bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation
Authors: Mosesson Michael 
Published: March 2018

SummaryThrombin substrate binding is mediated through fibrinogen recognition “exosite 1” in thrombin, resulting in fibrinopeptide cleavage to form fibrin. In addition, thrombin exhibits “non-substrate” binding to fibrin, an activity termed “Antithrombin I”. Antithrombin I (AT-I) is characterized by two classes of throm-bin binding sites, the first of “low affinity” in the fibrin E domain, and the other of high affinity, that is situated between C-terminal residues 414 and 427 of a variant γ chain termed γ’ 1-427L. Plasma fibrinogen molecules containing γ ’ chains (“fibrino-gen 2”) are virtually all heterodimers containing one γA chain (platelet-binding) and one γ’ chain. The remaining fibrinogen (~ 85%) is homodimeric, lacks high affinity thrombin-binding potential, and is termed “fibrinogen 1” (γA/γA). Thrombin generation in recalcified fibrinogen-depleted or congenital afibrinogenemic plasma is increased. Repletion with fibrino-gen 1 has a modest effect in normalizing thrombin generation, whereas repletion with fibrinogen 2 (γA/γ’) has a more marked effect. A post-translational γ’ chain derivative, γ’ 1-423P, accounts for 3%-34% of the γ’ chain population, lacks thrombin binding potential, and arises by proteolytic processing at the expense of γ’ 1-427L chains. Little is known about its effect on plasma AT-I activity under normal or pathological circumstances. In summary, fibrin formation (Antithrombin I) inhibits throm-bin generation in clotting blood by sequestering thrombin, and “high-affinity” thrombin-binding (i.e., via γ’ chains) plays a dominant role in this process. AT-1 should be considered when assessing the pathogenesis of thromboembolic disease.
Inherited Macrothrombocytopenia with Distinctive Platelet Ultrastructural and Functional Features
Authors: Ranelletti Franco , Maggiano Nicola , Ciabattoni Giovanni , De Cristofaro Raimondo , Landolfi Raffaele , Rocca Bianca 
Published: March 2018

SummaryWe report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65X109/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder.Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were approximately 2 standard deviations above control values, in spite of a reduced maximal biosynthetic capacity of thromboxane from giant platelets assessed in vitro during whole blood clotting.This inherited platelet disorder shows structural and functional features which allow to distinguish it from other syndromes associated with giant platelets. We also propose to include ultrastructural and cytoskeletal studies in the diagnosis as well as in the classification of inherited giant platelet disorders.
A Comparison of INRs Determined with a Whole Blood Prothrombin Time Device and two International Reference Preparations for Thromboplastin
Authors: van den Besselar A. M. H. P. 
Published: March 2018

SummaryOral anticoagulant therapy is usually monitored with the prothrombin time (PT) on citrate plasma samples. In recent years instruments have been developed for measurement of the PT in non-citrated whole blood. In the present study, the manufacturer’s calibration of one type of device (CoaguChek®) in terms of the international normalized ratio (INR) was evaluated by one laboratory. Three subsequent lots of test strips for the CoaguChek were investigated using blood samples from 56 coumarin-treated patients. Citrated plasma samples from the same patients were analysed with two international reference preparations for thromboplastin (IRP), i. e., rTF/95 (recombinant human) and RBT/90 (rabbit brain). There were statistically significant INR differences between CoaguChek and the international reference preparations (p <0.001), but the mean relative deviation of the INR was not greater than 0.104. Clinically relevant criteria were used to assess the agreement between the CoaguChek and the IRP results. Standard agreement ranged from 82% to 95%. It is concluded that these test strips achieved a clinically acceptable level of accuracy. Further studies of patient management with these strips are justified.
Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression
Authors: Lamfers M. L. M. , Wijnberg M. J. , Grimbergen J. M. , Huisman L. G. M. , Aalders M. C. , Cohen F. N. B. , Verheijen J. H. , van Hinsbergh V. W. M. , Quax P. H. A. 
Published: March 2018

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.
Desensitization of the Platelet Aggregation Response to ADP: Differential Down-regulation of the P2Y1 and P2cyc Receptors
Authors: Baurand Anthony , Eckly Anita , Bari Nadège , Léon Catherine , Hechler Béatrice , Cazenave Jean-Pierre , Gachet Christian 
Published: March 2018

SummaryPlatelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y1 receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADP β S. Platelets incubated for 1 h at 37° C with 1 mM ADP β S and resuspended in Tyrode’s buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADP β S treated platelets loaded with fura2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT2A receptor while adrenaline had no such effect. These results suggested that the refractory state of ADP β S treated platelets was entirely due to desensitization of the P2Y1 receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y1 and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y1 and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y1 sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y1 receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y1 receptor, where ADP β S treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y1 and P2cyc receptors are differently regulated during platelet activation.
Binding of von Willebrand Factor to Collagen Type III: Role of Specific Amino Acids in the Collagen Binding Domain of vWF and Effects of Neighboring Domains
Authors: van der Plas R. , Gomes Lucio , Marquart J. , Vink Tom , Meijers Joost C. , de Groot Philip , Sixma Jan , Huizinga Eric 
Published: March 2018

SummaryBinding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the threedimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain.In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD, indicating that the A2 domain somehow modulates binding of vWF to collagen type III.Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.
Early Embryonic Expression of Murine Coagulation System Components
Authors: Ong Kelly , Horsfall Wendy , Conway Edward , Schuh Andre 
Published: March 2018

SummaryThe development of the embryonic coagulation system, and its contribution to the maintenance of vascular integrity during the formation of embryonic blood vessels, remain poorly understood. We have characterized the temporal expression patterns of 27 hemostasis-related genes during murine development. We show that, although most coagulation and fibrinolysis-related factors are expressed coordinately b 7.5 dpc, several, including FIX, FXII and PAI2, are not detectable until later developmental timepoints. The expression of hemostasis-specific genes prior to the formation of a functional circulatory system supports the view that some coagulation factors have additional non-hemostatic functions during development. In addition, the discordant expression of some factors suggests that the embryonic hemostatic system may be distinct from that of the adult. These analyses will help to elucidate the regulation of hemostasis during embryonic/vascular development, and will provide a framework to facilitate the interpretation of gene inactivation studies.
Regulation of Murine Protein C Gene Expression In Vivo: Effects of Tumor Necrosis Factor-α, Interleukin-1, and Transforming Growth Factor-β
Authors: Shimokawa Takayoshi , Kojima Tetsuhito , Loskutoff David , Saito Hidehiko , Yamamoto Koji 
Published: March 2018

SummaryProtein C is a precursor of the anticoagulant serine protease, activated protein C, which inhibits coagulation factors Va and VIIIa. Although the liver appears to be the primary site of protein C synthesis, we previously demonstrated that the kidney and male reproductive organs also expressed abundant protein C mRNA in the mouse. In the present study, we further investigated the effects of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and transforming growth factor-β (TGF-β) on the expression of protein C mRNA in the principal producing organs, i.e., the liver, kidney, and testis. Both quantitative reverse transcription-PCR assay and in situ hybridization analysis revealed that TNF-α decreased protein C mRNA expression in the liver, kidney, and testis. IL-1 also down-regulated protein C mRNA expression in the liver and testis, but not in the kidney. In contrast, TGF-β unchanged the expression level of protein C mRNA in these three organs. These observations suggest that TNF-α and IL-1 may contribute to an increase in the procoagulant potential by down-regulation of protein C synthesis in the tissues during inflammatory processes.
Rapid Release of Matrix Metalloproteinase (MMP)-2 by Thrombin in the Rat Aorta: Modulation by Protein Tyrosine Kinase/Phosphatase
Authors: Fernandez-Patron Carlos , Zhang Yunlong , Radomski Marek , Hollenberg Morley , Davidge Sandra 
Published: March 2018

SummaryMatrix metalloproteinase-2 (MMP-2, gelatinase A) and thrombin contribute to many long-term (patho)physiological processes requiring the proteolytic breakdown of the vascular extracellular matrix (e.g., normal tissue repair, remodeling, tumor invasion, atherosclerosis plaque rupture). Thrombin (10 to 1000 nM, 0.5 to 50 U/ml) induced a rapid secretion of MMP-2 from freshly isolated rat aortic tissue (detectable after 1 min of thrombin exposure). This secretion was mediated by an unidentified thrombin receptor, distinct from the proteinase activated receptors (PAR)-1 and -2. Protein tyrosine kinase/phosphatase activity differentially modulated the basal and the thrombin-induced release of MMP-2. The inhibitors of protein tyrosine kinase, herbymicin A, genistein, and tyrphostin 1288 (1 to 100 μM), enhanced the basal release of MMP-2 but did not affect the thrombin-induced secretion of MMP-2. The inhibitor of phosphotyrosine phosphatases, vanadate (100 μM), selectively inhibited the thrombin-induced, but not the basal, release of MMP-2. Rapid release of vascular MMP-2 by thrombin could contribute to short-term processes where thrombin is involved such as the regulation of platelet aggregation and vascular reactivity. Vascular tyrosine kinase/phosphatase likely modulates this action of thrombin to prevent exaggerated platelet aggregation, thrombosis, and vasospasm.
RPR120844, a Novel, Specific Inhibitor of Coagulation Factor Xa Inhibits Venous Thrombosis in the Rabbit
Authors: Bentley Ross , Morgan Suzanne , Brown Karen , Chu Valeria , Ewing Richard , Spada Alfred , Pauls Henry , Perrone Mark , Dunwiddie Christopher , Leadley Robert , Bostwick Jeffrey 
Published: March 2018

SummaryThe in vivo antithrombotic activity of RPR120844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p <0.05 vs saline-treated animals) by RPR120844 at 30 and 100 μg/kg/min. At doses of 10, 30 and 100 μg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8 ± 0.3, 1.5 ± 0.9 and 2.4 ± 0.6 μM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.
Antiplatelet Activity of Clopidogrel in Coronary Artery Bypass Graft Surgery Patients
Authors: Limet R. , David J. L. 
Published: March 2018

SummaryClopidogrel is a recently introduced platelet ADP receptor antagonist, belonging to the thienopyridine derivatives, like its analogue ticlopidine. Its potential advantage is to be safer than ticlopidine. At 75 mg/od clopidogrel significantly inhibits platelet aggregation in ambulatory patients with symptomatic atherosclerotic disease and it prevents the recurrence of ischemic events more efficiently than aspirin. Its adequate dose in more acute situations remained to be determined. Therefore, sixty two patients with coronary artery disease were randomly assigned in four groups treated, within 24 h after coronary artery bypass graft, by clopidogrel 50 mg/od, 75 mg/od or 100 mg/od or by ticlopidine 250 mg/bid which was considered as the reference. The tolerance of clopidogrel was fairly good during the whole period of the study. Bleeding time and ex-vivo platelet aggregation induced by ADP 2 μM and 5 μM were performed at day -1, +9 and +28 after surgery. Like ticlopidine, the three dose levels of clopidogrel significantly inhibited ex-vivo platelet activity and prolonged the bleeding time at day 28. However, unlike ticlopidine, the inhibitory effects of clopidogrel were not significant at day 9, especially with 75 mg/od, a dose which was found to significantly protect patients in a chronic situation. Hence, although the clinical outcome for patients included in this limited study was the same in the four groups, these results suggest that the dose regime of clopidogrel should be more extensively investigated during the early period following coronary artery bypass graft, facing an overproduction of young and hyperreactive platelets. By analogy, the dose regime should be also investigated in other situations with an acute risk of arterial thrombotic occlusion.
Haematological Abnormalities in Early Abstinent Alcoholics Are Closely Associated with Alterations in Thrombopoietin and Erythropoietin Serum Profiles
Authors: Schmitt Marc , Gleiter Christoph , Nichol Janet , Pralle Lars , Hasselblatt Martin , Poser Wolfgang , Ehrenreich Hannelore 
Published: March 2018

SummaryNumerous reports exist on haematological pathology in alcoholism. However, no data are available regarding a potential involvement of haematopoietic growth factors in the recovery from alcohol-induced haematological abnormalities upon abstinence. Therefore, thrombopoietin (TPO) and erythropoietin (EPO) serum levels along with haematological and other routine laboratory parameters were closely followed in 14 thoroughly characterized male alcoholic patients over one to five months of controlled abstention from alcohol. Haematological changes in these early abstinent alcoholics consisted predominantly of (a) the well known rebound surge of platelets, (b) an early reticulocyte peak, and (c) persistently low haematocrit levels over months without signs of recovery. Observations on EPO and TPO during early abstinence can be summarized as follows: (1) Increased TPO levels precede the rebound thrombocytosis by several days, (2) both EPO and TPO concentrations are higher in anaemic than in nonanaemic alcoholics, with (3) nonanaemic subjects exhibiting levels of TPO in the range of healthy controls but levels of EPO below controls and (4) TPO concentrations show a stronger correlation with initial haematocrit values than with thrombocyte counts. To conclude, haematological recovery in early alcohol abstinence appears to be, at least in part, growth factor-driven, involving both TPO and EPO, and may reflect an intense interaction of erythro- and thrombopoiesis.
Comparison of Antiplatelet Effects of Two Nitric Oxide-donating Agents, FR146801 and FK409
Authors: Kato Yasuko , Fukuyama Shinichi , Ohno Mitsuko , Nishino Shigetaka , Kato Masayuki , Kita Yasuhiro , Hirasawa Yoshimi 
Published: March 2018

SummaryIn the present study, we examined the antiplatelet effects of the two nitric oxide (NO)-donating agents, (±)-N -[(E)-4-ethyl-3-[(Z)hydroxyimino]-6-methyl-5-nitro-3-heptenyl]-3-pyridinecarboxamide (FR146801), a more stable analog of FK409 ((±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide), and FK409 in in vitro and in vivo experiments. FR146801 and FK409 inhibited ADP- and collagen-induced platelet aggregation in human and rat platelet-rich plasma in a concentration-dependent manner, however, the inhibitory effect of FR146801 was weaker than that of FK409. In human washed platelets (WP), FR146801 and FK409 inhibited collagen-induced platelet aggregation in a concentration-dependent manner. The inhibitory effects of FR146801 and FK409 on platelet aggregation were closely reflected by the increase in the intraplatelet cGMP level. This intensely suggests that the antiplatelet activities of FR146801 and FK409 are due to NO-released from them. In the rat extracorporeal shunt model, FR146801 inhibited thrombus formation dose-dependently and its inhibition was significant at 10 mg/kg, p.o. FK409 suppressed thrombus formation significantly at 1.0 mg/kg, p.o., at which it induced significant hypotension, whereas FR146801 did not show any significant hypotensive effect even at 10 mg/kg, p.o. These results suggest that FR146801 has desirable antiplatelet effects both in vitro and in vivo and that its in vivo antiplatelet effect is more selective than its hypotensive effect, while FK409 does not show a selective antiplatelet effect in vivo.
Phospholipase A2 Modification Enhances Lipoprotein(a) Binding to the Subendothelial Matrix
Authors: Khaitan Alka , Fless Gunther , Hoover-Plow Jane 
Published: March 2018

SummaryLipoprotein(a), Lp(a), is found in the extracellular matrix in athero-sclerotic plaques, but with a different localization than LDL. A two-compartment system, with a monolayer of endothelial cells forming a barrier, was used to compare the transport, cell binding, and retention of Lp(a) and LDL into the subendothelial matrix. Baseline values for transport and retention of Lp(a) and LDL were not significantly different. Incubation with lipoprotein lipase or sphingomyelinase caused modest and similar increases in transport and retention of the two lipo-proteins. In contrast, incubation with phospholipase A2 (PLA2) resulted in a marked (4-fold) increase in retention of Lp(a) on the subendothelial matrix, but a lesser (2-fold) increase in LDL retention. Moreover, PLA2 treatment of Lp(a) enhanced its binding to individual matrix proteins (fibronectin, laminin, or collagen) by 4-10 times above that of LDL. The enzymatic activity of PLA2 was responsible for its effect on Lp(a) binding. The lysine binding sites of Lp(a) contributed to the increased binding of PLA2-modified Lp(a) to the matrix, and the enhanced lysine binding functions of PLA2-modified Lp(a) was demonstrated by two independent approaches. Thus, PLA2 modification leads to enhanced interactions of lipoproteins with the extracellular matrix, and this effect is more pronounced with Lp(a).