bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryHuman 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

SummaryPrevious 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

SummaryA 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

SummaryThe 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

SummaryResistance 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

SummaryArdeparin 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

SummaryCD36 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

SummaryAn 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

SummaryResistance 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

SummaryAs 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

SummaryChanges 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

Summaryα-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

SummaryType 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.