bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells
Authors: Kokame Koichi , Miyata Toshiyuki , Sato Naoaki , Kato Hisao
Published: March 2018

Thrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.
The European Concerted Action on Anticoagulation (ECAA) Evaluation of a Set of Lyophilized Normal Plasmas to Establish the Normal Prothrombin Time for Coagulometer Systems
Authors: Barrowcliffe T. W. , van den Besselaar A. M. H. P. , Jespersen J. , Tripodi A. , Houghton D. , Poller L. ,   ECAA by the above steering committee
Published: March 2018

Establishing the mean normal prothrombin time (MNPT) from fresh samples for prothrombin ratios and INR often presents difficulty in selection and collection of donors. A set of seven lyophilized normal plasmas has therefore been prepared at the ECAA Central Facility and studied at 143 laboratories in sixteen European states using coagulometers in serial field exercises. All centres tested either the high ISI ECAA rabbit or low ISI ECAA human reference thromboplastin. The MNPT of fresh plasmas and means of the lyophilized samples were closely comparable with most routine rabbit thromboplastins. Using human thromboplastins means with the lyophilized normals were marginally but significantly longer and with the bovine Thrombotest significantly shorter than the MNPT of fresh plasmas causing alterations in INR. There was no appreciable effect on INR of 2.5 and 3.5 when lyophilized normals were substituted for fresh normals with the rabbit reagents.
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

We 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

A 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

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

Stromelysin-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

Platelet 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

Calin 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

In 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

10C12, 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

Anti-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

Plasminogen 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

It 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

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

Gestational 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.
A Randomised, Controlled Study of the Effects of Aerobic Exercise and Dietary Fish on Coagulation and Fibrinolytic Factors in Type 2 Diabetics
Authors: Mori Trevor , Puddey Ian , Beilin Lawrie , Burke Valerie , Morton Alan , Stanton Kim , Dunstan David
Published: March 2018

Type 2 diabetes is associated with disturbances in coagulation and fibrinolysis. Prospective studies show that increased tissue plasminogen activator (tPA) antigen increases the risk of cardiovascular mortality. The present study examined the hypothesis that combining a regime of moderate aerobic exercise with one daily fish meal as part of a low-fat diet (30% total energy) would improve coagulation and fibrinolytic factors in dyslipidaemic type 2 diabetic patients. In a randomised, controlled, 8-week trial, 55 sedentary type 2 diabetic subjects with serum triglycerides >1.8 mmol/l and/or HDL-C <1.0 mmol/l were randomly assigned to a low-fat diet (30% daily energy intake) with or without one fish meal daily (3.6 g ω3 fatty acids/day) and further randomized to a moderate (55-65% VO2max) or light (heart rate <100 bpm) exercise program. Plasma levels of fibrinogen, coagulation factor VIIc, tPA and plasminogen activator inhibitor (PAI-1) antigen were measured before and after intervention. In the 49 subjects who completed the study, the fish diet alone, moderate exercise alone and the combination of fish and moderate exercise all led to significant reductions in tPA antigen concentrations (-2.1 ng/ml, p = 0.02, -1.9 ng/ml, p = 0.03, -2.0 ng/ml, p = 0.01, respectively) compared to controls. In multivariate regression, changes in fasting blood glucose (positively) and erythrocyte ω3 fatty acid composition (inversely) were independent predictors of the change in tPA antigen. The fish diet alone contributed to a significant rise in coagulation factor VIIc compared to controls (4.9%, p = 0.02), which was prevented by moderate exercise. No significant effects on PAI-1 antigen and fibrinogen were seen. In view of recent epidemiological findings, the reduction in tPA antigen with both fish and moderate exercise in these dyslipidaemic type 2 diabetic patients could reflect a reduced thrombotic potential and decreased cardiovascular risk. Furthermore, a small, albeit significant, increase in coagulation factor VIIc associated with fish can be prevented by a concomitant programme of moderate exercise.
Antibodies to Factor XII Associated with Lupus Anticoagulant
Authors: Gallimore M. J. , Harris S. L. , Winter M. , Jones D. W.
Published: March 2018

Falsely low levels of factor XII (FXII) have been documented in patients who are lupus anticoagulant positive (LA+). In addition, we have previously noted a surprisingly high incidence (20.9%) of apparently true FXII deficiency in patients who were LA+. We have hypothesised that this may be partly due to the presence of antibodies to FXII.

The aim of the present study was to investigate whether LA+ patient plasmas contain antibodies directed either against FXII or FXII in association with phospholipids. Plasma samples from 60 blood donors, all LA negative, and 51 LA+ patients were tested using ELISA assays employing purified FXII, phosphatidylserine (PS) and phosphatidylethanolamine (PE). We have identified seven patients whose plasma contained either IgG or IgM that reacted with purified FXII in the absence of PS or PE. When PS was included in the assay system four additional patient plasmas were shown to contain either IgG or IgM that reacted with FXII. The plasma of one patient contained IgG that reacted with FXII both in the presence and absence of PS. There was no reactivity to FXII with either IgG or IgM when PE was included in the assay system. Affinity purified IgG from three patients whose plasma reacted with FXII in the ELISA assay in the absence of PS, gave a positive reaction in an immunoblot assay. These results suggest that FXII antibodies are present in a significant proportion of LA+ patients and may lead to an erroneous diagnosis of FXII deficiency.
Contribution of Erythrocytes to Thrombin Generation in Whole Blood
Authors: Peyrou V. , Lormeau J. C , Hérault J. P. , Gaich C. , Pfliegger A. M. , Herbert J. M.
Published: March 2018

Thrombin generation (TG) initiated by diluted tissue-factor was investigated in whole human blood, in platelet-rich plasma (PRP), in platelet-poor plasma (PPP), and in PPP supplemented with red blood cells (RBCs). TG was characterized by the lag time preceding the thrombin burst and by the endogenous thrombin potential (ETP). RBCs at normal haematocrit were found to influence the lag time to the same extent as platelets. When TG was carried out in PRP or in PPP + RBCs, both the ETP and lag time were dependent on the platelet count or on the haematocrit, but the shapes of the dose-response curves were different. The inhibition of TG in PPP+ RBCs by two direct thrombin and factor Xa inhibitors: hirudin and DX 9065A, and two antithrombin III (AT)-dependent anticoagulants: heparin and SR 90107A was found to be similar to that previously described in PPP and in PRP: hirudin and DX 9065A only delayed TG whereas heparin and SR 90107A both delayed and decreased TG. FACscan analysis following labelling with FITC-annexin V or with phycoerythrin-labelled anti-glycophorin A of samples taken in the course of TG initiated in PPP + RBCs showed that no significant haemolysis occurred and revealed that 0.51 ± 0.075% (mean ± sem, n = 3) of RBCs steadily exposed procoagulant phospholipids on their outer surface throughout the TG course. Furthermore, incubation of factors Xa and Va with washed RBCs sampled during TG in PPP +RBCs resulted in a significant and constant prothrombinase activity.

Taken together, these data show for the first time that normal RBCs may participate in the haemostatic process through exposure of procoagulant phospholipids.
Hormone Replacement Therapy and Circulating ICAM-1 in Postmenopausal Women
Authors: Alhenc-Gelas Martine , Oger Emmanuel , Plu-Bureau Geneviève , Scarabin Pierre-Yves
Published: March 2018

Hormone replacement therapy may reduce the risk of coronary heart disease but underlying mechanism has not been adequately explained. Recent data suggest that intercellular adhesion molecule 1 (ICAM-1) plays a critical role in early stage of atherosclerosis and may serve as a molecular marker for the development of arterial disease. We investigated the effects of oral and transdermal cyclic oestradiol combined with progesterone on plasma concentration of soluble ICAM-1 (sICAM-1). Thirty-seven healthy postmenopausal women were randomly assigned to receive either oral estradiol valerate or transdermal estradiol both combined with micronized progesterone or no hormonal treatment. Plasma sICAM-1 was assayed at baseline and after a 6-month period. Oral but not transdermal estradiol regimen significantly decreased mean value of sICAM-1 compared with no treatment. Differences in sICAM-1 levels between active treatments were significant. There were no significant changes in mean values of fibrinogen between the three groups. Our results show a favorable effect of oral estrogen plus progesterone on a soluble marker of vascular inflammation and may provide plausible explanation for a cardioprotective effect of hormone replacement therapy among healthy postmenopausal women.
Increased Levels of Factor VIII and Fibrinogen in Patients with Venous Thrombosis Are not Caused by Acute Phase Reactions
Authors: Kamphuisen Pieter , Eikenboom Jeroen , Vos Hans , Pablo Renee , Sturk Auguste , Rosendaal Frits , Bertina Rogier
Published: March 2018

Factor VIII activity (factor VIII:C) levels ≥150 IU/dl are associated with a 5- to 6-fold increased risk of venous thrombosis compared to levels <100 IU/dl, and fibrinogen levels ≥5.0 g/l increase the thrombosis risk 4-fold. These high levels are present in 25% resp. 3% of the patients with a first episode of venous thrombosis. These findings were based on measurements after the thrombotic event, so the factor VIII and fibrinogen levels in thrombosis patients may have been influenced by acute phase reactions or ongoing inflammatory responses. In the present study we measured plasma C-reactive protein (CRP) as a sensitive marker of an acute phase reaction in 474 thrombosis patients and 474 age- and sex-matched healthy controls, that were part of the Leiden Thrombophilia Study (LETS). Mean and median CRP levels were higher in thrombosis patients than in the controls, suggesting inflammation in some patients. CRP affected both factor VIII and fibrinogen levels, in patients and controls alike. After adjustment for the effect of CRP, high factor VIII:C levels still increased the thrombosis risk 6-fold and high fibrinogen levels 4-fold, which is for both very similar to the risk before correction for CRP levels. These results show that although systemic inflammation may be present in some of the patients, elevated levels of factor VIII:C and fibrinogen were in general not caused by acute phase reactions. This further supports a causal relationship between both high factor VIII:C and fibrinogen levels and venous thrombosis.
The Risk of Recurrent Venous Thromboembolism in Carriers and Non-carriers of the G1691A Allele in the Coagulation Factor V Gene and the G20210A Allele in the Prothrombin Gene
Authors: Schulman Sam , Sten-Linder Margareta , Wiman Björn , Egberg Nils , Johnsson Hans , Lindmarker Per ,   and the DURAC Trial Study Group
Published: March 2018

The results concerning the risk of recurrent venous thromboembolism (VTE) in carriers of the G1691A mutation in the coagulation factor V gene are not consistent and this risk in carriers of the G20210A polymorphism in the prothrombin gene has hitherto not been reported. We followed 534 patients for 48 months after their first episode of objectively documented VTE. The prevalence of the G1691A allele in 467 (87.5%) of the patients and in 207 controls was 25.3% and 8.2%, respectively, in heterozygote form and 2.4% and 0.5%, respectively, in homozygote form. The adjusted odds ratio (OR) for the first VTE was 4.4 (95% CI 2.6-7.8). The risk of recurrent VTE in heterozygotes was not statistically different from non-carriers (17.8% vs 17.6%), with 85% power to detect a hazard ratio of 2.35. Homozygotes had a significantly increased risk (p = 0.036) of recurrent VTE. The prevalence of the G20210A allele in 456 patients and 207 controls was 6.1% and 1.4%, respectively. The adjusted OR was 4.6 (95% CI 1.6-19.3) for the first VTE in 28 carriers of this polymorphism. The risk of recurrent VTE for these was not statistically different from non-carriers with an OR of 0.9 (95% CI 0.2-2.9).
Predictive Value of Coagulation Markers Concerning Clinical Outcome 90 Days after Anterior Myocardial Infarction
Authors: Kontny Frederic , Abildgaard Ulrich , Dempfle Carl-Erik
Published: March 2018

To study the predictive value of coagulation markers concerning clinical outcome, prothrombin fragment F1.2 (F1.2), fibrin monomer antigen (FM), D-Dimer (DD), and fibrinogen were measured in plasma samples drawn 2 and 7 days after acute myocardial infarction (AMI) in 314 consecutive patients randomized in a clinical trial of low molecular weight heparin (Dalteparin) (the FRAMI trial). Placebo-treated patients suffering death or new AMI within 90 days had significantly higher levels at day 2 of FM (Enzymun-Test FM), and DD (TINAquant D-dimer) (p = 0.001 and 0.02, respectively), but not F1.2 (Enzygnost F1.2 micro), relative to those without serious clinical events. At day 7 all three coagulation activation markers were significantly higher in patients with subsequent adverse clinical outcome. The Dalteparin group had significantly lower levels of these markers as compared to the placebo group. Left ventricular (LV) thrombus formation was not associated with changes in coagulation activation. However, patients with thrombus had significantly higher fibrinogen levels than those without thrombus (p = 0.004 day 2), independent of treatment group. Thus, markers of coagulation activation may be useful in stratification of patients when estimating risk for adverse clinical outcome after AMI. Furthermore, elevated fibrinogen levels are associated with increased risk of LV thrombus formation.
Elevated Levels of Plasmin-α2 Antiplasmin Complexes in Unstable Angina
Authors: Bayés-Genís Antoni , Guindo Josep , Oliver Artur , Badimon Lina , Fiol Miquel , Mateo Jose , Carles Souto Joan , Dominguez Jose , Fontcuberta Jordi , Bayés de Luna Antoni
Published: March 2018

The evidence of elevated levels of several biochemical markers of prothrombotic state in patients with unstable angina suggests that thrombus formation and lysis play a pivotal role in acute coronary syndromes. The clinical syndrome of unstable angina encompasses a variety of clinical presentations of transient episodes of myocardial ischemia. This study was designed to assess plasmin generation in different settings of unstable angina. Evidence of plasmin generation in patients with unstable angina was measured by circulating plasmin-α2 antiplas-min complexes (PAP). A second objective was to identify whether PAP levels had a prognostic value to predict outcome. Eighty-five patients admitted to the coronary care unit for unstable angina were classified into three groups. Group A included 26 patients with postinfarction angina; group B comprised 26 patients with new onset angina; and group C included 33 patients with crescendo angina. Mean PAP levels were higher in the three groups compared to healthy controls. A significant correlation was found between levels of PAP and D-dimer, particularly in postinfarction angina (r = 0.6; p <0.0005). This trial adds new insights into the pathophysiology of unstable angina. It demonstrates that plasmin is generated in the different settings of unstable angina but particularly in postinfarction angina patients where a fibrin-rich thrombus is responsible of the symptoms. However, in this series PAP levels do not predict an uneventful outcome neither in the acute phase nor at long term (6 months).

Abbreviations and acronyms: CABG: coronary artery bypass graft; CCU: coronary care unit; ECG: electrocardiogram; IABP: intraaortic balloon pump; MI: myocardial infarction; PAP: plasmin-α2 antiplasmin complexes; PTCA: percutaneous transluminal coronary angioplasty
Start of UK Confidential Haemophilia A Database: Analysis of 142 Patients by Solid Phase Fluorescent Chemical Cleavage of Mismatch
Authors: Waseem Naushin , Bagnall Richard , Green Peter , Giannelli Francesco ,   and the Haemophilia Centres
Published: March 2018

A national strategy for optimising genetic services in haemophilia A has been initiated in the UK. Solid phase fluorescent chemical cleavage of mismatch is used to screen the entire coding region of factor VIII in six segments: four amplified from the trace of mRNA in blood lymphocytes and two from genomic DNA for the 3.4 kb exon 14 and flanking intron sequences. These segments are analysed in two threefold multiplexes so that the genes of 18 patients can be screened in a single ABI 377 gel. The promoter and polyadenylation signal region are amplified and sequenced directly. We have analysed 142 unrelated patients and identified 141 factor VIII mutations and one Normandy type von Willebrand homozygote. The former mutations include 89 missense, 10 nonsense, 5 frameshift, one 24 bp deletion and one splice signal defect. These comprise 71 different changes, of which 39 have not been previously observed.
Age- and Sex-related Differences of Plasma Activated Factor VII Levels in Children
Authors: Sakata Toshiyuki , Yamamura Taku , Kario Kazuomi , Katayama Yoshiaki , Matsuyama Tatsuo , Kato Hisao , Yamamoto Akira , Miyata Toshiyuki
Published: March 2018

Although the majority of factor VII (FVII) circulates in the zymogen form, low levels of activated factor VII (FVIIa) exist in plasma and play an important role in the initiation of tissue factor-induced coagulation. It has been reported that the concentrations of hemostatic components in children and adults are different. Here, we investigated the age- and sex-related differences of FVIIa and FVII antigen (FVIIag) levels in children at age of 10 (n = 123; males: n = 62; females: n = 61), and 13 (n = 105; males: n = 47; females: n = 58), and compared these levels with those of young adults (mean ± SD of age: 29.6 ± 6.0 years). In males, there was not a significant difference between the FVIIa levels of 10-year-old children and 13-year-old children, but the FVIIa levels in adult controls were significantly higher than those in children. In females, the FVIIa levels in 13-year-old children were significantly higher than that in 10-year-old children, but there was not a significant difference between 13-year-old children and adults. Thus, our study showed that plasma FVIIa levels were lower in children than in adults, and the increase of FVIIa level was earlier in females than in males.
A Common 4G Allele in the Promoter of the Plasminogen Activator Inhibitor-1 (PAI-1) Gene as a Risk Factor for Pulmonary Embolism and Arterial Thrombosis in Hereditary Protein S Deficiency
Authors: Zöller Bengt , Frutos Pablo García de , Dahlbäck Björn
Published: March 2018

Reduced fibrinolytic capacity due to increased plasminogen activator inhibitor-1 (PAI-1) activity in plasma is a common finding in patients with coronary heart disease or venous thromboembolism, although its clinical significance is debated. Recently, a dimorphism in the PAI-1 promoter (4G-5G) has been reported and homozygosity for the 4G allele is associated with increased transcription and higher PAI-1 levels. Homozygous 4G genotype has been suggested to be a risk factor for myocardial infarction. In the present study, the 4G-5G dimorphism was determined in 349 individuals from 21 thrombophilic families with hereditary protein S deficiency and in 140 unrelated healthy controls. Among the 143 protein S deficient individuals, there was no relationship between deep or superficial venous thrombosis and the PAI-1 dimorphism. However, 26% (12/46) of individuals having protein S deficiency combined with homozygosity for the 4G allele had suffered pulmonary embolism as compared to 7% (7/97) of protein S deficient individuals carrying at least one 5G allele (p = 0.0019). In protein S deficient individuals, arterial thrombosis was found to be associated with smoking and 4G homozygosity. No association was found between the PAI-1 dimorphism and arterial or venous thromboembolism in family members without protein S deficiency. In conclusion, the PAI-1 genotype affects the phenotypic expression of thrombophilia in protein S deficient individuals.
Thrombomodulin Modulates the Mitogenic Response to Thrombin of Human Umbilical Vein Endothelial Cells
Authors: Lafay Monique , Laguna Reyes , Le Bonniec Bernard , Lasne Dominique , Aiach Martine , Rendu Francine
Published: March 2018

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 μM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.
Effect of a Synthetic Factor Xa Inhibitor, YM-60828, on Blood Vessel Patency in Combination with a Thrombolytic Agent and on Blood Loss from the Operation Site in a Rat Model of Arterial Thrombosis
Authors: Sato Kazuo , Hirayama Fukushi , Koshio Hiroyuki , Taniuchi Yuta , Matsumoto Yuzo , Kawasaki Tomihisa
Published: March 2018

We examined the adjunctive effect of a novel factor Xa inhibitor, YM-60828, on vessel patency and blood loss from the operation site after successful thrombolysis with a modified tissue-type plasminogen activator (moPA) in an electrically-induced carotid artery thrombosis model in rats. Five minutes after the induction of occlusive thrombus, a test drug (YM-60828, argatroban, heparin or saline) was administered by i.v. bolus injection followed by continuous infusion. Thrombolysis was induced with moPA by i.v. bolus injection at a dose of 650,000 IU/ kg. YM-60828 at 1 mg/kg i.v. followed by 3 mg/kg/h significantly prevented reocclusion, increased the duration of patency, and improved vessel patency after successful thrombolysis without any significant increase in blood loss from the operation site. Argatroban at 1 mg/kg i.v. followed by 3 mg/kg/h and heparin at 300 U/kg i.v. followed by 150 U/kg/h also significantly improved these parameters, but were accompanied by a significant increase in blood loss. These results suggest that the factor Xa inhibitor YM-60828 may be a potent and useful adjunctive agent with a lower risk of bleeding complications than argatroban and heparin in thrombolytic therapy.