bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Rat Glomerular ATP Diphosphohydrolase Activity during Reperfusion Injury Is Associated with Oxidative Stress Reactions
Authors: Candinas Daniel , Koyamada Nozomi , Miyatake Tsukasa , Siegel Jonathan , Hancock Wayne W , Bach Fritz H , Robson Simon C 
Published: July 2018

SummaryEndothelial cell ATP diphosphohydrolases or ATPDases degrade extracellular inflammatory mediators ATP and ADP, thus inhibiting the formation of platelet thrombi, but the modulation of these ecto-enzymes during vascular injury remains largely undetermined. Renal glomerular ATPDase levels were determined in the rat following ische-mia-reperfusion or systemic complement activation, by direct biochemical methods and histochemistry. Ischemia followed by reperfusion times over 30 min were associated with loss of glomerular ATPDase activity. Cobra Venom Factor (CVF) inhibited ATPDase activity and potentiated the deleterious effects of reperfusion. Treatment with either soluble complement receptor type 1 (sCRl), an inhibitor of complement activation, or antioxidants prior to the ischemia-reperfusion was largely protective. Expression of rat glomerular ATPDase activity appears susceptible to the inflammatory injury associated with systemic complement activation and ischemia/reperfusion processes. Oxidative stress could, at least in part, result in the loss of ATPDase activity and thus thrombotic consequences of vascular injury.
Heparin Stimulation of the Inhibition of Activated Protein C and other Enzymes by Human Protein C Inhibitor - Influence of the Molecular Weight of Heparin and Ionic Strength
Authors: Aznar Justo , España Francisco , Estellés Amparo , Royo Montserrat 
Published: July 2018

SummaryThe ability of unfractionated (UF) heparin and low-molecular-weight heparin (LMWH) to potentiate the inhibition of fibrinolytic and coagulation factors by protein C inhibitor (PCI) was studied. Inhibition of activated protein C (APC), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), thrombin, factor Xa (Xa), factor XIa (XIa) and plasma kallikrein (KK) by PCI was found to be dependent on the size of the polysaccharide. In general, maximal stimulation was reached with UF heparin, except in the case of KK. Differences in heparin stimulation were more pronounced for thrombin, APC, uPA, tPA and XIa, whereas inactivation of Xa by PCI was less dependent on the presence of heparin, and kallikrein showed higher potentiation with LMWH than with UF heparin. The second-order rate constants for enzyme inhibition by PCI were strongly dependent on the ionic strength, and, in general, with an ionic strength higher than 0.15 the heparin stimulation of the inhibition reactions was drastically reduced. These results may explain the large discrepancies in the literature on the effect of heparin on the stimulation of enzyme inhibition by PCI. They also show that LMWH is less efficient in stimulating the PCI inhibition of APC, uPA and tPA, which could contribute to the antithrombotic effect of these enzymes.
Differential Association of Protein Ser/Thr Phosphatase Types 1 and 2A with the Cytoskeleton upon Platelet Activation
Authors: Toyoda Hideki , Nakai Keiji , Omay Serdar B , Shima Hiroshi , Nagao Minako , Shiku Hiroshi , Nishikawa Masakatsu 
Published: July 2018

SummaryThe association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PPl-like activity was present in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold increase in PPl-like activity and a 7-fold increase in PP2A-like activity, respectively, in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed that thrombin treatment increased association of PP1 catalytic subunit isozymes (PPlα, PPlβ, PP1γ) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even in the absence of aggregation. Co-extraction of protein kinase C and myosin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results suggest that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoskeletal proteins may regulate phosphorylation levels together with protein kinases in platelets.
Detection of Platelet Adhesion/Aggregation to Immobilized Ligands on Microbeads by an Aggregometer
Authors: Minamoto Yoko , Hato Takaaki , Nakatani Shingo , Fujita Shigeru 
Published: July 2018

SummaryPlatelet aggregation is believed to follow platelet adhesion to vascular injury sites. We have developed a turbidimetric assay for platelet aggregation following platelet adhesion to immobilized ligands using an aggregometer. The addition of polystyrene beads coated with von Willebrand factor (vWF) or fibrinogen (Fg) to platelet suspensions caused prompt aggregation of beads and platelets, which was detected as an increase in light transmission. Electron microscopic analysis revealed that platelets adhered to the bead surfaces and that additional platelets adhered to already adhering platelets, leading to the formation of platelet aggregates. vWF-coated beads induced larger aggregates than Fg-coated beads. The interaction of vWF-coated beads with platelets was abolished by both GPIb and GPIIb-IIIa blockers, while that of Fg-coated beads was abolished by GPIIb-IIIa blockers. vWF-coated beads induced modest secretion of granules from platelets but no thromboxane B2 synthesis. Fg-coated beads induced neither reaction. However, pleckstrin phosphorylation and protein tyrosine phosphorylation was induced by both types of bead. Platelet aggregation following platelet adhesion to both types of bead was inhibited by ADP scavengers, a protein kinase C inhibitor and a tyrosine kinase inhibitor, but not by aspirin. These findings suggest that vWF- and Fg-coated beads can induce platelet aggregation following platelet adhesion through specific ligand-receptor interactions and intracellular signaling. Our simple assay using these beads may represent a useful test for immobilized ligand-induced platelet adhesion and aggregation.
Species Specific Immunoassays to Measure Blood Platelet and Coagulation Activation in the Rat
Authors: Ravanat C , Freund M , Schuhler S , Grunert P , Meyer L , Cazenave J-P 
Published: July 2018

SummaryThe purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thromboplastin (90 to 2400 μl/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.
Thrombin Activity Associated with Indwelling Central Venous Catheters
Authors: Francis Charles W , Felcher Andrew H , White Janice , Braaten Julie V , Goss Rachel 
Published: July 2018

SummaryThrombotic complications are frequent with indwelling central venous catheters and result in catheter dysfunction, vascular obstruction and may also contribute to catheter-associated infections. The pathogenesis of catheter thrombosis is not well characterized but may involve vessel damage, local stasis and catheter-associated thrombin formation. We have, therefore, measured the thrombin activity associated with central venous catheters removed from patients and have also determined the ability of hirudin to inactivate catheter-associated thrombin. We obtained 48 catheters from 46 patients and removed 1 cm portions for study. These were taken from the distal end, 5 cm proximal, and 15 cm proximal from the end. Following washing, thrombin activity was measured with a chromogenic assay. Thrombin was associated with 40 of 48 catheters and with 100 of 144 segments with a mean activity of 132 ± 27 μU/cm with a range of 0 to 2,160 μU/cm. Incubation in hirudin reduced the activity from a mean of 122 ± 33 μU/cm to 18 ± 6 μU/cm (p <.001). Scanning electron microscopy of selected catheters showed that some had areas of fibrin deposition which was not apparent visually. The findings indicate that indwelling central venous catheters frequently have associated thrombin activity which can be inhibited by a direct-acting thrombin inhibitor such as hirudin.
Fibrinolytic and Coagulant Responses to Regional Limb Perfusions of Tumor Necrosis Factor, Interferon-γ, and/or Melphalan
Authors: Merryman Paula , Tannenbaum Susan H , Gralnick Harvey R , Yu Kelvin , Arnold W Scott , Alexander H Richard , Fraker Douglas , Horne McDonald K 
Published: July 2018

SummaryRegional limb perfusion with antineoplastic agents stresses the local vasculature in a variety of ways. However, by monitoring the perfusates from limbs treated with melphalan alone or with melphalan plus tumor necrosis factor (TNF) and interferon-γ (IFN-γ), we were able to distinguish the effect of the cytokines on the observed coagulant and fibrinolytic responses. We collected samples of effluent from a series of lower extremities that were perfused with the cytokines and/or melphalan as treatment for localized melanoma. Both regimens produced statistically significant evidence of coagulant and fibrinolytic activation. However, limbs receiving cytokines in addition to the melphalan responded with a sharper rise in tissue plasminogen activator (tPA) and plasmin (plasmin-antiplasmin complexes [PAP]) than limbs treated with melphalan alone. Evidence of thrombin formation (prothrombin fragment 1+2 [FI+2], thrombin-antithrombin complexes [TAT]) was also greater when the cytokines were included, although the response was delayed and less consistent than the fibrinolytic activation.
The Effect of Progesterone on the Haemostatic Mechanism
Authors: Blombäck M , Landgren B-M , Stiernholm Y , Andersson O 
Published: July 2018

SummaryThe hemostatic effect of progesterone administered vaginally at a dose of 100 mg twice a day throughout one menstrual cycle was investigated and compared with the coagulation factors in one untreated normal menstrual cycle in 15 women. The progesterone treatment resulted in a 20-fold progesterone rise in the early follicular phase from 1.2 nmol/1 in the pretreatment control cycle to levels between 26 and 29 nmol/1 during treatment. Ovulation was completely suppressed in seven women while eight women showed a slight rise in progesterone on treatment days 20 to 25 not compatible with the rise which could have been expected if ovulation had occurred. The effects found on haemostasis during progesterone treatment varied with the menstrual cycle and were so small that they could as well be due to chance and not to treatment.
Lateral Mobility of Integrin αIIbβ3 (Glycoprotein IIb/IIIa) in the Plasma Membrane of a Human Megakaryocyte
Authors: Schootemeijer Annelies , van Willigen Gijsbert , van der Vuurst Hans , Tertoolen Leon G J , De Laat Siegfried W , Akkerman Jan-Willem N 
Published: July 2018

SummaryThe migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin αIIbβ3 (glycoprotein Ilb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the P3 subunit. The diffusion of P3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 X10'9 cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 μM) or α-thrombin (10 U/ml) at 22° C induced transient decreases in both parameters reducing D to 0.21 X 10‘9 cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, whereas cytochalasin D completely abolished the decrease in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile αIIbβ3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.
Isolation and Regulation of the cGMP-lnhibited cAMP Phosphodiesterase in Human Erythroleukemia Cells
Authors: Sheth S B , Brennan K J , Biradavolu R , Colman R W 
Published: July 2018

SummaryThe predominant cAMP phosphodiesterase in human platelets is the low Km cGMP-inhibited phosphodiesterase (PDE 3A). We have isolated native PDE3A from platelets and human erythroleukemia (HEL) cells and studied its kinetics. The platelet and HEL cell enzymes hydrolyze cAMP with Km = 0.5 μM. Incubation of cell supernatant with cAMP dependent protein kinase resulted in a rapid increase in activity within minutes, which resulted from a 2-fold decrease in Km with no increase in Vmax. HEL cells grown for 24 h in the presence of 50 (iM forskolin, an adenylate cyclase activator, demonstrate further increase in PDE3A of 274% of control (p = 0.03). Cells incubated with forskolin and cycloheximide or actinomycin D demonstrated no increase suggesting that cAMP stimulates PDE3A synthesis by transcriptional regulation. The results indicate that cAMP affects both the short and long-term regulation of PDE3A. The latter effect may play a role in the developing hematopoietic cell and the cardiovascular system to regulate cAMP levels.
Mortality and Causes of Death in Inherited Antithrombin Deficiency
Authors: van Boven H H , Vandenbroucke J P , Westendorp R G J , Rosendaal F R 
Published: July 2018

SummaryTo assess the contribution of inherited antithrombin deficiency to mortality, we investigated the causes of death in 14 families with inherited antithrombin deficiency. Between 1830 and 1994, 86 of 266 family members who had a probability of 0.5 or more for heterozygosity died. The causes of death were obtained for 58 of 66 deaths occurring between 1940 and 1994. Standardized mortality ratios (SMR) were calculated using mortality rates from the general population adjusted for age, sex and calendar period.The overall SMR was 0.90 from 1830 to 1994 (95% C.1.0.72-1.11). From 1940 until 1994 44 men and 22 women died (SMR = 1.09, 95% C.I. 0.84-1.39; SMR men = 1.20, 95% C.I. 0.87-1.61; SMR women = 0.92,95% C.I. 0.58-1.39). No excess mortality compared to the general population was found for cancer (14 deaths) or circulatory diseases (28 deaths). A slightly increased mortality caused by respiratory diseases (7 deaths, SMR = 1.68,95% C.I. 0.68-3.47) seemed due to pneumonia (4 deaths, SMR = 2.86, 95% C.I. 0.78-7.32). Venous thromboembolic complications were listed once in association with a risk situation, and one other death could be attributed to fatal pulmonary embolism. Cerebral hemorrhages were listed three times. It could not be verified whether these hemorrhages were related to anticoagulant therapy; the frequency was slightly higher than the expected population figure (SMR = 1.49,95% C.I. 0.31-4.36). The mean age of death for all causes was 64 years; the two fatal thromboembolic episodes occurred at age 20 and 30 years.The data show that antithrombin deficiency is associated with a normal survival and a low risk of fatal thromboembolic events. The use of long-term anticoagulant treatment in asymptomatic individuals should be considered carefully in view of the greater risk of fatal bleeding associated with long-term anticoagulant prophylaxis.
A Novel Chimaeric Derivative of Saruplase, rscu-PA-40kDA/Hir, Binds to Thrombin and Exerts Thrombus-specific Fibrinolysis in Arterial and Venous Thrombosis in Dogs
Authors: Schneider J , Hauser R , Hennies H-H , Korioth J , Steffens G , Wnendt S 
Published: July 2018

SummaryThe chimaeric molecule rscu-PA-40kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino acid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. 125I-radiolabelled M23 (0.03 µM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 |xM) and hirudin (0.001-10 µµM) concentra-tion-dependently displaced 125I-M23 from its binding to thrombin. Saruplase (up to 10 (iM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 ± 4 vs 25 ± 11 min) and maximal height of reperfusion blood flow (98 ± 21 vs 108 ± 15 % of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated 125I-fibrin radioactivity in thrombosed saphenous veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of 125I-fibrin in the venous thrombosis model was 91 ± 1 % in M23-and 88 ± 5 % in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and a2-antiplasmin levels were slightly reduced (-27 ± 3 %) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 ± 19 %) and a2-antiplasmin (-75 ±11%) plasma levels. Template bleeding times virtually did not differ before (2.8 ± 0.3 min) and 60 min after bolus injection of M23 (3.1 ± 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 ± 0.2 min to 28 ± 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin.
The Urokinase-receptor (CD87) Is Expressed in Cells of the Megakaryoblastic Lineage
Authors: Wohn Karl-Dieter , Kanse Sandip M , Deutsch Varda , Schmidt Thomas , Eldor Amiram , Preissner Klaus T 
Published: July 2018

SummaryMegakaryocytopoiesis is governed in the bone marrow microenvironment by cellular interactions that include various adhesion receptor systems and pericellular proteolysis for proper regulation of cell motility and differentiation. In order to define the role of cell surface molecules required for these processes, we searched for protease receptors on these cells. In an in vitro system utilizing different cell lines of the megakaryoblastic lineage (MEG-01, Dami), low level surface expression of the urokinase (uPA) receptor was noted. Following stimulation with phorbolester (PMA), a 3-6 fold higher expression of uPA receptor over a period of up to 5 days could be observed by fluorescent activated cell-sorting as well as by direct ligand-binding of amino-terminal fragment of uPA or vitronectin. Together with elevated expression of αIIbβ3-integrin (glycoprotein IIb/IIIa complex), double immuno-fluorescence staining of stimulated cells confirmed the increased cell surface localization of uPA receptor. Semi-quantitative RT-PCR, ligand blot analysis and measurement of cell-bound proteolytic activity revealed a differentiation-dependent upregulation of the uPA receptor expression in megakaryoblastic cell lines as in monocytic cells. Due to its glycolipid anchorage, incubation with phosphatidyl-inositol-specific phospholipase C reduced uPA receptor-mediated ligand binding by about 60%. uPA receptor mRNA was expressed in cultured megakaryocytes derived from bone marrow, whereas no uPA receptor mRNA was detectable in platelets. These results indicate a differentiation-dependent increase in the expression of uPA receptor in megakaryoblastic cells. The characteristics of surface expression and functionality of the receptor on megakaryocytic cells may influence their maturation by regulating cellular communication in the bone marrow micro-environment.
ADP-mimicking Platelet Aggregation Caused by Rugosin E, an Ellagitannin Isolated from Rosa rugosa Thunb
Authors: Teng Che-Ming , Kang Ya-Fei , Chang Ya-Ling , Ko Feng-Nien , Yang Shu-Chen , Hsu Feng-Lin 
Published: July 2018

SummaryAmong the nine ellagitannins, rugosin E was the most potent platelet aggregating agent with an EC50 of 1.5 ± 0.1 µM in rabbit platelets and 3.2 ±0.1 µM in human platelets. The aggregations caused by rugosin E and ADP were inhibited by EGTA, PGE1, mepacrine, sodium nitroprusside and neomycin, but not by indomethacin, verapamil, TMB-8, BN52021 and GR32191B. Rugosin E-induced thromboxane formation was suppressed by indomethacin, EGTA, PGE,, verapamil, mepacrine, TMB-8 and neomycin. ADP-scavenging agents, such as CP/CPK and apyrase inhibited concentration-dependently ADP (20 εM)-, but not rugosin E (5 εM)-induced platelet aggregation. In thrombin (0.1 U/ml)-treated and degranulated platelets, rugosin E and ADP still caused 63.5 ± 3.0% and 61.2 ± 3.5% of platelet aggregation, respectively. Selective ADP receptor antagonists, ATP and FSBA inhibited rugosin E- and ADP-induced platelet aggregations in a concentration-dependent manner. Both rugosin E and ADP did not induce platelet aggregation in ADP (1 mM)-desensitized platelets. In contrast to ADP, rugosin E did not decrease cAMP formation in washed rabbit platelets. Both rugosin E and ADP did not cause phosphoinositide breakdown in [3H]myo-inositol-labeled rabbit platelets. In fura-2/AM- load platelets, both rugosin E and ADP induced increase in intracellular calcium concentration and these responses were inhibited by ATP and PGEj. All these data suggest that rugosin E may be an ADP receptor agonist in rabbit platelets.