bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decreased Platelet Membrane Fluidity Due to Glycation or Acetylation of Membrane Proteins
Authors: Winocour Peter D , Watala Cezary , Perry Dennis W , Kinlough-Rathbone Raelene L 
Published: July 2018

SummaryPlatelets from diabetic subjects and animals are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function and previously we observed reduced membrane fluidity in platelets from diabetic patients associated with hypersensitivity to thrombin. We previously reported that decreased fluidity of isolated platelet membranes from diabetic patients is associated with increased glycation of platelet membrane proteins, but not with any change in the cholesterol to phospholipid molar ratio. We have now examined in vitro whether incubation of platelet membranes in a high glucose medium causes sufficient glycation to reduce membrane fluidity. Incubation of platelet membranes from control subjects in a high glucose (16.1 mM) medium for 10 days at 37° C led to an increase in the extent of glycation of membrane proteins and a decrease in membrane fluidity (indicated by an increase in steady state fluorescence polarization); most of the changes occurred within the first 3 days of incubation. Incubation of platelet membranes with 5.4 mM glucose had less effect. In contrast, incubation of platelet membranes with the same concentrations of 1–0-methylglucose did not cause a change in either the extent of glycation of proteins or membrane fluidity. We also determined if acetylation by aspirin or acetyl chloride of the sites available for glycation on platelet membrane proteins leads to a similar reduction in membrane fluidity. Pretreatment of platelet membranes with aspirin or acetyl chloride diminished the extent of glycation that occurred when platelet membranes were subsequently incubated with glucose, but membrane fluidity was reduced even in the absence of glucose; subsequent incubation with glucose caused no further reduction in membrane fluidity. Similar results were obtained when red blood cells were incubated with high concentrations of glucose or methyl glucose either with or without pretreatment with aspirin or acetyl chloride. Further experiments using platelet membranes showed that the reduction in membrane fluidity due to aspirin was independent of its acetylating effect on platelet cyclo-oxygenase. Ingestion of aspirin also caused a reduction in membrane fluidity of platelets. Therefore, glycation of platelet membrane proteins reduces membrane fluidity, but the effect results from occupation of the sites available for glycation and not the presence of glucose moieties per se at these sites. Acetylation of platelet membrane proteins either in vitro or in vivo also reduces membrane fluidity; this effect is not associated with platelet hypersensitivity to thrombin.
Quantitation of Platelet Glycoprotein IV (CD36) in Healthy Subjects and in Patients with Essential Thrombocythemia Using an Immunocapture Assay
Authors: Thibert Valérie , Bellucci Sylvia , Edelman Lena , Tandon Narendra N , Legrand Chantal 
Published: July 2018

SummaryGlycoprotein IV (GPIIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 µg/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 ± 0.05 (mean ± SD, n = 15) µg per 100 µg of platelet proteins and a mean value of 27,440 ± 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean ± SD = 81,850 ± 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean ± SD = 26,810 ± 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.
Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain
Authors: Yahara Hitoshi , Matsumoto Keiji , Maruyama Hiroyuki , Nagaoka Tetsuya , Ikenaka Yasuhiro , Yajima Kazuyoshi , Fukao Hideharu , Ueshima Shigeru , Matsuo Osamu 
Published: July 2018

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.
The Residues AGDV of Recombinant γ Chains of Human Fibrinogen Must Be Carboxy-Terminal to Support Human Platelet Aggregation
Authors: Hettasch Joann M , Bolyard Mark G , Lord Susan T 
Published: July 2018

SummaryThe carboxy-terminus of the γ chain of fibrinogen contains a sequence which is believed to be one of the domains that interacts with glycoprotein (GP) IIb/IIIa to support platelet aggregation. A normal variant of fibrinogen exists in which the four carboxy-terminal amino acids are replaced by 20 amino acids. This variant, known as γ’, has been reported to bind less effectively to platelets. The purpose of the present study was to engineer novel proteins to determine what differences in amino acid sequence between the γ and γ’ chains influence the interaction of the carboxyterminus with GPIIb/IIIa. In this regard, the γ chain cDNA in a bacterial plasmid expression vector was modified by oligonucleotide-directed mutagenesis to produce recombinant γ chains with amino acid changes in the carboxy-terminus which reflect the differences between γ and γ’. The recombinant γ chain with an unmodified carboxy-terminus supported adenosine diphosphate (ADP)-induced platelet aggregation to the same extent as intact fibrinogen. In contrast, the ability of γ’ 427 (the recombinant γ’ variant) and γ 427 (where the 16 amino acid γ’ extension [412–427] was added to the carboxy-terminus of γ) to support platelet aggregation was markedly reduced. In addition, the extent of ADP-induced platelet aggregation was decreased in the presence of γ’ 411 (where amino acids 408–411 in γ were replaced with amino acids 408–411 in γ’), while γ 407 (where the four carboxy-terminal amino acids were deleted) was not capable of supporting aggregation. These findings demonstrate that the four residues AGDV are not only required but must be carboxy-terminal to support platelet aggregation.
Vitamin E Binds to Specific Binding Sites and Enhances Prostacyclin Production by Cultured Aortic Endothelial Cells
Authors: Kunisaki Makoto , Umeda Fumio , Inoguchi Toyoshi , Nawata Hajime 
Published: July 2018

SummaryWe evaluated the effect of d-α-tocopherol (vitamin E) on the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells. Vitamin E at physiological doses significantly enhanced the production of PGI2 by aortic endothelial cells when added to the culture simultaneously with histamine, the Ca2+ ionophore A23187 (A23187), plasma-derived serum (PDS), or arachidonic acid. This effect was found to occur in a time- and dose-dependent manner, and the maximal enhancement was produced by 9.28 µM of vitamin E for 1 h incubations. Significantly lower amounts of lipid peroxides were measured in endothelial cells stimulated by 10% PDS with 9.28 µM of vitamin E than in those stimulated without vitamin E for over 24 h, although the stimulation during the initial 1 to 12 h period did not have a significant effect on lipid peroxide formation in cultured aortic endothelial cells.We also demonstrated that bovine aortic endothelial cells have specific binding sites for [3H]vitamin E that exhibited time- and temperature-dependent saturability. At 4° C, the nonspecific binding was 8–12% of the total binding, and the specific binding reached equilibrium by 2 h. Specific binding increased with the concentration of [3H]vitamin E and became saturated at concentrations between 1.5 µM and 2.0 µM per 2.0 × 105 cells. Raising the unlabeled vitamin E concentration from 97.7 nM to 1,000 µM reduced the specific binding of 2.0 µM [3H]vitamin E. The Scatchard plot of [3H]vitamin E binding to the endothelial cells shows two classes of binding sites: one with a high affinity {K
a1 2.48 ± 0.32 × 107 NT-1, n = 6} and a low capacity {n
1 1.20 ± 0.34 × 107 sites/cell} and the other with a low affinity {K
a2 1.18 ± 0.32 × 105 M–1} and a high capacity {n
2 3.39 ± 0.53 × 109 sites/cell}.Our results suggest that the endothelial cells binding sites for vitamin E may play some roles in vascular homeostasis in vivo, and that vitamin E may prevent the development of atherosclerotic changes due in part to the enhancement of PGI2 production by the vascular wall and its action as an antioxidant in vascular endothelial cell.
The Formation of the Haemostatic Plug – A Special Case of Platelet Aggregation
Authors: Lüscher Ernst F , Weber Susanne 
Published: July 2018

SummaryThe formation of the haemostatic plug is an extremely fast process. This excludes, at least in its first phase, the involvement of soluble activating agents released from or produced by the platelets. An experiment with ADP-activated, formaldehyde-fixed platelets shows that platelets with activated fibrinogen receptors will bind inactive platelets in the presence of fibrinogen and Ca2+-ions. A survey of the literature shows that platelet activation is accompanied by the clustering of the fibrinogen receptors. The surface of an activated platelet, which makes part of the growing haemostatic plug therefore is covered with patches of tightly packed fibrinogen. This allows the multisite combination with the statistically distributed low affinity receptors of the newly arriving platelets. Tightly packed fibrinogen, as present on clusters of the activated GP IIb/IIIa receptors as well as when absorbed to artificial surfaces acts as an activator of platelets. Thus, the propagation of the activation process is possible without a requirement for other, external activators. Such agents, which are released from platelets and, finally, thrombin formation, are nonetheless of vital importance, not for the formation but for the consolidation of the haemostatic plug.
Treatment with Tranexamic Acid during Pregnancy, and the Risk of Thrombo-Embolic Complications
Authors: Lindoff Claes , Rybo Göran , Åstedt Birger 
Published: July 2018

SummaryTranexamic acid (AMCA) is an inhibitor of fibrinolysis used to treat fibrinolytic bleeding (e.g., menorrhagia and gastro-intestinal haemorrhage), and to prevent bleeding at surgery, in cases of abruptio placentae and general haemorrhage. As AMCA stabilises preformed clots and prolongs their dissolution, it has been debated whether treatment with AMCA might predispose to thrombosis by depressing the fibrinolytic system. Pregnant women constitute a group with low fibrinolytic capacity and an increased frequency of thrombosis further increased after Caesarean section, and are thus more likely to be susceptible to antifibrinolytic therapy. We therefore carried out a retrospective analysis of the case records of 2,102 patients with various bleeding disorders during pregnancy. Of the 256 patients treated with AMCA (mean duration of treatment, 46 days), 169 were delivered by Caesarean section. Of the remaining 1,846 patients (i.e., controls), 443 were delivered by Caesarean section. The relationship between the use of AMCA and the occurrence of thrombo-embolism was calculated with 95% confidence limits.Of the AMCA treated group (n = 256), two patients – one of whom belonged to the Caesarean section subgroup (n = 168) – had pulmonary embolism. Of the controls (n = 1,846), three patients had deep vein thrombosis and one had pulmonary embolism, all four cases belonging to the Caesarean section subgroup (n = 443). Thus, the findings in this high risk group of women with complicated pregnancies, frequently entailing delivery by Caesarean section, provided no evidence of any thrombogenic effect of AMCA.
Effect of Three Fibrate Derivatives and of Two HMG-CoA Reductase Inhibitors on Plasma Fibrinogen Level in Patients with Primary Hypercholesterolemia
Authors: Branchi Adriana , Rovellini Angelo , Sommariva Domenico , Gugliandolo Angelo G , Fasoli Angelo 
Published: July 2018

SummaryIn order to evaluate the effects of hypocholesterolemic drugs on plasma fibrinogen concentration, six groups of subjects with primary hypercholesterolemia have been put on treatment with diet alone or diet plus fenofibrate (100 mg t.i.d.), slow release bezafibrate (400 mg once a day), gemfibrozil (600 mg b.i.d.), simvastatin (20 mg once a day) or pravastatin (20 mg once a day) respectively. After 1 month of therapy, plasma fibrinogen significantly decreased by 9% and 15% in fenofibrate and bezafibrate groups respectively and increased by 19% in gemfibrozil treated patients. After 4 months of therapy the changes were −16% with fenofibrate, −10% with bezafibrate and +20% with gemfibrozil. No significant changes were observed in patients treated with diet alone, simvastatin or pravastatin. The fibrinogen lowering effect of fenofibrate and bezafibrate does not seem to be related to the hypolipidemic activity of the drugs.
A Variant of t-PA (T103N, KHRR 296-299 AAAA) that, by Bolus, Has Increased Potency and Decreased Systemic Activation of Plasminogen
Authors: Refino Canio J , Paoni Nicholas F , Keyt Bruce A , Pater Cheryl S , Badillo Julie M , Wurm Florian M , Ogez John , Bennett William F 
Published: July 2018

SummaryIn the accompanying paper, we reported that the properties of decreased plasma clearance rate, increased fibrin specificity, and resistance to inactivation by PAI-1 could be effectively combined in the t-PA variant T103N, KHRR 296-299 AAAA. In the current study we evaluated the in vivo efficacy of this variant as well as variants containing the individual mutations T103N and KHRR 296-299 AAAA. Plasma clearance and in vivo lysis of whole blood and platelet-rich clots were determined in a rabbit arterio-venous shunt model. The T103N containing variants were administered as an intravenous (i.v.) bolus. KHRR 296-299 AAAA and t-PA were infused i.v. over 90 min. The clearance rate of the KHRR 296-299 AAAA variant was similar to t-PA. However, the clearance of the T103N and T103N, KHRR 296-299 AAAA variants were 8 and 6-fold reduced, respectively. Potency of the variants relative to t-PA on whole blood clots ranged from 0.9 (T103N, KHRR 296-299 AAAA) to 1.7 (T103N). Relative potency on platelet-rich clots ranged from 2.4 (T103N) to 4.2 (T103N, KHRR 296-299 AAAA). Fibrinogen concentrations in rabbits 120 min after dosing with a 2.5 mg/kg bolus were: 24, 16, 82, and 77% of initial for t-PA; T103N; KHRR 296-299 AAAA; and T103N, KHRR 296-299 AAAA treatment groups, respectively. These results suggest that the T103N, KHRR 296-299 AAAA variant of t-PA, given as a bolus, could result in greater efficacy, particularly on refractory platelet-rich clots, without inducing the severe systemic lytic state produced by a bolus of a less fibrin specific variant.
Anticardiolipin Antibodies Block the Inhibition by β2-Glycoprotein I of the Factor Xa Generating Activity of Platelets
Authors: Shi Wei , Chong Beng H , Hogg Philip J , Chesterman Colin N 
Published: July 2018

SummaryAntiphospholipid antibodies, defined either by lupus anticoagulant (LA) activity or positive anticardiolipin immunoabsorbent assay (ACA) are associated with a predisposition to thromboses, recurrent fetal loss or thrombocytopenia. The mechanisms for these predispositions remain undefined. We have enriched immunoglobulin fractions from two patient plasmas to obtain antibodies with LA activity but no ACA, or conversely, with ACA positivity but no LA, in order to investigate in vitro characteristics which might explain a thrombotic propensity. β2-glycoprotein I (β2-GPI), the plasma cofactor required for ACA binding to negatively charged phospholipid, has previously been shown to inhibit prothrombinase generation in the presence of activated platelets (8). We now report that β2-GPI, at physiological concentrations, inhibits the generation of factor Xa in the presence of activated gel-filtered platelets. Further, ACA interferes with this inhibition, resulting in protracted, unopposed factor Xa generation. This interference with β2-GPI, a natural anticoagulant component of plasma, is potentially prothrombotic. LA immunoglobulins behave differently and inhibit factor Xa generation in a manner similar to β2-GPI. These findings provide the basis for a previously unsuspected mechanism for thrombosis in patients with aPL.
Von Willebrand Factor Release and P-Selectin Expression Is Stimulated by Thrombin and Trypsin but not IL-1 in Cultured Human Endothelial Cells
Authors: Collins Peter W , Macey Marion G , Cahill Mary R , Newland Adrian C 
Published: July 2018

SummaryThe stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation.We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.
Optimal Duration of Oral Anticoagulant Therapy: A Randomized Trial Comparing Four Weeks with Three Months of Warfarin in Patients with Proximal Deep Vein Thrombosis
Authors: Levine Mark N , Hirsh Jack , Gent Michael , Turpie Alexander G , Weitz Jeffrey , Ginsberg Jeffrey , Geerts William , LeClerc Jacques , Neemeh Jean , Powers Peter , Piovella Franco 
Published: July 2018

SummaryThe optimal duration of oral anticoagulant therapy for patients with acute proximal deep vein thrombosis (DVT) is uncertain. Based on the hypothesis that a normal impedance plethysmogram (IPG) following DVT defines a group of patients at low risk of recurrent venous thromboembolism (VTE), a trial was conducted to evaluate the efficacy of only four weeks of warfarin. Patients with venographically confirmed acute proximal DVT who had received four weeks of warfarin after initial heparin and whose four week IPG was normal were allocated to either continue warfarin (targeted International Normalized Ratio 2.0 to 3.0) for a further eight weeks or receive placebo. Patients with an abnormal four week IPG received warfarin for a further eight weeks. Based on clinical characteristics at the time of the qualifying thrombosis, all patients were classified as having either continuing or transient risk factors for recurrent VTE. During the eight weeks following randomization, nine (8.6%) of the 105 placebo patients developed recurrent VTE compared to one (0.9%) of the 109 warfarin patients, P = 0.009. Over the entire 11 months of follow-up, 12 placebo patients developed recurrence compared to seven warfarin patients, P = 0.3. Nineteen of the 192 patients with an abnormal four week IPG experienced recurrence during the nine months after discontinuing warfarin.In the 301 patients who received three months of warfarin in the randomized trial or in the cohort study, all 26 recurrent events were in the 212 patients with continuing risk factors.In conclusion, an IPG four weeks after proximal DVT is not a useful predictor for recurrent VTE; whereas the presence of continuing risk factors is a very strong predictor. Four weeks of oral anticoagulants may be all that is required in patients without continuing risk factors. Patients with continuing risk factors may require more than three months of oral anticoagulants.
Determining Survivals of Platelets and Fibrinogen Using HAP an Interactive Hemostasis Analysis Computer Program
Authors: Abramson Mark J , Stein Sidney F , Goldstein J H 
Published: July 2018

SummaryAn interactive computer program is described which permits computation of platelet survivals based on the gamma function model of Murphy. An additional feature permits the treatment of other labelled proteins, e. g. fibrinogen, disappearing via an exponential process. The program requires no prior computer background and typical determinations of survivals can be effected in about five min, representing a substantial enhancement to the gamma function program as currently provided by the International Committee for the Standardization of Hematology. Provision has also been made for modifications to meet individual needs. The program is described in sufficient detail, including sample calculations, to permit evaluation for possible applications.
A Native Whole Blood Assay for Blood-Materials Interaction
Authors: Dodds W Jean , DiNovo Jean M , Bergeron John A 
Published: July 2018

SummaryA convenient, accurate, and reproducible method was developed and characterized to test the effects of particulate solids and soluble agents on native whole blood. The effects of citrate anticoagulation and of variation in flow and column parameters were characterized by ancillary experiments. The study utilized a relatively homogeneous strain of large rabbits that had received intravenous [C14]-serotonin as a platelet radiolabel. The assay included a 4 min contact time of either the test sample or saline control with freshly drawn arterial blood and was followed by a series of hemostatic measurements, both before and after passage of the blood through a standard 1 g glass bead column. Changes induced by soluble (ADP, ellagic acid, aspirin) and particulate (collagen) agents in the reactivity of this blood to the subsequent challenge of a large area of reactive surface revealed reproducible quantitative effects on platelets and clotting. Generally comparable results were obtained with native whole human blood when the glass bead surface was adjusted (2 g) for the species difference in coagulability and platelet reactivity.