bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of pretreatment thrombocytosis on survival in primary breast cancer
Authors: Salat Andreas , Gnant Michael , Kwasny Werner , Mlineritsch Brigitte , Menzel Rainer-Christian , Schmid Marianne , Smola Michael , Stierer Michael , Tausch Christoph , Galid Arik , Steger Günther , Jakesz Raimund , Taucher Susanne
Published: March 2018

Platelet count has been reported to have predictive value in various cancer entities. In the case of breast cancer, evidence about involvement of platelets is still incomplete. Our objective was to assess the influence of pretreatment thrombocytosis on survival and establish its prognostic relevance for breast cancer patients.

We performed a retrospective, multivariate analysis of 4,300 patients with early-stage breast cancer. All subjects participated in one of five prospective, randomized, multicenter trials conducted by the Austrian Breast and Colorectal Cancer Study Group. Thrombocytosis was defined as a platelet count exceeding 400 G/L. Median follow-up was 52 months. Univariate and multiple Cox regression models were calculated for overall survival (OS), breast cancer-related survival and disease-free survival (DFS). Pretreatment thrombocytosis was observed in 161 patients (3.7%). Estimated median OS, breast cancer-related survival and DFS for patients with versus those without thrombocytosis was 71.0 versus 99.5, 72.0 versus 100.9, and 80.4 versus 88.4 months, respectively (p = 0.0054, p = 0.0095, p = 0.0199). A multiple Cox regression model including tumor and nodal status, grading, age, hormone receptor status and pretreatment thrombocytosis identified pretreatment thrombocytosis as an independent predictive factor for OS (p = 0.0064) and breast cancer-related survival (p = 0.0162). Multivariate analysis failed to identify pretreatment thrombocytosis as an independent risk factor for DFS (p = 0.1355).

In our retrospective study, elevated platelet counts at time of diagnosis were associated with poor prognosis in breast cancer. We hypothesize that platelets may contribute to the patho-physiology of hematogenous metastasis.
V34I and V34A substitutions within the factor XIII activation peptide segment (28-41) affect interactions with the thrombin active site
Authors: Trumbo Toni , Maurer Muriel
Published: March 2018

In the blood coagulation cascade, thrombin helps activate Factor XIII by cleaving the Factor XIII Activation Peptide at the R37-G38 peptide bond. The common polymorphism V34L yields a Factor XIII that is more easily activated than the wild-type enzyme. Peptides based on the Factor XIII (28-41) ( 28TVELQGVVPRGVNL41) sequence serve as an important model system for evaluating how to regulate thrombin activation of Factor XIII and subsequently fibrin clot character. Kinetic and NMR (1D proton line broadening and 2D transferred NOESY) studies have revealed that the P4-P1 region of the activation peptides is critical for binding to the thrombin surface. These results have led to an interest in exploring two new mutations at the P4 position including V34I and V34A. The V34I peptide was found to have the lowest Km of the peptides studied. However, unlike the V34L peptide, there are no P4 to P2 interactions observed in the transferred NOESY spectrum. The Leu thus promotes a bound conformation that cannot be achieved with the similar amino acid Ile. The V34A peptide exhibited a decrease in Km but also a substantial decrease in kcat. With this smaller amino acid, 1D proton line broadening NMR studies indicate that further contact of the Q32 residue with the thrombin surface is now possible. From these studies, valuable kinetic and structural information is being obtained to characterize interactions between thrombin and the Factor XIII activation peptide.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Formation of platelet microaggregates correlates with adverse clinical outcome in patients with coronary artery disease
Authors: Miyamoto Shinzo , Kawano Hiroaki , Sakamoto Tomohiro , Soejima Hirofumi , Kajiwara Ichiro , Shimomura Hideki , Kojima Sunao , Hokamaki Jun , Sugiyama Seigo , Hirai Nobutaka , Yoshimura Michihiro , Ozaki Yukio , Ogawa Hisao
Published: March 2018

Platelet activation plays a pivotal role in the pathogenesis of acute coronary syndromes. Laser-light scattering in a platelet aggregometer was used to evaluate aggregate size and number quantitatively. Small platelet aggregates ultimately develop into medium and then large platelet aggregates. Thus the measurement of small platelet aggregates is important in the evaluation of thrombus formation.

We examined the relationship between small platelet aggregates and the occurrence of subsequent cardiovascular events. We followed-up 204 patients (149 men and 55 women, mean age 68 ± 9 years) with coronary artery disease (CAD) for 48 months. Blood sampling to determine platelet aggregation was performed on the day of hospital discharge.

The degree of small platelet aggregates [relative risk 4.34, 95% confidence interval (1.62-11.7), p = 0.004] and low left-ventricular ejection fraction [relative risk 2.88, 95% confidence interval (1.23-6.73), p = 0.015] were independent predictors of the occurrence of cardiovascular events in multivariate Cox hazard analysis. In Kaplan-Meier analysis, the degree of small platelet aggregates correlated with the probability of cardiovascular event occurrence.

In patients with CAD, an increase in small platelet aggregates is closely correlated with the future occurrence of cardiovascular events
Blood Coagulation Factor X Deficiency Causes Partial Embryonic Lethality and Fatal Neonatal Bleeding in Mice
Authors: Dewerchin Mieke , Liang Zhong , Moons Lieve , Carmeliet Peter , Castellino Francis , Collen Désiré , Rosen Elliot
Published: March 2018

Mice with a total deficiency in blood coagulation Factor X (FX) were generated by targeted replacement of an 18-kb fragment of the FX gene, comprising all exons encoding the mature FX protein, with a neor
cassette. The genotype distribution among the offspring from heterozygous breeding pairs suggested that FX deficiency resulted in partial embryonic lethality, with approximately one-third of the FX
−/− embryos dying around embryonic day (E) 11.5-12.5. Two of 44 non-resorbed FX
−/− embryos analyzed at these stages showed signs of massive bleeding, one of which into the brain ventricles, but no histological defects in the vasculature of these embryos or their yolk sac were observed. The remainder of the FX
−/− embryos appeared normal and survived to term, but the majority of neonates (90%) died within 5 days, most frequently from intraabdominal bleeding. The remaining FX
−/− animals succumbed between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. The lethal phenotype of the FX
−/− mice illustrates the importance of FX function in embryonic and postnatal survival and demonstrates that these mice serve as effective models of the bleeding disorders observed in severe FX deficiency in humans.
Comparison of a Clinical Probability Estimate and Two Clinical Models in Patients with Suspected Pulmonary Embolism
Authors: Lijmer Jeroen , Mac Gillavry Melvin , Turkstra Franktien , Prins Martin , Büller Harry , Sanson Bernd-Jan ,   the ANTELOPE-Study Group
Published: March 2018

Recent studies have suggested that both the subjective judgement of a physician and standardized clinical models can be helpful in the estimation of the probability of the disease in patients with suspected pulmonary embolism (PE). We performed a multi-center study in consecutive in- and outpatients with suspected PE to compare the potential diagnostic utility of these methods. Of the 517 study patients, 160 (31%) were classified as having PE. Of these patients, 14% had a low probability as estimated by the treating physician, while 25 to 36% were categorized as having a low clinical probability with the use of two previously described clinical models. The objectively confirmed prevalence of PE in these three low probability categories was 19%, 28% and 28%, respectively. The three methods yielded comparable predictive values for PE in the other probability categories. We conclude that a physician’s clinical judgement alone and two standardized clinical models, although comparable, perform disappointingly in categorizing the pre-test probability in patients with suspected PE.
Outpatient Treatment of Pulmonary Embolism with Dalteparin
Authors: Anderson D. , Morrow B. , Gray L. , Touchie D. , Wells P. S. , Kovacs M. J.
Published: March 2018

Pulmonary embolism is a common complication of deep vein thrombosis. It has been established that low molecular weight heparin may be used to treat deep vein thrombosis or pulmonary embolism and randomized studies have established that outpatient management of deep vein thrombosis with low molecular weight heparin is at least as effective as in-hospital management with unfractionated heparin.

This was a prospective cohort study of eligible patients with pulmonary embolism managed as outpatients using dalteparin (200 U/kg s/c daily) for a minimum of five days and warfarin for 3 months. Outpatients included those managed exclusively out of hospital and those managed initially for 1-3 days as inpatients who then completed therapy o out of hospital. Reasons for admission included hemodynamic instability; hypoxia requiring oxygen therapy; admission for another medical reason; severe pain requiring parenteral analgesia or high risk of major bleeding. Patients were followed for three months for clinically apparent recurrent venous thromboembolism and bleeding.

Between three teaching hospitals, a total of 158 patients with pulmonary embolism were identified. Fifty patients were managed as inpatients and 108 as outpatients. Of the outpatients, 27 were managed for an average of 2.5 days as inpatients and then completed dalteparin therapy as outpatients. The remaining 81 patients were managed exclusively as outpatients with dalteparin. For all outpatients the overall symptomatic recurrence rate of venous thromboembolism was 5.6% (6/108) with only 1.9% (2/108) major bleeds. There were a total of four deaths with none due to pulmonary embolism or major bleed.

This prospective study suggests that outpatient management of pulmonary embolism is feasible and safe for the majority of patients.
Inherited Thrombophilia as a Risk Factor for the Development of Ischemic Stroke in Young Adults
Authors: Voetsch Barbara , Damasceno Benito , Camargo Erica , Massaro Ayrton , Bacheschi Luiz , Scaff Milberto , Annichino-Bizzacchi Joyce , Arruda Valder
Published: March 2018

Several recent studies have analyzed a possible effect of thrombophilia risk factors such as factor V Leiden, the prothrombin variant (allele 20210 A), and homozygosity for thermolabile methylenetetrahydrofolate reductase (MTHFR-T) on the development of ischemic stroke (IS). In the present study, we determined the role of these prothrombotic polymorphisms in the early onset of arterial IS or cerebral venous thrombosis (CVT) in a group of young Brazilian adults of Caucasian and African descent.

We conducted a cross-sectional study of 167 survivors of IS (153 patients with arterial IS and 14 cases of CVT; 66 men: 101 women; 124 of Caucasian and 43 of African origin; median age: 32.6 years; range: 15 to 45 years) and compared the prevalence of inherited thrombophilia risk factors with a control group of 225 sex and age matched individuals of the same ethnic background. To determine the interaction with atherogenic risk factors, the following diagnoses were considered: hypertension, hyperlipoproteinemia, diabetes mellitus, smoking status and use of oral contraceptives.

In the arterial IS group, no significant variation was found between patients and controls of Caucasian origin regarding the prevalence of factor V Leiden (P = 0.92), the prothrombin variant (P = 0.13) or homozygosity for MTHFR-T (P = 0.61). Among Brazilians of African descent, 10.3%were homozygous for MTHFR-T, which was significantly elevated, odds ratio of 5.9 (95%CI: 0.88 to 49.15). In the CVT group, two Caucasian patients (20%) were heterozygous for the prothrombin variant, odds ratio of 9.7 (95%CI: 0.95 to 89.71) and one patient was carrier of factor V Leiden (P = 0.49). No prothrombotic polymorphism was identified in patients with CVT of African descent. All women in the CVT group were in use of oral contraceptives or in the post-partum state

Inherited thrombophilia risk factors were not found to increase the risk of arterial IS among young patients of Caucasian descent. However, a potential role of homozygosity for MTHFR-T was observed in a small group of patients of African origin. The analysis of patients with CVT revealed an increased risk due to the prothrombin gene variant or oral contraceptive use. Further studies including all incoming patients with IS are necessary to evaluate the impact of inherited thrombophilia risk factors on early mortality.
Fast and Efficient Mutation Detection Method Using Multiplex PCR and Cycle Sequencing
Authors: Costa J. M. , Ernault P. , Vidaud D. , Vidaud M. , Meyer D. , Lavergne J. M.
Published: March 2018

A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR).
The Influence Exerted by a Restricted Phospholipid Microenvironment on the Expression of Tissue Factor Activity at the Cell Plasma Membrane Surface
Authors: Kunzelmann-Marche Corinne , Toti Florence , Zhang Youming , Nawroth Peter , Morrissey James , Freyssinet Jean-Marie , Satta Nathalie
Published: March 2018

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km
, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death.

Abbreviations: TF: tissue factor, FX: factor X, FVII(a): factor VII(a), PhtdSer: phosphatidylserine, PhtdCho: phosphatidylcholine, PhtdEth: phosphatidylethanolamine, BSA: bovine serum albumin, dcTF: des-cyto tissue factor (TF1-244: membrane-anchored TF lacking the cytoplasmic tail)
Thrombotic Variables and Risk of Idiopathic Venous Thromboembolism in Women Aged 45-64 Years
Authors: Woodward Mark , Vessey Martin , Rumley Ann , Gough Parimala , Daly Edel , Lowe Gordon
Published: March 2018

Hormone replacement therapy (HRT) has been shown to increase the relative risk of idiopathic venous thromboembolism (VTE) about threefold in several observational studies and one randomised controlled trial. Whether or not this relative risk is higher in women with underlying thrombophilia phenotypes, such as activated protein C (APC) resistance, is unknown. We therefore restudied the participants in a case-control study of the relationship between the use of HRT and the occurrence of idiopathic VTE in women aged 45-64 years. After protocol exclusions, 66 of the cases in the original study and 163 of the controls were studied. Twenty haematological variables relevant to risk of VTE were analysed, including thrombotic states defined from the literature. The relative risk of VTE showed significant associations with APC resistance (OR 4.06; 95% CI 1.62, 10.21); low antithrombin (3.33; 1.15, 9.65) or protein C (2.93; 1.06, 8.14); and high coagulation factor IX (2.34; 1.26, 4.35), or fibrin D-dimer (3.84; 1.99, 7.42). HRT use increased the risk of VTE in women without any of these thrombotic states (OR 4.09; 95% CI 1.26, 13.30). A similar effect of HRT use on the relative risk of VTE was also found in women with prothrombotic states. Thus for example, the combination of HRT use and APC resistance increased the risk of VTE about 13-fold compared with women of similar age without either APC resistance or HRT use (OR 13.27; 95% CI 4.30, 40.97).

We conclude that the combination of HRT use and thrombophilias (especially if multiple) increases the relative risk of VTE substantially; hence women known to have thrombophilias (especially if multiple) should be counselled about this increased risk prior to prescription of HRT. However, HRT increases the risk of VTE about fourfold even in women without any thrombotic abnormalities: possible causes are discussed.
Analysis of Prothrombotic Effects of two Human Monoclonal IgG Antiphospholipid Antibodies of Apparently Similar Specificity
Authors: von Landenberg Christine , Barlage Stefan , Schmitz Gerd , Lackner Karl
Published: March 2018

Two human monoclonal antiphospholipid antibodies (APA) of the IgG type, HL-5B and RR-7F have been generated from a patient with primary antiphospholipid syndrome and recurrent cerebral microemboli (H.L.) and from a patient with SLE without evidence of recurrent thrombosis (R.R.). Both monoclonal APA have similar characteristics in ELISA tests. To further analyse the prothrombotic potential, their effect on human monocytes and platelets, and bovine aortic endothelial cells (BAEC) was investigated. Monocytes were isolated from buffy coats by standard techniques. They were incubated either with the respective monoclonal APA in different concentrations, or a control monoclonal IgG of the same subtype, or plasma of the patients, from whom the antibodies were isolated. Incubation with LPS served as positive control. BAEC were grown to confluence, and then incubated with the appropriate agonists. Procoagulant activity (PCA) was determined by a single stage clotting assay. PCA expression after incubation is given as the ratio of the coagulation times observed with media only divided by that observed with the agonist. A PCA ratio >1 indicates the induction of PCA by the agonist. At 1 µg/ml HL-5B yielded a PCA ratio of 1.63 ± 0.16 while RR-7F induced no significant rise to 1.06 ± 0.18. Dose response curves showed that RR-7F can induce PCA at higher concentrations. However, its effect is approx. 1/50 of HL-5B based on equimolar antibody concentration. Further analysis indicates that the majority of the PCA induced by monoclonal APA can be inhibited by a specific tissue factor antibody. Neither monoclonal antibody induced PCA in BAEC. Sera from both patients were able to induce PCA in monocytes. However, the PCA ratio of serum from H.L. was higher (1.78) than that of R.R. (1.44). Neither monoclonal APA had an effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with 5 µM ADP or 15 µM TRAP-6. Similarly, there were no differences in platelet aggregation to different stimuli including submaximal activation. In summary, these data provide further evidence that induction of tissue factor in monocytes is one of the procoagulant effects of APA. Furthermore, the binding specificity of APA is perhaps not suited to predict the biological effects of the antibodies.
Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets
Authors: Ido Masaru , Hayashi Tatsuya , Gabazza Esteban , Suzuki Koji
Published: March 2018

Stimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PAR1 (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A2 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.
Aspirin Potentiates LPS-induced Fibrin Formation (FPA) and TNF-α-synthesis in Whole Blood
Authors: Haug K.B. , Joø G.B. , Westvik Å.-B. , Øvstebø R. , Kierulf P. , Osnes L.T.N.
Published: March 2018

The effect of aspirin on LPS-incubation of whole blood was investigated. Aspirin induced a concentration dependent increase (2.5–5-fold at 5 mM aspirin) in LPS-induced appearance of TNF-α and fibrinopeptide A (FPA) in plasma, despite the concomitant increase in the inhibitory cytokine IL-10. Aspirin substantially raised the levels of LPS-induced TF-mRNA and TNFα-mRNA in monocytes isolated from whole blood. The median ratio for TF-/β-actin mRNA increased from 1.5 ± 0.44 in the presence of LPS-alone, to 2.5 ± 0.51 when 5 mM aspirin was added. The TNFα/β-actin mRNA ratios were 1.8 ± 0.4 and 5.5 ± 2.7 respectively. Addition of exogenous PGE2 before incubation nearly abrogated the effect of aspirin on TNF-α, substantiating the role of PGE2 as a regulator of TNF-α synthesis, whereas the effect on FPA was small. Thus, in the presence of LPS in this whole blood model, aspirin apparently had a pro-inflammatory rather than an anti-inflammatory effect.
Platelets Release Thrombopoietin (Tpo) upon Activation: another Regulatory Loop in Thrombocytopoiesis?
Authors: Folman Claudia , Linthorst Gabor , van Mourik Jan , van Willigen Gijsbert , de Jonge Evert , Levi Marcel , de Haas Masja , von dem Borne Albert
Published: March 2018

Thrombopoietin is produced at a constant rate by the liver and kidney and is removed from the circulation upon binding and subsequent uptake via the Tpo receptor, c-Mpl, expressed by platelets and megakaryocytes. Apart from uptake, this study shows that platelets can also function as a storage pool for Tpo.

Upon stimulation with various platelet agonists, full-length biologically active Tpo was released by platelets. Platelet fractionation experiments indicated that this Tpo most likely is contained in the granules. When platelets were preincubated with Tpo-peptide mimetic or truncated Tpo prior to maximal activation, a three- to fivefold increment in Tpo release was seen, whereas, the release of other granule proteins such as vWF-propeptide or serotonin remained unchanged. Therefore, the Mpl agonists might compete with Mpl-bound Tpo, thereby releasing Tpo into the platelet supernatant.

Intravascular release of Tpo by platelets might occur in patients with massive platelet activation, as occurs in patients with disseminated intravascular coagulation. The Tpo concentration in these patients is elevated (p <0.01) and correlates with markers for thrombin generation, TAT complexes and Fl+2 (rp =0.8 and 0.9; p <0.01). This suggests that the increment in Tpo concentration was attributed to Tpo release by activated platelets in vivo, which might be instrumental in subsequent stimulation of thrombocytopoiesis.
Heparin Induces Synthesis and Secretion of Tissue Factor Pathway Inhibitor from Endothelial Cells
In Vitro
Authors: Svensson Birgit , Olsen Randi , Ezban Mirella , Østerud Bjarne , Paulssen Ruth , Hansen John-Bjarne
Published: March 2018

TFPI is a potent inhibitor of the extrinsic coagulation system constitutively synthesized by endothelial cells. A major portion of intravascular TFPI is stored associated with endothelial cells, and administration of unfractionated heparin (UFH) in vivo causes a prompt mobilization of TFPI into the circulation. The present study was conducted to investigate how UFH affected the synthesis, secretion and anticoagulant potency of TFPI in endothelial cells in vitro. A spontaneously transformed immortal endothelial cell line was used (ECV304). Stimulation of ECV304 cells with UFH caused a prompt dose-dependent (0-5 IU UFH/ml) release of TFPI to the medium accompanied by no change of TFPI at the surface membrane assessed by immunocytochemical methods. Northern blot analysis revealed two mRNA transcripts for TFPI with a molecular size of 1.4 kb and 4.4 kb, respectively. Stimulation of ECV304 cells for 24 hrs with various concentrations of UFH caused a dose-dependent increase of TFPI in the medium (6.2-29.6 ng/106 cells within the concentration range 0-10 IU/ml). A similar dose-dependent increase in the expression of both TFPI mRNA species was observed. Long-term incubation of ECV304 cells with 5.0 IU/ml UFH caused a 5-10 fold increase in the TFPI concentration accumulated in the medium over 48 hrs. The increased TFPI mRNA expression induced by UFH appeared already after 10 min, peaked after 2-4 hrs, remained augmented throughout the entire period of UFH exposure, and preceeded the synthesis-dependent increase in TFPI release by 2-4 hrs. The procoagulant activity of the cells was downregulated by 36 % and the contribution of TFPI to the anticoagulant potency of ECV304 cells was moderately increased after 24 hrs heparin stimulation. It is suggested that these mechanisms are of major importance for the anticoagulant function of heparins.
Increased Fibrinolytic Activity during Use of Oral Contraceptives Is Counteracted by an Enhanced Factor XI-independent down Regulation of Fibrinolysis
Authors: Middeldorp Saskia , Tekelenburg Winnie , van den Ende Abraham , Tans Guido , Prins Martin , Rosing Jan , Büller Harry , Bouma Bonno , Meijers Joost
Published: March 2018

The effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 µg ethinylestradiol, 150 µg levonorgestrel) or third generation OC (30 µg ethinylestradiol, 150 µg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-α2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.
Plasminogen Binding Properties of Macrophage Inflammatory Protein (MIP)-2α
Authors: Arza Begoña , Fábregas Pere , Laroche Yves , Collen Désiré , Lijnen Roger , Félez Jordi
Published: March 2018

The chemokine macrophage inflammatory protein (MIP)-2α was identified as a plasminogen binding protein by phage display analysis. MIP-2α and a truncated form lacking 5 lysine residues in the COOHterminal region (mut-MIP-2α) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2α but not mut-MIP-2α bound specifically to plasminogen, with KA of 3.7×105 M−1 for the interaction of plasminogen with surface-bound MIP-2α. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2α. Activation of plasminogen bound to surface-associated MIP-2α by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency kcat/KM of 0.1 µM−1s−1, as compared to 0.04 µM−1s−1). In contrast, binding of plasminogen to MIP-2α in solution was very weak, as evidenced by the absence of competition of MIP-2α with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2α did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissuetype plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by α2-antiplasmin. Thus, association of MIP-2α with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.
Antiphospholipid and Antiprotein Syndromes in Non-thrombotic, Non-autoimmune Women with Unexplained Recurrent Primary Early Foetal Loss
Authors: Quéré Isabelle , Sanmarco Marielle , Boutiere Brigitte , Mercier Eric , Amiral Jean , Hubert Anne-Michèle , Ripart-Neveu Sylvie , Hoffet Méderic , Tailland Marie-Laure , Rousseau Olivier , Monpeyroux Françoise , Dauzat Michel , Sampol José , Daures Jean-Pierre , Berlan Jacques , Marès Pierre , Gris Jean-Christophe
Published: March 2018

Various antiphospholipid and/or antiprotein antibodies have been suspected to be associated with recurrent early foetal loss in absence of any habitual aetiology. We conducted a hospital-based case control study on women with no antecedent of thromboembolic or autoimmune disease. We studied 3 groups of 518 women: patients with unexplained primary recurrent early foetal loss, patients with explained episodes and mothers with no previous obstetrical accident. Matching the 3 groups was carried out on the basis of age, number or pregnancies and time elapsed since the end of the last pregnancy. Significant biological markers were then prospectively tested.

The various antibodies were shown to be dependent on parity and on the presence of previous foetal loss: cut-off values were thus calculated using data obtained from the group of explained accidents, and adjusted for parity. Only anti-phosphatidylethanolamine IgM [odds ratio: 6.0, 95% confidence interval (2.3–15.7), p = 0.0003], anti-β2-glycoprotein I IgG [4.4, (1.6–11.7), p = 0.0035] anti-annexin V IgG antibodies [3.2 (1.2–8.1), p = 0.015] and lupus anticoagulant [3.0, (1.3–6.8), p = 0.009], were found to be independent retrospective risk factors for unexplained early foetal loss. These four markers were subsequently found to be, during the following pregnancy, associated with a significant risk of foetal loss despite a low-dose aspirin treatment.

In non-thrombotic, non-auto-immune women with unexplained primary reccurent early foetal loss, subgroups of patients with positive anti-phosphatidylethanolamine IgM antibodies, or positive anti- β2-glycoprotein-I IgG antibodies, or positive anti-annexin V IgG anti-bodies or lupus anticoagulant must be particularised. This should allow therapeutic trials to be carried in well-defined patients.
Modulation of Vascular Human Endothelial and Rat Smooth Muscle Cell Growth by a Fucosylated Chondroitin Sulfate from Echinoderm
Authors: Drouet B. , Matou S. , Mourão P. A. S. , Bros A. , Letourneur D. , Fischer A. M. , Tapon-Bretaudière J.
Published: March 2018

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 ± 5 µg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 µg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 ± 7.2%) and 27% (126.9 ± 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 µg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 ± 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 ± 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form, +98% (198.2 ± 25.%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.
Human Carotid Artery Smooth Muscle Cells Rarely Express αvβ3 Integrin at Sites of Recent Plaque Rupture
Authors: Yee Karen , Hatsukami Thomas , Schwartz Stephen , Ikari Yuji
Published: March 2018

Fibrin(ogen) is the major matrix ligand for β3 integrins. If αvβ3 is the major receptor for fibrin(ogen) on intimal smooth muscle cells, we might expect to see this integrin associated with fibrin(ogen). Eightyfour specimens obtained from endarterectomies of 14 patients were studied. Fibrin was frequently observed in carotid intima even at the non-atherosclerotic areas. As for β1 and β3 integrins, β1 was predominant in intima. The αvβ3 integrin expression was less frequent than α5β1, another receptor for fibrin(ogen), in diffuse intimal thickening, fibrous cap and advanced plaques. Furthermore, αvβ3 was generally negative on smooth muscle cells in recent plaque ruptures. In conclusion, we suggest more attention should be paid on abundant fibrin matrix in intima. Histologically, the α5β1 integrin rather than the αvβ3 is the major receptor for fibrin in intimal smooth muscle cells.
Predicting Adverse Outcome in Patients with Acute Pulmonary Embolism: A Risk Score
Authors: Wicki Jacques , Perneger Thomas , Bounameaux Henri , Junod Alain , Perrier Arnaud
Published: March 2018

Reliable prediction of adverse outcomes in acute pulmonary embolism may help choose between in-hospital and ambulatory treatment. We aimed to identify predictors of adverse events in patients with pulmonary embolism and to generate a simple risk score for use in clinical settings. We prospectively followed 296 consecutive patients with pulmonary embolism admitted through the emergency ward. Logistic regression was used to predict death, recurrent thromboembolic event, or major bleeding at 3 months. Thirty patients (10.1%) had one or more adverse events during the 3-month follow-up period: 25 patients (8.4%) died, thromboembolic events recurred in 10 patients (3.4%), and major bleeding occurred in 5 patients (1.7%). Factors associated with an adverse outcome in multivariate analysis were cancer, heart failure, previous deep vein thrombosis, systolic blood pressure <100 mmHg, arterial PaO2 <8 kPa, and presence of deep vein thrombosis on ultrasound. A risk score was calculated by adding 2 points for cancer and hypotension, and 1 point each for the other predictors. A score of 2 best identified patients at risk of an adverse outcome in a receiver operating characteristic curve analysis. Of 180 low-risk patients (67.2%) (score ≤2), only 4 experienced an adverse outcome (2.2%), compared to 23 (26.1%) of 88 high-risk patients (score ≥3). A simple risk score based on easily available variables can accurately identify patients with pulmonary embolism at low risk of an adverse outcome. Such a score may be useful for selecting patients with pulmonary embolism eligible for outpatient care.
Characterization of Cleaved Plasma Protein S with a Monoclonal Antibody-based Assay
Authors: Morbœuf Olivier , Borgel Delphine , Gaussem Pascale , Vincenot Anne , Pittet Jean-Louis , Aiach Martine , Gandrille Sophie
Published: March 2018

A monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i. e. the carboxyterminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.
Histidine-rich Glycoprotein (HRG) Tokushima 2: Novel HRG Deficiency, Molecular and Cellular Characterization
Authors: Yoshida Hidemasa , Kanagawa Yasuhiko , Satoh Koichi , Wakabayashi Sadao , Matsumoto Toshio , Koide Takehiko , Shigekiyo Toshio
Published: March 2018

The proband, a 76-year-old woman, suffered from dural arteriovenous fistula. Her plasma histidine-rich glycoprotein (HRG) level was 50% of the normal level. A low level of plasma HRG was also found in her third daughter. A single nucleotide substitution of T to C was found at nucleotide position 11,438 in exon 6 of the HRG gene from the proband, converting Cys223 to Arg in the second cystatin-like domain. The same mutation was also identified in her third daughter, but not in the other four family members having normal HRG levels or in 50 unrelated healthy Japanese individuals. Expression studies in BHK cells showed that substantial intracellular degradation of the mutant occurred and only about 40% of the recombinant HRG mutant was secreted. These results indicate that congenital HRG deficiency caused by a substitution of Cys223 to Arg is hereditary in this family.
Third Generation Oral Contraceptives and Heritable Thrombophilia as Risk Factors of Non-fatal Venous Thromboembolism
Authors: Andersen Birthe Søgaard , Olsen Jørn , Nielsen Gunnar Lauge , Steffensen Flemming Hald , Sørensen Henrik Toft , Baech John , Gregersen Henrik
Published: March 2018

Third generation oral contraceptives (OCs) are apparently stronger risk factors for venous thromboembolism (VTE) than other OCs, however, the increased risk may be due to confounding by indication related to differences in prescription behaviour.

We estimated the risk of VTE associated with use of OCs with and without the presence of Factor V Leiden mutation, protein C-, protein S- or antithrombin deficiency.

Sixty-seven cases with VTE were compared with 134 controls. The risk of VTE in the presence of thrombophilia was of the same magnitude for third generation OC users as for users of other OCs; OR: 52.5 (95% CI: 3.7-738.1) and OR: 63.3 (95% CI: 6.2-648.4), respectively.

It is unlikely that confounding by indication entirely explains the risk of VTE associated with third generation OCs since the combined effect exceeds what could be explained if this source of error was the only determinant of the association.
D-dimer Strategy in Thrombosis Exclusion
Authors: Trillaud H. , Labrouche S. , Gauthier P. , Javorschi S. , Bernard P. , Grenier N. , Freyburger G.
Published: March 2018

DD are now recognized as a valuable tool to screen patients suspected of deep venous thrombosis or pulmonary embolism before carrying out a gold standard radiologic examination. The newest methods available claim to be able to ascertain the absence of thrombosis, but they have yet to prove their efficiency. We compared the performances of 3 reference ELISA methods (D-DI Asserachrom™ Stago, D-dimer Enzygnost™ Behring and Dimertest GOLD EIA™ Agen), 5 recent rapid methods (VIDAS D-Dimer™ bioMérieux, Instant IA™ Stago, Simplired™ Agen, Nycocard D-dimer™ Nycomed and Accuclot D-Dimer™ Sigma Diagnostics) and two routine latex methods (Dimertest™ American Diagnostica and FDP-Slidex™ bioMérieux) in 100 patients. One of the rapid quantitative methods was demonstrated to have a level of efficiency comparable to that of ELISA methods. Finally, the cost and efficiency of different strategies were evaluated, the association of a routine latex method with the VIDAS D-Dimer™ bioMérieux being proven to be the most efficient.
Thrombophilic Genotypes in Subjects with Idiopathic Antiphospholipid Antibodies – Prevalence and Significance
Authors: Tommasino Catello , D’Andrea Giovanna , Iannaccone Luigi , Brancaccio Vincenzo , Margaglione Maurizio , Ames Paul
Published: March 2018

To evaluate the significance of common thrombophilic genotypes in subjects with idiopathic antiphospholipid antibodies (aPL) we determined the methylenetetrahydrofolate reductase C677→ (MTHFR) and factor V A506→ G (FV Leiden) polymorphisms in 49 subjects with idiopathic aPL (57% of whom suffered spontaneous vein thrombosis), in 70 subjects with a history of spontaneous vein thrombosis and in 193 healthy subjects. The prevalence of MTHFR C677→+/+ (homozygotes) was 25%, 18% and 17% respectively amongst aPL thrombotics, non aPL thrombotics and controls and that of MTHFR C677→+/– (heterozygotes) was 53%, 59% and 53% respectively in the same groups. The prevalence of FV Leiden was higher in aPL thrombotics (14%) and in non aPL thrombotics (18%) than in controls (4%) (p ≤ = 0.05). APL thrombotics with MTHFR C677→+/+ had a lower mean age at first thrombotic event (22 ± 6 years) than aPL thrombotics with MTHFR C677→+/– and non mutated considered together (38 ± 14 years, p = 0.0004) and than non aPL thrombotics with MTHFR C677→+/+ (38 ± 14 years, p = 0.003). FV Leiden may con tribute to the hypercoagulability of a small, albeit significant proportion of thrombotic aPL subjects, whereas the association between MTHFR C677→+/+ and aPL may have an impact on age at first occlusive event and suggests a possible pathogenetic interaction.
Clinical Significance of Neutrophil Adhesion Molecules Expression after Coronary Angioplasty on the Development of Restenosis
Authors: Sakai Yoshihiko , Fujito Tsuneo , Hoshi Kazuhiro , Hayashi Terumi , Takayanagi Kan , Morooka Shigenori , Inoue Teruo
Published: March 2018

To investigate the neutrophil activation process following percutaneous transluminal coronary angioplasty (PTCA), we examined the expressions of Mac-1 (CD11b/CD18), L-selectin (CD62L), and sialyl-LewisX (SLX) on the surface of neutrophils after the PTCA procedure, by flow cytometric analysis. Twenty-nine patients with single vessel coronary artery disease of the left anterior descending artery who underwent elective PTCA were enrolled. In the 17 patients without restenosis at the follow-up angiography, the mean channel fluorescence intensity (MFI) for CD18, CD62L and SLX did not change after PTCA. Only the CD11b level was increased at 48 h after the PTCA. In the remaining 12 patients who developed restenosis, the MFI values for CD18 and CD11b were increased at 24 h and 48 h after the PTCA. The MFI value for CD62L was decreased and that for SLX was increased at 48 h after the PTCA. These changes were more prominent in the coronary sinus blood samples than in those of the peripheral blood samples.

Our data indicate the down-regulation of L-selectin, probably by shedding, as well as the up-regulations of Mac-1 and sialyl-LewisX, especially in patients with restenosis. It is suggested that neutrophil activation by an interaction between the selectin family and carbohydrate ligands after PTCA may play a role in the development of restenosis, as does the integrin family.