bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thrombotect: An Optimized Sample Collection System For The Inhibition Of In Vitro Platelet Release
Authors: Workman E F , Clark D M 
Published: July 2018

Platelet activation occurs in a variety of thrombotic disease states and may be monitored via measurement of Platelet Factor 4 (PF4) levels in plasma. In order for PF4 levels to reflect in vivo activation, care must be taken to eliminate in vitro α -granule release during the collection and preparation of blood specimens. Thrombotect is an evacuated blood collection tube containing an anticoagulant (EDTA) and two platelet inhibitors (2-chloroadenosine and procaine-HCl). This system is effective in preventing in vitro release under a variety of centrifugation conditions. Subsequent to a 30 minute icing step, whole blood samples may be centrifuged at room temperature and one of three centrifugation speeds - 2500 × g for 20 minutes,1500 × g for 30 minutes, and 1000 × g for 40 minutes. Plasma specimens from normal individuals were assayed after being prepared under the above conditions. PF4 levels were as follows:	2500 × g, *2.79 ± 1.00 (73); 1500 × g, 3.84 ±1.78 (20); 1000 × g, 3.35 ± 1.65 (20). Variations in the temperature of centrifugation had little effect on the ability of Thrombotect to inhibit PF4 release, whereas other commonly used blood collection cocktails became progressively less effective with increasing temperature.*(Mean PF4 in ng/ml ± 1 S.D., “n” values in parenthesis). Results similar to those obtained for EDTA/Theophylline were obtained when EDTA was used in conjunction with adenosine and/or aspirin. The Thrombotect system has been employed in an examination of PF4 levels in a variety of thrombotic disease states. It also provides an excellent collection system for use with other “platelet-release” assays.
Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient
Authors: Rendu F , Lebret M , Caen J P 
Published: July 2018

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.
Albumin And Platelets
Authors: Forestier F , Cherbit G , Solé Y , Tersarkissian M H 
Published: July 2018

We set up to point a double antibody radio-immuno-assay to quantify platelet albumin content.We used commercial reagents such as an antibody of rabbit reacting against human albumin and a highly purified human albumin antigen, labelled with 125I./Chloramine.The main results were:
-	109 platelets contained approximately 1 mg of albuminZonal ultra-centrifugation (105.000 g) in saccharose gradients showed that albumin was located in non-lysosomal α granules.The release of albumin occurred at the same time as β Thrombo-Globulin and Platelet Factor 4.We studied the serum albumin (SA), platelet albumin (PA) and the number of platelets on 144 normal individuals aged from 20 to 90 years.Our data show that:
-	PA decreases in correlation with the platelet countthe ratio PA/SA increases with agea correlation between the level of SA and the platelet counts exists.These data suggested a regulation mechanism between serum albumin and platelet levels. On the other hand our findings proved an active transport of albumin through the platelet membrane.
The Effect Of Metal Ions On Human Factor IX Antigenicity
Authors: Lewis R M , Reisner H M , Abels B C , Roberts H R 
Published: July 2018

Affinity chromatography of an inhibitor to human factor IX (F.IX) separated the antibody into two populations. The ion dependent population of antibodies had an absolute divalent cation (Me++) binding requirement. The non-ion dependent population bound F.IX equally in the presence or absence of Me++. The concentration of Me++ required for ½ the maximum ion dependent antibody binding (½ max) was (in nM) Ca++ 0.40, Mn++ 0.05, Sr++ 0.70 and Mg++ 0.65.Ca++ potentiated the binding of antibody in the presence of excess Mg++. In addition, the ½ max for Ca++ was reduced about four fold. These observations are consistent with separate binding sites on the F.IX molecule for Ca++ and Mg++ and potentiation of Ca++ binding by Mg++. Scat- chard analysis of ion dependent antibody binding indicates about a 10 fold greater affinity of antibody in the presence of Ca++ than Mg++. In the presence of both cations, affinity was at least as high as in the presence of Ca++ alone supporting the presence of separate ion binding sites on the F.IX molecule.
In Vivo And In Vitro Comparisons Of Anti-Inhibitor Coagulant Concentrates (AICC) For Hemophilic Therapy
Authors: Pancham N , Fournel M A , Coan M H 
Published: July 2018

In FVIII:C deficient persons, who also have circulating antibodies to the molecule, replacement therapy with FVIII concentrates elevates the antibody level and is of limited or no value. Treatment with prothrombin complex concentrates, however, has had varying degrees of success in these patients depending on the significance of the bleed.Currently available Anti-Inhibitor Coagulant Concentrates (AICC), containing factors II, VII, IX, and X in their nonactivated as well as activated forms, have been shown to be effective in controlling bleeds in some of these patients.Two commercially available AICC products and two research preparations were evaluated using conventional in vitro clotting techniques. In vivo assays were also performed in animal systems using the Wessler venous stasis assay. Despite similarities by in vitro assays, striking differences exist in the results obtained using the animal systems. As shown by the Wessler venous stasis assay for in vivo testing, the materials are much more active (at least an order of magnitude) than currently manufactured factor IX concentrates.In vitro clotting assays that are now available (namely factors II, VII, IX, X, IIa, Xa, NAPTT, FVIII correctional activity, and FVIII inhibitor bypassing activity assays) do not necessarily predict the in vivo efficacy of these AICC products. Furthermore, there is no correlation between the FVIII correctional activity and the FVIII inhibitor bypassing activity assays which are now being used to monitor these preparations.
Platelet Activation During Exercise Induced Myocardial Ischaemia
Authors: Hughes Andrew , IIsley Charles , Wilkinson Linda 
Published: July 2018

Twenty six normal subjects (aged 18-52) and eighteen patients with ischaemic heart disease (aged 38-67) underwent maximum 12 lead ECG treadmill exercise testing. Coronary angiography was performed on sixteen of the patients. In vivo platelet activation was assessed by measuring plasma levels of the platelet-specific released protein beta-thromboglobulin (B-tg).The mean B-tg levels pre and post exercise in the normal subjects were 47 (SD:26-85) ng/ml and 48 (29-78) ng/ml respectively, and in the patient group 68 (43-107) ng/ml and 70 (45-109) ng/ml. There was a significant difference between the two groups (p < 0.025), but there was no change in either following exercise. The mean B-tg level in those patients with positive exercise stress tests (≤0.1 mV ST depression at 80 ms post J point) was 75 (52-109) ng/ml, but only 46 (26-82) ng/ml in those with negative tests (p < 0.05). B-tg levels showed no change with increasing exercise ischaemia as judged by ST segment depression, but there was a positive correlation with the extent of angiographically demonstrated coronary disease (r = 0.497, p < 0.05).We conclude that in vivo platelet activation is present at rest in patients with coronary artery disease, and whilst related to the presence and extent of such disease is unrelated to induced ischaemia.
Cerebrovascular Disease - Results With Antithrombotic Therapy : Some Observations
Authors: Sandok B A 
Published: July 2018

Antithrombotic therapy is now widely used in the treatment of patients with ischemic cerebrovascular disease. Governmental approval will increase use and further convince many that the issues of therapy are now resolved. They are not. The following approved professional labelling guidelines serve to highlight the many clinical and investigational problems that remain:
(1)	There is evidence that aspirin is safe and effective for reducing the risk of stroke in men who have symptoms due to fibrin-platelet emboli.(2)	Patients should have a complete medical and neurologic evaluation.(3)	The recommended dosage is 1300 mgm/day.The evidence for effectiveness, while of statistical significance, becomes clinically less important when one notes that in the Canadian study, a disturbing 12.4% of patients under treatment with aspirin, developed subsequent stroke. This should not be unexpected since similar cerebral ischemic symptoms may be produced by differing pathophysiologic mechanisms (not all related to fibrin-platelet embolization); and treatment for one may be illogical and ineffective for another. Accurate diagnosis is imperative for selecting appropriate therapy. In the clinical situation, in spite of “complete” evaluation, uncertainty of the underlying mechanism often remains. Questions also remain about the dosage recommendations, and other treatment alternatives for both women and men.Subsequent clinical trials will need to consider each of the above. Until then, patients with cerebral ischemic symptoms must be evaluated individually. Some may benefit from antithrombotic therapy-many will not.
Biological Activities Of Two Plasmin Preparations: Differential Binding To Fibrin And α2-antiplasmin
Authors: Philo R D , Gaffney P J 
Published: July 2018

The intention to replace the present International Reference Preparation (IRP) for plasmin (a preparation auto activated from plasminogen in glycerol) with a freeze-dried urokinase (UK-activated plasmin (PL)) has prompted a comparison of the interactions of these two plasmins with fibrin and α2-antiplasmin (α2_APL). On a molar basis UK-PL was shown to digest fibrin about twice as rapidly as IRP-PL and these differential rates of fibrin lysis are reflected by the different binding affinities of these plasmins for forming fibrin.The titration curves of plasma α2-APL with these two plasmins were constructed from data obtained when the residual PL was measured both by S-2251 and fibrin hydrolysis. With increasing amounts of plasma α2-APL, both plasmins were neutralised to near completion when a clot lysis assay was used. However when S-2251 was used the IRP plasmin was only about half neutralised.The titration curve obtained when plasmin was neutralised with plasma α2-APL was similar in shape to an exponential decay whereas that for the reaction of purified α2-APL with UK-PL was a straight line. The IRP-PL reaction with purified α2-APL could be analysed as two straight lines.The differential binding of these plasmins to fibrin and α2-APL suggested that the IRP plasmin is made up of at least two populations of plasmin molecules. The exponential binding curve found with plasma α2-APL could be explained by the presence of the histidine-rich glycoprotein in plasma which is known to inhibit the plasmin-antiplasmin reaction.Polyacrylamide-detergent gel electrophoresis was used to 2onfirm the differences in functional plasmin/α2-APL data for the UK- and IRP-plasmins. These data could imply that a population of ‘plasmin’ molecules exists in the IRP which, because of the loss of certain strategic kringle regions in the heavy (A) chain, cannot bind to either fibrin or α2-APL.
Kinetics Of Plasminogen Activation By Tissue Plasminogen Activator. Role Of Fibrin
Authors: Hoylaerts M , Rijken D C , Lijnen H R , Collen D 
Published: July 2018

The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.
Venous Volumetry In The Assessment Of The Post-Thrombotic Limb
Authors: Ruckley C V , Crawshaw H M , Seaton J 
Published: July 2018

The reproducibility of venous volumetry has been evaluated in 20 normal legs (N), 20 legs with primary varicose veins (W) and 30 with stable post-thrombotic chronic venous insufficiency (CVI).The test has shown significant differences between the groups in mean relative expelled volume (EV rel, ccs): N 1.65 ± SD 0.66; W 1.01 ± 0.40; CVI 0.64 ± 0.39 and in half refilling time (T3 secs): N 23.9 ± 11.5; W 11.8 ± 7.4; CVI 3.6 ± 1.8. The percent coefficients of variation in tests repeated at intervals of up to a month were as follows.The reproducibility of the test confirms its value as a non-invasive means of measuring the severity of postthrombotic venous disease and thus as a means of assessing response to treatment. Serial volumetric tests on 25 patients before and for up to 12 months after Linton operations for chronic venous insufficiency show significant early improvements in expelled volume. But in many of these patients the refilling time tends to remain short indicating residual valvular incompetence and the possibility of clinical relapse in the long term.
Human Platelet Activation In The Absence Of Aggregation: A Calcium-Dependent Phenomenon Independent Of Thromboxane Formation
Authors: Levy-Toledan S , Maclouf J , Bryon P A , dela Baume H , Savariau E , Hardisty R , Caen J P 
Published: July 2018

In response to the ionophore A 23187, thrombasthenic platelets undergo a change in light transmission (LT) using either PRP or washed platelets. This change was accompanied by a normal release of 14C serotonin (5HT) and thromboxane (TX) synthesis in absence of aggregation; lactic dehydrogenase determinations showed no evidence of platelet lysis. Ultrastructural qualitative electron microscopy revealed the formation of the central gel mass and the central apposition of organelles but the platelets were loosely packed. This ultrastructural change which was accompanied by biochemical phenomena was quantified; this includes an increase of the elongation coefficient, a decrease of the circularity coefficient as well as a decrease of the percentage of granules and surface connecting system in comparison with the total volume of the cell.The change in light transmission was not inhibited by non-steroidal anti-inflammatory drugs (aspirin, indometha- cin, flurbiprofen) suggesting a dissociation between a normal release of 14C-5HT and an absence of TX formation. Moreover it was not inhibited by creatine phosphate/ creatine phosphokinase, prostaglandin E1 or cytochalasine and/or colchicine; it was then not dependent on ADP, cAMP or the integrity of microfilaments and microtubules. However chlorpromazine, TMB8 and dibucaine, drugs which interfere with intracellular membrane transport of calcium ions inhibited this platelet activation (change in LT, 14C-5HT release or TX synthesis).The stimulation of internal calcium fluxes by the ionophore was sufficient to induce platelet activation in the absence of aggregation; it was found to be independent of TX formation.
Involvement Of Calmodulin In Platelet Reaction
Authors: Kösaki G , kambayshi J , Ohno H , Morimoto K , Hakata H 
Published: July 2018

Calmodulin is a ubiquitous Ca2+-binding protein, modulating a number of Ca2+-dependent cellular reactions. In blood platelets, involvement of calmodulin has been demonstrated in the activation of myosin light chain kinase and of phospholipase A2. In the present study, involvement of this protein in platelet reaction was thoroughly investigated, utilizing phenothiazines(chlorpromazine and trifluoperazine) known as selective inhibitors of calmodulin.Phenothiazines in micromolar concentration completely inhibited platelet aggregation and secretion induced by ADP epinephrine, collagen, thrombin or Ca2+-ionophore. They also completely inhibited the aggregation triggered by arachido- nate or by the mixture of thromboxane A2 and prostaglandin endoperoxides, which is probably due to the inhibition of activation of myosin light chain kinase. As another possible involvemnt of calmodulin in the reaction, the effect of phenothiazines on the release of arachidonate from phospholipids was studied in stimulated platelets to which radioactive arachidonate had been incorporated. The release was dose-dependently inhibited by either of phenothiazines. Then, their effect on the hydrolysis of phosphatidylinositol by platelet sonicates was investigated, using 3H-myoinositol phosphatidylinositol as substrate. Either of phenothiazines suppressed the hydrolysis in the micromolar concentration, indicating their inhibition on phosphatidylinositol specific phospholipase C.These observations suggest involvement of calmodulin in at least two steps of platelt reaction: one is the step of the release of arachidonate from phospholipids (the activation of phospholipase C in addition to phospholipase A2) and the other is the step of contraction of platelet actomyosin after the formation of thromboxane A2 (the activation of myosin light chain kinase).
Effect Of TicloPidine On Platelet Deposition In Gore-Tex And Autologous Vein Grafts
Authors: Murphy K P , Dewanjee M K , Fuster V , Didisheim P , Kaye M P 
Published: July 2018

To quantitatively study platelet deposition on arterial prostheses and the effect of platelet inhibitory therapy with ticlopidine, 14 dogs underwent bilateral femoral artery excision and replacement with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Five dogs received 60 mg/kg/day of ticlopidine orally on four consecutive days prior to surgery. Nine untreated dogs served as controls. Collagen-induced platelet aggregometry studies were performed at the end of day 4. Autologous indium-111-labeled platelets were injected 24 hours prior to surgery to serve as aplatelet marker. All animals received heparin (1 mg/kg I.V.) 5 minutes prior to excision of the arterial segment. Grafts were removed 1 hour following resumption of blood flow and gently flushed with 30 cc normal saline. Radioactivity per unit weight of Gore-Tex, vein and blood was determined with a gamma counter. The relative radioactivity with respect to the internal reference standard, blood, is tabulated below.Ticlopidine treated dogs showed a 15-fold reduction in platelet deposition on Gore-Tex grafts and a 3-fold reduction on venous grafts. Platelet deposition was reduced significantly in Gore-Tex grafts (P<0.001) to a level comparable to that in autologous veins. Platelet inhibitory effect was also observed in collagen-induced platelet aggregometry.According to this “in vivo” data, ticlopidine appears to be a promising platelet inhibiting agent.
Immunoradiometric Assay Of VIII:CAG, A Potential Tool To Detect Human Anti-VIII:C Antibodies
Authors: Hellings J A , Over J , van Leeuwen F R , van Mourik J A 
Published: July 2018

A modification of a two-site, solid-phase immunoradiometric assay (IRMA) for Factor VIII coagulant antigen (VIII: CAg) has been evaluated for its potential to detect antibodies against Factor VIII coagulant activity (VIII: C) in patient plasma samples. For this purpose the assay system comprised four steps: 1) coating of test tubes with human anti —VIII:C , 2) incubation with normal Factor VIII— VWF complex, 3) incubation with test sample, and 4) binding of radiolabeled human anti–VIII: C as marker.Of eight hemophilic plasma samples containing antibodies against VIII: C (as detected in a clotting assay) five were able to prevent binding of radiolabel partially and three prevented this completely. One of these three (which actually was the antibody used in the IRMA), was effective even at very high dilutions. Two hemophilic plasma samples without detectable antibodies in the clotting assay, a severe Von Willebrand’s disease plasma and normal plasma samples showed no significant interference with binding of radiolabeled human anti-VIII: C. Also, a plasma sample containing a high titer of spontaneous human antibody to VIII: C as well as a heterologous antiserum against Factor VIII—VWF complex did not interfere with binding of radio-activity.It is concluded that the test system described is a sensitive tool to detect antibodies of the same specificity as those used in the IRMA. It may also detect antibodies of differing specificity. The lack of crossreactivity with some antibodies points to interindividual differences in specificity of antibodies against VIII: C.
Increased Circulating Immune Complex Levels In Hemophilia Patients Post-Infusion
Authors: Hilgartner M W , Inman R D , Miller C H 
Published: July 2018

Elevated levels of circulating immune complexes (CIC) have been reported in a significant proportion of hemophilia patients tested by Raji cell, Clq binding, and staphylococcal binding (SBA) methods. The clinical significance of these complexes is unclear, as is their relationship to transfused material. By SBA, we found elevated CIC levels (>10 ug/ml) in 90% of severe hemophiliacs and 75% of multiply-transfused controls, all more than 72 hours post-transfusion. This was in contrast to our previous finding elevated CIC in 50% of hemophiliacs and 29% of controls by Raji cell assay.Using the SBA, seven patients were tested at intervals after Factor VIII concentrate infusion. Five showed increased CIC levels. The peak level occurred after the Factor VIII clotting activity had peaked and was declining. Two subjects showed rapid clearance of the complexes, while the others remained elevated at 24 hours. Factor VIII-related antigen was detected in isolated complexes from 2 patients. Our previous finding of HBsAg in such complexes suggests heterogeneity and perhaps different clearance patterns.