bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Resveratrol-mediated Suppression of Tissue Factor Gene Expression
Authors: Pendurthi Usha , Meng Feng , Mackman N. , Rao L. Vijaya
Published: March 2018

Tissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis. A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality. Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM. Arterioscler Thromb Vasc Biol 1999; 19: 419-426). In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell. Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol. The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner. Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA. However, resveratrol suppressed the transcription of cloned human TF promoter. Further experiments revealed that resveratrol reduced κB- but not AP-1-driven transcriptional activity. Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation. In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-κB/Rel proteins but inhibits NF-κB/Rel-dependent transcription by impairing the transactivation potential of p65.
The Upward Spiral of Drug Costs: A Time Series Analysis of Drugs Used in the Treatment of Hemophilia
Authors: Guirguis Hany , Lipton Richard , Seremetis Stephanie , DiMichele Donna , Agnew George , Karpatkin Margaret , Barish Robert , Jones Robert , Bianco Celso , Knothe Barbara , Lee Myung-Soo , Rogoff Edward
Published: March 2018

Hemophilia is an expensive disease because its treatment is heavily dependent on costly clotting factor drugs. Over the last nine years, a consortium of three Comprehensive Hemophilia Treatment Centers and other hospitals, which purchased clotting factors for their patients, has seen treatment costs escalate on average 17% annually. Currently, new, even more expensive drugs are entering the market.

This study analyzes 3,244 purchases that were made over a nine-year period totaling nearly 500 million units of clotting factor, representing every product on the market. Purchases were made both apart from and under the Federal Public Health Service (PHS) discount pricing rules.

The main cause of the increases was the move to newer, more expensive products. The average price of existing products increased less than 2% per year, but new products were priced, on average, 47% higher than existing products. Overall consumption increased by an average of 5% per year, likely reflecting prophylactic treatment modalities that require greater amounts of clotting factor. Government pricing programs, such as the PHS program, were ineffective or counterproductive at reducing costs.There is a notable absence of competition in this market, with a few dominant companies having a functional monopoly in the largest segments of the market. Prices of older products are not lowered, even when new products are brought to market. A few products that serve small patient groups have had their prices increased substantially.

This escalation is likely to continue as new, more expensive clotting factor drugs are developed. Since these new products are not proven to be any safer or more effective than the current products, this situation creates a risk of intervention by government and insurers to address both treatment costs and exhaustion of patients’ insurance caps. Drug companies are not serving the patients by pricing new, but often very similar, products so aggressively. The trends seen in this patient group will likely be seen in other patient groups in the future. Ultimately, doctors and patients will lose treatment options and health care availability unless collaborative strategies are developed to reduce costs.
Phosphorotioated Oligonucleotides Trigger Synthesis of Human Coagulation Serine Proteases
Authors: Hellstrand Kristoffer , Tarkowski Andrej , Bokarewa Maria
Published: March 2018

CpG containing phosphorotioated oligonucleotides (ODN) are efficient adjuvants able to enhance macrophage and B cell activities. Their impact in the generation of coagulation and fibrinolytic factors has not been analysed.

Production of coagulation and fibrinolytic proteins by human peripheral blood mononuclear cells (PBMC) treated with ODN was assessed.

ODN induced in vitro generation of tissue factor (TF), thrombin and plasminogen, by PBMC. Synthesis of TF and thrombin occurred mostly in monocytes, while plasminogen was produced by both monocytic and lymphocytic cell populations. Generation of these proteins stimulated by CpG was totally blocked by cycloheximide, indicating the requirement of ongoing protein synthesis. Protein synthesis was equally pronounced at stimulation with cytosine-phosphate-guanosine (CpG)- and GpC- containing ODN, and dependend on the presence of the phosphorotioate moiety backbone in the ODN. Plasminogen, synthesized by monocytes and lymphocytes, was shown to be the primary product of ODN activation, leading subsequently to the expression of TF and thrombin generation.

Our findings should be taken into consideration when assessing advantages and drawbacks of immunotherapy and gene therapy.
Characterization of a Ligand-attenuated Binding Site on Glycoprotein IIb/IIIa
Authors: Quinn Martin , Fullard John , Kerrigan Steven , Harriott Patrick , Cox Dermot , Fitzgerald Desmond
Published: March 2018

We describe an epitope on the platelet integrin, GPIIb/IIIa, identified by the monoclonal antibody, 4F8, which is attenuated by small-molecule GPIIb/IIIa ligands. 4F8 did not bind to the ligand binding pocket as it did not compete with a radiolabelled antagonist, 3H-SC-52012. This indicates that the 4F8 epitope behaves as a ligand-attenuated binding site (LABS). Ligand-induced attenuation of 4F8 was an active process as it was prevented by pretreating platelets with cytochalasin D and reduced by prostaglandin E1 or inhibition of protein kinase C. Disappearance of the epitope was required for full platelet activation as 4F8 prevented platelet aggregation without inhibiting fibrinogen binding. These results suggest a model where disappearance of the 4F8 epitope is a secondary event required for full “outside-in” signaling through GPIIb/IIIa.
F11-Receptor (F11R/JAM) Mediates Platelet Adhesion to Endothelial Cells: Role in Inflammatory Thrombosis
Authors: Babinska Anna , Ahmed Tahir , Batuman Olcay , Ehrlich Yigal , Hussain M. , Kedees Mamdouh , Athar Humra , Kornecki Elizabeth
Published: March 2018

The F11 receptor (F11R) is a cell adhesion molecule (CAM), member of the immunoglobulin superfamily found on the surface of human platelets, and determined to play a role in platelet aggregation, secretion, adhesion and spreading. The same molecule is present also at tight junctions of endothelial cells (EC) where it is known as JAM and acts as a CAM through homophilic interactions. The role of F11R/JAM in the interaction of platelets with endothelial cells was investigated in the current studies. We report here that washed human platelets adhere specifically to a matrix made of immobilized, recombinant sF11R. Furthermore, platelets adhere to cytokine(TNF-α, INF-γ) stimulated human umbilical vein endothelial cells (HUVEC), and approximately 40-60% of the adhesive force is exerted by homophilic interactions between the F11R of platelets and EC. This is evidenced by the inhibition of platelet adhesion to endothelial cells by recombinant soluble form of the F11R, and by two F11R peptides with amino acid sequences of the N-terminal region, and in the 1st Ig fold of the F11R, respectively. This study suggests a role for F11R in the adhesion of platelets to cytokine-inflamed endothelial cells and thus in thrombosis and atherosclerosis induced in non-denuded blood vessels by inflammatory processes. Agents that block the F11R-mediated adhesion of platelets to EC may be of therapeutic value in controlling thrombosis and preventing heart attacks and stroke.
Ultrasound Improves Tissue Perfusion in Ischemic Tissue through a Nitric Oxide Dependent Mechanism
Authors: Suchkova Valentina , Baggs Raymond , Sahni Sanjeev , Francis Charles
Published: March 2018

Ultrasound accelerates enzymatic fibrinolysis in vitro and in animal models and may be used as an adjunct to thrombolytic therapy. Ultrasound can also affect vascular tone directly, and we have now investigated the effect of ultrasound on tissue perfusion in a rabbit model of acute muscle ischemia to characterize the magnitude and temporal course of vasodilation and determine its mechanism. After ligation of the femoral artery of rabbits, tissue perfusion in the gracilis muscle as determined using a laser Doppler probe declined by 53% from 13.7 ± 0.3 U to 6.4 ± 0.2 U. The tissue became acidotic as pH declined from normal to 7.05 ± 0.2. Application of 40 kHz ultrasound at intensities from 0.25 to 0.75 W/cm2 progressively improved perfusion over 60 min and reversed acidosis, but these effects were both completely blocked by pre-treatment with the nitric oxide synthase inhibitor LNAME. Nitric oxide synthase activity in muscle was measured using an assay based on the conversion of radiolabeled L-arginine to L-citrulline and demonstrated an increase of 3.6-fold following ultrasound exposure. This effect was greatest at locations close to the transducer and declined progressively away from it. Histologic examination showed greater capillary circumference in ultrasound exposed muscle compared to unexposed tissue with no other histologic changes. We conclude that the application of 40 kHz at low intensity improves perfusion and reverses acidosis in acutely ischemic muscle through a nitric oxide dependent mechanism.
Diacylglycerol Kinases: Regulation and Signaling Roles
Authors: Prescott Stephen , Topham Matthew
Published: March 2018

Diacylglycerol kinases (DGKs) are thought to attenuate diacylglycerol signals by converting diacylglycerol to phosphatidic acid. The nine mammalian diacylglycerol kinases that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. The activity of DGKs is regulated by mechanisms that can modify their access to diacylglycerol, affect their activity, or alter their ability to bind to other proteins. Although little is known of the specific function of DGKs in platelets, they likely influence actin reorganization and other signaling events requiring diacylglycerol.
Multicentre Evaluation of IL Test™ Free PS: A Fully Automated Assay to Quantify Free Protein S
Authors: Sales Miquel , Chitolie Andrew , Domènech Pere , Rossi Edoardo , Borrell Montserrat , Dahlbäck Björn , Serra Josep
Published: March 2018

Deficiency of the anticoagulant vitamin K-dependent protein S (PS) is associated with increased risk of venous thrombosis. In human plasma, PS circulates in two forms: as free protein (free PS) and PS bound to C4b-binding protein (C4BP), a regulator of the complement system. Assays for free PS have higher sensitivity and specificity for protein S deficiency than assays for total protein S. We have extensively evaluated the analytical performance of a novel assay for free PS, the IL Test™ Free Protein S, which takes advantage of the affinity of C4BP for free PS, and compared its performance to existing methods. IL Test™ Free Protein S is a rapid, fully automated turbidimetric assay consisting of two reagents: a C4BP coated latex and an anti-PS monoclonal antibody coated latex. The test range, precision and linearity were adequate and the assay tolerated high concentrations of interfering substances of clinical significance. The reference range agreed with previously published studies. The analysis of 903 patient samples belonging to 20 different clinical categories with the new assay yielded free PS results that agreed well with those obtained using the assays established in the participating laboratories. The study demonstrated the IL Test™ Free Protein S to be rapid, reliable and easy to perform.
Pathogenic Effects of Anti-Glycoprotein Ib Antibodies on Megakaryocytes and Platelets
Authors: Alimardani Gulie , Guichard Josette , Fichelson Serge , Cramer Elisabeth
Published: March 2018

Antibodies directed against the glycoprotein (GP) Ib have been identified as the potential cause of various platelet disorders: Immune thrombocytopenic purpura (ITP) may be caused by such autoantibodies; Anti-thrombotic drugs targeting GPIb also induce thrombocytopenia. In order to elucidate the potential mechanism(s) of the anti-GPIb effects, we have examined by electron microscopy (EM) the effect of several antibodies directed against GPIb and GPIIb-IIIa on human culture megakaryocytes (MK). Virtually all antibodies to GPIb enhanced the interaction of newly formed platelets with MK when compared to other antibodies. These effects were retrieved when antibodies were tested on platelets. We conclude that antibodies to GPIb can potentially inhibit platelet release by MK, and can also induce homotypic platelet adhesion. These results may have implications in the pathophysiology of thrombocytopenia and platelet recovery in ITP, and shed light on the pathological effect of anti-GPIb antibodies used as antithrombotic drugs.
Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma
Authors: Leurs Judith , Wissing Britt-Marie , Nerme Viveca , Schatteman Katinka , Björquist Petter , Hendriks Dirk
Published: March 2018

Carboxypeptidase U (CPU, TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this study was to investigate the time course of the generation of CPU activity during coagulation and fibrinolysis using an in vitro clot lysis model in human plasma. A first peak of CPU activity appeared after initiation of the coagulation phase and a second rise in CPU activity was observed during the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation during coagulation but not during fibrinolysis. Addition of the plasmin inhibitor aprotinin during fibrinolysis resulted in a decrease in CPU activation during the lysis phase. These results demonstrate that proCPU was activated during coagulation by thrombin and during fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.
Prospective assessment of the natural history of positive D-dimer results in persons with acute venous thromboembolism (DVT or PE)
Authors: Kuruvilla John , Wells Phil , Morrow Bev , MacKinnon Karen , Keeney Michael , Kovacs Michael
Published: March 2018

The natural history of initially positive D-dimers for venous thromboembolism is not known. If it returns to negative in the majority of patients, it would be potentially helpful to diagnose a recurrence. In this study, we prospectively measured D-dimer levels in outpatients with a diagnosis of venous thrombo-embolism. There were a total of 152 patients with an average age of 57. D-dimer results were performed at baseline and repeated at one week, one month and three months.

At baseline 120 of 152 (79%) had a positive D-dimer result. Of those with an initially positive result, 80% were still positive at one week and 39% were still positive at one month. Finally at three months, 13% remained positive. Seven patients had recurrent events and all had persistently elevated D-dimers at one month. This study suggests that a persistently positive D-dimer result after one month of treatment may indicate a higher risk of recurrent venous thromboembolism. D-dimer testing for the diagnosis of recurrence of venous thromboembolism deserves further study.
Direct thrombin inhibitor melagatran followed by oral ximelagatran in comparison with enoxaparin for prevention of venous thromboembolism after total hip or knee replacement
Authors: Agnelli Giancarlo , Cohen Alexander , Dahl Ola , Mouret Patrick , Rosencher Nadia , Eskilson Christina , Nylander Ingela , Frison Lars , ögren Mats , Eriksson Bengt ,   on behalf of the METHRO III Study Group* (* see Appendix for METHRO III study participants)
Published: March 2018

We evaluated whether a postoperative regimen with melagatran followed by oral ximelagatran, two new direct thrombin inhibitors, was an optimal regimen for thromboprophylaxis in major orthopaedic surgery. In a double-blind study, 2788 patients undergoing total hip or knee replacement were randomly assigned to receive for 8 to 11 days either 3 mg of subcutaneous melagatran started 4-12 h postoperatively, followed by 24 mg of oral ximelagatran twice-daily or 40 mg of subcutaneous enoxaparin once-daily, started 12 h preoperatively. Ximelagatran was to be initiated within the first two postoperative days. The primary efficacy endpoint was venous thromboembolism (deep-vein thrombosis detected by mandatory venography, pulmonary embolism or unexplained death). The main safety endpoint was bleeding. Venous thromboembolism occurred in 355/1146 (31.0%) and 306/1122 (27.3%) patients in the ximelagatran and enoxaparin group, respectively, a difference in risk of 3.7% in favour of enoxaparin (p = 0.053). Bleeding was comparable between the two groups.
Effect of fibrinogen concentration and platelet count on the inhibitory effect of abciximab and tirofiban
Authors: Renda Giulia , Rocca Bianca , Crocchiolo Roberto , De Cristofaro Raimondo , Landolfi Raffaele
Published: March 2018

Glycoprotein (Gp) IIb/IIIa blockers are powerful antithrombotic agents, but display a wide variability of their effect using weight-adjusted dosing. We investigated whether some hemostatic variables affected in vitro platelet inhibition exerted by the antibody abciximab and the peptidomimetic tirofiban. High fibrinogen concentrations reduced platelet inhibition by tirofiban, assessed in whole blood (r = –0.85, n = 10, p <0.01) and in platelet rich plasma (r = –0.89, n = 10, p <0.01) through PFA 100 and Born’s aggregometry assay, respectively. Both drugs were unaffected by von Willebrand factor levels, while platelet count was inversely related to their inhibitory effect (tirofiban: r = –0.9, n = 7, p <0.01; abciximab: r = –0.81, n = 9, p <0.01). Analysis of GpIIb-IIIa blockade showed that receptor occupancy at a fixed abciximab dose was inversely related to platelet counts. These experimental data were in agreement with the classical model of receptor saturation by a tight binding inhibitor.

A role for fibrinogen and/or platelet count in influencing the antithrombotic properties of platelet GpIIb-IIIa antagonists can be hypothesized.
Blood coagulation and fibrinolytic factors in 105 patients with hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillar in Santa Catarina, Southern Brazil
Authors: Zannin Marlene , Lourenço Dayse , Motta Guacyara , Dalla Costa Luis , Grando Margaret , Gamborgi Geni , Noguti Maria , Chudzinski-Tavassi Ana
Published: March 2018

Haemostatic disorders caused by Lonomia obliqua caterpillars has reached epidemic proportions in southern Brazil. Here we evaluated coagulation and fibrinolysis in 105 patients after accidental contact with Lonomia obliqua caterpillars. Global coagulation tests were prolonged in most cases and patients were divided into 3 groups according to fibrinogen (Fg) level: 0.5 g/l (group A); 0.51–1.5 g/l (group B), >1.5 g/l (group C). There was a significant reduction of factors V, XIII, VIII and prekallikrein in group A, with no change in factors X, II and von Willebrand factor. Thrombin-antithrombin and prothrombin F1+2 were elevated in most patients. Antithrombin and protein S were not changed whereas protein C levels were reduced in group A. Plasminogen and alfa2-antiplasmin levels were significantly reduced in group A and D-Dimer levels were extremely high in all groups, showing that fibrinolysis had been activated, possibly secondary to fibrin production. Levels of t-PA were normal and PAI-1 was mildly elevated in group A. The platelet count remained above 150 × 109 platelets/ml in 97% of cases. In summary, our results suggest that Lonomia obliqua envenoming is characterized by a consumption coagulopathy and secondary fibrinolysis.
The preparation and phospholipid binding property of the C2 domain of human factor VIII
Authors: Takeshima Kazuya , Smith Christina , Tait Jonathan , Fujikawa Kazuo
Published: March 2018

The C2 domain of human factor VIII was expressed in a yeast secretion system and its binding properties were studied. A cDNA coding the C2 domain sequence of human factor VIII with a N-terminal six amino acids extension (C-C2) was constructed, transformed into Pichia pastoris cells and expressed. The product was purified by ammonium sulfate fractionation and anion exchange chromatography. It emerged as a single peak from both ion exchange and gel filtration columns, indicating C-C2 is a homogenous monomer. The binding activity of C-C2 to phosphatidylserine-containing phospholipid vesicles was measured by competitive binding with annexin V. The values of IC50were approximately 70nM for both factor VIII and its light chain, but were about 7000nM for C-C2. These results indicated C-C2 has 100-fold less binding affinity than factor VIII or the light chain. Direct binding to solidified phosphatidylserine-containing phospholipids also showed that C-C2 has ~50-fold less binding affinity than does the light chain. C-C2 poorly inhibited Xase activity. These results together clearly show that the C2 domain alone does not have full membrane binding activity, and suggest that the other light chain domains, A3 and/or C1, are also involved in the phospholipid binding activity of factor VIII.
Evidence of prolonged disturbances in the haemostatic, hemorheologic and inflammatory profiles in transmural myocardial infarction survivors
Authors: Saldanha Carlota , Monteiro José , Perdigão Carlos , eSilva João Martins , Sargento Luis
Published: March 2018

Haemostatic, hemorheologic and inflammatory disturbances have been associated with acute coronary syndromes. Most knowledge is reported in cross sectional studies and are without time dependent evolution of these profiles.

The aim of this study was to evaluate, during the first year, the evolution of the haemostatic, hemorheologic and inflammatory profiles determined at hospital discharge in survivors with transmural myocardial infarction (MI).

Eighty eight (79 male; 9 female) mean age of 58 ± 11 years, survivors of a transmural MI were prospectively studied at discharge, 6 months and one year after the event. Haemostatic (protein C, antithrombin III and plasminogen activator inhibitor 1), hemorheologic (blood fluidity and components) and inflammatory profiles (polymorphonuclear elastase and leukocyte count) were determined using standard methodology.

The results of the study can be summarized as follows: (1) Protein C decreased (p < 0.05) over time while PAI-1 only varied significantly until 6th month. (2) Plasma viscosity and fibrinogen (p < 0.001) decrease over time, while erythrocyte aggregation (p < 0.001) and haematocrit increased. Whole blood viscosity did not vary. (3) Leukocyte decreased (p < 0.001) and elastase did not (4). Those patients with cardiovascular events (n = 7) had higher PAI-1 concentration (p<0.05) and leukocyte count (p < 0.01), at discharge (5) Left ventricle ejection fraction correlated significantly with plasma viscosity (r = 0.35 p < 0.05). The results of this longitudinal study show dynamic modifications of the haemostatic, hemorheologic and inflammatory profiles during the first year of a transmural myocardial infarction. In addition, there are interrelations between them and the clinical profile that could help to explain the clinical evolution of this group of patients.
Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway
Authors: Tkachuk Natalia , Haller Hermann , Dumler Inna , Kiian Ioulia
Published: March 2018

Urokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.
Independence of growth factor induced plasminogen activator inhibitor type-1 (PAI-1) expression from the 4G/5G polymorphism of the PAI-1 gene
Authors: Eschenfelder Esther , Peter Karlheinz , Sobel Burton , Bode Christoph , Nordt Thomas
Published: March 2018

Increased PAI-1 expression has been implicated in accelerating atherogenesis. Increases under some conditions are modulated by growth factors. Genetic factors such as the 4G/5G poly-morphism in the promoter of the PAI-1 gene play a role under certain circumstances. The present study was designed to delineate for the first time interactions between growth factors and the 4G/5G polymorphism with respect to PAI-1 expression in human arterial smooth muscle cells (HASMC).

HASMC were genotyped and exposed to growth factors. PAI-1 gene and protein expression were induced consistently by TGF-β (up to 4.0-fold), PDGF (2.1-fold),TNF-α (1.7-fold), and thrombin (2.3-fold). Results were similar regardless of which genotype (4G/4G [n=9], 4G/5G [n=13], and 5G/5G [n=7]) was present.

The induction of increased PAI-1 expression in human arterial smooth muscle cells by growth factors implicated in accelerated atherogenesis is independent of the PAI-1 4G/5G polymorphism. Accordingly, modulation of PAI-1 expression is likely to be influenced predominantly by environmental factors acting on, rather than genetic factors intrinsic to the PAI-1 promoter.
Association between thrombin-activatable fibrinolysis inhibitor (TAFI) and clinical outcome in patients with unstable angina pectoris
Authors: Brouwers Geert-Jan , Tanck Michael , Jukema J. Wouter , Kluft Cornelis , de Maat Moniek , Leebeek Frank
Published: March 2018

Decrease of fibrinolytic potential is considered to be a risk factor for arterial thrombosis. The recently described thrombinactivatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis by cleaving of the C-terminal lysine residues from fibrin, thereby inhibiting tPA mediated plasminogen activation. The role of plasma TAFI antigen (Ag) levels and gene polymorphisms in arterial thrombosis is still not elucidated. In this prospective study, the association between plasma TAFI Ag levels and the TAFI gene polymorphisms, Ala147Thr, Thr325Ile and -438A/G, with refractory unstable angina pectoris (UAP) was determined. The study population consisted of 209 patients with UAP of whom 76 were refractory and 133 non-refractory to medical treatment. In the same study population the contribution of these polymorphisms to plasma TAFI Ag levels was determined.

Plasma TAFI Ag levels were significantly higher in non-refractory patients compared to refractory patients (geometric mean 114.4 and 105.6 U/dl respectively, p=0.042). Plasma TAFI Ag levels in the lowest quartile resulted in a 2.6 fold (95% confidence interval 1.2-5.9) increased risk for refractory UAP compared to plasma TAFI Ag levels in the upper quartile. The three studied TAFI polymorphisms had an independent and additive effect on plasma TAFI Ag levels. However, no significant association between the individual TAFI polymorphisms and refractiveness was observed.

In conclusion, in this study population plasma TAFI Ag levels are significantly correlated with refractiveness in patients with UAP. Furthermore, all three polymorphisms contribute independently to plasma TAFI Ag levels, but not to refractiveness.

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.
Synergistic effect of stromelysin-1 (matrix metalloproteinase-3) promoter 5A/6A polymorphism with smoking on the onset of young acute myocardial infarction
Authors: Liu Ping-Yen , Li Yi-Heng , Wu Hua-Lin , Shi Guey-Yueh , Chen Jyh-Hong
Published: March 2018

The objective of this study was to elucidate the association between the polymorphism of stromelysin-1, also called matrix metalloproteinases-3 (MMP-3), and smoking in the pathogenesis of young acute myocardial infarction (MI).

Plaque rupture is well established as a critical factor in the pathogenesis of acute MI. MMP-3 can degrade extracellular matrix and are identified extensively in human coronary atheroslcerotic plaques, and may contribute to the weakening of the cap and subsequent rupture.

We studied 150 consecutive patients with acute MI onset at age under 45 years (84% men) and 150 sex- and age-matched control subjects. 5A/6A genotype in the stromelysin-1 promoter was determined using polymerase chain reaction and direct sequencing.

Results show that the frequency of the 5A allele and the prevalence of 5A/5A + 5A/6A genotypes were both significantly higher in the young MI than the control group (35.0% vs.20.0%, odds ratio [OR] 2.15,95% confidence interval [CI] 1.30 to 6.80, p<0.001; 44.7% vs. 27.4%, OR 2.19, 95% CI 1.21 to 3.98, p=0.009). Multiple logistic regression analysis showed that the 5A allele was an independent risk factor (OR 2.36, 95% CI 1.24 to 5.90, p=0.008) as were as smoking (OR 3.92, 95% CI 1.75 to 9.21, p=0.001), diabetes mellitus (OR 3.51, 95% CI 1.41 to 6.32, p=0.0068) and hypertension (OR 1.85, 95% CI 1.35 to 4.33, p=0.0025) for the premature onset of MI. Compared to 6A/6A subjects, among patients who did not smoke, the 5A allele poly-morphism was associated with a higher risk of MI at a young age (OR 2.69, 95% CI 1.3 to 8.6), but smoking carriers of the 5A allele had a significantly 10-fold higher risk of MI (OR 9.98, 95% CI 2.3 to 12.5).

We can conclude that there was a significant association between the 5A/6A polymorphism in the promoter region of stromelysin-1 gene and young MI in Taiwan. A synergistic effect between smoking and this polymorphism for the premature onset of MI had been shown in this study.
The G20210A Prothrombin Polymorphism Is not Associated with Increased Thromboembolic Risk in a Large Protein C Deficient Kindred
Authors: Hasstedt Sandra , Callas Peter , Valliere Julia , Scott Bruce , Bauer Kenneth , Long George , Bovill Edwin
Published: March 2018

Likelihood analysis was used to test the effect of the G20210A prothrombin mutation and the His107Pro protein C mutation (resulting from a C insertion) on thrombosis status and prothrombin level in a large kindred of French Canadian descent with type I protein C deficiency. Genotypes were available on 279 pedigree members or their spouses. Of this total, 36 pedigree members were heterozygous for the G20210A variant and one pedigree member was homozygous for G20210A, while 64 were heterozygous for the His107Pro protein C mutation. The factor V Leiden mutation (Arg506Gln) was observed in only one of 181 tested family members. Objectively verified thrombosis was present in 26 of the 279 pedigree members. Thrombosis was suspected in an additional 19 pedigree members. The transmission disequilibrium test of Spielman, 1996, as extended to pedigrees, was used to test for excess transmission of G20210A or His107Pro to thrombosis cases, with transmission of 0.5 specifying no effect. Although the His107Pro mutation was over transmitted (0.837 ± 0.075 p <0.001) to thrombosis cases in this pedigree, the G20210A variant was not (0.491 ± 0.130 NS).

Measured genotype analysis was used to examine a total of 184 individuals for the relationship between prothrombin level and both the G20210A variant and thrombosis. The G20210A variant increased prothrombin level from 97 ± 2% to 124 ± 4% (p <0.0001), but thrombosis status was not associated with any additional increase in prothrombin level. Thus, in a large thrombophilic, protein C deficient kindred, with the G20210A variant present in a proportion (13%) far higher than the general Caucasian population (∼2%), neither the presence of the variant nor the plasma concentration of prothrombin were associated with increased risk for thrombosis. These findings contrast with those of others who have established the G20210A variant as a thrombophilic risk factor; and emphasize the complex nature of the multigenic pathogenesis of thrombophilia.
Effect of the Factor V Leiden Mutation on the Clinical Expression of Severe Hemophilia A
Authors: Walker I.R. , Teitel J. , Poon M.-C. , Ritchie B. , Akabutu J. , Sinclair G. D. , Pai M. , Wu J. W. Y. , Reddy S. , Carter C. , Growe G. , Lillicrap D. , Lam M. , Blajchman M. A. , Lee D.H.
Published: March 2018

To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.
Minor Events and the Risk of Deep Venous Thrombosis
Authors: Eekhoff E. M. W. , Vandenbroucke J. P. , Rosendaal F. R.
Published: March 2018

Background Deep venous thrombosis is a common disease, with genetic and acquired risk factors. Many patients have a history of minor events (short periods of immobilisation such as prolonged travel, short illness, minor surgery or injuries) before onset of venous thrombosis. However, the role of these minor events has received little formal study. Also, we do not know how minor events might interact with the presence of genetic prothrombotic defects (factor V Leiden mutation, factor II mutation, protein C, S and antithrombin deficiency). Patients and Methods On the basis of case-control data from a thrombosis service in the Netherlands, we added a follow-up period for a casecross-over analysis of minor events as risk factors, and a case-only analysis for the interaction with factor V Leiden. A total of 187 patients with first, objectively diagnosed venous thrombosis of the legs, aged 15–70, without underlying malignancies and without major acquired risk factors entered the study. For the analysis of minor events in the case-cross-over analysis, we used a matched odds ratio; in the caseonly analysis, we used the multiplicative synergy index. Results In 32.6% of the 187 patients with deep venous thrombosis who did not have major acquired risk factors, minor events were the only external risk factors. Minor events increased the risk of thrombosis about 3-fold, as estimated in the case-cross-over analysis (odds ratio 2.9, 95% confidence interval 1.5–5.4). The synergy index between minor events and factor V Leiden mutation in the case-only analysis was 0.7 (95% confidence interval 0.3–1.5). Therefore, persons with factor V Leiden mutation who experience a minor event will have an estimated risk increase of about 17-fold, which exceeds the sum of the individual risk factors. Conclusions Minor events are likely to play an important role in the development of deep venous thrombosis, especially in the presence of genetic prothrombotic conditions.
Absence of Correlation between X Chromosome Inactivation Pattern and Plasma Concentration of Factor VIII and Factor IX in Carriers of Haemophilia A and B
Authors: Scheibel Elma , Ingerslev Jørgen , Schwartz Marianne , Ørstavik Karen
Published: March 2018

Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (≥80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells.
The Effect of Flow on Lysis of Plasma Clots in a Plasma Environment
Authors: Rijken Dingeman , Sakharov Dmitry
Published: March 2018

Fibrinolysis initially generates channels in an occluding thombus which results in blood flow through the thrombus. Since the impact of flow along the surface of a thrombus on thrombolysis has not been investigated in detail, we studied in vitro how such a flow affects lysis. Compacted and noncompacted plasma clots were used as model thrombi. With compacted clots, fibrin-specific lysis induced by alteplase in the outer plasma was accelerated about 2-fold by strong flow (arterial shear rate). Non-fibrin-specific lysis induced either by a high concentration of alteplase or by streptokinase was slow, was accompanied by rapid depletion of plasminogen in the outer plasma, and was only slightly accelerated by flow. With noncompacted clots, similar acceleration factors were documented, when mild flow (venous shear rate) was applied. Strong flow further accelerated fibrin-specific lysis, up to 10-fold as compared to lysis without flow, but paradoxically retarded non-fibrin-specific lysis. The data suggest that flow accelerates lysis by enhancing transport of plasminogen from the outer plasma to the surface of the clot. Both opposite effects of the strong flow were mediated by forceful intrusion of the outer plasma into the noncompacted clot due to flow irregularities. In the case of non-fibrin-specific lysis this resulted in the replacement of the plasminogen-containing milieu by plasminogen-depleted outer plasma in certain areas of the clot turning them into virtually unlysable fragments. This flow-enforced “plasminogen steal” may contribute to the relatively high percentage of incomplete thrombolysis (TIMI-2 grade flow) documented in a number of trials for non-fibrin-specific thrombolytic agents. In the case of fibrin-specific lysis, the effect of flow on the speed of fibrinolysis is always beneficial.
Superoxide Anion and Hydroxyl Radical Release by Collagen-induced Platelet Aggregation - Role of Arachidonic Acid Metabolism
Authors: Caccese Daniela , Praticò Domenico , Ghiselli Andrea , Natoli Silvia , Pignatelli Pasquale , Sanguigni Valerio , Iuliano Luigi , Violi Francesco
Published: March 2018

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2
−) and hydroxyl radical (OH°) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2
− and OH°, respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2
− and OH° was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2
− and OH° formation. To further analyze this relationship, O2 and OH° formation was measured if other oxidant species, namely O2
− and OH°, contribute to the during AA-stimulated platelet aggregation (PA); we observed that O − and OH° release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen-induced platelet O− and OH° release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH° and O2
− respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2
− and OH° formation, which is dependent upon AA release and analyzed if O2
− and OH° are released during aggregation induced by metabolism.
The Lupus Ratio Test – An Interlaboratory Study on the Detection of Lupus Anticoagulants by an APTT-based, Integrated, and Semi-quantitative Test
Authors: Barna-Cler L. , Taylor J. M. , Triplett D. A. , Wisløff F. , Jacobsen E. M.
Published: March 2018

The Lupus Ratio (LR) test for lupus anticoagulants integrates screening, mixing with normal plasma and confirmation procedures into one assay. The sensitivity and reproducibility of the APTT based version of this assay was tested in an interlaboratory study that was part of the Fifth International Survey of Lupus Anticogulants (ISLA-5).

One LA negative plasma containing heparin, six LA positive plasmas and a normal pooled plasma (NP) were distributed to 31 laboratories world-wide together with two APTT reagents, one with a high and one with a low phospholipid concentration. The laboratories performed two APTTs, one with each reagent, on 1 : 1 mixtures of test plasma and NP. The ratio between the two clotting times was divided by the corresponding ratio for the NP. This final ratio is the LR of that plasma. The overall sensitivity was found to be 95.1%, and the normal, heparin-containing sample was reported to be negative by all the laboratories.

When the results were grouped in low, medium and high positive plasmas, a “consensus” regarding the strength of each plasma was easily found. 85.0% of the results were in agreement with this consensus.

This study shows that with the LR test, it is possible to obtain high interlaboratory agreement regarding the presence or absence of LA as well as the semi-quantification of this inhibitor.

Appendix, please see p. 708.
Vitronectin and Substitution of a β-Strand 5A Lysine Residue Potentiate Activity-neutralization of PA Inhibitor-1 by Monoclonal Antibodies against α-Helix F
Authors: Schousboe Susanne , Egelund Rikke , Kirkegaard Tove , Preissner Klaus , Rodenburg Kees , Andreasen Peter
Published: March 2018

Some monoclonal antibodies against plasminogen activator inhibitor-1 (PAI-1) are able to inhibit its reaction with its target proteinases. We have characterized the effect on PAI-1 of two monoclonal antibodies, Mab-2 and Mab-6, with overlapping epitopes in a sequence encompassing α-strand 1A, α-helix F, and the loop connecting α-helix F and β-strand 3A (the hF/s3A loop). Mab-2 reduced the inhibitory activity of wild type PAI-1 and almost totally abolished the inhibitory activity of a PAI-1 variant harboring an Ala substitution of Lys 325 (335 in the α1-proteinase inhibitor template residue numbering) in α-strand 5A. In both cases, the neutralizing effect of the antibody was strongly potentiated by vitronectin. Mab-6 had no effect on wild type PAI-1, but reduced the inhibitory activity of the K325A variant. The effect of Mab-6 was not potentiated by vitronectin. With both Mab-2 and Mab-6, the neutralization of PAI-1 activity was associated with PAI-1 substrate behaviour. Mab-2, but not Mab-6, prevented vitronectin from rescuing PAI-1 from cold-induced substrate behaviour. We propose that the antibodies act by weakening the anchoring of α-helix F to the adjacent structures, resulting in an increased flexibility of α-strand 5A and the hF/s3A loop and a changed conformational response to the binding of vitronectin in the α-helix E region. The potentiating effect of vitronectin on neutralization of PAI-1 by antibodies is a novel concept in the development of compounds for neutralizing PAI-1 in vivo.