bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity
Authors: Jennings Lisa K , White Melanie M , Mandrell Timothy D 
Published: July 2018

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.
Fast and Reproducible Vascular Neointima Formation in the Hamster Carotid Artery: Effects of Trapidil and Captopril
Authors: Matsuno H , Stassen J M , Hoylaerts M F , Vermylen J , Deckmyn H 
Published: July 2018

SummaryNeointima formation was induced in the hamster carotid artery by mechanical intraluminal injury with a catheter covered with roughened dental cement. Neointimal thickening occurred as early as 7 days after denudation and further increased during the next 1 to 2 weeks. Proliferation indices of smooth muscle cells (SMCs) showed the highest proportion of proliferating cells in the media and neointima respectively 1 and 5 days after the vascular injury. Transmission and scanning electron microscopy of damaged carotid artery sections as well as immuno-histochemical stainings of von Willebrand factor (vWF) confirmed that reendothelialization was progressive and already complete on day 14, at which time the neointima formation was almost complete.In order to pharmacologically characterize this model further, the effects on neointima formation of trapidil (triazolopyrimidine), a platelet-derived growth factor (PDGF) antagonist, and captopril, an angiotensin converting enzyme inhibitor, were investigated. Trapidil administered orally twice daily at total doses of 25, 50 and 100 mg/kg/day, started 3 days prior to infliction of injury and up to 7 or 14 days after the catheterization, significantly reduced neointima formation. Captopril administered orally three times daily at a total dose of 100 mg/kg/day, equally reduced neointima formation, with 100 mg/kg/day trapidil being more effective than 100 mg/kg/day captopril 7 days after injury. When the treatment by either one of these drugs was arrested on day 7, neointima formation resumed quickly.The hamster appears to be a small, reproducible and fast model for the study of SMC proliferation, requiring only relatively small amounts of experimental drugs. The model furthermore is sensitive to substances known to reduce neointima formation in other animal models.
Quantitative Comparison of Fibrin Degradation with Plasmin, Miniplasmin, Neurophil Leukocyte Elastase and Cathepsin G
Authors: Kolev Krasimir , Komorowicz Erzsébet , Owen Whyte G , Machovich Raymund 
Published: July 2018

SummaryThe relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.
Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa
Authors: Tokita Shigeru , Arai Morio , Yamamoto Naomasa , Katagiri Yasuhiro , Tanoue Kenjiro , Igarashi Koji , Umeda Masato , Inoue Keizo 
Published: July 2018

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.
Primary Binding Domain of Bovine von Willebrand Factor Fragment Expressed in E. coli
Authors: Bakhshi Meenakshi R , Sinha Dipali , Vora Renu K , Budzynski Andrei Z , Kirby Edward P 
Published: July 2018

SummaryBovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.
Elevated Fibrinopeptide A and B Levels during Thrombolytic Therapy: Real or Artefactual?
Authors: Weitz Jeffrey I 
Published: July 2018

SummaryThere is good evidence that thrombolytic therapy induces a procoagulant state that retards the lytic process and triggers reocclusion. Thus, both in experimental animal systems and in humans, potent inhibitors of platelets and antithrombin Ill-independent thrombin inhibitors have been shown to be better than heparin at accelerating thrombolysis and preventing reocclusion. These in vivo observations have lent credence to the concept that elevated FPA levels that are suppressed by heparin reflect systemic activation of coagulation during the thrombolytic process. However, this concept may not be correct. Thus, when heparin is given in conjunction with plasminogen activators only modest increases in FPA levels are found, and our data suggest that this is due, at least in part, to the activity of enzymes other than thrombin. In the absence of concomitant heparin, thrombolytic therapy causes a much greater increase in the FPA values. These high FPA values are rapidly reduced by heparin, even though heparin has limited antithrombotic activity in the setting of pharmacologic thrombolysis. Based on these considerations, we believe that plasma FPA and desarginine FPB levels should not be used as specific markers of thrombin activity during the course of thrombolytic therapy and suppression of elevated FPA values by heparin should not be accepted as evidence that heparin is effective in this clinical setting.
Infusion of Phospholipid Vesicles Amplifies the Local Thrombotic Response to TNF and Anti-Protein C into a Consumptive Response
Authors: Taylor F B , He S E , Chang A C  K , Box J , Ferrell G , Lee D , Lockhart M , Peer G , Esmon C T 
Published: July 2018

SummaryInflammation often is considered a contributing factor to both thrombosis and disseminated intravascular coagulation. The molecular mechanisms that dictate which of these clinical manifestations will result from the inflammatory stimulus remain obscure. Bacterial infection and certain tumors are common initiators of the disseminated intravascular coagulant response. Complement activation resulting from bacterial infection shares with selected tumors the capacity to generate or release membrane particles that lack functional adhesion receptors and hence could circulate to amplify a disseminated intravascular coagulant response. We developed a model of venous thrombosis that resulted in localized thrombus formation without disseminated intravascular coagulation. The model involves infusion of tumor necrosis factor, blockade of protein C and a partial decrease in venous flow caused by ligation of the superficial femoral vein without obstruction of the deep femoral vein. Infusion of phospholipid vesicles into this model resulted in amplification of a localized thrombotic response into a consumptive response.Seven different groups of animals were studied. The first three groups established the conditions necessary to produce deep vein thrombosis. The second four groups established the conditions necessary to produce disseminated intravascular coagulation. The infusion of phospholipid vesicles plus tumor necrosis factor and anti-protein C antibody resulted in consumption of fibrinogen, the production of thrombin/antithrombin complexes, a fall in platelet count, and venous thrombosis. Without ligation and catheterization phospholipid vesicles failed to produce the consumptive response. We conclude, therefore, that phospholipid vesicles can amplify a local thrombotic response into a consumptive response, and that vesiculation accompanying inflammation is one means by which localized coagulant activity may be amplified to produce disseminated intravascular coagulation.
Characterization of Fcγ Receptors on Human Megakaryocytes
Authors: Wu Zhanhe , Markovic Boban , Chesterman Colin N , Chong Beng H 
Published: July 2018

SummaryMegakaryocyte and platelet Fcγ receptors (FcR) are of importance in the pathophysiology of immune complex-mediated thrombocytopenias such as heparin-induced thrombocytopenia. In this study, FcγR proteins and mRNAs in normal human megakaryocytes were examined. FcγR proteins were studied with immunocytochemical staining, dual colour flow cytometry and immunoprecipitation using monoclonal antibodies against FcγR I, FcγR II and FcγR III. FcγR mRNAs were measured with biotinylated cDNA or oligonucleotide probes using a novel quantitative in situ hybridization technique. Using these techniques, FcγR II protein and mRNA, but not FcγR I and FcγR III proteins and transcripts were detected in megakaryocytes. Further, transcript analysis showed that megakaryocytes contain only the transcript of FcγR IIA gene but no transcripts of FcγR IIB nor FcγR IIC genes; FcγR IIA transcripts with and without the transmembrane (TM) exon are present in approximately equal proportions. In contrast, neutrophils and macrophages also contain FcγR IIA transcript but FcγR IIA transcript with the TM exon predominates suggesting cell lineage-specific FcγR IIA expression. FcγR IIA transcript lacking the TM exon predicts the presence of a potential soluble form of FcγR in platelets and megakaryocytes which may have a physiological role as it can compete with the membrane-bound FcγR IIA for binding of IgG-containing immune complexes and thus protect these cells from excessive binding and injurious effects of immune complexes.
Fibrinogen Promotes Adhesion of Monocytic to Human Mesothelioma Cells
Authors: Shetty Sreerama , Kumar Anuradha , Pueblitz Siegfried , Emri Saleh , Gungen Yucel , Johnson Alice R , Idell Steven 
Published: July 2018

SummaryAdhesion between monocytic and mesothelioma or pleural meso-thelial cells influences stromal remodeling in pleural neoplasia. We found that cultured monocytic cells (U937) adhere to either human pleural mesothelioma (MS-1) or mesothelial (MeT5A) cells in vitro. 125I-fibrinogen bound specifically and saturably to either cell line, and specific fibrinogen binding increased upon stimulation of these cells with proinflammatory agents such as phorbol myristate (PMA), lipo-polysaccharide (LPS) or tumor necrosis factor (TNF-±). We purified the fibrinogen receptor protein from a membrane fraction of MS-1 cells and identified it by immunoprecipitation as intercellular adhesion molecule (ICAM-1). Anti-ICAM-1 antibody or antisense oligonucleotides inhibited fibrinogen-mediated cell adhesion and binding of 125I-fibrinogen to mesothelioma or mesothelial cells. Cultured monocytic cells adhere to either mesothelioma or mesothelial cells, and the interaction is promoted by fibrinogen binding ICAM-1 at the cell surface. ICAM-1 is expressed by mesothelioma cells and CD 1 lb by macrophages in the fibrinous mesothelioma tumor stroma. The data suggest a common mechanism by which monocytic cells could adhere to either malignant mesothelioma cells or the mesothelial surface in pleural neoplasia.
Reduced Effect of Aspirin on Thrombus Formation at High Shear and Disturbed Laminar Blood Flow
Authors: Marius Barstad R , Ørvim Una , Hamers Maria J A.G , Tjønnfjord Geir E , Brosstad Frank R , Sakariassen Kjell S 
Published: July 2018

SummaryAspirin is the most commonly used antithrombotic drug in primary and secondary prophylaxis against cardio- and cerebrovascular disease. In previous studies from our laboratory it was demonstrated that the effect of aspirin on collagen-induced thrombus formation in a parallelplate perfusion device with laminar blood flow is shear rate dependent. Although aspirin did not affect collagen-induced thrombus formation at 650 s-1 (medium sized arteries), a significant inhibition of thrombus formation by approximately 38% at 2,600 s-1 (moderately stenoses in medium sized arteries) was observed. At present we have extended these studies to thrombus formation at the apex of eccentric stenoses in a parallel-plate perfusion chamber device. The stenoses reduced the cross-sectional area of the blood flow channel of the perfusion chambers by 60 or 80%, introducing disturbed laminar flow and apex wall shear rates of 2,600 and 10,500 s-1, respectively. The corresponding wall shear stresses were 80 and 315 dynes/cm2, respectively.Aspirin reduced the platelet thrombus volume at the 60% stenosis by 45% (p <0.03), and the fibrin deposition by 70% (p <0.004). However, none of these parameters were affected by aspirin at the 80% stenosis. These observations may at least partly explain why aspirin has a limited clinical effect in preventing arterial thrombus formation in atherosclerotic vessels at high shear and disturbed blood flow. In contrast, thrombus formation in blood from one patient with Glanzmann’s thrombasthenia and two patients with von Willebrand disease subtype 2M was almost abolished at this blood flow condition. Thus, blocking the function of either von Willebrand factor or glycoprotein IIb/IIIa may represent better antithrombotic approaches for such critical events than blocking the prostaglandin metabolism by aspirin. The lack of effect of aspirin on thrombus formation at the 80% stenosis may reflect shear-induced platelet activation at the stenosis inlet region, since shear-induced platelet aggregation in rotational viscometers is not affected by aspirin at shear stresses exceeding 100 dynes/cm2.
Evidence for Cultured Human Vascular Smooth Muscle Cell Heterogeneity: Isolation of Clonal Cells and Study of their Growth Characteristics
Authors: Benzakour Omar , Kanthou Chryso , Kanse Sandip M , Scully Michael F , Kakkar Vijay V , Cooper David N 
Published: July 2018

SummaryThe monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth of HVSMC in semi-solid medium was studied. Platelet-derived growth factor-BB (PDGF-BB) and to a lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.
Reliable Rapid Blood Test for the Exclusion of Venous Thromboembolism in Symptomatic Outpatients
Authors: Turkstra Franktien , van Beek Edwin J R , ten Cate Jan W , Buller Harry R 
Published: July 2018

SummaryIn this study we assessed the reliability of a rapid bed-side whole blood D-dimer assay prospectively in patients with clinically suspected venous thromboembolism, referred to the Academic Medical Centre, Amsterdam. In consecutive outpatients with clinically suspected deep vein thrombosis or pulmonary embolism we measured the sensitivity, specificity and negative predictive value of the assay compared to the outcome of standard diagnostic tests and 3-month follow-up. A total of 234 patients were included; the prevalence of venous thromboembolism was 29%. A sensitivity, specificity and negative predictive value of 100% (95% Cl: 95% -100%), 58% (95% Cl: 50% - 65%) and 100% (95% Cl: 96% -100%), respectively, were obtained. The exclusion rate was 41% of all referred patients. Conclusion: The SimpliRED whole blood D-dimer assay appears to be a simple and reliable method for the exclusion of venous thromboembolism in symptomatic outpatients.
A Pilot Study on Bolus Administration of Recombinant Staphylokinase for Coronary Artery Thrombolysis
Authors: Vanderschueren S , Collen D , Van de Werf F 
Published: July 2018

SummaryRecombinant staphylokinase (Sak) is a highly fibrin-specific thrombolytic agent but the optimal dose and mode of administration remain to be defined. Intravenous (i.v.) infusion over 5 min of 20 mg Sak in 12 patients with acute myocardial infarction induced complete coronary patency (TIMI perfusion grade 3) in 7 patients (58%) within 60 min. In 3 of the 5 patients with no or suboptimal flow (TIMI grade 0,1 or 2) at 60 min, an additional 10 mg i.v. bolus of Sak resulted in TIMI grade 3 flow at 90 min. No major treatment-related complication occurred. Residual fibrinogen and α2-antiplasmin levels at 90 min were 110 ± 6.0% and 98 ±4.1% (mean ± SEM) of baseline, respectively. Median antibody-related Sak-neutralizing activity was low at baseline (0.0 μg/ml) and after 1 week (0.5 μg/ml) but increased from day 10 on (to 4.0 μg/ml).Thus, bolus thrombolysis with Sak may induce efficient coronary artery recanalization while preserving circulating fibrinogen and α2-antiplasmin. Comparative trials of coronary thrombolysis with double-bolus Sak appear to be warranted.
Effect of Thrombomodulin on the Molecular Recognition and Early Catalytic Events in Thrombin-Protein C Interaction
Authors: De Cristofaro Raimondo 
Published: July 2018

SummaryA viscosity perturbation method allowed to compute the second order rate constant, k±15 for the formation of thrombin-Protein C complex, both in the absence and presence of thrombomodulin (TM) at pH 8.00 and 37° C. In the absence of TM the second order rate constant was found equal to 7.9 ± 0.6 × 103 M-1 sec-1, whereas it was enhanced to 9.9 ± 0.4 × 104 M-1 s-1 by a saturating (100 nM) TM concentration. Addition of 5 mM Ca++ to solution containing 100 nM TM induced a further increase of k+1 value up to 7.3 ± 0.5 × 105 M-1 s-1. Moreover, it was demonstrated that the thrombin-PC complex undergoes the acy-lation reaction more rapidly than it dissociates to form free thrombin and substrate (stickiness ratio = 2.4 ± 0.9). This tendency is even favored when thrombin is bound to TM both in the absence and presence of Ca++ (stickiness ratio = 9 ± 6 in the absence of Ca++ and 16 ± 10 in the presence of Ca++). Altogether these results demonstrate that TM is able to positively affect both the molecular encounter and the kinetics of the early catalytic events of the thrombin-Protein C interaction.