bluuebunny/crossref_metadata_2025_split huggingface dataset:
Is Phospholipase A2 (PLA2) Involved In PAF-Acether Formation By Platelets
Authors: Chignard M , Vargaftig B B , Le Couedic J P , Benveniste J 
Published: July 2018

PAF-acether (platelet-activating factor) has been recently identified as l-O-alkyl-2-acetyl-sn-glyceryl-phosphorylcholine, and later chemically synthetized. Platelets form PAF-acether upon stimulation with the calcium ionophore A 23187 or with more physiological stimuli such as thrombin or collagen. By contrast, arachidonic acid (AA) and adenosine diphosphate (ADP) do not trigger formation of PAF-acether. Since 1) PAF-acether is a phospholipid derivative and 2) aggregating agents which trigger PAF-acether formation are potent platelet PLA2 stimulators, we speculated that PLA2 could be implicated in its formation.Rabbit washed platelets were incubated at 37°C in the presence of thrombin (2.5 U/ml) or of ionophore A 23187 (2.5 uM) for 7 min and ethanol (80 % final) was added. After centrifugation, the supernatant was evaporated and concentrated. The extract was tested for its aggregating property on rabbit washed platelets preincubated with a cyclo-oxygenase inhibitor (aspirin) and an ADP scavenging system (creatine phosphate and creatine phosphokinase).In the presence of calcium chelating agents such as EDTA (5 mM) and EGTA (5 mM) most of the synthesis of PAF-acether was suppressed (93 % and 100 % of inhibition respectively). Dibutyryl cyclic AMP (5 mM) also suppressed PAF-acether formation from platelets challenged by thrombin or by the ionophore A 23187 (100 % and 62 % inhibition respectively). Bromophenacyl bromide (0.1 mM) and compound CB 874 (0.1 mM) proved also to be very potent inhibitors of PAF-acether synthesis (100 % inhibition both). All these drugs are well-known platelet PLA2 inhibitors. Upon stimulation platelets also form a deacetylated PAF-acether (lyso- PAF-acether) which could be the direct precursor of PAF-acether. The release of lyso-PAF-acether and the blockade of PAF-acether formation by various molecules having in common a PLA- inhibitory activity lead us to conclude that a PLA2 may be implicated in PAF-acether formation from platelets. Alternative explanations include the possibility that the various inhibitors act on other membrane-related sites.
Enhanced Phospholipase A2 Activity In Cholesterol-Enriched Platelets From Rabbits Fed A Cholesterol-Enriched Diet
Authors: McLeod A J , Johnson M , Sucklino K E , Walton P 
Published: July 2018

Phospholipase A2(PLA2) could be the rate-limiting enzyme in the metabolism of arachidonic acid (AA) derived from membrane phospholipid to thromboxane A2 (TXA2). Mal- ondialdehyde (MDA) production, which is considered to be an index of TXA2 synthesis, is increased in platelets which have been enriched in cholesterol by incubation with cholesterol-rich phospholipid dispersions in vitro.Rabbits were fed a diet supplemented with 0.5% w/w cholesterol for 4 weeks after which serum cholesterol was determined and the platelets examined and compared with rabbits fed a control diet. The cholesterol:phospholipid molar ratio (c/p) in the platelets, MDA production (stimulated by AA (imM) and basal) and PLA2 activity were estimated. PLA2 activity was estimated by measuring the % inversion by resuspended washed platelets of 1-acyl-2-(l- 14C)arachidonyl phosphatidylcholine to (1- 14C)arachidonic acid on stimulation with collagen (2μg/ml). Serum cholesterol in the cholesterol-fed group (n=9) was 488 ± 104 mg/ 100ml compared with the control group (n=3) which was 34 ± 4.7 mg/l00ml. Platelets from the cholesterol-fed rabbits showed a 20% increase in C/P (p<0.05); basal and AA stimulated MDA production was increased by 40% and 27% respectively compared with platelets from the control group. PLA2 activity was 1.26% conversion to products in the cholesterol-enriched platelets compared with 0.10% in the control platelets. This increase in activity was significant (p < 0.05).These results suggest that increased AA metabolism in cholesterol-enriched platelets may in part be due to increased PLA2 activity. This may reflect a physical effect of cholesterol on the platelet membrane predisposing arachidonyl phosphatidylcholine to PLA2 catalysed hydrolysis.
Assay And Occurrence Of Two Molecular Forms Of α2-Anti- Plasmin In Plasma
Authors: Kluft C , Los P , Jie A F H 
Published: July 2018

A procedure to assay a plasminogen-binding (PB) and a non-plasminogen-binding (NPB) form of α2-antiplasmin in plasma has been developed. The procedure involves agarose electrophoresis in a medium containing lys-plasminogen resulting in specific retardation of the PB form. Both forms are quantified by crossed immuno-electrophoretic techniques.The presence of both forms in vitro appears not to be an artefact as evidenced by its normal presence irrespective of the use of an inhibitor cocktail, different anticoagulants, treatment with various enzymes or aging of plasma.In a normal plasma pool 65% of α2-antiplasmin-related-antigen appears as the PB form. In the thirteen individuals constituting the pool, values range from 61-71%. In a case of homozygous α2-antiplasmin deficiency both forms were absent; in heterozygotes with approximately half normal plasma levels both forms were found in the normal ratio.On coagulation of blood part of α2-antiplasmin becomes irreversibly bound to fibrin by the action of factor XIIIa. In the remaining serum the amount of the PB form in particular is reduced, suggesting that this form is preferentially bound to fibrin. Associated with the consequent change in ratio between PB and NPB forms in serum, compared to plasma, a clear discrepancy between immunochemical assay and functional (immediate plasmin inhibition) assay is evoked. This discrepancy can be explained by assuming different expression of both forms in the two assays.It is concluded that the study and assay of α2-antiplasmin in blood has to take into account the occurrence of this inhibitor in two forms, which apparently behave differently in reactivity towards plasmin and in binding to fibrin. (Supported by Praeventiefonds, projectnumber 28-443).
Changes In The Primary Fibrinolysis Inhibitor (α2-Antiplasmin) After Cardio-Pulmonary Bypass
Authors: Bagge L , Nyström S-O , Saldeen T 
Published: July 2018

The blood level of the primary fibrinolysis inhibitor (PFI) has been reported to be relatively unchanged in most clinical conditions. However, we have found a strong increase 2-7 days after trauma in the form of this inhibitor (PFIα) with high affinity to plasminogen whereas the form with no or weak affinity to plasminogen (PFIβ) is not increased. The increase of PFIα may be due to increased synthesis in the liver. The method used for the measurement of the two forms is a two-step affinity chromatography using lysine- and plasminogen-Sepharose, respectively.In the present investigation we have studied the serum level of the two forms before and after the cardio-puhnonary bypass in 14 patients operated upon for cardiac surgery. Four patients had a blood loss of more than 1000 ml during the first 16 hours after bypass (group 1)6 patients lost between 500-1000 ml (group II) and 4 patients lost less than 500 ml (group III).PFIα was significantly lower and PFIβ was significantly higher in group I than in groups II and III. One patient with the largest blood loss showed the strongest decrease (30 % of normal value) in PFIα in the whole material and the strongest increase in PFIβ (160 %). This patient also had the lowest level of plasminogen activators and plasminogen.The reason for the decreased level of PFIα in the patients with heavy bleeding may be a consumption and the increase in PFIβ suggests that this form may be a degradation product of PFIα.
Biochemical Characteristics And Inactivation Studies Of An Inhibitor Of Urokinase, From Leucocytes
Authors: Kopitar M , Drobnič-Košorok M , Babnik J , Turk V 
Published: July 2018

Leucocyte cells are known to contain the acid, thiol and many types of neutral proteinases; among the latter also plasminogen activator. During the last five years appeared also many reports on the endogenous inhibitors of proteinases. In the preceeding publications we reported the isolation and characterization of two inhibitors of proteinases, from leucocytes (of thiol proteinases and of serino proteinases).In the present work we present the biochemical characteristics as well as the inactivation (by cathepsin D) of an urokinase inhibitor isolated from pig blood.Leucocytes were isolated from the peripheral pig blood by dextran procedure. Inhibitor of urokinase could be isolated either from cytoplasm (post granular supernatant) or from the extract of nuclei. Further purification of inhibitor was performed by cation exchange chromatography, by gel filtration on Sephadex G-100 and G-150 and by affinity chromatography on urokinase-Sepharose 4B and on antibody (of urokinase inhibitor )-Sepharose 4B. Inhibitor was isolated in the homogenous form, in a single polypeptide chain with molecular weight of 68 000, designated (leucocyte) Inhibitor-3. This inhibitor has an isoelectric point from 4.4-4.5 and is stable in buffer solutions from pH 3-8, and belongs to the type of so called fast reacting inhibitors. We also established that cathepsin D inactivates this inhibitor by hydrolysis of the inhibitor molecule. The conversion of active inhibitor into inactive protein proceeds catalytically. These last inactivation studies suggest a new pathway by which the active urokinase inhibitor in leucocyte cells may be inactivated.
Changes In The Antithrombin III Activity At The Interface Plasma-Phospholipids
Authors: Josso F , Béguin S 
Published: July 2018

The role of antithrombin III (AT III) on kinetics of thrombin activity was studied in an inhibitor free coagulation system in which purified AT III was added at various concentrations. Coagulation was initiated either by kaolin or by Stypven in the presence of phospholipids.
Kinetics of the respective activities of factor Xa and thrombin were followed on chromogenic substrates S-2222 and S-2238 and on fibrinogen for the latter.These experiments gave the following results :
(i) kinetics of thrombin formation as well as the peak level of thrombin activity were strongly dependent on AT III concentration ; (ii) Xa formation was unaffected by AT III level ; moreover the decrease of Xa activity was much slower than that of thrombin and was negatively correlated with phospholipid concentration.These results illustrate the impact on blood coagulation of minor deviations from normal of AT III level. This assumption was confirmed by AT III infusion to normal subjects. On the other hand our findings cast doubt on the physiological importance of the inhibitory activity of AT III on serine-proteases other than thrombin. At the interface piasma/phospholipids, when complexed with phospholipids and protein cofactors (i.e. factors V and VIII) the affinity of the enzymes for the inhibitor becomes negligible compared to their affinity towards their respective zymogen substrates. In a complete coagulation system, inhibition of thrombin formation by AT III seems to result mainly from the inhibition of the cooperative action of thrombin on its own formation.
Serial Platelet Survival Half-Life (PS) In Peripheral Vascular Disease And Diabetes Mellitus
Authors: Kazmier F J , Fuster V , Chesebro J H , O’Fallon J H , Palumbo P J 
Published: July 2018

Mayo Clinic, Mayo Foundation, Rochester, MinnesotaPlatelets are believed to play an important role in the pathogenesis of atherosclerosis and thrombotic occlusive arterial disease. As part of a prospective study of peripheral occlusive arterial disease in diabetes mellitus, platelet survival half-life (PS) was determined in years 1 and 2 from the disappearance pattern of 51cr-labeled platelets obtained by measuring radioactivity in serial samples over 8 days in four groups of patients age 50-70. Using computer assisted least-square analysis, a single exponent is fitted to the data and half-life determined.Groups included (1) normal subjects (NC); (2) subjects with clinical and objective evidence (transcutaneous doppler ultrasound and treadmill exercise) of peripheral arterial occlusive disease (AS0); (3) subjects with diabetes mellitus without AS0 (DM); (A) subjects with both diabetes mellitus and AS0 (DM+AS0). 112 subjects were tested in year 1 and 75 of these were available for retesting in year 2.In year 1, there is a difference in the distribution of platelet survival (PS) for the four groups (p=.002). PS is significantly short in groups AS0 and DM+AS0 compared with controls (NC), Year 2 values for PS (75 subjects) were compared to year 1. Using the t test on mean differences PS for each group was not significantly different year 2 vs year 1; mean percent differences year 2 vs year 1 were 1.2% (NC), 0.2% (ASO), 3.2% (DM), and -9.3% (9-3% lower than year 1 for DM+ ASO).Platelet survival half-life (PS) does distinguish popula tion groups and is reproducible when repeated.
Systemic Thromboemboli In Mitral Starr-Edwards Prosthesis: A Long-Term Followup (10-19 Years)
Authors: Fuster V , Chesebro J H 
Published: July 2018

The study comprised 170 consecutive patients (pts) seen at Mayo Clinic between 1962 and 1970 who underwent mitral valve replacement with Starr-Edwards prosthesis (model 6000 in 35% of pts, model 6120 in 65% of pts) because of significant chronic mitral incompetence (functional Class III-IV). All patients were on oral anticoagulants and were followed until January 1980 with a followup of 10 to 19 years (yrs) (median 15 yrs). We analyzed (1) the cumulative incidence of systemic emboli, their location and whether or not they left residual deficit; and (2) possible risk factors that might have been predictive, including in the analysis: age, sex, functional class, presence or absence of atrial fibrillation, degree of cardiomegaly, type of prosthesis and degree of anticoagulation.At 12 yrs of followup, 48% of pts had at least one episode of emboli (among these pts, 12% had two episodes); the rate of emboli was maximal within 1 yr postoperatively but persisted over the total followup at a rate of about 3% per yr. Of all emboli, 94% occurred in the cerebral circulation and 66% left neurological deficit. Regarding predictive factors, the incidence of emboli was slightly higher in pts with the prosthesis model 6000 (p<0.05) and in pts considered to be poorly anticoagulated (p<0.05).This unique long-term followup study on pts with mitral Starr-Edwards prosthesis reveals that systemic emboli is a persistent and significant problem. Preliminary data indicates that the addition of a platelet inhibitor to the oral anticoagulant may be beneficial.
Endothelial Alterations During The Platelet Activating Factor (PAF) Respiratory Distress Syndrome
Authors: Lewis J C , O’Flaherty J T , McCall C M , Wykle R L 
Published: July 2018

PAF (l-0-alkyl-2-0-acetyl-sn-gylcero-3-phosphocholine) induces polymorphonucelar leukocyte and platelet stasis in rabbit lung capillaries in vitro and produces a model of acute respiratory disease. Since PAF mediates anaphylactic reactions, a study was done to determine the ultrastructural effects of PAF treatment. Mature rabbits were treated by intravenous administration of either PAF (0.15-10 μg/kg: 8 animals) or BSA (the PAF carrier: 3 animals) prior to sacrifice and intraventricular perfusion with 0.1 M phosphate buffered (pH 7.2) glutaraldehyde (2.5%). Animals (n=5) injected with a high concentration of PAF (3-10 μg/kg) and sacrificed within 15 minutes of PAF administration had grossly contracted lungs, the vasculature of which (as observed by scanning electron microscopy) contained numerous marginated leukocytes and platelet aggregates. Animals (n=3) given PAF in the concentration range 0.15-2.4 μg/kg had less consistent lung contraction and fewer platelet aggregates within capillaries. Luminal surfaces of capillary endothelial cells in all PAF treated animals (when observed by transmission electron microscopy [TEM]) were dramatically altered. In contrast to the uniformly smooth surfaces in control animals, vessels in the PAF treated animals had tortuous surfaces with plasma membrane discontinuities and protrusion of plasma membrane fragments into the capillary lumen. Morphometric analysis of TEM micrographs substantiated statistically significant (p<0.01) increases in the number and size of plasmalemmal vesicles.These observations clearly document a cytotoxic effect for capillary endothelium. This cytotoxic action may in part explain the clinical effect of PAF.
Group Analysis Of Abnormal Factor X Molecules
Authors: Fair D S , Edgington T S 
Published: July 2018

Factor X lies central in blood coagulation being the point of convergence of the intrinsic and extrinsic pathways and in its activated form is the initial enzyme in the final stages of clot formation. The interaction of Factor X with its activators, cofactors and substrates suggest that there are multiple structural loci present on the surface of this molecule available for these molecular associations. Hence, abnormal molecules may reflect different specific structural defects resulting in decreased function. We analyzed 28 abnormal Factor X plasmas by four parameters: 1) antigen (Ag) measured in radioimmunoassay and activity as measured by: 2) prothrombin time (PT), 3) Russell’s viper venom time (RVV), and 4) activated partial thromboplastin time (APTT). Abnormal Factor X plasmas were from congenital (16), transient (2) and amyloid associated (10) deficiencies. In order to objectively determine groups of abnormal molecules we evaluated their distributions in the following manner: (1) PT activity vs Ag concentration; (2) RVV activity vs PT/Ag; and (3)APTT activity vs RVV/(PT/Ag).A non-random distribution of plasmas was observed which could be grouped into ten classes and subclasses of abnormal Factor X molecules. These data suggest that the groups of abnormal molecules may reflect common structural defects shared among its members located at specific structural loci involved in either the activation or the function of these Factor X molecules.
Sialic Acid Content And The Biological Activity Of Stuart Factor (F X)
Authors: Engel A M 
Published: July 2018

Substantial release of sialic acid (SA) from purified F X was obtained with sialidase from Clostridium perfringens. The total SA content of F X was found to be 1.7%, in agreement with previous reports. After almost complete removal of SA, aliquots were examined by polyacrilamide disc electrophoresis and by polyacrilamide in sodium dodecy1 sulphate (SDS) and the gels were stained for proteins and for glycoproteins. When SA was progressively removed from F X its clotting activity was slightly increased while its electrophoretic mobility was retarded, as expected. The tryptic activation of F X was affected by the removal of 60-70% of SA, yielding more clotting activity than the native F X. No difference was observed between the esterase activity of regular F X and asialo-X as measured by the hydrolysis of N-alpha-acetyl glycyl-L-lysy1-methyl ester acetate (AGLMe).When asialo-X was interacted with 25% sodium citrate the kinetics were accelerated and the maximal clotting and esterase activities were obtained at 6 hours of activation while the native F X reached the peack of both activities at 24 hours. Both activation mixtures were analyzed, at the period of maximal activity, for liberation of peptidic material by thin-layer electrophoresis (TLE). TLE patterns of asialo-X varied according to the activation system used; they also differed from the ones obtained with native F X.The data indicate that SA influences the kinetic of activation of F X and the nature and distribution of the peptidic material released during activation.
Interaction Of Platelets With Soluble, Polymerizing And Polymerized Fibrin In Human Plasma
Authors: Brosstad F , Kierulf P , Godal H C , Eika C 
Published: July 2018

Previous studies on platele/fibrin interactions find polymerizing fibrin capable of inducing platelet aggregation but are controversial as to the same ability of soluble fibrin. It has also been controversial wether the fibrin-induced platelet aggregation is mediated via the platelet release or not. Since previous studies have utilized fibrin prepared With soluble phase thrombin or reptilase, it was of interest to reinvestigate the problem with fibrins prepared with insoluble phase reptilase (des-AA) and thrcmbin (des-AABB). Urea-solubilized des-AA or des-AABB fibrin monomers were added to hunan, citrated PRP (ci-PRP) in the aggregcmeter either in soluble anoints (8% of ci-PRP fibrinogen cone) or in amounts resulting in the formation of visible fibrin (50% of the ci-PRP fibrinogen level, or added as polymerized fibrin which had been homogenized.The increase in light transmission and beta-trcmboglobulin releas (β-TG) were recorded. Soluble or polymerized, homogenized fibrin had no effect on either parameter, whilst fibrin added in amounts resulting in visible fibrin formation (polymerizing fibrin) caused both platelet aggregation and β-TG release comparable to that of collagen (100 ug/ml) Fibrin monomer solvent (urea) had no such effects. In experiments with EDTA-PRP, visible aggregation was not observed but βTG confirmed that platelet release had taken place . With KCN-PRP and Acetylosalicylic-PRP,neither aggregation, norβ-TG ,was observed. It is concluded that: 1. Soluble or polymerized fibrin does not aggregate platelets. 2. Polymerizing fibrin aggregates platelets. 3. Platelet aggregation observed with polymerizing fibrin is mediated via the platelet release reaction. 4. Des-AA and des-AABB fibrin are equally potent in inducing platelet aggregation.
Platelet-Associated IgG Quantitation By A Simplified Rapid Nephelometric Technique
Authors: Morse B S , Giuliani D , Nussbaum M 
Published: July 2018

The complexity of tests used to measure PAIgG has diminished its effectiveness as a measure of platelet bound antibody. We recently reported a simplified radial immunodiffusion technique to quantify PAIgG using washed solubilized platelets and low level IgG immunoplates (Behring-Calbiochem). Three days were required for diffusion to reach completion. Normal values ranged from 1.6 - 7.0 fg IgG/platelet. 92% of patients with immune thrombocytopenia (ITP) had values which exceeded the mean by greater than 2 S.D. units. To further simplify and hasten the measurement, we have developed a nephelometric method. 0.25 × 108 platelets, obtained from 10-30 ml of blood collected in EDTA are solubilized using 1% triton X-100 plus sonication. Nephelometric buffer containing polyethylene glycol is added followed by anti-human IgG. Relative light scattering units thus generated can be converted to ng of IgG by comparison with standards. Results of PAIgG levels can thus be made available within several hours of blood collection.These results are in excellent agreement with PAIgG determined by radial immunodiffusion. Our cumulative experience indicates a mean normal control value of 3.9 ± 1.9 fg IgG/platelet. 90% (40/45) of patients with immune thrombocytopenia exceed the mean by greater than 2 S.D. units. Elevated values are also seen in SLE patients with (50%) or without (25%) thrombocytopenia, 50% of patients with sepsis and thrombocytopenia, 80% of patients with IgG myeloma, and 20% of patients with thrombocytopenias of diverse etiologies.