bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets
Authors: Siddiqua Ashia , Wilkinson Michael , Kakkar Vijay , Patel Yatin , Rahman Salman , Authi Kalwant 
Published: March 2018

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.
Gender Differences in the Relation between Fibrinogen, Tissue-type Plasminogen Activator Antigen and Markers of Insulin Resistance: Effects of Smoking
Authors: Balkau Beverley , Charles Marie-Aline , D’Hour Alain , le Mauff Jean-Michel ,   the D.E.S.I.R. Study Group, Mennen Louise 
Published: March 2018

SummaryA stronger positive association between fibrinogen or tissue-type plasminogen activator antigen (tPA-ag) and fasting insulin is observed in women than in men. We investigated whether this effect could be explained by a difference in smoking habits.The relations between markers for insulin resistance [fasting insulin and insulin resistance as estimated by the homeostasis model assessment (HOMA-IR)] and fibrinogen and tPA-ag were examined cross-sectionaly in 4976 (582 for tPA-ag) subjects from the D.E.S.I.R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) study. The modifying effect of smoking habits were evaluated.Seventeen percent of women and 27% of men were smokers. Fibrinogen concentrations were higher in smokers than in non-smokers, in men only. Female smokers had lower concentrations of tPA-ag than non-smokers. In both women and men, fibrinogen was positively associated with fasting insulin [women: β = 0.33 mg/U (95% confidence interval: 0.29, 0.37); men: β = 0.15 mg/U (0.11, 0.19)] and with HOMA-IR [women: β= 0.17 μg/μU mol/l (0.15, 0.19); men: β = 0.06 (0.04, 0.08)]. For tPA-ag these associations were for insulin β = 0.76 mg/U (0.54, 0.98) and β = 0.89 mg/U (0.67, 1.11), and for HOMA-IR β = 0.47 μg/μU mol/l (0.33, 0.61) and β = 0.45 μg/μU mol/l (0.33, 0.57), women and men respectively. The associations of fibrinogen and tPA-ag with insulin and HOMA-IR were sharply reduced in male smokers compared to male non-smokers, however the strength of the associations in male non-smokers did not reach that in women.Fibrinogen and tPA-ag are independetly related with markers of insulin resistance, with the relation with fibrinogen being stronger in women than in men. The strong modifying effect of smoking habits does not completely explain this gender difference.
The 4G4G Genotype of the Plasminogen Activator Inhibitor 4G/5G Gene Polymorphism Is Associated with Coronary Atherosclerosis in Patients at High Risk for this Disease
Authors: Lohre Jana , Katz Norbert , Tillmanns Harald , Hehrlein Friedrich Wilhelm , Haberbosch Werner , Gardemann Andreas 
Published: March 2018

Summary
Background. Disturbances in fibrinolytic activity, such as increase in plasminogen activator inhibitor (PAI) activity, have been linked with an increased risk for coronary artery disease (CAD) and myocardial infarction (MI). Since 4G4G homozygotes of an insertion/deletion (4G/5G) gene variation in the promoter of PAI-I have been shown to have increased levels of PAI-I, we analysed the relation of this gene polymorphism to CAD and MI in a population of 2565 participants who underwent coronary angiography for diagnostic purposes.
Results. In the total sample, the PAI-I 4G/4G genotype was associated with the presence, but not with the extent of CAD. However, in a subgroup of former and present smokers (n = 1782) or of individuals with a BMI above the mean value of 26.9 kg × m-2 (n = 1269), the PAI-I 4G4G genotype was not only associated with the presence, but also with the extent of CAD, defined either by the number of diseased vessels or by the CHD score according to Gensini. This observation also applied to other high-risk groups of individuals with high BMI and hypertension (n = 869), of subjects with high fibrinogen plasma levels (> 3.53 g × 1-1, mean value) and hypertension (n = 599) and of former and present smokers with high fibrinogen and hypertension (n = 452). An association of the gene variation with MI was not detected.
Conclusions. The present data indicate that the 4G/4G genotype of the PAI-I gene polymorphism is an independent risk factor for coronary artery disease and that the additional presence of major cardiovascular risk factors accelerates the risk for this disease.
Modulation of Cell-associated Plasminogen Activation by Stromelysin-1 (MMP-3)
Authors: Ugwu F. , Lemmens G. , Collen D. , Lijnen H. R. 
Published: March 2018

SummaryStromelysin-1 (MMP-3) cleaves a 55 kDa kringle 1-4 fragment, containing the lysine-binding site(s) involved in cellular binding, from 92 kDa plasminogen and removes a 17 kDa NH2-terminal fragment, containing the cellular receptor-binding site, from 45 kDa urokinase (u-PA), but a potential role of MMP-3 in the regulation of cellular fibrinolytic activity by affecting binding and/or activation of plasminogen and/or single-chain u-PA has not been established. Human plasminogen (input concentration 100 nM for 4 × 106 cells per ml) was shown to bind specifically to human monocytoid THP-1 cells, to murine MMP-3 deficient smooth muscle cells (SMC) and fibroblasts (1.9, 0.92 and 1.0 × 106 molecules per cell, respectively). Treatment with MMP-3 (final concentration 0-50 nM) of cells saturated with bound plasminogen (about 25 nM), overnight at 37° C, resulted in a dose-dependent reduction of the amount of u-PA activatible plasminogen (reduction to 25-40% of the value in the absence of MMP-3). Immunoblotting with specific monoclonal antibodies and autoradiography of eluates of the cells treated with MMP-3 revealed cleavage of plasminogen into the 55 kDa fragment and miniplasminogen (kringle 5 plus the proteinase domain).Binding of human single chain u-PA (scu-PA) to human THP-1 and HT 1080 cells amounted to 2.5 × 106and 7.1 × 106 molecules per cell, respectively. Treatment with MMP-3 (final concentration 0-25 nM) of cell-bound u-PA (about 17 nM for THP-1 and 47 nM for HT1080 cells), overnight at 37° C, did not alter cell-associated u-PA activity, measured in a direct chromogenic substrate assay or in a plasminogen-coupled chromogenic substrate assay (residual u-PA activity always ≥85% of that without MMP-3 treatment). Autoradiography of 125I-labeled u-PA moieties, removed from the cells by treatment with acid or with phosphatidylinositol phospholipase C, confirmed that u-PA remained essentially intact after MMP-3 treatment.These data indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity.
Platelet Compatibility of an Artificial Surface Modified with Functionally Active Heparin
Authors: Videm Vibeke , Christiansen Dorte , Bergseth Grethe , Riesenfeld Johan , Hovig Torstein , Mollnes Tom Eirik 
Published: March 2018

SummaryPlatelet compatibility after coating an artificial material with functionally active heparin was investigated. Blood was circulated in uncoated or heparin coated PVC tubing. In one hour platelet counts decreased from 155 (113-184) ×109/l to 124 (100-148) ×109/l with uncoated compared to 164 (132-192) ×109/l with heparin coated tubing (intergroup p = 0.02). β-thromboglobulin increased from 116 (80-148) μg/l to 1039 (757-1298) μg/l with uncoated and to 352 (229-638) μg/l with heparin coated tubing (intergroup p = 0.005). Platelet counts and β-thromboglobulin correlated closely with complement activation. Solid-phase enzyme immunoassay demonstrated substantial deposition of CD42a/GPIbIX and CD61/GPIIIa on uncoated, but not on heparin coated tubing (intergroup p <0.0005). Scanning electron microscopy demonstrated activated platelets and aggregates on uncoated in contrast to heparin coated tubing, where scattered, unactivated platelets were found. Changes in P-selectin and microparticles were minor. In conclusion, this heparin surface substantially improved platelet compatibility. Markers of choice for in vitro evaluation were platelet counts, β-thromboglobulin and platelet deposition.
The Collagen-binding Leech Products rLAPP and Calin Prevent both von Willebrand Factor and α2β1 (GPIa/IIa)-I-domain Binding to Collagen in a Different Manner
Authors: Deckmyn H. , Depraetere H. , Kerekes A. 
Published: March 2018

SummaryCalin and rLAPP are two natural inhibitors that are able to inhibit the vWF-binding and platelet adhesion to collagen both under static and flow conditions. In this study we demonstrate that both rLAPP and Calin prevent α2I-domain binding to human collagen type I with an IC50 of 5 μg/ml. However, although both vWF and α2I-domain binding to collagen is prevented by rLAPP and Calin, the latter two do not bind to the same collagen site since Calin only partially could compete with rLAPP for binding to collagen. Also vWF and the α2I-domain were unable to compete completely with each other for the binding to collagen. So the following hypothesis can be made: the binding sites of vWF and of the α2I-domain on human collagen type I are different but close to each other since rLAPP could inhibit both interactions, and thus should bind to an overlapping epitope. The Calin preparation on the other hand may still contain two active principles, one interfering with vWF-binding, the other with the α2I-domain-binding to collagen.
Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells
Authors: de Leeuw Hubert , Wijers-Koster Pauline , van Mourik Jan , Voorberg Jan 
Published: March 2018

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.
A Human Antibody that Binds to the γ-carboxyglutamic Acid Domain of Factor IX Is a Potent Antithrombotic In Vivo
Authors: Himber Jacques , Burcklen Louis , Moran Paul , Peek Mark , Suggett Shelley , Devaux Brigitte , Kirchhofer Daniel , Refino Canio 
Published: March 2018

Summary10C12, a human antibody F(ab’)2, which specifically binds to the Gla domain of factor IX, interfered with all known coagulation processes that involve factor IX/IXa. These include the function of the intrinsic Xase complex and the activation of zymogen factor IX by factor XIa and by the tissue factor:factor VIIa complex. Furthermore, 10C12 potently inhibited activated partial thromboplastin clotting times (APTT) in plasma of guinea pig and rat, thus enabling in-vivo evaluation. In guinea pigs, a bolus administration of 10C12 (10 μg/kg) prevented cyclic flow variations in damaged carotid arteries without affecting coagulation or bleeding parameters. At a 100-fold higher dose, 10C12 had no effect on normal hemostasis as assessed by the cuticle bleeding time. At this dose, 10C12 was also efficacious in a rat arterial thrombosis model, substantially reducing clot weight and duration of vessel occlusion while prolonging ex-vivo APTT only 1.2-fold. The dose of heparin required to produce comparable antithrombotic effects prolonged the APTT by 12-fold and increased the tail bleeding time (TBT) by 8-fold. In contrast, 10C12 had no effect on TBT. However, rat tails showed a tendency for rebleeding which 10C12 exacerbated. In conclusion, the antithrombotic potency of the 10C12 antibody in two species provides evidence for an important role of F.IX, and its Gla domain in particular, during thrombogenesis under arterial flow conditions. The relative safety at effective doses of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.
Abbreviations
Gla: γ-carboxyglutamic acid; CDRs: complementarity determining regions; CVFs: cyclic flow variations; TF:F.VIIa: tissue factor:factor VIIa; APTT: activated partial thromboplastin time; PT: prothrombin time; ACT: whole blood activated clotting time; CBT: cuticle bleeding time; TBT: tail bleeding time
dRVVT Is more Sensitive than KCT or TTI for Detecting Lupus Anticoagulant Activity of Anti-β2-glycoprotein I Autoantibodies
Authors: Biasiolo A. , Rampazzo P. , Brocco T. , Pengo V. 
Published: March 2018

SummaryAnti-β2-glycoprotein I (β2-GPI) antibodies behave as classical Lupus Anticoagulants (LA), as they inhibit phospholipid-dependent coagulation reactions and their activity disappears in the presence of excess exogenous phospholipids (PLs). We have recently shown that a certain amount of PLs in the dilute Russell Viper Venom Time (dRVVT) test system is required to express LA activity of anti β2-GPI antibodies. We have now extended this observation to two other tests,
i.e., Kaolin Clotting Time (KCT) in which PLs are not added, and Tissue Thromboplastin Inhibition test (TTI) in which PLs are extremely diluted. In fact, affinity-purified antibody preparations from 5 patients with antiphospholipid syndrome did not express or only weakly expressed anticoagulant activity in both tests; the mean ratios of coagulation times obtained with purified antibodies and that of control buffer were 1.11 and 1.0 for KCT and TTI, respectively. On the contrary, the mean ratios in dRVVT were 1.31 and 1.49 at a PLs dilution of 1:8 and 1:64, respectively. Therefore, the presence of LA activity due to autoantibodies to β2-GPI is characterized by a positive dRVVT and negative or only weakly positive KCT and TTI.
Effect of Pulse Pressure on Vascular Smooth Muscle Cell Migration: The Role of Urokinase and Matrix Metalloproteinase
Authors: Cahill Paul , Hirsch Mindy , Wang Yi-Ning , Sitzmann James , Okada Steve , Redmond Eileen 
Published: March 2018

SummaryPlasminogen activator (PA) expression plays an important role in smooth muscle cell (SMC) migration and may therefore contribute to mechanical force-induced arterialization of vein grafts. The aim of this study was to determine whether pulse pressure due to pulsatile flow modulates SMC migration via urokinase (u-PA)-dependent mechanisms. Using a perfused transcapillary culture system, human umbilical vein SMC were exposed to pulse pressures (0-56 mmHg), in the absence or presence of human umbilical vein endothelial cells (EC) by varying pulsatile flow rates (0 ml/min to 25 ml/min). SMC cultured in the absence of EC increased their migration following exposure to increased pulse pressure (248 ± 14%). Both u-PA and matrix metalloproteinase 1 (MMP-1) expression was significantly elevated in SMC exposed to pressure as compared to static controls. The role of proteases in the pulse pressure-induced enhancement of SMC migration was confirmed following pretreatment with aprotinin, an anti u-PA antibody and metalloproteinase inhibitors (181 ± 14% for aprotinin vs. 256 ± 25% for control, 108 ± 4% for anti-u-PA antibody vs. 233 ± 17% for non-immune IgG, and 114 ± 9% for BB-94, 105 ± 7% for BB-3103 vs. 222 ± 5% for control). Using SMC derived from u-PA gene knockout mice, the SMC migratory response to increased pulse pressure was completely inhibited despite a significant increase in MMP expression in these cells. These results suggest that pulse pressure due to pulsatile flow induces SMC migration in vitro via u-PA and MMP-dependent mechanisms. Moreover, u-PA gene deletion results in blunting of pressure-induced SMC migration despite the endogenous upregulation of metalloproteinase. Modulation of u-PA expression by pressure may thus represent an important mechanism whereby hemodynamic forces regulate smooth muscle cell migration.
Thrombomodulin Gene Defects in Families with Thromboembolic Disease – A Report on Four Families
Authors: Marlar Richard , Öhlin Ann-Kristin 
Published: March 2018

SummaryIt has been suggested that an impaired thrombomodulin (TM) function could constitute an abnormality leading to thromboembolic disease (TED). The TM gene from 51 unrelated American patients with TED and 100 American blood donors was screened for mutations. Four heterozygous point mutations in the TM gene were detected. The mutations are distributed throughout the TM gene and predict amino acid changes 1) Pro483 to Leu, 2) Gly61 to Ala, 3) Asp468 to Tyr (earlier described) and 4) a silent mutation not predicting any amino acid change at Glu163. Family studies reveal that the occurrence of the different TM mutations is associated with a history of TED, but there are indications of multiple risk factors and no perfect co-segregation of the TM defects and TED. Among the controls, three individuals carried heterozygous TM variants predicting either a Pro477-Ser mutation (two cases) or an Asp468-Tyr mutation. Our results thus demonstrate that a previously undocumented abnormality in the protein C anticoagulant pathway, a defect in the TM gene, to a certain extent co-segregates with familial thrombophilia. Further studies are needed to prove the causality of these TM mutations.
Is the Prevalence of the Factor V Leiden Mutation in Patients with Pulmonary Embolism and Deep Vein Thrombosis Really Different?
Authors: Karemaker Rosa , Kuijer Philomeen , Prins Martin , Büller Harry , Turkstra Franktien 
Published: March 2018

Summary
Introduction. Previous investigations have suggested a lower prevalence of the factor V Leiden mutation in patients with pulmonary embolism, as compared to patients with deep leg vein thrombosis. Methods. We studied unselected patients with pulmonary embolism, in whom we also assessed the presence of deep vein thrombosis by ultra-sonography. We assessed the prevalence of heterozygosity for the factor V Leiden mutation and compared the outcome of patients with a normal ultrasound (primary pulmonary embolism) to those with an abnormal ultrasound (combined form of venous thromboembolism). Furthermore, we performed a literature search to identify all articles regarding the prevalence of heterozygous factor V Leiden mutation in patients with primary deep vein thrombosis, primary pulmonary embolism and a combined form of venous thromboembolism. We calculated a (common) odds ratio for these 3 manifestations of venous thromboembolism, including the current findings. Results. In 92 patients with proven pulmonary embolism, 25 (27%) had also an abnormal ultrasound. In these patients, the prevalence of the factor V Leiden mutation was 24% (95% CI 9%-45%), whereas the mutation was present in 5 of 67 patients with primary pulmonary embolism (7%; 95% CI 2%-16%). The literature analysis indicated the common odds ratio for the presence of heterozygous factor V Leiden mutation in patients with primary deep vein thrombosis, primary pulmonary embolism and the combined form of venous thromboembolism to be 7.9 (95% CI 5-12), 3.5 (95% CI 2-6) and 6.8 (95% CI 3-14), respectively. Conclusion. In patients with primary pulmonary embolism the prevalence of the factor V Leiden mutation appears to be half of that reported in patients with primary deep vein thrombosis. The mechanism remains unclear.
Prothrombotic Genetic Risk Factors and the Occurrence of Gestational Hypertension with or without Proteinuria
Authors: Margaglione Maurizio , Colaizzo Donatella , Cappucci Giuseppe , Sciannamé Natale , Montanaro Sergio , Paladini Dario , Martinelli Pasquale , Minno Giovanni , Grandone Elvira 
Published: March 2018

SummaryGestational hypertension with or without proteinuria is a multi-factorial disease in which the presence of a hypercoagulable state has been suggested. The prothrombin G20210A, the Factor V (FV) Leiden mutations, and the C677T 5-10 methylenetethrahydrofolate reductase (MTHFR) polymorphism were investigated in 140 women with gestational hypertension and in 216 normotensive women from Southern Italy. Nine controls (4.1%) and 16 cases (11.4%; OR: 2.96, 95% CI: 1.27-6.91) carried the prothrombin A20210 allele. FV Leiden mutation was observed in 4 controls (1.8%) and 11 cases (7.9%; OR: 4.53, 95% CI: 1.41-14.53). The TT MTHFR genotype was found in 36 controls (16.6%) and 34 cases (24.4%; OR: 1.61, 95%CI: 0.96-2.74). The impact of potential confounding variables was evaluated using a logistic regression analysis. Nulliparity, Factor V Leiden and prothrombin A20210 carrier status resulted to be independent risk factors of having gestational hypertension with or without proteinuria. Imbalance of haemostasis, through prothrombotic genetic factors, may predispose to the occurrence of gestational hypertension.