bluuebunny/crossref_metadata_2025 huggingface dataset:
15 Deoxy Δ12,14 PGJ2 Induces Procoagulant Activity in Cultured Human Endothelial Cells
Authors: Xie Shaoping , O’Regan David , Kakkar Vijay , Scully Michael
Published: March 2018

15 deoxy Δ12,14 PGJ2 (15d-PGJ2), a high affinity ligand of peroxisome proliferator-activated receptor γ (PPAR γ has been proposed to act as a negative feedback regulator of the inflammatory response. We investigated the effect of 15d-PGJ2 on the anticoagulant property of endothelial cells. 15d-PGJ2 stimulated a moderate but sustained increase in tissue factor (TF) activity in HUVECs and EA.hy926 cells while causing a partial loss of thrombomodulin (TM) activity. When cells were co-treated with 15d-PGJ2 and TNF-α, the subsequent elevation of TF activity was synergistically increased over that of cells treated with TNF-α, alone and the decline of TF activity after 24 h was less marked than TNF-α, alone. The induction of TF by 15d-PGJ2 alone and in combination with TNF-α, was reduced in the presence of PD 98059, suggesting the participation of the MEK/ERK pathway. The thiazolidinedione PPAR γ agonist ciglitazone had no effect on TF levels but reduced the expression of endothelial protein C receptor. The ability of 15d-PGJ2 to enhance a procoagulant phenotype arising from TNF-α, suggests a pro-inflammatory role for the prostaglandin.
Adipocyte Hypertrophy in Stromelysin-3 Deficient Mice with Nutritionally Induced Obesity
Authors: Van Hoef B. , Frederix L. , Rio M.-C. , Collen D. , Lijnen H. R.
Published: March 2018

Several matrix metalloproteinases (MMPs), including the stromelysins MMP-3 and MMP-11, are expressed in adipose tissue. To investigate a potential role of MMP-11 (stromelysin-3) in adipose tissue development, five-week-old male wild-type mice (MMP-11+/+) or mice with deficiency of MMP-11 (MMP-11−/−) were fed a high fat diet (HFD, 42% fat) for 15 weeks. Haematologic parameters, including white and red blood cells, platelets, haemoglobin and haematocrit, and metabolic parameters including glucose, triglycerides and total cholesterol were not different for both genotypes. At the time of sacrifice, the body weight of the MMP-11-/mice was higher than that of the MMP-11+/+ mice (36 ± 1.4 g versus 29 ± 0.9 g, p = 0.0002). The weight of the isolated subcutaneous (SC) and gonadal (GON) fat deposits was also higher in MMP-11−/−mice (620 ± 150 mg versus 280 ± 28 mg for SC fat, and 970 ± 180 mg versus 430 ± 62 mg, p < 0.05, for GON fat). Adipocytes in MMP-11−/−adipose tissue were hypertrophic as compared to MMP-11+/+ adipocytes (volume of 57 ± 12 × 103 µm3 versus 31 ± 2.4 × 103 µm3 for SC fat, and 100 ± 18 × 103 µm3 versus 57 ± 7.6 × 103 µm3 for GON fat; both p < 0.06). In nutritionally induced obesity models in mice a potential role of the fibrinolytic system was suggested in adipocyte hypertrophy. The hypertrophy observed in this model is, however, not related to changes in fibrinolytic parameters, as suggested by our finding that levels of t-PA, u-PA and PAI-1 antigen as well as t-PA and u-PA activity were not different in SC or GON adipose tissue extracts of both genotypes. As the main biological function of MMP-11 remains unknown, it is not clear whether the adipocyte hypertrophy in MMP-11−/−adipose tissue is directly related to the deficiency or to other pathways affected by MMP-11.
No Association of Plasma Prothrombin Concentration or the G20210A Mutation with Incident Cardiovascular Disease
Authors: Smiles Adam , Jenny Nancy , Tang Zhonghua , Arnold Alice , Cushman Mary , Tracy Russell
Published: March 2018

Prothrombin is a key factor in blood clotting, a process intimately involved in thrombotic disease. We assessed prothrombin levels and G20210A genotype in a case-control study within the Cardiovascular Health Study. Cases included angina, myocardial infarction, stroke, and the presence of MRI-detectable infarcts (n ≈ 250 each). Populationbased controls free of clinical cardiovascular disease (CVD) (n ≈ 500) and a subset free of clinical and subclinical CVD (n ≈ 250) were used for comparison. The 20210 A allele, frequency 2.9%, was associated with higher mean prothrombin levels: 166.3 vs. 139.5 µg/ml (P <0.001). Significant correlates of prothrombin included gender, plasma lipids, other vitamin K-dependent proteins, and inflammatory markers, but not race, smoking, hypertension, diabetes, measures of subclinical CVD, or markers of procoagulant activity. Compared to controls, neither genotype nor prothrombin level was associated with any CVD case group. We conclude that, in the elderly, neither prothrombin level nor 20210 genotype were associated with either CVD risk factors or events. This is consistent with the lack of association of prothrombin levels with measures of underlying CVD or procoagulant markers.
Antibodies to Heterologous Proteins in Hemophilia A Patients Receiving Recombinant Factor VIII (Recombinate™)
Authors: Ingerslev Jørgen , Christiansen Kirsten , Ravn Hanne , Gomperts Edward , Bray Gordon ,   The Recombinate Study Group
Published: March 2018

As a consequence of the manufacturing process, trace quantities of Chinese hamster ovary cell protein, bovine serum albumin and murine immunoglobulin G are present in Recombinate™ recombinant human factor VIII (rhFVIII). The development of antibodies (Abs) to these heterologous proteins was evaluated during long-term rhFVIII therapy of hemophilia A in 68 previously treated and 73 previously untreated patients. Ab prevalence was also assessed in 157 non-hemophilic subjects. Abs against heterologous proteins could be detected in varying percentages of patients and non-hemophilic subjects. Abs arose in patients sporadically, and levels were typically low. There were no adverse events associated with development or presence of anti-heterologous protein Abs. These data indicate that sustained immune responses to trace levels of heterologous proteins are very infrequent during long-term rhFVIII therapy.
Nonsense Mutation in Exon-19 of GPIIb Associated with Thrombasthenic Phenotype. Failure of GPIIb(Δ597-1008) to Form Stable Complexes with GPIIIa
Authors: Arias-Salgado Elena , Tao Jianming , González-Manchón Consuelo , Butta Nora , Vicente Vicente , Ayuso Matilde , Parrilla Roberto
Published: March 2018

We report the molecular genetic analysis of a patient with thrombasthenic phenotype. The lack of surface platelet GPIIb-IIIa complexes and the presence of GPIIIa suggested it was a case of type I Glanzmann’s thrombasthenia due to a mutation in GPIIb. Single stranded conformational polymorphism analysis (SSCP) of exon-19 of GPIIb showed polymorphic DNA bands. The DNA sequence of exon-19 revealed the presence of a homozygous C1882T transition that changes residue R597 to STOP codon. Since no other mutations were found in either GPIIb or GPIIIa it is concluded that the C1882T substitution in GPIIb is responsible for the thrombasthenic phenotype of the patient. The lack of platelet GPIIb-mRNA in the proband indicates instability of the [C1882T]GPIIb-mRNA. Coexpression of normal GPIIIa and GPIIb(Δ597-1008) in CHO cells failed to show surface expression of GPIIb(Δ597-1008)-IIIa complexes. Immunoprecipitation analysis demonstrated that GPIIb(Δ597-1008) may indeed complex GPIIIa; however, the association is either unstable or incapable of progressing along the secretory pathway.
Release from Intracellular Stores Is Responsible for Calcium Signaling with von Willebrand Factor in Human Platelets
Authors: Milner Elizabeth , Zheng Qi , Kermode John
Published: March 2018

Interaction of von Willebrand factor (VWF) with the platelet promotes hemostasis upon vascular injury. Such interaction raises intracellular free calcium concentration ([Ca2+]i) and induces platelet activation. The platelet [Ca2+]i increase is generally attributed to influx across the plasma membrane. The present study defined the contribution of intracellular calcium stores. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of VWF transiently elevated [Ca2+]i after a lag phase. Studies with 63 healthy donors consistently revealed a VWF-induced platelet [Ca2+]i signal in the absence of extracellular calcium; there was only modest enhancement with extracellular calcium. Blockade of plasma membrane calcium channels did not diminish the signal, whereas depletion or blockade of the intracellular calcium stores abolished it. These findings imply that release from intracellular stores is responsible for the VWF-induced platelet [Ca2+]i increase. Influx across the plasma membrane plays no more than a minor role, probably representing “capacitative entry” to refill the intracellular stores.
Identification of Critical Residues for Ligand Binding in the Integrin β3 I-domain by Site-directed Mutagenesis
Authors: Yamanouchi Jun , Tamura Tatsushiro , Fujita Shigeru , Hato Takaaki
Published: March 2018

To define the structural basis of ligand recognition by αIIb β3, we conducted site-directed mutagenesis of residues located on the top surface of the β3 I-domain that is homologous to the I-domain of several α subunits and contains a putative ligand binding site. Here we identify D158 and N215 in β3 as novel residues critical for ligand binding. Alanine substitution of D158 or N215 abolished binding of a ligand-mimetic antibody and fibrinogen to αIIb β3 induced by different types of integrin activation. CHO cells expressing recombinant αIIb β3 bearing D158A or N215A mutation did not adhere to fibrinogen. These mutations had the same effect on ligand binding to another β3 integrin, αV β3. Compared to the αI-domain structure, the βB-βC loop containing D158 in the β3 I-domain is quite different in length and sequence. These results suggest that the structure for ligand recognition is different in the βI- and αI-domains.
Prothrombin G20210A is a Bifunctional Gene Polymorphism
Authors: Carter Angela , Sachchithananthan Mythily , Stasinopoulos Stan , Maurer Fabienne , Medcalf Robert
Published: March 2018

The G20210A polymorphism has been shown to alter the efficiency of prothrombin mRNA processing. Here we show that the G20210A mutation also alters prothrombin mRNA stability. Three-fold more prothrombin protein and mRNA were produced in NIH-3T3 cells transfected with the prothrombin cDNAs containing the 20210A variant compared to cells expressing the 20210G variant. mRNA stability assays using chimeric globin transcripts harboring the G or A variant of the 97 nt prothrombin 3’-UTR indicated that the 20210G variant conferred greater instability to the globin reporter transcript than the A variant in transfected HepG2 cells. Both variants of the prothrombin 3’-UTR were shown to provide binding sites for a number of cellular proteins including HuR, an RNA binding protein associated with mRNA stability. Our results indicate that the G20210A is a bifunctional polymorphism, as it not only alters the efficiency of mRNA processing, but also the decay rate of prothrombin mRNA.
The Parasitic Hematophagous Worm Haemonchus contortus Inhibits Human Platelet Aggregation and Adhesion: Partial Purification of a Platelet Inhibitor
Authors: Crab Annick , Noppe Wim , Pelicaen Conny , Van Hoorelbeke Karen , Deckmyn Hans
Published: March 2018

Blood sucking parasites elaborate mechanisms to counteract the hemostatic system of their victim. Haemonchus contortus worms use several mechanisms directed against the normal platelet hemostatic function. Platelet adhesion onto collagen and fibrinogen, and the ristocetin-mediated interaction of von Willebrand Factor with glycoprotein (GP) Ib were inhibited by the protein extract of adult worms. Also platelet aggregation induced by collagen, thrombin, ADP, ristocetin or A23187 was inhibited. Although we obtained evidence for interference with fibrinogen binding to GPIIb/IIIa, the strongest inhibition was seen when the agonists collagen or thrombin were used. A small multisubunit inhibitor of collagen-induced platelet aggregation was partially purified using anion exchange chromatography, gelfiltration and RP-HPLC. The inhibitor has a pI between 4 and 6.5, elutes with a molecular weight of 23,800 Da after gelfiltration, and is part of the elaborate broad-spectrum antiplatelet activity that results in the potent synergistic anti-hemostatic cocktail produced by H. contortus.
Tissue Factor Pathway Inhibitor Reduces Experimental Lung Metastasis of B16 Melanoma
Authors: Meyer Todd , Chang Jen-Yea , Amaya Mildred , Siddiqui Farooq , Desai Hina , Francis John , Amirkhosravi Ali
Published: March 2018

The importance of tissue factor (TF) in tumor biology has been highlighted by studies suggesting its involvement in cell signaling, metastasis and angiogenesis. Since many animal studies have shown that anticoagulant therapy can reduce experimental metastasis, we studied whether the natural inhibitor of TF-mediated blood coagulation, Tissue Factor Pathway Inhibitor (TFPI), might be similarly effective. Using a murine experimental model, we found that intravenous injection of recombinant murine TFPI immediately before introduction of tumor cells reduced metastasis by 83% (P <0.001). B16 murine melanoma cells stably transfected with a TFPI expression vector exhibited reduced lung seeding following intravenous injection by 81% (P <0.001) compared with controls. No difference in primary tumor growth was observed between TFPI+ and control cells. Mice receiving intravenous somatic gene transfer of sense TFPI expression vector developed 78% fewer lung nodules than controls (P <0.05). We conclude that TFPI has significant anti-metastatic activity in this experimental model.
Randomised, Double Blind, Multicentre, Placebo Controlled Study of Sulodexide in the Treatment of Venous Leg Ulcers
Authors: Scondotto Gaetano , Agnelli Giancarlo , Aloisi Daniele , Palazzini Ernesto , Zamboni Villiam , Coccheri Sergio ,   for the venous arm of the SUAVIS (Sulodexide Arterial Venous Italian Study) Group
Published: March 2018

Sulodexide, a highly purified glycosaminoglycan, was investigated for treatment of venous leg ulcers. Patients (n = 235) undergoing local treatment including wound care and compression bandaging, were randomised to receive either sulodexide or matching placebo for three months. Primary study endpoint was complete ulcer healing after 2 months; secondary endpoints were ulcer healing at 3 months and the time-course changes of ulcer areas.

The proportion of patients with complete ulcer healing was higher with sulodexide at 2 months (p = 0.018) and 3 months. The “number needed to treat” to obtain one additional patient healed with sulodexide was 7 at 2 months and 5 at 3 months. The changes in ulcer surface area with time were significant for sulodexide only (p = 0.004). Fibrinogen significantly decreased in sulodexide patients (p = 0.006).

In conclusion, sulodexide associated with local treatment proved to be effective and well tolerated in the management of venous leg ulcers.
Tissue Factor Expression on Monocyte Subpopulations during Normal Pregnancy
Authors: Wallace Julie , Gilmore W. S. , McFaul P. , Alexander H. D. , Holmes Valerie
Published: March 2018

Pregnancy is often referred to as a hypercoagulable state due to changes in the haemostatic system. Tissue factor (TF) is the initiator of blood clotting in vivo. The effect of pregnancy on monocyte TF expression was determined in a longitudinal case control study (89 pregnant, 39 non-pregnant). Using whole blood flow cytometry and CD14 as a monocyte marker, TF expression was measured on all CD14 positive, CD14Bright and CD14Dim cells. TF expression was significantly lower in pregnant women than in non-pregnant control subjects, on all CD14 positive cells at 20 and 35 weeks, on CD14Bright cells at 12 and 35 weeks and on CD14Dim cells at 20 weeks. Additionally, we report that a higher percentage of CD14Dim than CD14Bright cells express TF. These results suggest that, in order to maintain homeostasis in haemostasis in an otherwise hypercoagulable state, monocyte TF expression is reduced during normal pregnancy.
Heterogeneous Detection of A-antigen on von Willebrand Factor Derived from Platelets, Endothelial Cells and Plasma
Authors: Collins Peter , Bowen Derrick , Brown Simon
Published: March 2018

The exact function of the carbohydrate component of von Willebrand factor (VWF) is unknown. ABO blood group antigens are present as integral structures on the oligosaccharide side chains and it has long been recognised that ABO blood group is a determinant of VWF levels. The mechanism for this is not known. Using a monoclonal antibody against the A-antigen, we investigated the presence of this antigen on VWF from plasma, platelets, human umbilical vein endothelial cells (HUVEC) and saphenous vein endothelial cells. Initial studies on plasma VWF revealed that 23.5% of samples appeared to be negative for the A-antigen. This was shown to correlate with the A2 subtype of the A-antigen (p <0.01). Analysis of intracellular VWF from saphenous vein endothelial cells revealed low levels of A-antigen to be present in comparison to the corresponding plasma VWF. In contrast, VWF from platelets and HUVEC gave no detectable A-antigen. However, within 1 h of administration of DDAVP to type 1 VWD patients, there was a >2-fold increase in the A-antigen/VWF:Ag ratio for VWF in the plasma. In vitro experiments with serum N-acetlygalactosaminyltransferase failed to demonstrate any addition of A-antigen to platelet or HUVEC VWF. These data are consistent with heterogeneity in the content of A-antigen on VWF from different physiological compartments. Also, they are consistent with either a change in the A-antigen content of VWF after release from the intracellular compartment or a difference in the intracellular addition of A-antigen to VWF by endothelium from different vascular beds.
In Vivo Plasminogen Deficiency Reduces Fat Accumulation
Authors: Ellis Jill , Yuen Lawrence , Hoover-Plow Jane
Published: March 2018

Obesity and non-insulin dependent diabetes are associated with a decrease in fibrinolysis, which is mediated by the plasminogen system. The purpose of the current study was to investigate the role of the plasminogen system in the reduced body weight of the plasminogen deficient (Plg−/−) mice. In this study we have found that the reduced body weight in Plg−/− mice is due to a reduced rate of the adipose tissue (25% less) and whole body fat (30% less) accumulation during growth in Plg−/− compared to wild-type (WT) littermates. When the mice are fed a high fat-lipogenic diet, adipose tissue accumulation increases in the Plg−/− mice indicating that the capacity for lipid filling of cells was not blocked. In addition, glycerol phosphate dehydrogenase, a marker of late differentiation, was not different in the depots from WT and Plg−/− mice. The number of stromal cells (number × 105/g adipose tissue), isolated from inguinal (Plg−/− 3.4 ± 1.2, n = 6; WT 0.17 ± 0.07, n = 7, p < 0.02) and gonadal (Plg−/− 11.0 ± 0.4, n = 6; WT 3.1 ± 0.7, n = 7, p < 0.05) fat depots, was markedly higher in the depots from the Plg−/− mice than WT mice. Differentiation of stromal cells in culture from the Plg−/− mice was reduced compared to cells from WT mice. These results suggest that differences in the stromal cell population are responsible for the reduced adipose tissue accumulation in the Plg−/− mice, and that the plasminogen system plays an important role in adipose tissue accumulation.
An Additional Mechanism of Action of Abciximab: Dispersal of Newly Formed Platelet Aggregates
Authors: Marciniak Stanley , Furman Mark , Michelson Alan , Jakubowski Joseph , Jordan Robert , Marchese Peter , Frelinger Andrew , Mascelli Mary
Published: March 2018

The ability of abciximab to prevent fibrinogen binding to activated platelets indicates it may also promote dissolution of platelet-rich thrombi. The present study examined the capacity of abciximab to reverse platelet aggregation in vitro.

Experiments were performed on blood from healthy non-medicated donors. Platelet aggregate formation and disaggregation were monitored turbidimetrically. Platelet-bound fibrinogen was measured by flow cytometry. For disaggregation studies, platelets were first stimulated with either ADP or the 11-mer thrombin receptor activating peptide (TRAP), then varying amounts of abciximab were added at periodic intervals after agonist addition. Platelet disaggregation was detected by comparing the extent of light transmittance at 4 min after addition of either abciximab or saline to PRP. ATP release was simultaneously monitored by chemi-luminescence. When added 1 min after low concentrations of ADP, abciximab rapidly (<1 min) dispersed platelet aggregates in a dose-dependent manner, with complete disaggregation observed with 6.25 µg/mL of the β3 antagonist. In contrast, equivalent concentrations of abciximab did not induce appreciable disaggregation to platelets stimulated with TRAP (10 µM). Platelet counts of samples that had undergone complete disaggregation, as assessed by aggregometry, were equivalent to baseline, indicating dispersal of aggregates to single cells. Concentrations of abciximab that produced complete disaggregation induced partial displacement of platelet-bound fibrinogen (52 ± 8% inhibition of fibrinogen binding at 12.5 µg/ml abciximab). The disaggregation effectiveness of abciximab decreased as the time between ADP and subsequent abciximab addition widened, and as the amount of both dense granule release and agonist stimulation increased. However, pre-treatment of platelets with acetylsalicylic acid (ASA) did not potentiate platelet disaggregation induced by abciximab.

These data indicate that abciximab facilitates the dispersal of newly formed platelet aggregates in vitro, by partially displacing fibrinogen from activated GPIIb/IIIa receptors. In vivo, abciximab may destabilize coronary thrombi by preventing aggregate formation and dispersing mural thrombi.
Selective Inhibition of Protease-activated Receptor 4-dependent Platelet Activation by YD-3
Authors: Wu Chin-Chung , Hwang Tsong-Long , Liao Chang-Hui , Kuo Sheng-Chu , Lee Fang-Yu , Lee Chun-Yann , Teng Che-Ming
Published: March 2018

In the present study, the antiplatelet effect and its mechanism of a new synthetic compound YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] were examined. YD-3 inhibited the aggregation of washed human platelets caused by protease-activated receptor (PAR) 4 agonist peptide GYPGKF (IC50 = 0.13 ± 0.02 µM), but had no or little effect on that by thrombin, PAR1 agonist peptide SFLLRN, collagen or U46619. YD-3 produced a parallel, rightward shift of the concentration-response curve for GYPGKF without decreasing of the maximum platelet aggregation, indicating a competitive antagonism. In contrast to human platelets, both thrombin- and GYPGKF-induced mouse platelet shape change and aggregation were completely inhibited by YD-3. YD-3 also selectively prevented GYPGKF-induced intracellular Ca2+ mobilization in human platelets. Furthermore, in the PAR1-desensitized human platelets, thrombin induced a relatively slow rise and decay of calcium mobilization that was significantly inhibited by YD-3. In addition, the synergistic effect of SFLLRN and GYPGKF on platelet activation was prevented by YD-3. YD-3 also inhibits both fMLP-stimulated neutrophil- and purified cathepsin G-induced platelet aggregation, which has been demonstrated to be PAR4-dependent. Taken together, our results suggest that YD-3 selectively inhibits PAR4-dependent platelet activation through blockade of PAR4. To the best of our knowledge, it is the first non-peptide PAR4 antagonist.
Glanzmann’s Thrombasthenia: Identification of 19 New Mutations in 30 Patients
Authors: D’Andrea Giovanna , Colaizzo Donatella , Vecchione Gennaro , Grandone Elvira , Di Minno Giovanni , Margaglione Maurizio ,   the GLAnzmann’s Thrombasthenia Italian Team (GLATIT)
Published: March 2018

Glanzmann’s thrombasthenia (GT) is a genetically heterogeneous autosomal recessive syndrome associated with a bleeding tendency. To elucidate molecular basis of GT we have screened for mutations 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the αIIb and 4 in the β3 gene have been found. Only two (αIIb Pro145Ala and IVS3(−3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in αIIb and β3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of αIIb and β3 genes.
The Expression of Tissue Factor and Tissue Factor Pathway Inhibitor in Aortic Smooth Muscle Cells Is Up-regulated in Synthetic Compared to Contractile Phenotype
Authors: Ghrib Farida , Brisset Anne-Cécile , Dupouy Dominique , Terrisse Anne-Dominique , Navarro Chantal , Cadroy Yves , Boneu Bernard , Sié Pierre
Published: March 2018

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes.

Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs.

A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.
No association between thrombosis and factor V gene polymorphisms in Chinese Han population
Authors: Yanqing Hu , Qinzhi Xie , Zaifu Jian , Guangping Wang , Xiaoxia Zuo , Xiaoqun Pu , Fangping Chen ,   Xiaobo
Published: March 2018

Activated protein C resistance (APCR) is the most common hereditary condition of thrombosis in Western countries. And it is significantly linked to a single nucleotide polymorphisms (SNPs) in the coagulation factor V gene that results in the mutations at R506, R306 and HR2 alleles. To determine the prevalence of APCR and its association with the factor V gene SNPs in Chinese Han thrombotic patients, we investigated a total of 346 Chinese thrombotic patients and 140 normal controls for APCR using the APTT-based assays, according to manufacturer’s instructions, APC ratio ≦2 indicated APC resistance. Mutations of factor V at R506, R306, HR2 allele were detected by PCRMnl/I, Bst/NI, Rsa/I digestion as described before respectively. The results showed that the incidence of APC resistance were 12.0% (12 of 100 cases) in acute cerebral thrombosis (ACT) patients (P <0.05), 13.5% (13 of 96 cases) in acute myocardial infarction (AMI) patients (P <0.05), 16.7% (10 of 60 cases) in deep venous thrombosis (DVT) patients (P <0.05), 15.6% (14 of 90 cases) in systemic lupus erythematosus (SLE) patients (P <0.05) and 5.0% (7 of 140 cases) in normal controls. APCR is associated with thrombotic events. But no factor V R506Q mutation (FV Leiden) was found in all 5 groups. Only one AMI patient and one DVT patient revealed heterozygous R306G mutation, which was confirmed by direct sequencing PCR products. Additionally, two SLE patients showed to be heterozygous HR2 allele for the first time in the Chinese Han population. We concluded that APC resistance in the Chinese Han population might not be associated with mutations of factor V at R506, R306 and HR2 polymorphisms. Some other factors might contribute to APC resistance in the Chinese Han population.
Rise of circulating thrombopoietin following cardiothoracic surgery is potentiated in patients with coronary atherosclerosis: correlation with a preceding increase in levels of interleukin-6
Authors: Cotton James , Hong Ying , Hawe Emma , Mathur Anthony , Humphries Steve , Brown Angie , Martin John , Erusalimsky Jorge
Published: March 2018

Thrombopoietin (TPO) is the major regulator of platelet production. Plasma levels of TPO are thought to be regulated by its binding to platelets and megakaryocytes. Here we have used a model of cardiac surgery with cardiopulmonary bypass (CBP) to test the possibility that changes in TPO levels are influenced by the presence of coronary artery disease (CAD) and by changes in interleukin-6 (IL-6). After surgery patients with CAD (n = 22) or with normal coronary arteries (n = 11) showed a significant thrombocytopaenia followed by a reactive thrombocytosis. The platelet recovery was preceded by a significant rise in TPO (from 62.6 ± 9.4 pg/ml at baseline to 129.2 ± 19 pg/ml at 60 h, P <0.001), which in turn was preceded by, and was positively correlated with, a marked increase in circulating IL-6 (from 1.5 ± 0.3 pg/ml at baseline to 269.3 ± 30.6 pg/ml at 3-12 h, P <0.001). The rise of both IL-6 and TPO was significantly larger in patients with CAD. No correlation was found between the post-operative drop in platelet mass and changes in either the TPO or IL-6 levels. These findings suggest that in man circulating TPO levels, besides being controlled by changes in platelet mass, are influenced by inflammatory processes, including the presence of coronary atherosclerosis.
2MHz ultrasound enhances t-PA-mediated thrombolysis: comparison of continuous versus pulsed ultrasound and standing versus travelling acoustic waves
Authors: Pfaffenberger Stefan , Devcic-Kuhar Branka , El-Rabadi Karem , Gröschl Martin , Speidl Walter , Weiss Thomas , Huber Kurt , Benes Ewald , Maurer Gerald , Wojta Johann , Gottsauner-Wolf Michael
Published: March 2018

In addition to fibrinolytic enzymes, ultrasound has the potential to enhance thrombolysis. High frequency ultrasound has the advantage that a combination of diagnostic and therapeutic ultrasound with only one device is possible. Therefore, we investigated the optimal high frequency (2 MHz) ultrasound field characteristics and application mode in vitro. Continuous ultrasound significantly enhanced rt-PA mediated thrombolysis: in a travelling wave field thrombolysis was augmented by 49.0 ± 14.7% and in a standing wave field by 34.8 ± 7.3%. In an intermittent application mode (1Hz, 10Hz, 100Hz, 1kHz) most efficient results were obtained for both wave fields using 1 Hz (46.4 ± 10.7% and 39.1 ± 6.6%, respectively). Referring to a possible in vivo application our in vitro data suggests that an intermittent application of a 2 MHz high frequency ultrasound using a travelling wave field would be the most potent application for lysing blood clots.
Posttranscriptional regulation of PAI-1 gene expression
Authors: Dlakic Wendy , Gelehrter Thomas , Heaton Joanne
Published: March 2018

The plasminogen activator-plasmin cascade is involved in multiple physiological and pathological processes including fibrinolysis, wound healing, fibrosis, angiogenesis, embryo implantation and tumor cell invasion and metastasis. Plasminogen activator-inhibitor type 1 (PAI-1) is the major physiological regulator of plasminogen activation. PAI-1 is expressed in a variety of mammalian cells and is regulated by growth factors, cytokines and hormones, including agents that elevate cAMP levels. Although cyclic nucleotide regulation of PAI-1 is observed in diverse cell types in various species, including human, limited studies have addressed the mechanism of this regulation. Here we review our work on the regulation of PAI-1 mRNA degradation in HTC rat hepatoma cells, describing the cis-acting cAMP-responsive sequence in the transcript and a novel RNA binding protein that interacts with it. Potential mechanisms by which this RNA-binding protein may be involved in cyclic nucleotide regulation of mRNA stability are discussed and cAMP regulation of PAI-1 in other systems is summarized.

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.
Platelet P-selectin expression: requirement for protein kinase C, but not protein tyrosine kinase or phosphoinositide 3-kinase
Authors: Libersan Danielle , Merhi Yahye
Published: March 2018

P-selectin is translocated from the α-granules to the surface of activated platelets where it participates in thrombosis and inflammation. We investigated the signaling pathways involved in thrombin-induced human platelet P-selectin expression. Assessed by flow cytometry, inhibition of protein kinase C (PKC) with chelerythrine reduced P-selectin expression by 66%, platelet/neutrophil binding, GPIIb/IIIa activation and aggregation (p<0.05). Gö 6976, an inhibitor of the conventional PKCs (α and β), did not alter P-selectin expression. However, rottlerin inhibited by 50% its expression (p<0.05), but only at doses that interfere with the novel (є, η) and atypical (ζ) PKCs. Inhibition of protein tyrosine kinase (PTK) and phosphoinositide 3-kinase (PI3-K) did not significantly affect P-selectin expression. In conclusion, thrombin-induced P-selectin expression is PKC-sensitive, but PTK and PI3-K-insensitive. The novel є and η and atypical ζ, but not the conventional α and β and the novel θ PKCs, may be involved in this process.
Increased inflammatory status and higher prevalence of three-vessel coronary artery disease in patients with concomitant coronary and peripheral atherosclerosis
Authors: Piscione Federico , Silvestro Antonio , Galasso Gennaro , Di Donato AnnaMaria , Oliva Gabriella , Scopacasa Francesco , Chiariello Massimo , Brevetti Gregorio
Published: March 2018

The aim of this study was to determine whether patients with coronary artery disease (CAD) and concomitant peripheral arterial disease (PAD) have a greater inflammatory status than those with CAD alone. To this aim, we evaluated PAD (ankle/brachial pressure index <0.9), and measured plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6) and the soluble forms of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in 234 patients who underwent coronary angiography. Median levels of CRP, IL-6 and sICAM-1 were higher in the CAD without PAD (n=134) and CAD+PAD (n=40) groups than in 60 patients without either disease (“controls”). Median CRP values were higher in patients with CAD+PAD than in patients with CAD alone (4.7 mg/L [1.5;7.6] vs 2.4 mg/L [0.9;3.8], p < 0.01). Three-vessel CAD was diagnosed in 60% of CAD+PAD patients and in 21% (p< 0.01) of CAD only patients. After adjustment for confounding factors, only PAD was independently associated with three-vessel CAD (p<0.001). This association was maintained after adjustment for IL-6, the only inflammatory parameter significantly associated with three-vessel CAD at univariate analysis (p<0.01). In conclusion, in CAD the coexistence of PAD is associated with a greater inflammatory status and more widespread coronary atherosclerosis. These results could help to explain the high cardiovascular risk of patients with concomitant CAD and PAD and suggest that PAD be included among the variables used to identify CAD patients for further diagnostic evaluation.
The ADAMs family of proteins: from basic studies to potential clinical applications
Authors: Lynn David , Lloyd Andrew , O’Shea Caroline , Duffy Michael
Published: March 2018

The ADAMs are a family of membrane proteins possessing a disintegrin and metalloprotease domain. Currently,34 members are known to exist. Approximately 50% of the ADAMs contain a metalloprotease-like domain and some of these have been shown to possess protease activity. Most of the protein substrates identified to date for ADAMs are either integral membrane or extracellular matrix (ECM) proteins. In addition to hydrolysing proteins, a number of ADAMs bind to integrins. The attachment to integrins occurs via the disintegrin domain. Since the ADAMs can play a role in both proteolysis and adhesion, they have been implicated in a variety of biological processes such as sperm-egg fusion, somatic cell-cell adhesion, ecto-domain shedding, myoblast fusion and development. Altered expression of certain ADAMs has been associated with a number of diseases including asthma, arthritis, Alzheimer’s disease, atherosclerosis and cancer.

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.
Measurement of tissue factor messenger RNA levels in leukocytes from patients in hypercoagulable state caused by several underlying diseases
Authors: Sase Tomohiro , Wada Hideo , Nishioka Junji , Abe Yasunori , Gabazza Esteban , Suzuki Koji , Nakamura Sin , Nobori Tsutomu , Shiku Hiroshi
Published: March 2018

In a preliminary study, we have demonstrated that tissue factor (TF) is immunohistochemically stained in monocytes from patients with disseminated intravascular coagulation (DIC) but not from healthy volunteers, and that leukocytes from DIC patients induce enhanced activated factor X (FXa) generation in the presence of a mixture of FVIIa, FX and Ca2+. Then, TF mRNA levels in leukocytes were measured to evaluate the role of TF in the pathophysiology of various diseases. TF mRNA levels in leukocytes were low in healthy volunteers but they were significantly increased in various diseases, especially in patients with infectious diseases, solid cancer, anemd hematopoietic tumors. TF mRNA levels in leukocytes were significantly higher in patients with high levels of C reactive protein (CRP) than in those with low CRP. TF mRNA levels were significantly higher in patients with DIC than in those without DIC. TF mRNA levels were well correlated with TF antigens in plasma and leukocytes. These findings suggest that the expression of TF mRNA in leukocytes is increased in various diseases and that this may play an important role in hypercoagulability or DIC.
Store-mediated calcium entry in the regulation of phosphatidylserine exposure in blood cells from Scott patients
Authors: Munnix Imke , Harmsma Marjan , Giddings John , Collins Peter , Feijge Marion , Comfurius Paul , Heemskerk Johan , Bevers Edouard
Published: March 2018

Scott syndrome is a bleeding disorder, characterized by impaired surface exposure of procoagulant phosphatidylserine (PS) on platelets and other blood cells, following activation with Ca2+-elevating agents. Since store-mediated Ca2+ entry (SMCE) forms an important part of the Ca2+ response in various blood cells, it has been proposed that deficiencies in Ca2+ entry may relate to the impaired PS exposure in the Scott syndrome. Here, we have tested this hypothesis by investigating the relationship between Ca2+ fluxes and PS exposure in platelets as well as B-lymphoblasts derived from the original Scott patient (M.S.), a newly identified Welsh patient (V.W.) with similar bleeding symptoms, and two control subjects. Procoagulant activity of V.W. platelets in suspension, measured after stimulation with collagen/thrombin or Ca2+-ionophore, ionomycin, resulted in 52% or 17%, respectively, compared to that of correspondingly activated control platelets. Procoagulant activity of V.W. erythrocytes treated with Ca2+-ionophore resulted in less than 6% of the activity of control erythrocytes. Single-cell Ca2+ responses of M.S. and V.W. platelets, adhering to collagen, were similar to those of platelets from control subjects, while PS exposure was reduced to 7% and 15%, respectively, compared to controls. Stimulation of non-apoptotic B-lymphoblasts derived from both patients and controls with Ca2+-ionophore or agents causing Ca2+ mobilization and SMCE, resulted in similar Ca2+ responses. However, in lymphoblasts from M.S. and V.W. Ca2+-induced PS exposure was reduced to 7% and 13% of the control lymphoblasts, respectively. We conclude that i. patient V.W. is a new case of Scott syndrome, ii. Ca2+ entry in the platelets and lymphoblasts from both Scott patients is normal, and iii. elevated [Ca2+]i as caused by SMCE is not sufficient to trigger PS exposure.
Helicobacter pylori causes gastrointestinal hemorrhage in patients with congenital bleeding disorders
Authors: Rehnberg Ann-Sofie , Hein Marju , Hegedus Olga , Lindmarker Per , Hellström Per , Schulman Sam
Published: March 2018

Helicobacter pylori (H. pylori) infection is associated with peptic ulcer disease and gastric cancer. The eradication of H. pylori is of special interest in patients with congenital bleeding disorders, for whom treatment of gastrointestinal hemorrhage with factor concentrates is costly. The prevalence of H. pylori varies between different populations and identification of high-risk subgroups may allow for more targeted screening and eradication of the infection. We performed a 5-year retrospective study of gastrointestinal bleeding, combined with screening and treatment for H. pylori and a long-term prospective follow-up in 168 Swedish and 23 Estonian patients with hemophilia or von Willebrand disease. The prevalence of seropositivity was lower in Sweden than in Estonia (28 versus 48%, p = 0.03), lower in native Swedes than in non-Nordic immigrants to Sweden (20 versus 76%, p = 0.0001) and lower in patients less than 40 years of age than older patients (16 versus 38%, p = 0.002). The incidence of gastrointestinal hemorrhages among the 35 Swedish patients with active H. pylori infection, confirmed by a urea breath test, was 6.0 per 100 patient-years before eradication therapy versus 1.7 during the prospective followup. A negative urea breath test one month after therapy always remained negative after one year. Screening, followed by treatment of all infected patients, yielded a reduction of direct costs over a 5-year period of 130 US-$ per screened patient. We conclude that screening and eradication therapy for infection with H. pylori in patients with congenital bleeding disorders is an effective and economic strategy.
New molecular defects in the γ subdomain of fibrinogen D-domain in four cases of (hypo)dysfibrinogenemia: fibrinogen variants Hannover VI, Homburg VII, Stuttgart and Suhl
Authors: Franke Kathrin , Richter Walter , Steiniger Frank , Seyfert Ulrich , Schenk Joachim , Treuner Jörn , Haberbosch Werner , Eisert Roswit , Barthels Monika , Meyer Michael
Published: March 2018

Four new molecular abnormalities in the γ subdomain of the D domain elucidated in three unrelated thrombophilic patients and in one asymptomatic case of hypofibrinogenemia are reported: fibrinogen Suhl, γ 326,Cys →Tyr, fibrinogen Hannover VI, γ 336 Met →Ile, fibrinogen Stuttgart, γ 345, Asn→Asp and fibrinogen Homburg VII, γ354,Tyr→Cys. In all cases, fibrin polymerization in plasma is impaired. In the case of fibrinogen Suhl, there was a normalization of fibrin polymerization in plasma at higher Ca2+ concentration. The protective effect of Ca2+ on plasmic degradation of fibrinogen was incomplete with all three variants. The fibrinogen molecules in variants Homburg VII and Suhl contain covalently bound albumin. Fibrin clot structure was abnormal in case of variant Homburg VII, with finer and more branched fibers forming a less porous clot. Experimental data indicate possible effects of the molecular abnormalities on Ca2+-binding, D-E interaction and lateral association of protofibrils.
Enhanced nutritionally induced adipose tissue development in mice with stromelysin-1 gene inactivation
Authors: Maquoi Erik , Demeulemeester Diego , Vörös Gabor , Collen Désire , Lijnen H.
Published: March 2018

To investigate a potential role of stromelysin-1 (MMP-3) in development of adipose tissue, 5 week old male MMP-3 deficient mice (MMP-3-/-) and wild-type (MMP-3+/+) controls were kept on a high fat diet (HFD) for 15 weeks. MMP-3-/- mice were hyperphagic and gained more weight than the MMP-3+/+ mice. At the time of sacrifice, the body weight of the MMP-3-/- mice was significantly higher than that of the MMP-3+/+ mice, as was the weight of the isolated subcutaneous (SC) and gonadal (GON) fat deposits. Significant adipocyte hypertrophy was observed in the GON but not in the SC adipose tissue of MMP-3-/- mice. Fasting plasma glucose and cholesterol levels were comparable in both genotypes, whereas triglyceride levels were significantly lower in MMP-3-/- mice. Staining with an endothelial cell specific lectin revealed a significantly higher blood vessel density and larger total stained area in the GON adipose tissues of MMP-3-/- mice. Thus, in a murine model of nutritionally induced obesity, MMP-3 impairs adipose tissue development, possibly by affecting food intake and/or adipose tissue-related angiogenesis.

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.
Genetic Polymorphisms and Coronary Artery Disease in the South of France
Authors: Canavy I. , Henry M. , Morange P. E. , Tiret L. , Poirier O. , Ebagosti A. , Bory M. , Juhan-Vague I.
Published: March 2018

Vascular disease is a multifactorial disease that involves atherosclerotic and thrombotic factors. Genetic polymorphisms have been associated with myocardial infarction and angina pectoris. The aim of the present study was to assess the relationship between some genetic polymorphisms and myocardial infarction (MI) or vasospastic angina pectoris in a population from southern France. Genetic polymorphisms of the renin angiotensin system (the D/I polymorphism of the ACE gene and the A1166C polymorphism of the angiotensin II type 1 receptor [AT1R]) and of haemostatic factors (the -675 4G/5G polymorphism of the plasminogen-activator inhibitor 1 [PAI-1] gene, and the G to T common point mutation in exon 2, codon 34 of the Factor XIII A-subunit gene) were examined.

We assessed the genotype distribution in consecutive coronary artery disease (CAD) patients with MI (n = 201) and vasospastic angina pectoris (n = 43) and in 244 healthy controls comparable in age, sex, body mass index and total cholesterol level.

The genotype distribution of AT1R polymorphism was significantly different between controls and patients, the prevalence of the C allele carriers being higher in patients with MI after the age of 45 than in control individuals (61 vs 45%, p <0.01), leading to an odds ratio (OR) of 2 (CI: 1.2-3.4). When looking at the group of patients with vasospastic angina the difference was even higher (76 vs 45%, p <0.01) yielding an OR of 4.3 (CI: 1.4-17.4). Genotype distributions of ACE, PAI-1 and Factor XIII polymorphisms were similar in patients and in controls.

This study is in favor of a role of AT1R gene polymorphism in myocardial infarction and vasospastic angina.