An Assay for the Discrimination of Increased VWF Clearance in VWD.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3478-3478
Author(s):  
Michelle A Stapleton ◽  
Kristi Ladvienka ◽  
Elizabeth Wuitschick
Keyword(s):  
Half Life ◽  
Limit Of Detection ◽  
Performance Testing ◽  
Grey Zone ◽  
Antigen Level ◽  
Factor Activity ◽  
Operating Characteristics ◽  
Patient Sample ◽  
Bleeding History

Abstract Abstract 3478 Poster Board III-415 The quantitative deficiency in von Willebrand Factor (VWF) levels observed in Type 1 VWD can be caused by ineffective synthesis and storage or by a decrease in the half-life of the VWF in the circulation. To date, a number of different point mutations in VWF have been shown to cause a reduced VWF half-life. Clinically it is important to recognize this enhanced clearance phenotype because the increased clearance of VWF can reduce the efficacy of desmopressin treatment in these patients. The VWF propeptide is synthesized as part of a pro-VWF protein and is subsequently cleaved, stored and secreted in an equi-molar ratio with mature VWF. The level of VWF propeptide in the circulation can be used as a marker of VWF synthesis. In individuals with low VWF synthesis, the propeptide level is similarly decreased yielding a propeptide: VWF ratio near 1.0. In individuals with normal levels of VWF synthesis and decreased survival of VWF in circulation, an increased propeptide:VWF ratio is observed. GTI Diagnostics, Inc. (Waukesha, Wisconsin) has developed a fluorescent ELISA for the quantitative measurement of VWF levels and VWF propeptide levels in plasma and for the calculation of a propeptide:VWF ratio. All reagents necessary to run the assay are included in the kit as well as an analysis workbook for easy calculation of results. Assay incubation steps are only 15 minutes, therefore the assay can be completed in 90 minutes. In performance testing the VWF & Propeptide Assay showed excellent within-run, between-run and total imprecision. The limit of detection is 0.02 IU/dL for VWF and 0.02 U/dL for propeptide. The assay range varies depending on the calibrator stock included in the kit however the assay range is at least 1 – 273 IU/dL VWF or U/dL propeptide. No patient sample conditions tested were shown to interfere with the assay. Clinical studies were conducted to evaluate if the VWF & Propeptide Assay can be used to distinguish Type 1 VWD patients with mutations known to cause an increased VWF clearance phenotype (Type 1C) from those without these mutations. One hundred-fifteen Type 1 VWD patients diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, and past bleeding history were tested and 24 Type 1 VWD patients with increased clearance of VWF (Type 1C) diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, past bleeding history, and the presence of a point mutation previously shown to cause increased clearance of VWF. Patients with the following increased clearance mutations were included in the study: R1205H, S2179F, and W1144G (although the majority of the samples contained the R1205H mutation). Using the VWF & Propeptide Assay VWF levels, propeptide levels, and propeptide:VWF ratios were determined for each patient sample. The propeptide:VWF ratios were used to determine an appropriate diagnostic cutoff by receiver operating characteristics (ROC) curve analysis. From the ROC analysis, a propeptide:VWF ratio cutoff value of ≥3.3 provided optimal clinical sensitivity and specificity when distinguishing between Type 1 VWD and Type 1 VWD with increased VWF clearance (Type 1C). Using the ratio cutoff of ≥3.3 yielded 100.0% sensitivity, characterizing all known Type 1C patients correctly and yielded 97.4% specificity, where 3 Type 1 patients were characterized as Type 1C. Two of the 3 mischaracterized patients had Type O blood and the blood group of the third sample was unknown. Since it has been demonstrated that patients with a Type O blood generally have a lower VWF level and correspondingly a slightly elevated propeptide:VWF ratio, we suggest the use of the following grey zone. Propeptide:VWF ratios of 3.3 – 4.1 may be due to increased VWF clearance or the result of a Type 1 VWD patient with Type O blood. Ratios of ≥ 4.2 are indicative of Type 1C VWD. Disclosures: Stapleton: GTI Diagnostics: Employment. Ladvienka:GTI Diagnostics: Employment. Wuitschick:GTI Diagnostics: Employment.

α2-Antiplasmin: Functional Characterization and Metabolism in a Heterozygote Deficient Patient

1986 ◽  
Vol 55 (03) ◽  
pp. 375-378 ◽  
Author(s):  
E A R Knot ◽  
J W ten Cate ◽  
R J Lamping ◽  
Liem Kian Gie
Keyword(s):  
Half Life ◽  
Functional Characterization ◽  
Bleeding Tendency ◽  
Normal Range ◽  
Crossed Immunoelectrophoresis ◽  
Synthetic Rate ◽  
Catabolic Rate ◽  
Deficient Patient ◽  
Antigen Activity ◽  
Bleeding History

SummaryAn 81-year-old male with a mild life-long bleeding history and an α2-antiplasmin (α2-AP) plasma level of 55% biological activity and 41% antigen activity (normal range 80-140%) was studied. The ratio of plasminogen binding (PB): non-plasminogen binding (NPB) α2-AP assayed by modified crossed immunoelectrophoresis (CIE) was 7.3/2.7 (controls 6.3 ± 0.49 SD/3.7 ± 0.49 SD). The patient’s α2-AP showed decreased affinity for fibrin, i. e. 8.3% versus 32.4% of normal control α2-AP associated with fibrin during clotting of plasma. A metabolic study performed with human purified 125I-α2-AP(PB/NPB 7.7/2.3) showed a plasma radioactivity disappearance half-life of 72.9 h (n 60.1 ± 5.3 h) with a normal fractional catabolic rate and a reduced absolute catabolic (synthetic) rate of 0.70 mg/kg/day (n 2.10 ± 0.60 mg/kg/day). The exchange between the central and third compartment was increased. The increased α2-AP PB form and the increased plasma radioactivity disappearance half-life are suggestive of a slower conversion of the PB form into the NPB form and/or slower degradation of the PB form. The bleeding tendency in this patient could be explained by decreased synthesis of α2-AP and decreased binding to fibrin.

scholarly journals Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

2020 ◽  
Vol 58 (9) ◽  
pp. 1461-1468 ◽  
Author(s):  
Jean-Claude Alvarez ◽  
Pierre Moine ◽  
Isabelle Etting ◽  
Djillali Annane ◽  
Islam Amine Larabi
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Half Life ◽  
Limit Of Detection ◽  
Treated Patient ◽  
Internal Standard ◽  
Therapeutic Drug ◽  
Active Metabolites ◽  
Limit Of Quantification ◽  
Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

scholarly journals Isolation and Characterization of Replication-Competent Human Immunodeficiency Virus Type 1 from a Subset of Elite Suppressors

2006 ◽  
Vol 81 (5) ◽  
pp. 2508-2518 ◽  
Author(s):  
Joel N. Blankson ◽  
Justin R. Bailey ◽  
Seema Thayil ◽  
Hung-Chih Yang ◽  
Kara Lassen ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Highly Active Antiretroviral Therapy ◽  
Limit Of Detection ◽  
Isolation And Characterization ◽  
Immunodeficiency Virus ◽  
Elite Suppressors ◽  
Hiv 1

ABSTRACT Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who control viremia to levels below the limit of detection of current assays. The mechanisms involved in this control have not been fully elucidated. Several studies have demonstrated that some ES are infected with defective viruses, but it remains unclear whether others are infected with replication-competent HIV-1. To answer this question, we used a sensitive coculture assay in an attempt to isolate replication-competent virus from a cohort of 10 ES. We successfully cultured six replication-competent isolates from 4 of the 10 ES. The frequency of latently infected cells in these patients was more than a log lower than that seen in patients on highly active antiretroviral therapy with undetectable viral loads. Full-length sequencing of all six isolates revealed no large deletions in any of the genes. A few mutations and small insertions and deletions were found in some isolates, but phenotypic analysis of the affected genes suggested that their function remained intact. Furthermore, all six isolates replicated as well as standard laboratory strains in vitro. The results suggest that some ES are infected with HIV-1 isolates that are fully replication competent and that long-term immunologic control of replication-competent HIV-1 is possible.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

scholarly journals Type 1 TNF Receptor Forms a Complex with and Uses Jak2 and c-Src to Selectively Engage Signaling Pathways That Regulate Transcription Factor Activity

2008 ◽  
Vol 181 (2) ◽  
pp. 1288-1298 ◽  
Author(s):  
Roxana Pincheira ◽  
Ariel F. Castro ◽  
Osman Nidai Ozes ◽  
Prema S. Idumalla ◽  
David B. Donner
Keyword(s):  
Transcription Factor ◽  
Signaling Pathways ◽  
Tnf Receptor ◽  
Transcription Factor Activity ◽  
Factor Activity

scholarly journals Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1

2019 ◽  
Vol 20 (8) ◽  
pp. 1938 ◽  
Author(s):  
Voellenkle ◽  
Perfetti ◽  
Carrara ◽  
Fuschi ◽  
Renna ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Splicing ◽  
Mononuclear Cells ◽  
Circular Rnas ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Sequencing Data ◽  
Operating Characteristics ◽  
Peripheral Blood Mononuclear

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, which is the linking covalently of 3′- and 5′-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

scholarly journals Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.

1989 ◽  
Vol 109 (6) ◽  
pp. 3347-3354 ◽  
Author(s):  
A Picard ◽  
J P Capony ◽  
D L Brautigan ◽  
M Dorée
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Germinal Vesicle ◽  
Histone H1 ◽  
Specific Inhibition ◽  
Factor Activity ◽  
Microtubule Network ◽  
Germinal Vesicle Breakdown ◽  
M Phase

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.

Quantitation of Bleeding Symptoms in Children with von Willebrand Disease (VWD) or a Platelet Function Disorder: Use of a Standardized Pediatric Bleeding Score.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2137-2137 ◽  
Author(s):  
T.T. Biss ◽  
V.S. Blanchette ◽  
M. Bowman ◽  
D.S. Clark ◽  
M. Silva ◽  
...  
Keyword(s):  
Platelet Function ◽  
Dense Granule ◽  
Von Willebrand Disease ◽  
Number Of Patients ◽  
Kingston General Hospital ◽  
Bleeding Score ◽  
Sick Children ◽  
Bleeding History ◽  
Score Range

Abstract Obtaining an accurate bleeding history is an essential step in the diagnosis of a bleeding disorder, which is often made during childhood. A bleeding history is usually taken in an informal manner, with the interpretation of the responses dependent on the experience of the observer. The development of standardized bleeding questionnaires has improved objectivity and allowed the determination of quantitative scores according to the severity of mucocutaneous bleeding. In adults, using the International Society on Thrombosis and Haemostasis (ISTH) Bleeding Questionnaire for Diagnosis of Type 1 VWD, a bleeding score of >3 in males and >5 in females is considered abnormal (Rodeghiero et al., J Thromb Haemost2005:3;2619). Children have often not been exposed to hemostatic challenges and may have low scores despite significant bleeding disorders. Symptoms specific to childhood, such as post-circumcision bleeding, umbilical stump bleeding and cephalohematoma may be of greater significance in this patient group. Thus, we adapted the ISTH Bleeding Questionnaire for use in pediatrics, with a symptom-specific grade of -1 to 4 (Tosetto et al., J Thromb Haemost2006:4;766). Bleeding scores were determined by interview of parents/children for 80 children with a previous diagnosis of VWD or a platelet function disorder at The Hospital for Sick Children, Toronto, or Kingston General Hospital, Kingston. 62 children had a diagnosis of VWD and 18, a disorder of platelet function (Glanzmann thrombasthenia: 3; dense granule defect: 2; MYH9-related thrombocytopenia: 2; Hermansky-Pudlak syndrome: 1; unspecified: 10). 45 children were female and 35, male (median age: 10 yrs (range: 9 mo-17 yrs)), with a median age of females of 12 yrs (range: 0–17) and of males, 9 yrs (range: 1–17). Bleeding scores ranged from 0–28 (median: 7), with a median score in females of 7 and in males, 8. Bleeding scores according to diagnosis and age are shown in Tables 1 and 2, respectively. The most frequent reasons for a positive score of ≥2 were epistaxis (43% of patients), bleeding from minor wounds (38%), bleeding after dental extraction (31%) and excessive bruising (26%). Menorrhagia requiring treatment occurred in 47% of menstruating females. Bleeding from the umbilical stump, post-circumcision bleeding and cephalohematoma were reported in 10%, 6% and 4% of patients, respectively. In summary, we have used a standardized bleeding questionnaire, adapted for use in pediatrics, with an accompanying score to quantify bleeding symptoms in children with confirmed VWD or a platelet function disorder. Bleeding scores were lowest in the youngest age group (0–3 yrs), and were slightly higher in males than in females. Bleeding occurred early in childhood, i.e. post-circumcision bleeding and bleeding from the umbilical stump. This standardized pediatric bleeding questionnaire/score may be useful in clinical practice in the assessment of children presenting with symptomatic bruising and bleeding. Diagnosis Type 1 VWD Type 2 VWD Type 3 VWD Platelet function disorder Number of patients 48 8 6 18 Median bleeding score (range) 6 (0–18) 10 (0–17) 14 (9–28) 9 (1–19) Age (years) 0–3 4–6 7–9 10–12 13–15 16–18 Number of patients 7 13 18 16 14 12 Median bleeding score (range) 2 (0–9) 10 (0–16) 6 (0–19) 9.5 (1–28) 6 (2–18) 12 (6–21)

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