bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunological Quantitation of Rabbit Plasminogen Activator Inhibitor-1 in Biological Samples
Authors: Ngo Thu-Hoa , Declerck Paul 
Published: March 2018

SummaryTwo immunoassays for the specific quantitation of rabbit plasminogen activator inhibitor-1 (PAI-1) antigen and activity in biological samples were developed and applied for the evaluation of PAI-1 in rabbits. Levels of PAI-1 antigen in rabbit plasma were 9.8 ± 4.6 ng/ml (mean ± SD, n = 6), with a corresponding value of 20.5 ± 13.5 ng/ml for PAI-1 activity. In rabbit serum PAI-1 antigen was 11.8 ± 4.9 ng/ml (n = 6) and PAI-1 activity was 2.9 ± 2.0 ng/ml (n = 6). Endotoxin injection (20 μg/kg, iv) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rabbit plasma, eventually resulting in a 40- to 90-fold increase (p <0.0001 vs baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.90, n = 6, p <0.05) and in plasma from endotoxin-treated rabbits (r = 0.98, n = 20, p <0.001). Analysis of PAI-1 antigen and activity in lysates of washed rabbit platelets revealed the absence of PAI-1 (i.e. <0.03 ng/108 platelets).In conclusion, development of specific immunological assays allowed the quantitation of PAI-1 in rabbit samples. In striking contrast to other species (human, rat, mouse, pig) rabbit platelets lack detectable amounts of PAI-1 (i.e. >100-1000 fold lower vs other species studied). This observation may have important implications for the use of experimental rabbit models especially in studies on the role of platelets in various pathological conditions including thrombosis and atherosclerosis.
Lysophosphatidic Acid Activates Nuclear Factor Kappa B and Induces Proinflammatory Gene Expression in Endothelial Cells
Authors: Robson Simon , Nehls Volker , Palmetshofer Alois 
Published: March 2018

SummaryThe cellular phospholipid, lysophosphatidic acid (LPA), released by activated platelets and fibroblasts or, at high levels, from ovarian and cervical carcinomas is a powerful serum mitogen that may modulate several signaling pathways in endothelial cells (EC). Hence, LPA could function in a paracrine manner during EC-platelet interactions at sites of vascular injury.Here, we demonstrate activation of the transcription factor nuclear factor kappa B (NF-κB) in EC following exposure to LPA. EC activation was further characterized by increased levels of mRNA transcripts encoding E-selectin, Intercellular Adhesion Molecule-1, Interleukin-8 and Monocyte Chemoattractant Protein-1. These effects were inhibited by preincubating EC either in the presence of mepacrine (to block phospholipase A2) or of pertussis toxin (to increase ADP-ribosylation of Gi proteins). No inhibition was observed in the presence of putative LPA receptor antagonists suramin or thrombospondin.LPA induces a proinflammatory activation of endothelial cells that (i) involves Gi proteins; (ii) depends on phospholipase A2 activity; (iii) is associated with the activation of NF-κB and (iv) results in increased expression of proinflammatory genes. We propose that LPA release by activated platelets may directly modulate vascular inflammatory responses.
Abbreviations: EC, endothelial cells; LPA, lysophosphatidic acid; NF-κB, nuclear factor kappa B; PA, phosphatidic acid; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1, ICAM-1, intercellular adhesion molecule-1, vascular adhesion molecule-1; GAPDH, glycerol aldehyde phosphate dehydrogenase; MAPK, mitogen activated kinase, MEK, MAPK kinase, PAEC, porcine aortic EC.
Clinical Utility of Prothrombin Fragment 1+2, Thrombin Antithrombin III Complexes and D-dimer Measurements in the Diagnosis of Deep Vein Thrombosis following Total Hip Replacement
Authors: Cofrancesco E. , Corradi A. , Ravasi F. , Bertocchi F. , Cortellaro M. 
Published: March 2018

SummaryBackground: Measurements of prothrombin fragment 1+2 (F1+2), thrombin antithrombin III complexes (TAT) and D-dimer plasma levels have been proposed as non-invasive screening tests to exclude postoperative deep venous thrombosis (DVT). We investigated the diagnostic efficacy of these coagulation activation markers to rule out postoperative DVT in patients undergoing hip surgery under antithrombotic prophylaxis. Methods: In this substudy of a randomized double-blind thrombosis prophylaxis trial comparing three doses of desirudin (10, 15 or 20 mg b.i.d.) with unfractionated heparin (5000 IU t.i.d.) we used ELISA procedures to measure F1+2, TAT and D-dimer in 159 patients undergoing total hip replacement at baseline (day 0) and on postoperative days 1, 3 and 6. Bilateral venography was performed in all cases 8-11 days after surgery. Results: For the F1+2 assay sensitivity ranged from 73 to 83% in the three postoperative days investigated, and negative predictive value (NPV) from 68 to 74%. For TAT and D-dimer sensitivity ranged from 71 to 73% and from 71 to 83% and NPV from 61 to 65% and from 61 to 74% respectively. Interpretation: In terms of sensitivity and NPV F1+2 and D-dimer are equivalent and are superior to TAT. However, their accuracy is too low to rule out the presence of DVT after hip surgery under antithrombotic prophylaxis.
Factor V Leiden and Fatal Pulmonary Embolism
Authors: Bertina Rogier , Holmes Zandra , Spaargaren Caroline , van Krieken Joannes , Manten Bert , Vandenbroucke Jan , Reitsma Pieter 
Published: March 2018

SummaryTo investigate whether the factor V Leiden mutation increases the risk of fatal pulmonary emboli, we determined the presence of the factor V Leiden mutation in pathology material from two series of autopsies of patients from the Leiden University Hospital, The Netherlands. The first series consisted of consecutive autopsies in which pulmonary emboli were mentioned in the autopsy report; most patients of this series had major underlying disease. The second series consisted of autopsies in patients younger than age 70 in which pulmonary emboli were the sole cause of death and no major acquired risk factor for venous thrombosis was present. Extraction of DNA was done on newly prepared tissue from archival paraffin blocks. In the first series, the presence of factor V Leiden was determined in 44 patients, 1 of whom carried the mutation (2.3 percent; 95% confidence interval 0.06 to 12.0 percent). This prevalence is not different from the general population prevalence in The Netherlands. In the second series, factor V Leiden could be determined in 30 patients of whom 3 carried the mutation (10 percent; 95% confidence interval 2.1 to 26.5 percent), which would lead to a threefold relative risk. A large number of patients with diverse psychiatric diagnoses was present in the second series (eleven). We conclude that in the presence of severe illness, the factor V Leiden mutation plays no additional role in the development of pulmonary emboli. The relative risk of the very rare fatal pulmonary embolus that is the sole cause of death might also be less than the relative risk for deep-vein thrombosis in carriers of the factor V Leiden mutation.
Platelet cNOS Activity Is Reduced in Patients with IDDM and NIDDM
Authors: Bruno Guido , Trucco Fulvia , Zumpano Elena , Tagliabue Milena , Bisceglie Cataldo Di , Pescarmona Gianpiero , Martina Valentino 
Published: March 2018

SummarySeveral studies in vitro and in vivo suggest that the nitric oxide (NO) production is impaired in diabetes mellitus. Reduced levels of NO could contribute to vascular alteration facilitating platelet-vascular wall interaction, adhesion of monocytes to endothelium, vascular smooth muscle proliferation and by decreasing endothelium-dependent vasodilation. In this study we evaluated the activity of the constitutive nitric oxide synthase (cNOS) in platelets of patients with insulin-dependent diabetes mellitus (IDDM) and with non-insulin-dependent diabetes mellitus (NIDDM). When compared to that of normal subjects, cNOS activity is significantly lower in patients with IDDM and with NIDDM (1.57 ± 0.25 vs. 0.66 ± 0.10 fmol/min/10 9 PLTs and 1.57 ± 0.25 vs. 0.67 ± 0.08, respectively; p <0.005). These data demonstrate that the platelet cNOS activity is decreased in diabetes mellitus.
Lonomia obliqua Caterpillar Spicules Trigger Human Blood Coagulation via Activation of Factor X and Prothrombin
Authors: Donato José , Moreno Ronilson , Hyslop Stephen , Duarte Alaor , Antunes Edson , Le Bonniec Bernard , Rendu Francine , Nucci Gilberto de 
Published: March 2018

SummaryIn southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.
Inhibition of Rat Platelet Aggregation by Mycalolide-B, a Novel Inhibitor of Actin Polymerization with a Different Mechanism of Action from Cytochalasin-D
Authors: Asai Fumitoshi , Saito Shinya , Ozaki Hiroshi , Fusetani Nobuhiro , Karaki Hideaki , Koike Hiroyuki , Sugidachi Atsuhiro 
Published: March 2018

SummaryIn vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 μM) but was inhibited by higher concentrations (3 and 10 μM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 μM). Potentiation of ADP-induced aggregation by MB (0.3 μM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 μM). In contrast, cytochalasin-D (CD) at 3 μM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 μg/ml) or ADP (10 μM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 μM) and CD (3 μM) abolished both ADP (10 μM)- and collagen (3 μg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 μM) had no effects on intracellular Ca2+ concentrations in ADP (10 μM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 μM) was inhibited by MB (0.3-3 μM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 μM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.
Enhancement of Tissue-type Plasminogen Activator-induced Thrombolysis and Prevention of Reocclusion by Combination with a Humanized Anti-glycoprotein IIb/IIIa Monoclonal Antibody, YM337, in a Rhesus Monkey Model of Coronary Thrombosis
Authors: Sato Kazuo , Suzuki Ken-ichi , Sakai Yumiko , Taniuchi Yuta , Kaku Seiji , Yano Shinya , Inagaki Osamu , Tomioka Kenichi , Masuho Yasuhiko , Yanagisawa Isao , Takenaka Toichi , Kawasaki Tomihisa 
Published: March 2018

SummaryWe examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 μg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.
A Glanzmann Thrombasthenia-like Phenotype Caused by a Defect in Inside-Out Signaling through the Integrin αIIbβ3
Authors: Shiraga Masamichi , Kinoshita Seiji , Ambo Hironobu , Kurata Yoshiyuki , Matsuzawa Yuji , Kunicki Thomas , Tomiyama Yoshiaki 
Published: March 2018

SummaryActivation of the platelet integrin αIIbβ3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of αIIbβ3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient‘s αIIbβ3„ while an activating anti-β3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient‘s αIIb and β3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced αIIbβ3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate αIIbβ3.
Fibrin Deposition in Squamous Cell Carcinomas of the Larynx and Hypopharynx
Authors: Bárdos Helga , Juhász Attila , Répássy Gábor , Ádány Róza 
Published: March 2018

SummaryExtravascular fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumor growth. The distribution of fibrin deposits was investigated in squamous cell carcinomas representing different stages of tumor progression of the larynx (n = 25) and hypopharynx (n = 9) by immunofluorescent techniques. Double and treble labelings were used to detect fibrinogen and fibrin in combination with marker antigens for tumor cells (cytokeratin), endothelial cells (von Willebrand factor), macrophages (recognized by KiM7), as well as factor XIII subunit A (FXIIIA) and tenascin (an embryonic extracellular matrix protein newly expressed during tumorigenesis). All tissue samples showed specific staining for fibrinogen/fibrin. Fibrin deposition was localized almost exclusively in the connective tissue compartment of tumors with characteristic accumulation at the interface of connective tissue and the tumorous parenchyma. In certain tumor samples showing highly invasive characteristics, fibrin deposits were observed in close association with tumor blood vessels in the tumor cell nodules. The overlapping reactions with polyclonal antibody to fibrinogen/fibrin and monoclonal antibody to fibrin indicate the activation of the coagulation cascade resulting in in situ thrombin activation and fibrin formation. Fibrin was crosslinked and stabilized by FXIIIA as revealed by urea insolubility test. Accumulation of phagocytozing macrophages detected by Ki M7 monoclonal antibody could be seen in areas of fibrin deposition. The blood coagulation factor XIIIA was detected in and around the cells labeled with Ki M7 antibody. Tenascin and fibrin deposits were found in the same localization in the tumor stroma and in association with tumor blood vessels within the tumor cell nodules. Neither fibrin nor tenascin were detected in the histologically normal tissue adjacent to tumors. The close association between fibrin deposits and macrophage accumulation strongly suggests the active participation of tumor-associated macrophages in the formation of stabilized intratumoral fibrin that facilitates tumor matrix generation and tumor angiogenesis.
Recombinant Antitrypsin Pittsburgh Undergoes Proteolytic Cleavage during E. coli Sepsis and Fails to Prevent the Associated Coagulopathy in a Primate Model
Authors: Taylor F. B. , Cadena R. A. DeLa , Courtney M. , Colman R. W. , Carrell R. W. , Harper P. L. 
Published: March 2018

SummaryDuring severe sepsis there is dramatic activation of both contact pro-teases and the coagulation pathway. These processes contribute to the development of shock and disseminated intravascular coagulation (DIC) respectively. The Pittsburgh mutant of antitrypsin (358Met-Arg) is a novel protease inhibitor with activity against both thrombin and the contact proteases and should therefore prove beneficial as a therapeutic agent in the management of septic shock. This hypothesis was supported by an earlier study in a pig model where recombinant antitrypsin Pittsburgh (rAT Pittsburgh) at a concentration of 1 μM alleviated some of the features of shock, but did not improve survival. In order to reduce the lethal effects of E. coli sepsis we postulated that a higher concentration of antitrypsin Pittsburgh would be necessary. To test this hypothesis we used rAT Pittsburgh in a primate model. This was chosen in preference to another species as E. coli sepsis in the primate has been well characterised and closely resembles the changes seen in man.Surprisingly this treatment did not alleviate the features of shock and unexpectedly appeared to exacerbate the associated coagulopathy. We propose two possible mechanisms for this unforeseen outcome. The first results from the broad spectrum of activity of antitrypsin Pittsburgh. As well as inhibiting thrombin and the contact proteases, the Pittsburgh mutant also inhibits activated protein C. Inhibition of the protein C system is known to exacerbate septic shock. Secondly, a significant quantity of inactive antitrypsin Pittsburgh, cleaved at the reactive centre, was detected in the plasma of the treated animals. Proteolytically altered serpins, including antitrypsin, have been shown to enhance the inflammatory process. Therefore the accumulation of cleaved rAT Pittsburgh might be expected to exacerbate septic shock.
Plasminogen Activator Inhibitor 1 Contains a Cryptic High Affinity Receptor Binding Site that Is Exposed upon Complex Formation with Tissue-type Plasminogen Activator
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Authors: Horn Ivo , van den Berg Birgit , Moestrup Søren , Pannekoek Hans , Zonneveld Anton-Jan van 
Published: March 2018

SummaryThe low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (KD) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in KD values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (KD values of 6.1, 3.7, 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the KD for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both 125I-t-PA:PAI-1 K69A and 125I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of 125I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation.
Plasma TAFI Levels Influence the Clot Lysis Time in Healthy Individuals in the Presence of an Intact Intrinsic Pathway of Coagulation
Authors: von dem Borne Peter , Meijers Joost , Bouma Bonno , Mosnier Laurent 
Published: March 2018

SummaryThrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.Abbreviations: The abbreviations used are: TAFI, Thrombin Activatable Fibrinolysis Inhibitor; TAFIa, activated TAFI; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; tPA, tissue-type plasminogen activator.