bluuebunny/crossref_metadata_2025_split huggingface dataset:
Salicylate, Sulphinpyrazone, Indomeihacin And Aspirin May Interact With Platelet Cyclooxygenase At A Site Different From The Substrate Binding Site
Authors: Cerletti C , Livio M , Rajtar G , de Gaetano G 
Published: July 2018

In vitro studies have shown that salicylate(SA), inactive by itself, prevents the inhibitory effect of aspirin (ASA)on platelet prostaglandin production. The purpose of this study was to verify this interaction in vivo and the ability of other non-steroidal anti-inflammatory drugs to counter ASA effect on platelet cyclooxygenase. Groups of male CD-COBS rats received one of the following drugs : SA (25-200 mg/kg i.p.),sulphinpyrazone(200 mg/kg p.o.), indomethacin (0.5 mg/kg i.p.) or the corresponding vehicles, followed after 30 min by ASA(1-50 mg/kg i.p.)as its soluble lysine salt. One or 24 hours later platelet-rich plasma was prepared and MDA (spec- trophotanetric assay) or TxB2 (RIA) generated on stimulation with 0.4-1 mM Na arachidonate were measured. All 3 compounds blunted ASA inhibitory action. In particular: 1. The preverting effect of SA was dose-dependent and could be overcome by increasing the dose of ASA. At a dose ratio (SA:ASA) of 20 the effect of ASA was reduced by about 50%, but no interaction was apparent at a ratio below 5. 2. Sulphinpyrazone had a greater preventing effect when it was administered 6 h (100%) than 3 h or 30 min (60%) before ASA (5 mg). 3. Indo-methacin reduced by 50% the effect of 5 mg of ASA but not of 10 mg. Tests were made 24 h after ASA administration, when the inhibitory effect of indcmethacin was no longer detectable. 4. 200 mg/kg SA reduced by 50% the inhibitory effect of indcmethacin.This data suggests that the 4 drugs interact with the same enzyme and that competition does not necessarily involve the substrate arachidonic acid. This supports the existence of a supplementary binding site which regulates cyclooxygenase activity, but is not directly involved with the substrate site. The clinical implications of these results include the interference of ASA with its hydrolysis product SA and a reappraisal of the pharmacological basis for the association of sulphinpyrazone and ASA in thrombosis prevention trials.
Tissue Factor-Like Activity Of The Human Monocytic Tumor Cell Line U937
Authors: Hudig D , Rapaport S I , Bajaj S P 
Published: July 2018

Cells of the human monocytic cell line U937, derived from a patient with histiocytic lymphoma (Sundstrom and Nilsson, Int. J. Cancer 17:565, 1976) have procoagulant activity similar to that of activated peripheral blood monocytes, although about 10-fold more U937 cells than monocytes are required for equivalent activity. Procoagulant activity of the cells is Ca2+ dependent and is not demonstrable in factor VII deficient or factor X deficient plasma. Culture with E. coli 0127:B8 1ipopolysaccharide increases the procoagulant activity of washed U937 cells two-fold. Exposure of U937 cells to lymphokines from normal lymphocytes does not induce further coagulant activity. The slope of the log/log plot of cells vs. clotting time parallels that of human brain thromboplastin. Other cell lines of myeloid or lymphoid origin, e.g., K562 cells, WI-L2 cells, do not have procoagulant activity. Thus, U937 cells have constitutive factor VII-dependent coagulant activity similar to the tissue factor activity induced by activation of normal monocytes.In further experiments, U937 cells were incubated with purified human factor VII in the presence or absence of Ca2+ and then repeatedly washed. When subsamples of the cells were then added to recalcified factor VII deficient plasma in the absence of added tissue factor, the following clotting times were obtained: for cells incubated with factor VII in the presence of Ca2+,45"; for cells incubated with factor VII in the absence of Ca2+, 150". These data suggest that U937 cells can bind factor VII in a reaction requiring Ca2+, which then enables the cells to express their tissue factor-like activity in factor VII deficient plasma.
Subcellular Localisation Of ADP-ASE In Cultured, Porcine, Endothelial Cells
Authors: Lieberman G E , Leake D S , Peters T J 
Published: July 2018

A role for vessel wall ADP-ase in the maintenance of haemostasis has been suggested by a number of studies. For such a role to be likely the enzyme ideally should be an ecto-enzyme. The purpose of this study was to localise the ADP-ase using the technique of subcellular fractionation on a sucrose-density gradient in conjunction with marker enzyme assays for the different organelles.Firstly, homogenates of cultured, pig aortic endothelial cells which had been harvested following the 5th-7th subculture were prepared. These were used to determine the kinetics of ADP-ase activity using the recently described rapid assay for ADP-ase which employs[β-s2P]ADP. The activity was found to be linear with respect to incubation time and protein concentration. The pH optimum was found to be 7.4 and the Km to be 45μM.Post-nuclear supernatants derived from these homogenates (controls) and those from cells which had first been incubated with digitonin which binds to the membrane cholesterol thus making membranes heavier, were fractionated. In the controls, the profile of ADP-ase activity throughout the gradient was dissimilar to lactate dehydrogenase (cytosol), malate dehydrogenase (mitochondria), neutral α glucosidase (endoplasmic reticulum) and N-acetyl β glucosaminidase (lysosomes). It most closely corresponded to the 5’ nucleotidase profile (plasma membrane). Following digitonin treat ment, both 5’NT and ADP-ase shifted to a denser zone in the gradient confirming their coincident localisation.Lastly, intact cells and sonicated cells were incubated with the enzyme inhibitor: diazotised sulphanalic acid which is a poorly permeant reagent. With intact cells, ADP-ase and 5’NT were inhibited whereas LDH was not, but with sonicated cells all three enzymes were inactivated.Thus ADP-ase is an ecto-enzyme on the plasma membrane of pig aortic endothelial cells.
Heat-Induced Fragmentation Of Human Plasma Fibronectin
Authors: Miekka Shirley I 
Published: July 2018

Human plasma fibronectin, purified by affinity chromatography on gelatin-Sepharose, undergoes fragmentation during heat treatment at 100°C prior to reduced SDS-PAGE. The electrophoretic patterns showed a progressive decrease in the intensity of the 230 kd doublet characteristic of reduced fibronectin, which correlated with the appearance of increasing amounts of smaller components having molecular weights ranging from 30-200 kd. Fragmentation was temperature dependent, being undetectable after 2 hr at either 22° or 60°C, but clearly evident after 30 min at 70°C and above. After 2 hr at 100°C the intact 230 kd monomer was no longer visible. The effect was also pH dependent; fibronectin samples heated for 20 min at 100°C in SDS solutions buffered at pH 2.5, 4, 7 and 10 exhibited maximum degradation at pH 4. When heated in the absence of SDS, the protein was partially aggregated to polymers which did not enter the gel, but fragments were still observed. Equivalent results were obtained whether mercaptoethanol was added before or after the heat incubation step.The fragmentation does not appear to be due to proteolysis. Pretreatment with various combinations of inhibitors of serine-, thiol- or metallo-proteases did not detectably diminish the effect. Sterile filtration through microporous membranes (0.22 or 0.45 uM) to remove possible cellular contaminants did not improve the stability. Furthermore, when mixtures of fibronectin and fibrinogen were heated (20 min, 100°C) the fibrinogen chains were not detectably altered while fibronectin was fragmented as usual.These observations suggest that heat-induced fragmentation of fibronectin reflects an intrinsic instability of the molecule at high temperatures, perhaps analogous to that observed with α2-macroglobulin and complement components.
The Source Of Thromboxane Formation Associated With Complement-Mediated Pulmonary Leukostasis In Sheep
Authors: McDonald J W D , Ali M , Cooper J D , Townsend E R 
Published: July 2018

The infusion of plasma containing Zymosan-activated complement (ZAC) into sheep produces leukopenia with pulmonary leukostasis and transient pulmonary arterial hypertension (PAH). Previous work has related PAH to elevations of plasma thromboxane B2 (TXB2) rather than to mechanical obstruction by sequestered leukocytes (WBC). We have investigated the source of the TXB2 formation in this model. Incubation of platelet-poor WBC preparations with arachi- donate resulted in negligible TXB2 formation. WBC-poor platelet preparations on the other hand formed significant amounts of TXB2 (approximately 6-18 ng/108 platelets). Incubation of whole sheep blood or plasma with ZAC failed to generate significant amounts of TXB2. Thus, WBC agglutination in vitro did not induce platelet TXB2 formation.Pretreatment of sheep with aspirin (ASA)(10 mg/kg IV) completely blocked TXB2 formation and PAH in response to infusion of plasma containing ZAC. The infusion of 10-50% nonnal platelets into sheep 4 hours after ASA pretreatment failed to restore TXB2 formation and pulmonary vascular response to subsequent challenge with ZAC. TXB2 formation during blood clotting ex vivo was restored by the platelet infusions. These experiments make it appear unlikely that platelets are the source of the TXB2. It is possible that the transfused platelets respond to thrombin but are unable to interact with sequestered leukocytes. Sheep lung and pulmonary artery were incubated in vitro with arachidonate. Lung formed 630 ng TXB2 and 39 ng 6-keto-PGF1α/g of wet tissue. Pulmonary artery formed 9 ng TXB2 and 180 ng 6-keto-PGF1α/g of wet tissue. The relative proportions of TXB2 and 6-keto-PGF1α formed by lung parenchyma but not pulmonary artery resemble the proportions observed in previous in vivo experiments with ZAC. It appears that lung tissue is the most likely source of TXB2 formation causing PAH in response to ZAC-mediated pulmonary leukostasis.
Dissociation Of Aprotinin-Human Plasmin Complex By Fibrin
Authors: Vairel E G , Thely M , Brouty-Boye H 
Published: July 2018

Aprotinin inhibits plasmin by forming an inactive complex which has a binding force of 3.108.When the complex, in a 0.15M buffer solution at pH 7.4, is percolated through a column of human fibrin balanced with the same buffer, all of the aprotinin appears promptly in the eluent and the fibrin column dissolves progressively from the top. If the buffer is replaced with a 0.15M epsilon amino-caproic acid solution as soon as lysis begins, the fibrinolysis is stepped and plasmin is eluted.If citrated human plasmin is used in a 1:1 solution with buffer, the same events take place but over a longer period of time ; the aprotinin appears slowly in the eluent and fibrinolysis begins later.It thus appears that fibrin is able to dissociate the aprotinin-plasmin complex, probably by forming a shortlived ternary complex. The complex binds to fibrin even in a plasma medium, and fibrinolysis occurs even with an excess oxsf plasmic inhibitors in the medium.Fibrinogen does not possess this property of fibrin. The complex can be detected in the circulating blood several hours after intravenous administration to rabbits.This property of fibrin suggests a possible use of the aprotinin-plasmin complex as specific fibrinolytic treatment.
The In Vitro Effect Of Ticlopidine On Fibrinogen And Factor VIII Binding To Human Platelets
Authors: Lee H , Paton R C , Ruan C 
Published: July 2018

Ticlopidine is an anti-aggregating drug whose mode of action is not yet fully understood. Its multiple effects on platelet function include prolonged bleeding time, reduction in primary and secondary waves of ADP-induced aggregation and inhibition of collagen and thrombin- induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as Factor VIII/vWF binding induced by ristocetin.
125I fibrinogen binding was measured in suspensions of freshly-washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-Factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline-induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the Factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19 %) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.
Retraction Of Fibrin Clots By Cultured Endothelial Cells
Authors: Barbieri B , Balconi G , Dejana E , Donati M B 
Published: July 2018

The interactions between endothelial cells (EC) and fibrin are still poorly understood. We approached this problem by studying the ability of cultured EC to induce in vitro the retraction of fibrin clots. EC were obtained from bovine aorta and human umbilical veins by collagenase treatment and grown in Eagle MEM. At the time of the test the cells were harvested from the flask by a short trypsin-EDTA treatment and resuspended in tyrode solution. The test system involved incubation of the cell suspension in a water-bath at 37°C in the presence of cell-free plasma which was clotted by thrombin. The course of retraction was followed by measuring the diameter of the clot with a microcaliper. Retraction values were expressed after calculation of percent activity by an appropriate formula. EC were found to induce the retraction of the fibrin clot to an extent which increased with the time (1-24 h) and with the number of cells in the system (l-4×l06/ml f.c.). Fibrin clot retractile (FCR) activity of EC could not be detected at 22°C or in presence of Na2-EDTA or using mechanically disrupted cells. Moreover, using batroxobin instead of thrombin as a clotting agent, no retraction occurred; FCR of EC thus showed many characteristics in common with platelet- and fibroblast- induced clot retraction.FCR activity of bovine EC increased with the number of subcultures, being very low in cells harvested from primary cultures. In contrast, human EC had high activity in primary cultures. Similarly to fibroblasts, EC with higher density in culture showed lower FCR, suggesting that con-fluency inhibits the cell contractile capacity.FCR could thus represent a simple in vitro test to further characterize the biology of EC and to evaluate their role in the development of fibrin thrombi.
Pathogenesis Of Clinical Events In Coronary Artery Disease. Clinical Observations
Authors: Maseri A 
Published: July 2018

Studies on angina at rest showing that an increase of myocardial demand beyond a fixed supply is not the only or even the most frequent cause of acute myocardial ischemia open a new avenue in our understanding of ischemic heart disease. Functional factors that transiently interfere with regional coronary blood supply, such as coronary spasm, seem to play a major role in determining the clinical events associated with coronary atherosclerotic heart disease. These functional factors appear to be the major cause of angina at rest, nocturnal, post-prandial and cold-induced angina and for variable threshold of exertional angina.Sudden death was also shown to be a possible consequence of coronary spasm which was shown to cause ventricular fibrillation and cardiac arrest during reversible ischemic episodes with or without anginal pain.Myocardial infarction consistently occurs in the same vascular bed shown to undergo transient ischemic episodes before the onset of the final, irreversible one. The transient episodes were shown to be caused by coronary spasm. The irreversible episode could not be relieved by intracoronary nitrates. A platelet mural thrombus was found at post mortem at the site of demonstrated persistent spasm.We suggest that coronary vasospasm and platelet aggregation may be responsible for the onset of acute myocardial infarction and, possibly, for the development of coronary thrombotic obstructions.Thus, the clinical manifestations of ischemic heart disease may largely depend on the presence and severity of functional factors, transiently and acutely interfering with flow, along with a variable degree of coronary atherosclerosis. In turn, prognosis may be determined by the severity of acute functional factors and the extent to which the coronary circulation and the myocardium are already chronically jeopardized by organic lesions.
A New Method To Determine The Binding Of Factor X To Phospholipid Bilayers
Authors: Tans G , Rosing J , Dieijen G V , Rijn J V , Hemker H C 
Published: July 2018

The binding parameters (dissociation constant and the number of binding sites) for factor X binding to phospholipid bilayers in the presence of Ca-ions can be determined using the factor X-activating protein present in Russel’s Viper Venom (RVV-X). It is shown that RVV-X is only able to activate factor X molecules that are not bound to the phospholipid bilayer. Therefore, from the observed rates of factor X activation by RVV-X measured in the presence and absence of phospholipid the amount of factor X not bound to the phospholipid bilayer can be calculated. When the concentration of factor X added is known the bound factor X can be calculated and the binding parameters for factor X are obtained from a Scatchard plot. The technique presented here offers an advantage over other techniques published thus far. It is rapid and simple as compared to the Hummel andDryer technique. There is also an advantage over techniques using light scattering. Since the technique presented here is not hampered by aggregation of vesicles it is possible to study the binding of factor X to vesicles containing a high mole fraction of PS and at high CaCl2 concentrations.The binding parameters for factor X binding to vesicles of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) are dependent on the amount of negatively charged phospholipid present. At increasing mole fractions of PS factor X binds more tightly to the vesicles and also the number of factor X binding sites increases. However, at high amounts of PS (60%) the number of sites present decreases dramatically which is likely due to aggregation of the vesicles. Binding to vesicles composed of a mixture of phosphatidylgly- cerol (PG) and PC is considerably weaker whereas the lumber of sites present is about the same. Prothrombin and factor X compete for the same binding site as was determined by competition experiments. Our data will be discussed with respect to the mode of interaction of vitamin-K dependent clotting factors with negatively charged phospholipid interfaces.
The Contribution Of Factor VIII In The Intrinsic Activation Of Human Factor X
Authors: Mertens K , van Wijngaarden A , van Tilburg N H , Bertina R M 
Published: July 2018

In the intrinsic pathway, factor X is activated by a complex consisting of activated factor IX, factor VIII, phospholipid and calcium ions. The contribution of each of this components has been estimated by recombination of purified human coagulation factors, and measurement of the initial rate of factor Xa formation using a spectrophoto- metric assay.In the absence of phospholipid and factor VIII, factor X is activated at a low, but significant rate. Addition of either phospholipid or activated factor VIII stimulates the initial rate of this reaction 250 and 500 fold, respectively; addition of both cofactors stimulates more than 100,000 fold. The activation of factor VIII by factor Xa or by thrombin is essential for the stimulating effect of factor VIII. Factor VIII complex, containing both VIIIR:WF and VIII:C, and purified factor VIII:C behave identically in this system; purified factor VIIIR:WF does not influence the rate of factor Xa formation.The same kinetic approach was used for the characterization of preparations of abnormal factor VIII, obtained from plasma of haemophilia A patients.
New Evidence For An Adp-Independent Mechanism Of Thrombin Induced Platelet Activation
Authors: Figures W R , Colman R F , Niewiarowski S , Morinelli T , Wachtfogel Y , Colman R W 
Published: July 2018

The adenosine nucleotide analog, 5’fluorosulfonylbenzoyl adenosine (5’FSBA) is a potent inhibitor of ADP induced platelet shape change, aggregation, and fibrinogen binding and covalently incorporates into a single membrane polypeptide (Mr=100,000). To evaluate the role of released intracellular ADP in thrombin induced aggregation and fibrinogen binding, the effects of 5’FSBA on these events were studied. Following incubation with 5’FSBA, the platelets became unresponsive to ADP but were aggregated by purified human thrombin (0.2 U/ml). However, the aggregation was slower and less extensive than that of untreated control platelets. Binding of 125I-fibrinogen to control platelets occured after exposure to ADP or after exposure to thrombin in the presence of hirudin to inhibit clotting. Binding of 125I-fibrinogen was absent in platelets treated with 5’FSBA after exposure to ADP and diminished after incubation with thrombin (0.2 U/ml, 5 min.).The effect of thrombin on the single platelet membrane polypeptide modified by 5’FSBA was examined by SDS-PAGE. Thrombin treatment (0.5 U/ml, 30 min.) of cells labeled with (H)-5’FSBA resulted in partial cleavage of this polypeptide as evident by a decrease in the (H) FSBA-modified polypeptide in the thrombin treated samples as compared to controls. Unlike chymotrypsin activation of platelets, no fragment of the protein at 75,000 daltons was associated with the membranes of thrombin treated cells. The results of these experiments indicate that thrombin induced platelet aggregation and fibrinogen binding depend on both ADP binding which is blocked by 5’FSBA and the proteolytic cleavage of the same 100,000 dalton membrane polypeptide cleaved by chymotrypsin.
Association Of α-Actinin With The Plasma Membrane Of Human Blood Platelets
Authors: Sixma Jan J , Schiphorst Marion E , Jockusch Brigitte H 
Published: July 2018

The association of α-actinin with the plasma membrane of human blood platelets was investigated using immuno- adsorbed antibodies against α-actinin from porcine skeletal muscle and against actin from chicken gizzard. Platelet membranes enriched 10 times in radiolabelled wheat-germ- agglutinin were prepared utilizing the polylysine-bead technique of Cohen et al (1977). Immunofluorescent staining showed the presence of actin and α-actinin associated with these membranes.Platelets were ectolabelled with lactoperoxydase-iodina- tion. A crude membrane preparation was solubilized with Triton X 100, and incubated with anti α-actinin or appropriate control antibodies. The formed immune complexes were bound to Protein A-Sepharose beads, and subjected to 2-D- electrophoresis with i.e.f. in the first dimension, α-actinin from these membranes was not radiolabelled, had an apparent mol wt of 10U kdaltons and a pi of 6.7, similar to an α-actinin preparation from porcine skeletal muscle that was investigated in parallel. The location of α-actinin in the 2-D pattern was different from that of glycoprotein III.No specific glycoprotein was found bound to α-actinin under the experimental conditions. Treatment of membranes with non-ionic detergents and ‘protein perturbants’ indicated that α-actinin is an ‘extrinsic protein’. These data indicate that α-actinin as well as actin are closely associated with the plasma membrane, linked by electrostatic interaction. Both actin and α-actinin were not exposed on the outside in a resting platelet.