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and a-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.
Gel Filtration Patterns of Factor VIII Coagulant Antigen and Factor VIII Related Antigen in Normal and von Willebrand’s Disease
Authors: Chediak Juan , Peake Ian , Bloom Arthur 
Published: July 2018

SummaryGel filtration (sepharose 2B CL) patterns of factor VIII coagulant antigen (VIIIC :Ag) and factor VIII related antigen (VIIIR: Ag) were obtained using normal plasma and plasma from patients with von Willebrand’s disease. The latter group consisted of five individuals with normal mobility of factor VIIIR :Ag on cross-immunoelectrophoresis (Type I) and five others with abnormal (increased) mobility (Type II). Results showed that the elution of VIIIC: Ag was delayed in those subjects whose ratio of VIIIR :Ag to VIIIC :Ag was reduced. It has previously been reported that VIIIR :Ag exerts a stabilizing influence on the coagulant activity of factor VIII (VIII: C); our data suggests that when VIIIR :Ag is deficient, abnormal (low molecular weight) forms of VIIIC: Ag circulate.
The Ability of Fibrinogens, FI and FII, to Support ADP- Induced Platelet Aggregation
Authors: Phillips H M , Mansouri A , Perry C A 
Published: July 2018

SummaryFibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen Aa chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their Aα chains. The FI fraction contains intact Aα chains while FII and FIH-IV-V fractions have one and two partially degraded Aα chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the Aα chain does not play an important role in promoting ADP-induced platelet aggregation.
Functional and Antigenic Antithrombin III Values in Plasma and Serum from 115 Regular Blood Donors and Antithrombin III In Prediction of Postoperative Thrombosis
Authors: Nilsen D W T , Jeremic M , Weisert O K , Fønstelien E , Godal H C 
Published: July 2018

SummaryAntithrombin III (AT-III) was determined functionally using chromogenic substrate (S-2238) and immunologically using radial immunodiffusion (RID) in plasma and serum from 115 blood donors. There was a decrease in functional activity by 43.5% and in antigen concentration by 18.4% during in vitro coagulation when corresponding serum and plasma samples were compared. A positive correlation was found between the two methods in plasma (r=0.784) as well as in serum (r = 0.658). RID in serum correlated well with RID in plasma (r = 0.811), but the correlation for S-2238 in plasma and serum was poor (r = 0.411). Functional AT-III decrease during coagulation was uncorrelated with age and fibrinogen, and was not statistically affected by sex, smoking or blood groups.Preoperative functional AT-III activity was measured in 25 patients undergoing elective hip replacement, 60% of whom developed postoperative thrombosis. The functional activity in serum discriminated well between the thrombotic and nonthrombotic group of patients (p<0.025), whereas the activity in plasma showed only a minor difference.
1-O-Hexadecyl-2-Acetyl-SN-3-Glycerophosphoryl- choline (PAF): Some Effects on the Aggregation of Human Platelets by Thrombin or Collagen
Authors: Hornby Edward J , Perry Colin R 
Published: July 2018

Summary1-O-hexadecyl-2-acetyl-sn-3-glycerophosphorylcholine (PAF) was examined as a potential mediator of aggregation induced by thrombin or high concentrations of collagen in indomethacin treated platelet rich plasma by investigating the response to collagen and thrombin of platelets previously desensitised to PAF. No reduction in the response to collagen or thrombin was observed and it was concluded that PAF was not a mediator of non-prostanoid dependent aggregation in platelets exposed to thrombin or collagen.
Enhancement of the Release Reaction Not Accompanied with Enhanced Phosphatidylinositol Turnover in the Reserpinized Rabbit Blood Platelets
Authors: Watanabe Y , Soda M , Fukamachi N , Kobayashi B 
Published: July 2018

SummaryThrombin-induced platelet release reaction examined with secretion of calcium and N-acetylglucosaminidase was significantly enhanced in the platelets from reserpine-treated rabbits as compared with the control. On the other hand, 32P-incorporation into phosphatidic acid was suppressed in the reserpinized platelets in activated state. Thrombin induced phosphatidylinositol (PI)- breakdown, which was examined by decreases in radioactivity and content of PI, and an increase in diacylglycerol, was not enhanced in the reserpinized platelets as compared with the control. The phosphorylation of the specific protein coupled to thrombin- induced platelet PI-breakdown was not stimulated in the reserpinized platelets as compared with the control. In contrast to PI, PC-degradation by thrombin was significantly stimulated in the reserpinized platelets. Possible existence of pathway(s) other than that associated with an enhancement of Pl-tumover is conceivable as a mechanism involved in platelet release reaction.
Inactivation of Thrombin by the Aortic Endothelium
Authors: Swedenborg J , Dryjski M , Olsson P 
Published: July 2018

SummaryThe aim of the investigation was to clarify the uptake of thrombin on vascular endothelium and the inhibition of thrombin on the endothelium in the presence or absence of plasma. Segments of porcine aorta were used. Thrombin was labelled with 125I and its enzymatic activity was assayed amidolytically using a specific chromogenic substrate. After exposure to the endothelium both enzymatic activity and radioactivity disappeared from the thrombin solution and were recovered as surface bound activities.The enzyme activity confined to the endothelium rapidly disappeared in the presence of plasma but no activity was recovered in the plasma. The surface confined radioactivity, however, decreased slowly and was quantitatively recovered in the plasma. In the absence of plasma, i. e. in the presence of a balanced Ringer’s solution, only slow disappearance of thrombin enzymatic activity occurred although the rate of disappearance was higher than that of release of radioactivity.It is concluded that thrombin, taken up on the endothelium, is almost instantaneously inhibited by an interaction mechanism with plasma and then released in an inactive state. The endothelium itself, however, seems to slowly inhibit bound thrombin and then release it.
Standardization of Factor VIII – IV. Establishment of the 3rd International Standard for Factor VIII: C Concentrate
Authors: Barrowcliffe T W , Curtis A D , Thomas D P 
Published: July 2018

SummaryAn international collaborative study was carried out to establish a replacement for the current (2nd) international standard for Factor VIII: C, concentrate. Twenty-six laboratories took part, of which 17 performed one-stage assays, three performed two-stage assays and six used both methods. The proposed new standard, an intermediate purity concentrate, was assayed against the current standard, against a high-purity concentrate and against an International Reference Plasma, coded 80/511, previously calibrated against fresh normal plasma.Assays of the proposed new standard against the current standard gave a mean potency of 3.89 iu/ampoule, with good agreement between laboratories and between one-stage and two- stage assays. There was also no difference between assay methods in the comparison of high-purity and intermediate purity concentrates. In the comparison of the proposed standard with the plasma reference preparation, the overall mean potency was 4.03 iu/ampoule, but there were substantial differences between laboratories, and the two-stage method gave significantly higher results than the one stage method. Of the technical variables in the one-stage method, only the activation time with one reagent appeared to have any influence on the results of this comparison of concentrate against plasma.Accelerated degradation studies showed that the proposed standard is very stable. With the agreement of the participants, the material, in ampoules coded 80/556, has been established by the World Health Organization as the 3rd International Standard for Factor VIII :C, Concentrate, with an assigned potency of 3.9 iu/ampoule.