169,825 research outputs found
Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations
No full textBlood. 2002 Jun 1;99(11):3985-92.
Isolation and characterization of an antifactor V antibody causing activated
protein C resistance from a patient with severe thrombotic manifestations.
Kalafatis M, Simioni P, Tormene D, Beck DO, Luni S, Girolami A.
Department of Chemistry, Cleveland State University, The Cleveland Clinic
Foundation, Cleveland, OH 44115, USA. [email protected]
A 44-year-old woman with a history of severe thrombotic manifestations presented
with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA
sequencing of and around the regions encoding the APC cleavage sites in the
factor Va molecule excluded the presence of the factor VLeiden mutation and of
other known genetic mutations. No antiphospholipid antibodies were present in the
patient's plasma and both prothrombin time and activated partial thromboplastin
time were normal. The total immunoglobulin fraction was isolated from the
patient's plasma and found to induce severe APC resistance when added to normal
plasma and to factor V-deficient plasma supplemented with increasing
concentrations of factor V. Immunoblotting and immunoprecipitation experiments
with the total immunoglobulin fraction purified from the patient's plasma
demonstrated that the antibody recognizes factor V, is polyclonal, and has
conformational epitopes on the entire factor V molecule (heavy and light chains,
and B region). Thus, the immunoglobulin fraction interferes with the
anticoagulant pathway involving factor V. The inhibitor was isolated by
sequential affinity chromatography on protein G-Sepharose and factor V-Sepharose.
The isolated immunoglobulin fraction inhibited factor Va inactivation by APC
because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the
cofactor. The isolated immunoglobulin fraction was also found to inhibit the
cofactor effect of factor V for the inactivation of factor VIII by the
APC/protein S complex. Our data provide for the first time the demonstration of
an antifactor V antibody not related to the presence of antiphospholipid
antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.
PMID: 12010798 [PubMed - indexed for MEDLINE
Increased tissue factor-initiated prothrombin activation as a result of the Arg506-->Gln mutation in factor V Leiden.
No full textThe effect of the Arg506 --> Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 microM thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nM TFPI, 65 nM protein C, and 10 nM recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nM, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506 --> Gln mutation in factor VLEIDEN
An in vitro analysis of the combination of hemophilia A and factor V (Leiden)
No full textThe classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe ( Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A
A review of the relationships and impact of market orientation and market positioning on organisational performance
No full textThe debate concerning the importance of adopting a market orientation in collaboration with market positioning strategies has gone on for years. Nevertheless, on their own, market orientation and market positioning do not guarantee profitable firm performance unless marketers employ and integrate both on a long-term basis. Achieving this synergy is somewhat problematic owing to focus on short-term operational exigency, as well as the lack of research identifying the relationship between the two concepts. This review fills the gap in the literature by answering two questions: What is the relationship between firm market orientation capability and firm market positioning strategies? How does this relationship impact the performance of organisations? The article sheds light on these issues and contributes to the debate by proposing relationships between positioning strategy and market orientation. Furthermore, the researchers propose how positioning mediates the relationship between market orientation and business benefits
Platelet-derived factor V/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C
No full textWe investigated the role of the thrombin-activated platelet in modulating the rate and extent of activated protein C (APC)-catalyzed inactivation of platelet-derived factor Va and factor VaLeiden. Platelet-derived factor Va and factor VaLeiden were inactivated by APC at near identical rates; however, complete inactivation of the cofactors was never achieved. Greater residual cofactor activity remained when using thrombin-activated platelets compared with that observed with synthetic phospholipid vesicles and platelet-derived microparticles, suggesting that thrombin-activated platelets protect the cofactors from APC-catalyzed inactivation. This apparent protection was not due to (1) an insufficient number of membrane binding sites for APC or factor Va; (2) the destruction of these sites; or (3) the presence of a platelet-associated APC inhibitor. Results from a plasma-based clotting assay (with or without APC) with platelets or PCPS vesicles added to induce clot formation indicated that, even in the presence of high concentrations of APC, platelets offered protection of the cofactor by delaying cleavage at Arg506. This resulted in incomplete proteolysis of the heavy chain, suggesting that platelets can also protect plasma-derived factor Va from APC-catalyzed inactivation. However, additional experiments indicated that the plasma-derived cofactor, bound to thrombin-activated platelets, was completely inactivated by APC, suggesting that the plasma and platelet-derived cofactor pools represent different substrates for APC. Collectively, these results indicate that platelets sustain procoagulant events by providing a membrane surface that delays cofactor inactivation and by releasing a cofactor molecule that displays an APC resistant phenotype. Thus, at sites of arterial injury, the factor VLeiden mutation may not as readily predict arterial thrombosis, because the normal and variant platelet-derived cofactors are equally resistant to APC at the activated platelet surface
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma - Characterization of the dysfunctional protein C Padua(3) (Protein CR-1L/propeptide)
No full textAbstract:
A heterozygous G-->T transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia
Mitomycin C in highly myopic eyes - Author reply
No full textOphthalmology. 2005 Feb;112(2):208-18; discussion 219.
Mitomycin C modulation of corneal wound healing after photorefractive keratectomy in highly myopic eyes.
Gambato C, Ghirlando A, Moretto E, Busato F, Midena E.
SourceRefractive Surgery Service and Antimetabolite Therapy Research Unit, Department of Ophthalmology, University of Padova, Padova, Italy.
Abstract
PURPOSE: To evaluate the role of topical mitomycin C in corneal wound healing (CWH) after photorefractive keratectomy (PRK) in highly myopic eyes.
DESIGN: Prospective, double-masked, randomized clinical trial.
PARTICIPANTS: Seventy-two eyes of 36 patients affected by high (>7 diopters) myopia.
METHODS: In each patient, one eye was randomly assigned to PRK with intraoperative topical 0.02% mitomycin C application, and the fellow eye was treated with a placebo. Postoperatively, mitomycin C-treated eyes received artificial tears (3 times daily, tapered in 3 months), whereas the fellow eye was treated with fluorometholone sodium 2% and artificial tears (3 times daily, tapered in 3 months).
MAIN OUTCOME MEASURES: Uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA), contrast sensitivity, manifest refraction, and biomicroscopy. Contrast sensitivity was determined using the Pelli-Robson chart. Corneal confocal microscopy documented CWH.
RESULTS: Mean follow-up was 18 months (range, 12-36). No side effects or toxic effects were documented. At 12-month follow-up examination, UCVAs (logarithm of the minimum angle of resolution) were 0.4+/-0.48 and 0.5+/-0.53 (P = .03) in mitomycin C-treated eyes and corticosteroid-treated eyes, respectively. At 1 year, corneal haze developed in 20% of corticosteroid-treated eyes, versus 0% of mitomycin C-treated eyes. At 12, 24, and 36 months, corneal confocal microscopy showed activated keratocytes and extracellular matrix significantly more evident in untreated eyes (Ps = 0.004, 0.024, and 0.046, respectively).
CONCLUSION: Topical intraoperative application of 0.02% mitomycin C can reduce haze formation in highly myopic eyes undergoing PRK.
Comment in
Ophthalmology. 2006 Feb;113(2):357; author reply 357-8
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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