bluuebunny/crossref_metadata_2025 huggingface dataset:
Die Anwendung von Gelfiltration zur weiteren Reinigung von Rinderprothrombin
Authors: Mammen E , Ramien A
Published: July 2018

Prothrombin wurde aus Rinderplasma nach der von Seegers beschriebenen Methode (13, 19) isoliert. Durch Gelfiltration an Sephadex G-75 konnte die spezifische Aktivität (Einheiten pro Milligramm Tyrosin) um 27 bis 45% gesteigert werden. Die Ausbeute an Prothrombin nach der Filtration betrug 87 bis 97%. Als Elutionsmittel wurde 0,9% Kochsalzlösung mit verschiedenem pH verwandt, wobei pH 7,0 und pH 8,0 die besten Ergebnisse brachten. Das durch Sephadex filtrierte Prothrombin hat seine Aktivierungseigenschaften nicht geändert, in 25%iger Natriumzitratlösung entsteht Thrombin, und mit Thrombin entsteht Autoprothrombin II. Die mit Sephadex erhaltenen Ergebnisse werden mit den bislang angewandten Verfahren der weiteren Reinigung von Prothrombin verglichen.
On the Inhibiting Effect of ε-Amino Caproic Acid upon the Fibrinolytic System During „Spontaneous Activation”
Authors: Doleschel W , Auerswald W , von Lützow A
Published: July 2018

The effect of ε-ACA (ε-amino caproic acid) upon the factors of the fibrinolytic system contained in a human euglobulin preparation was studied during “spontaneous activation“. The results obtained with the bovine fibrin plate method, the clot lysis, and the casein tests demonstrated a strong inhibitory effect of ε-ACA on the formation of a serum plasminogen activator and on the conversion of plasminogen into plasmin by this activator resulting in a “potentiated inhibition“. On the other hand, no inhibition of plasmin could be found with ε-ACA in concentrations below 0.1 molar. In tests on human fibrin plates finally it became obvious that the human serum plasminogen activator contained in purified human plasmin preparations, having a much higher specificity towards the human plasminogen than the bovine one becomes inhibited by relatively low ε-ACA concentrations. The significance of these results for the inhibitory effect of ε ACA on the fibrinolytic system in vivo was discussed.
The Effects of Leguminous Seed Extracts on Blood Coagulation
Authors: Speer R. J , Porter J. L
Published: July 2018

The saline extracts of one hundred leguminous seed were screened for their effect on blood coagulation by the following tests: thrombin clot time, recalcification time, Quick prothrombin time, clot lysis and clot retraction. Nineteen of these extracts, when reacted with normal plasma, gave a prolonged clot time in at least one of two tests: recalcification time and Quick prothrombin time. The eight which gave a prolonged Quick prothrombin time were tested for inhibition in the following assay systems: Factor V, factor VII-complex, and prothrombin. All nineteen, including the seventeen which prolonged the re-calcification time and the two which did not, were tested for their inhibitory action in the following assay systems: Factor VIII, factor IX, and plasma thromboplastin antecedent.

The inhibition of prothrombin, factor V, and factor VII-complex was mild and not specific for any of these factors. Factor V activity was depressed more than the other two. By contrast, the inhibition of factor VIII, factor IX, and plasma thromboplastin antecedent was very strong and demonstrated a relatively high degree of specificity.
Three Dimensional Structure of Human Fibrinogen under Aqueous Conditions Visualized by Atomic Force Microscopy
Authors: Marchant Roger E , Barb Matthew D , Shainoff John R , Eppell Steven J , Wilson David L , Siedlecki Christopher A
Published: July 2018

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.
Soluble P-selectin in Atherosclerosis: A Comparison with Endothelial Cell and Platelet Markers
Authors: Blann Andrew D , Lip Gregory Y H , Beevers D Gareth , McCollum Charles N
Published: July 2018

von Willebrand factor and soluble thrombomodulin are established plasma markers of endothelial cell dysfunction, whilst beta thromboglobulin is an established plasma marker of platelet activity. Soluble P-selectin may be the product of either or both types of cell and levels of all four molecules have been previously found to be increased in atherosclerosis. To determine the relationship of soluble P-selectin to the endothelial cell and platelet products, we measured the four indices in a case control study of 55 patients with peripheral vascular disease and 55 age and sex matched controls, von Willebrand factor (p <0.0001), beta thromboglobulin (p = 0.0006), soluble P-selectin (p = 0.0021) and soluble thrombomodulin (p = 0.021) were all raised in the patients. Soluble P-selectin correlated with beta thromboglobulin (r = 0.34, p = 0.019) but failed to correlate with either endothelial cell marker. Co-culture of endothelial cells in vitro with bovine thrombin resulted in increased levels of von Willebrand factor in the supernatants but levels of soluble thrombomodulin and soluble P-selectin were not enhanced. Exposure of endothelial cell monolayers to elastase resulted in different patterns of release of von Willebrand factor, soluble thrombomodulin and soluble P-selectin. We suggest that soluble P-selectin is unlikely to arise from the endothelium and may be a new marker of platelet activation in atherosclerosis.
Positive Association of the β Fibrinogen H1/H2 Gene Variation to Basal Fibrinogen Levels and to the Increase in Fibrinogen Concentration during Acute Phase Reaction but not to Coronary Artery Disease and Myocardial Infarction
Authors: Gardemann Andreas , Schwartz Oliver , Haberbosch Werner , Katz Norbert , Weiß Tanja , Tillmanns Harald , Hehrlein Friedrich Wilhelm , Waas Wolfgang , Eberbach Andreas
Published: July 2018

Background: Fibrinogen has been demonstrated to be an independent risk factor of cardiovascular disease. The absence of the Haelll cutting site (H2 allele) of an H1/H2 gene variation in the promoter region of the (β fibrinogen gene was associated with increased levels of fibrinogen. Methods and Results: In the present study, the effects of the H1/H2 gene variation not only on plasma fibrinogen concentrations but also on coronary artery disease (CAD) and myocardial infarction (MI) were investigated in 923 individuals who underwent coronary angiography for diagnostic purposes. Relation of the H1/H2 genotype to fibrinogen plasma levels: A strong association was observed between the H1/H2 gene variation and fibrinogen levels. The differences in fibrinogen plasma levels between H2H2 and H1H1 homozygotes were almost threefold more pronounced within subjects with clinical chemical signs of an acute phase reaction (CRP ≥ 7.5 mg/1) than within a subgroup of subjects without these signs (CRP < 7.5 mg/1) (median of CRP distribution: 7.5 mg/1). In 207 patients who underwent aortocoronary bypass surgery plasma fibrinogen levels were almost identical directly after surgery. Two days after operation fibrinogen increased to clearly higher levels in H2H2 homozygotes than in H1H2 and H1H1 genotypes, whereas almost the same maximal increases in fibrinogen concentrations were reached 3-4 days after surgery in all individuals. Relation of the H1/H2 genotype to CAD and MI. Whereas in the total population the plasma fibrinogen concentrations were strongly associated with smoking, CAD and MI, an association of the H1/H2 gene variation to CAD and MI was not detected. However, mean age at first MI of H2H2 individuals (62.9 years) was clearly higher than of H1H2 genotypes (56.9 years) and of H1H1 subjects (56.4 years). In addition, in a subgroup of individuals with a higher risk of MI by either high apoB and/or low apoAl plasma levels the portion of MI patients was clearly smaller within H2H2 homozygotes than within H1H2 or H1H1 genotypes, although – also in these high risk groups – mean age at first MI of H2H2 individuals were higher than of the other two genotypes.

Conclusions: Obviously, the H2 allele of the fibrinogen H1/H2 genotype does not only influence basal fibrinogen concentrations, but particularly also the extent of fibrinogen level increase during acute phase reaction. Whereas the fibrinogen plasma level is positively associated with coronary artery disease and myocardial infarction, the H2 allele - although exhibiting an association with elevated fibrinogen levels - was not positively associated with CAD and MI.
The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S
Authors: Lu Deshun , Xie Rong-Lin , Rydzewski Andrzej , Long George L
Published: July 2018

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458’468’489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458→Gln, Ser460→Gly; Asn468→Gln, Thr470→Gly; Asn489→Gln, Thr491→Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of ~78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by α-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser460) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.
Effects of Heparin Therapy on Fibrinolysis in Patients with Pulmonary Embolism
Authors: Minnema M C , ten Cate H , Beek E J R van , Ende A van den , Hack C E , Brandjes D P M
Published: July 2018

Previous investigations suggested that heparin administration to humans enhances the tissue type plasminogen activator (tPA) levels in blood, but it remains uncertain whether this effect induces fibrinolysis. We studied the effect of therapeutic levels of heparinization on plasma markers for fibrinolysis in patients suspected of pulmonary embolism (PE). Blood samples were taken from 49 consecutive patients; 28 had confirmed PE, 21 had PE excluded.

On admission, the plasma levels of plasmin-α2antiplasmin complexes and D-dimer were significantly higher in the patient group with PE compared to those in whom PE was excluded. After heparinization the tPA levels increased in both groups, showing that this effect was not dependent on the initial level of activity of fibrinolysis. In spite of this increment in tPA levels, the concentrations of plasmin-α2antiplasmin complexes and D-dimer decreased.

In conclusion, although heparinization in patients with or without pulmonary embolism does lead to elevated tPA: Ag levels, this is not accompanied by enhanced fibrinolysis.
D-Dimer Determination to Assess Regression of Deep Venous Thrombosis
Authors: Janssen M C H , Verbruggen H , Wollersheim H , Hoogkamer B , van Langen H , Nováková I R O
Published: July 2018

A number of studies evaluating deep venous thrombosis (DVT) have demonstrated that plasma levels of thrombotic and fibrinolytic parameters change during treatment, but the relationship between thrombus regression and evolution of these markers remains unknown. The objective of the present study was to correlate levels of D-Dimer (DD) with thrombus regression as assessed by duplex scanning.

From 44 patients treated for acute DVT, DD were determined at diagnosis and at the end of initial heparin therapy of at least 5 days. Thrombus regression was measured by repeated duplex scanning at diagnosis and after 1 and 3 months.

DD significantly decreased during heparin treatment as compared with values at presentation. DD levels were significantly higher in the group of patients without normalization of the DVT after 3 months (p = 0.003). A ninefold excess tendency was seen for DD levels > 1200 ng/ml at the end of initial treatment to be associated with poor resolution of the DVT [odds ratio 9.0, 0.95 confidence interval (CI) 2.3-35.4]. When the patients with an established malignancy were excluded, the differences were even more significant (p = 0.0004 for DD levels after initial treatment and an odds ratio of 17.5, 0.95 CI 3.3-92.5).

These results suggest that increased DD levels after the initial phase of treatment are related to poor resolution of DVT after 3 months. These findings contribute to further insight into the process of thrombus regression. Furthermore high DD levels might help to identify the patients with a poor prognosis and could be useful to judge the efficacy of anticoagulant treatment.
Factor XI Activation by Meizothrombin: Stimulation by Phospholipid Vesicles Containing both Phosphatidylserine and Phosphatidylethanolamine
Authors: Borne Peter A Kr von dem , Mosnier Laurent O , Tans Guido , Meijers Joost C M , Bouma Bonno N
Published: July 2018

The activation of factor XI by meizothrombin was investigated using recombinant meizothrombin (R155A meizothrombin) that is resistant to autocatalytic removal of fragment 1. Meizothrombin was capable of activating factor XI at an activation rate similar to that of thrombin. Dextran sulphate and heparin, known cofactors of thrombin-mediated factor XI activation, did not stimulate the activation of factor XI by meizothrombin. However, the activation of factor XI by meizothrombin was markedly enhanced by vesicles containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas PC/PS or PC/PE vesicles only had a minor effect on the activation. Thrombin-mediated factor XI activation was not influenced by phospholipids.

The effect of PC/PS/PE and PC/PS vesicles was studied in a factor XI dependent clot lysis assay. In this assay, factor XI inhibits clot lysis by a feedback loop in the intrinsic pathway via thrombin-mediated factor XI activation. Removal of endogenous phospholipids in plasma by centrifugation resulted in an increased clot lysis, which could be restored to the pre-centrifugation level by the addition of PC/PS/PE vesicles, but not by PC/PS vesicles. When clot lysis was initiated by factor IXa in the presence of a factor XIa blocking antibody, there was no difference in inhibitory effect of PC/PS/PE or PC/PS vesicles. These data suggested that the differences in clot lysis inhibition observed between PC/PS/PE and PC/PS vesicles were caused by factor XI activation by meizothrombin. Meizothrombin-mediated factor XI activation may therefore play an important role in the antifibrinolytic feedback loop in the intrinsic pathway.
The VITA Project: Phenotypic Resistance to Activated Protein C and FV Leiden Mutation in the General Population
Authors: Tosetto Alberto , Rodeghiero Francesco , Missiaglia Edoardo , Gatto Elisabetta
Published: July 2018

Resistance to activated protein C (APC) has been recently identified as a common abnormality of the clotting system that significantly increases the risk of venous thromboembolism. The distribution of plasma response to APC in the general population and the variables potentially influencing it are however unknown. In this study, we analyzed the data from the first 4,000 subjects enrolled in the Vicenza Thrombophilia and Atherosclerosis (VITA) Project to identify the demographic and laboratory variables affecting the plasma response to APC. Plasma response to APC, expressed as APC-ratio, was significantly influenced not only by the presence of the FV Leiden mutation but also by the aPTT ratio, triglycerides, fibrinogen and cholesterol level and by pill use, ABO blood group, gender, smoke, body-mass index and age. The effect of these variables was independent of the presence of the FV Leiden mutation, and adjustment for their effect improved the discriminating efficiency of the APC-ratio for the presence of the FV Leiden mutation. Notwithstanding adjustment, the APC ratio was unsuitable for screening purposes in the general population (positive predictive value 82.7%).
Absolute and Comparative Subcutaneous Bioavailability of Ardeparin Sodium, a Low Molecular Weight Heparin
Authors: Troy Steven , Fruncillo Richard , Ozawa Tsunenori , Mammen Eberhard , Holloway Scott , Chiang Soong
Published: July 2018

Ardeparin sodium (Normiflo®, Wyeth-Ayerst) is a low molecular weight heparin undergoing clinical evaluation as an antithrombotic agent. The objective of this study was to evaluate the absolute and comparative bioavailability of ardeparin following subcutaneous administration of three different formulations [two formulations of ardeparin at 10,000 anti-factor Xa (aXa) U/ml, but with different preservatives, and a 20,000 aXa U/ml formulation]. The study was conducted using a randomized 4-period crossover design (three subcutaneous treatments and one intravenous treatment) in 24 healthy subjects, and the pharmacokinetics of ardeparin were characterized by plasma anti-factor Ila (alia) and anti-factor Xa (aXa) activities. The mean absolute bioavailability of ardeparin based on alia activity ranged from 62% to 64% and the mean absolute bioavailability based on aXa activity ranged from 88% to 97%. Based on bioequivalence testing criteria, the three ardeparin formulations were bioequivalent.
Thrombospondin Induces Dimerization of Membrane-Bound, but not Soluble CD36
Authors: Daviet L , Malvoisin E , Wild T F , McGregor J L
Published: July 2018

CD36 is a cell surface receptor that has been shown to interact with a large variety of ligands including thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes, apoptotic neutrophils, modified low density lipoproteins, anionic phospholipids and long chain fatty acids. A number of these CD36 ligands elicit the transduction of intracellular signals involved in cell activation and internalization of bound ligands. The engagement of CD36 possibly activates three cytosolic protein tyrosine kinases that are presumably associated with the C- terminal cytoplasmic tail of CD36. However, the mechanisms by which CD36 functions in ligand binding and signal transduction are poorly understood. In the present study, a membrane-bound and a truncated soluble form of CD36 were expressed in HeLa cells and analyzed by velocity-gradient centrifugation and chemical cross-linking. We show that membrane CD36 exists predominantly as a monomer but a homo- dimeric form is also found. In contrast, soluble CD36 sedimented in sucrose gradient as a monomer. However, when incubated with thrombospondin, the membrane form of CD36 predominantly sedimented as a dimer whereas soluble CD36 was monomeric. This study shows that thrombospondin has the ability to induce dimerization of CD36 and may be implicated in the signal transduction capacity of this adhesion molecule.
Application of Thrombin Based Fibrin Glue and Non-Thrombin Based Batroxobin Glue on Intact Human Blood Vessels: Evidence for Transmural Thrombin Activity
Authors: Dascombe William H , Dumanian Gregory , Hong Chull , Heil Brian V , Labadie Karyn , Hessel Birgit , Blombäck Birger , Johnson Peter C
Published: July 2018

An alternative method of uniting small diameter vessels to obtain tissue union while limiting the thrombogenic effect of suture placement at a vessel anastomosis involves the use of a thrombin based fibrin glue as a surgical sealant. This investigation addresses whether the in vitro application of a thrombin based glue (TG), or batroxobin glue (BG), a non-thrombin based glue made with the snake venom enzyme batroxobin, alters intravascular platelet deposition (PD) or cleaves blood fibrinogen, as measured by fibrinopeptide A (FPA) production, when the respective glue is applied to the external surface of an intact human placental artery or an artery with an anastomosis.

When TG was applied to the adventitial surface of an intact vessel or an anastomosis (n = 7) of control and experimental vessels, there was a significant increase in intraluminal platelet deposition, an effect not realized with BG (n = 12, intact vessel TG p = 0.01, BG p = 0.66, anastomosis TG p <0.01, BG p <0.01). Both TG and BG significantly increased FPA levels when human whole blood was perfused through both intact vessels or vessels containing an anastomosis when compared to control vessels (intact vessel TG and BG p <0.01, anastomosis TG and BG p <0.01). Labelled thrombin studies document the rapid passage of thrombin through an intact vessel wall or vessels with an anastomosis when TG was applied to the adventitial surface of the vessel.

The data suggest that TG and BG are drug delivery systems for their respective enzymes that either pass through or transfer a message across not only a surgically created anastomosis, but also an intact vessel wall.
Female Gender and Resistance to Activated Protein C (FV:Q506) as Potential Risk Factors for Thrombosis after Elective Hip Arthroplasty
Authors: Svensson P J , Benoni G , Fredin H , Bjӧrgell O , Nilsson P , Hedlund U , Nylander G , Bergqvist D , Dahlbäck B
Published: July 2018

Resistance to activated protein C (APC) caused by the R506Q mutation in factor V is the most common inherited risk factor for venous thrombosis. To elucidate whether APC-resistance is a risk factor for venous thrombosis after elective total hip replacement, the association between APC-resistance (presence of FV:Q506 allele) and postoperative thrombosis was investigated in patients (n = 198) randomised to received short (during hospitalisation, n = 100) or prolonged prophylaxis (three weeks after hospitalisation, n = 98) with low molecular weight heparin (LMWH). Among APC-resistant individuals receiving short prophylaxis, 7/10 developed thrombosis as compared to 2/12 receiving long prophylaxis (p <0.0179). Odds ratio for association between APC-resistance and thrombosis in the short prophylaxis group was 4.2 (CI 95% 1.02-17.5) (p <0.0465). Among those receiving prolonged, prophylaxis, there was no increased incidence of thrombosis associated with APC-resistance. Two unexpected observations were made. One was that APC-resistance was much more common in women (19/109) than in men (3/89) (p <0.001). The other was that even women without APC-resistance were much more thrombosis-prone than men. Thus, 24/48 of women with normal FV genotype and short prophylaxis developed thrombosis vs 8/42 among men, p = 0.002. The increased risk of thrombosis associated with female gender and APC-resistance was neutralised by the prolonged treatment. In conclusion, among patients receiving short prophylaxis, female gender was found to be a strong risk factor for venous thrombosis. Even though APC-resistance appeared to be a risk factor for postoperative thrombosis, the uneven distribution of APC-resistance between men and women, taken together with the increased risk of thrombosis among women, precluded valid conclusions to be drawn about the association between APC-resistance and an increased risk of thrombosis. Our results suggest that prolonged prophylaxis with LMWH after hip surgery is more important for women than for men.
Seasonal Variation in Fibrinogen in the Rotterdam Study
Authors: Bom J G van der , Maat M P M de , Bots M L , Hofman A , Kluft C , Grobbee D E
Published: July 2018

As evidence accumulates to implicate fibrinogen as a risk indicator for cardiovascular disease, it is of interest to study its seasonal variation. A population based cross-sectional study was performed among participants of the Rotterdam Study, a cohort of 7,983 men and women, aged 55 years and over. Fibrinogen levels were measured by the prothrombin time derived method in the first 2,325 participants of the study. Fibrinogen levels were considerably higher in winter. The seasonal difference was 0.34 g/1 (95% confidence interval 0.29,0.39) and was more pronounced in subjects aged 75 years and over than in subjects aged 55 to 75 years, 0.43 g/1 (0.34,0.52) and 0.29 g/1 (0.24,0.35), respectively. Additional adjustment for body mass index, systolic and diastolic blood pressure, and total and HDL cholesterol did not materially change the findings. After adjustment for seasons, outdoor temperature was not associated with fibrinogen. Adjustment for outdoor temperature did not change the seasonal variation of fibrinogen, seasonal difference 0.31 g/1 (0.24, 0.37). In conclusion, fibrinogen levels are highest in Winter. The seasonal variation of fibrinogen is more pronounced in the elderly. Outdoor temperature does not seem to play a role in the seasonal variation of fibrinogen. Seasonal variation of fibrinogen may partly explain the increased cardiovascular disease mortality in Winter.
Properties of Optical Data from Activated Partial Thromboplastin Time and Prothrombin Time Assays
Authors: Braun Paul J , Givens Thomas B , Stead Andrew G , Beck Lisa R , Gooch Sheila A , Swan Robert J , Fischer Timothy J
Published: July 2018

Changes in characteristics of optical transmittance data from coagulation assays were examined as a function of concentration of coagulation proteins or anticoagulants. Transmittance data were collected for activated partial thromboplastin time (APTT) and prothrombin time (PT) assays from: 1) plasmas prepared by mixing normal plasmas with deficient plasmas to give varying levels of coagulation proteins; 2) plasmas containing added heparin; and 3) 200 specimen plasmas that were also assayed for fibrinogen, coagulation factors, and other components. Optical profiles were characterized using a set of parameters describing onset and completion of coagulation, magnitude of signal change, rate of coagulation and other properties. Results indicated that parameters other than those typically reported for APTT and PT are associated with individual deficiencies, but that diagnosis of specimen status on the basis of optical data is complex. These results suggest possibilities for expanded interpretation of PT/APTT optical data for clinical or research applications.
Identification of an Epitope of α-Enolase (a Candidate Plasminogen Receptor) by Phage Display
Authors: Arza Begoña , Félez Jordi , Lopez-Alemany Roser , Miles Lindsey A , Munñoz-Cánoves Pura
Published: July 2018

α-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An α-enolase related molecule (a-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that a-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against a-ERM. This mAB reacted with both α-ERM and purified human α-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of a-enolase/a-ERM in permea bilized U937 cells. A phage display approach was employed to identify the a-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human α-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 en coded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human α-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that a-enolase and a-ERM share common amino acid sequences.
A Type 2b von Willebrand Disease Mutation (lle546→Val) Associated with an Unusual Phenotype
Authors: Federici A B , Mannucci P M , Stabile F , Canciani M T , Di Rocco N , Miyata S , Ware J , Ruggeri Z M
Published: July 2018

Type 2B von Willebrand disease (vWD) is typically characterized by enhanced ristocetin-induced platelet aggregation (RIPA) caused by increased von Willebrand factor (vWF) affinity for platelets. Furthermore, absence of larger vWF multimers in plasma is characteristic of the originally described type IIB patients, now considered a subgroup of type 2B. We describe here three affected members of a family presenting with prolonged bleeding time, thrombocytopenia, markedly enhanced RIPA and spontaneous platelet aggregation, but normal plasma vWF antigen and ristocetin cofactor activity. Larger plasma vWF multimers, albeit decreased, were present in relatively greater proportion than in other type IIB patients. Genetic studies performed in two of these patients resulted in the identification of a previously unreported A→G transition at nucleotide 4175 in the sequence of the pre-pro-vWF cDNA, corresponding to the substitution Ile546→Val in the mature vWF subunit. This mutation appears to be responsible for an unusual type 2B phenotype, with greater enhancement of the vWF platelet interaction than in typical cases but partial preservation of the larger vWF multimers in plasma.
Effects of Gemfibrozil and Ciprofibrate on Plasma Levels of Tissue-type Plasminogen Activator, Plasminogen Activator Inhibitor-1 and Fibrinogen in Hyperlipidaemic Patients
Authors: Kockx Maaike , de Maat Moniek P M , Knipscheer Haye C , Kastelein John J P , Kluft Cornelis , Princen Hans M G , Kooistra Teake
Published: July 2018

Evaluation of fibrate treatment in humans has focused primarily on its anti-lipidaemic effects. A potentially favourable haemostasis-modulating activity of fibrates has also been recognized but the data are not consistent. We sought to learn more about this variability by examining the effects of gemfibrozil and ciprofibrate on plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and fibrinogen in primary hyperlipidaemic patients after six and twelve weeks of treatment using different assay systems for PAI-1 and fibrinogen. Although both fibrates effectively lowered triglyceride and cholesterol levels, no effect on the elevated baseline antigen levels of t-PA and PAI-1 was observed after fibrate treatment. However, both fibrates influenced plasma fibrinogen levels, albeit in a different way. Fibrinogen antigen levels were elevated by 17.6% (p <0.05) and 24.3% (p <0.001) with gemfibrozil after six and twelve weeks, respectively, whereas with ciprofibrate there was no effect. Using a Clauss functional assay with either a mechanical end point or a turbidity-based end point, no significant change in fibrinogen levels was seen after six weeks of gemfibrozil treatment. However, after twelve weeks, gemfibrozil enhanced functional fibrinogen levels by 7.2% (p <0.05) as assessed by the Clauss mechanical assay, but decreased functional fibrinogen levels by 12.5% (p <0.0001) when a Clauss assay based on turbidity was used. After six or twelve weeks of ciprofibrate treatment, functional fibrinogen levels were decreased by 10.1% (p <0.001) and 10.5% (p <0.0001), respectively on the basis of Clauss mechanical and by 14.2% (p <0.001) and 28.2% (p <0.0001), respectively with the Clauss turbidimetric assay. A remarkable and consistent finding with both fibrates was the decrease in functionality of fibrinogen as assessed by the ratio of functional fibrinogen (determined by either of the two Clauss assays) to fibrinogen antigen.

Taken together, our results indicate that at least part of the variability in the effects of fibrates on haemostatic parameters can be explained by intrinsic differences between various fibrates, by differences in treatment period and/or by the different outcomes of various assay systems. Interestingly, the two fibrates tested both reduced the functionality of fibrinogen.
High Antibody Levels to Prothrombin Imply a Risk of Deep Venous Thrombosis and Pulmonary Embolism in Middle-aged Men
Authors: Palosuo Timo , Virtamo Jarmo , Haukka Jari , Taylor Philip R , Aho Kimmo , Puurunen Marja , Vaarala Outi
Published: July 2018

Antibodies against phospholipid-binding plasma proteins, such as β2-glycoprotein I (β2-GPI) and prothrombin, are associated with thromboembolic events in patients with systemic lupus erythematosus and also in subjects with no evident underlying diseases. We wanted to examine whether increased levels of antibodies to negatively-charged phospholipids (cardiolipin), to phospholipid-binding plasma proteins β2-GPI and prothrombin and to oxidised low-density lipoprotein (LDL) were associated with risk of deep venous thrombosis or pulmonary embolism in subjects with no previous thrombosis. The antibodies were measured in stored serum samples from 265 cases of deep venous thrombosis of the lower extremity or pulmonary embolism occurring during a median follow-up of about 7 years and from 265 individually matched controls. The study subjects were middle-aged men participating in a cancer prevention trial of alpha-tocopherol and beta-carotene and the cases of thromboembolic events were identified from nationwide Hospital Discharge Register.

The risk for thrombotic events was significantly increased only in relation to antiprothrombin antibodies. As adjusted for body mass index, number of daily cigarettes and history of chronic bronchitis, myocardial infarction and heart failure at baseline, the odds ratio per one unit of antibody was 6.56 (95% confidence interval 1.73-25.0). The seven highest individual optical density-unit values of antiprothrombin antibodies were all confined to subjects with thromboembolic episodes.

In conclusion, the present nested case-control study showed that high autoantibody levels against prothrombin implied a risk of deep venous thrombosis and pulmonary embolism and could be involved in the development of the thrombotic processes.
Effect of Oxidized Low Density Lipoprotein on Thrombomodulin Expression by THP-1 Cells
Authors: Oida Koji , Tohda Gen , Ishii Hidemi , Horie Shuichi , Kohno Mitsuyuki , Okada Eiko , Suzuki Jinya , Nakai Jsuguhiko , Miyamori Isamu
Published: July 2018

We have investigated the effects of oxidized low density lipoproteins (oxidized LDL) on the expression of TM by THP-1 monocytic cells. TM antigen levels and its cofactor activity for thrombin-dependent protein C activation were increased by oxidized LDL and accompanied by an increase in TM mRNA levels. Incubation of THP-1 cells with 300 μg/ml oxidized LDL for 24 h resulted in an 80% increase of cellular TM antigen levels. Native LDL and acetylated LDL did not affect the TM expression by these cells. The resultant aqueous phase after extraction of oxidized LDL by chloroform/methanol increased the TM antigen levels as well as oxidized LDL. Phorbol 12-myristate 13-acetate (PMA) also increased the TM antigen level 2.1 times the control and was accompanied by the adhesion of cells to plastic dishes and increasing macrophage cell surface antigen CD 14 levels. In contrast, oxidized LDL did not induce differentiation to the macrophage. The present results indicate that oxidized LDL increases cellular TM antigen without cellular differentiation and that up-regulation of TM by oxidized LDL in monocytes may have some implication in atherosclerosis.
An in vivo Model for the Assessment of Acute Fibrinolytic Capacity of the Endothelium
Authors: Newby David E , Wright Robert A , Ludlam Christopher A , Fox Keith A A , Boon Nicholas A , Webb David J
Published: July 2018

The effects on blood flow and plasma fibrinolytic and coagulation parameters of intraarterial substance P, an endothelium dependent vasodilator, and sodium nitroprusside, a control endothelium independent vasodilator, were studied in the human forearm circulation. At subsystemic locally active doses, both substance P (2-8 pmol/min) and sodium nitroprusside (2-8 μg/min) caused dose-dependent vasodilatation (p <0.001 for both) without affecting plasma concentrations of PAI-1, von Willebrand factor antigen or factor VIII:C activity. Substance P caused local increases in t-PA antigen and activity (p <0.001) in the infused arm while sodium nitroprusside did not. At higher doses, substance P increased blood flow and t-PA concentrations in the noninfused arm. We conclude that brief, locally active and subsystemic infusions of intraarterial substance P cause a rapid and substantial local release of t-PA which appear to act via a flow and nitric oxide independent mechanism. This model should provide a useful and selective method of assessing the in vivo capacity of the forearm endothelium to release t-PA acutely.
Testing for Occult Cancer in Patients with Idiopathic Deep Vein Thrombosis – A Decision Analysis
Authors: Barosi Giovanni , Marchetti Monia , Dazzi Luisella , Quaglini Silvana
Published: July 2018

Objective. To determine the effectiveness and cost-effectiveness of testing for occult cancer in idiopathic deep vein thrombosis (IDVT). Design. Threshold analysis was performed on the risk-adjusted cancer prevalence in a cost-effectiveness model of ideal testing for selecting cancers with potentially desirable utility (candidate cancers). Decision analysis was employed to compare different testing programs for candidate cancers with that of no testing. Life expectancy (LE) of early- and late-detected cancers and costs of testing were the dimensions of utility. Cost-effectiveness was expressed as marginal cost per year of life saved. The perspective of the third payer was adopted, and a discount rate of 3% was applied to both costs and benefits. Data sources. Risk of cancer in IDVT, testing policies, test characteristics, and LE were gathered from literature. Costs were provided from our hospital rate book and accounting service. Results. Ideal testing would support a gain of LE of 40 days or more for prostate, colon and bladder cancer in males and for colon, breast and endometrium cancer in females aged from 60 to 69 years. Testing females with colonoscopy and mammography in any sequence provides 70 days of life gained. Testing males with colonoscopy provides 27 days of life gained. Lower and older ages reduce testing effectiveness. The qualitative results are stable over plausible ranges of test characteristics, while variations in the value of benefit for early cancer diagnosis may modify the strategy. Incremental cost-effectiveness ranges from $ 1,789 to $ 6,979 per year of life gained. Conclusions. According to the effectiveness criterion adopted, the only worthwhile investigation strategy includes colon and breast cancer in females. Testing for colon cancer in males is desirable at a lower criterion of effectiveness. All the strategies are cost effective.
Endothelial Marker Proteins in Hyperhomocysteinemia
Authors: Jong Sylvia C de , Stehouwer Coen D A , Berg Michiel van den , Vischer Ulrich M , Rauwerda Jan A , Emeis Jef J
Published: July 2018

Hyperhomocysteinemia is associated with severe, premature atherosclerosis and thromboembolism. The mechanisms involved in the atherogenic and thrombotic complications of hyperhomocysteinemia are not understood. It has been suggested that hyperhomocysteinemia predisposes to atherosclerosis by injuring the vascular endothelium. Whether hyperhomocysteinemia is independently associated with changed endothelial function, either in the absence or the presence of clinically manifest atherosclerotic disease, is, however, not known. Therefore we investigated, both in patients with peripheral arterial occlusive disease and in healthy individuals, whether plasma protein markers of endothelial function differed between subjects with, and subjects without hyperhomocysteinemia. We studied 80 individuals under the age of 56 years: healthy individuals with (n = 20) and without (n = 20) hyperhomocysteinemia and patients with peripheral arterial occlusive disease with (n = 20) and without (n = 20) hyperhomocysteinemia. The following endothelium-derived proteins were measured as markers of endothelial cell function: von Willebrand factor (vWf) and von Willebrand factor propeptide (vWf: Agll), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), cellular fibronectin (cFN) and thrombomodulin ™. In addition we assessed C-reactive protein (CRP). vWf, vWf: Agll, tPA and CRP were significantly higher in the patients with peripheral arterial occlusive disease than in the healthy individuals. No differences in marker protein plasma levels were found between individuals with, and those without hyperhomocysteinemia, apart from vWf, which was significantly raised in hyperhomocysteinemic as compared to normohomocysteinemic patients. We did not find any evidence for an independent association between hyperhomocysteinemia and protein markers of endothelial cell function in healthy subjects.
A Multicenter Pharmacosurveillance Study for the Evaluation of the Efficacy and Safety of Recombinant Factor VIII in the Treatment of Patients with Hemophilia A
Authors: Aygören-Pürsün Emel , Scharrer Inge ,   German Kogenate Study Group
Published: July 2018

In this open multicenter study the safety and efficacy of recombinant factor VIII (rFVIII) was assessed in 39 previously treated patients with hemophilia A (factor VIII basal activity ≤15%).

Recombinant FVIII was administered for prophylaxis and treatment of bleeding episodes and for surgical procedures. A total of 3679 infusions of rFVIII were given. Efficacy of rFVIII as assessed by subjective evaluation of response to infusion and mean annual consumption of rFVIII was comparable to that of plasma derived FVIII concentrates. The incremental recovery of FVIII (2.4 ± 0,83%/IU/kg, 2.12 ± 0.61%/IU/kg, resp.) was within the expected range. No clinical significant FVIII inhibitor was detected in this trial. Five of 16 susceptible patients showed a seroconversion for parvovirus B19. However, the results are ambiguous in two cases and might be explained otherwise in one further case. Thus, in two patients a reliable seroconversion for parvovirus B19 was observed.
Antivasoconstrictor and Antiaggregatory Activities of Picotamide Unrelated to Thromboxane A2 Antagonism
Authors: Vezza Roberta , Spina Domenico , Tallarida Ronald J , Nathan Malevika , Page Clive P , Gresele Paolo
Published: July 2018

Picotamide is a dual thromboxane (Tx) A2 receptor antagonist/Tx synthase inhibitor although some observations suggest an anti-vasoconstrictor effect independent of TxA2 inhibition/antagonism. The aim of our study was to assess whether picotamide antagonises vascular contractions induced by different vasoactive substances in vitro. Picotamide inhibited competitively the contraction of rabbit aortic rings induced by the TxA2 mimetic U46619 (pA2 = 3.59) but also the contractions induced by phenylephrine (pA2 = 3.93) and serotonin (5-HT) (pA2 = 5.81) although in a not competitive way. Picotamide did not inhibit potassium-induced contractions, thus excluding aspecific effects on vascular smooth muscle. Picotamide inhibited 5-HT-induced platelet aggregation in vitro with an IC50 (212 μM) similar to that found when other aggregating stimuli are used, but it did not affect shape change (IC50> 1 mM) suggesting that the effects of picotamide can not be ascribed to 5-HT2-receptor antagonism; in the same experimental conditions neither a Tx-receptor antagonist (BM13.177) nor a dual Tx-receptor antagonist/synthase inhibitor (ridogrel) affected 5-HT-induced platelet responses.

Our studies demonstrate that picotamide exerts antivasoconstrictor and platelet inhibitory effects unrelated to TxA2 antagonism. This activity may contribute to the anti-thrombotic/anti-ischaemic effects of the drug in vivo.
Platelet Activation Markers in Patients with Peripheral Arterial Disease
Authors: Gresele Paolo , Catalano Mariella , Giammarresi Carlo , Volpato Raul , Termini Rosanna , Ciabattoni Giovanni , Nenci Giuseppe G , Davì Giovanni
Published: July 2018

Peripheral vascular disease (PVD) is an indicator of diffuse atherosclerosis and is associated with a greatly increased incidence of coronary heart and cerebrovascular disease. Although several studies have assessed whether in vivo platelet activation takes place in patients with PVD, no data are available comparing different platelet function tests in this patient population.

We have compared prospectively four tests for the measurement of in vivo platelet activation (plasma βTG, plasma PF4, intraplatelet (βTG and urinary excretion of 11-dehydro-TXB2) and one in vitro platelet function test (ADP-induced platelet aggregation) in 63 well-characterized patients with intermittent claudication and in 18 age- and sex- matched healthy volunteers.

No statistically significant difference was found between patients and controls for plasma βTG (20.0 ± 11.8 vs. 18.8 ± 9.0 ng/ml, respectively), plasma PF4 (5.2 ± 2.9 vs. 6.3 ± 3.5 ng/ml), βTG/PF4 ratio (4.0 ± 2.9 vs. 3.6 ± 1.8), intraplatelet pTG (4503 ± 1482 vs. 4059 ± 1065 ng/ml), and threshold aggregatory concentration of ADP (1.7 ± 0.72 vs. 1.45 ± 0.56 μM).

Urinary 11-dehydro-TXB2 was instead significantly higher in the PVD group (55.4 ± 27.5 vs. 26.7 ± 7.0 ng/h, p <0.001).

Our study shows that urinary 11-dehydro-TXB2 is a more sensitive index of in vivo platelet activation than the measurement of either platelet specific proteins or of in vitro platelet aggregation in patients with PVD.