Materials used in the different studies

Slides:



Advertisements
Similar presentations
General Approach of Haemostasis
Advertisements

PG-Seph. APCCB IDENTIFICATION OF MACRO- PROLACTIN WITH PROTEIN-G SEPHAROSE Jones GRD, Giannopoulos P. Departments of Chemical Pathology, St Vincent’s.
MIXING STUDIES General Approach of Haemostasis
Clinical diagnostic biochemistry - 8
Date of download: 5/27/2016 Copyright © The American College of Cardiology. All rights reserved. From: RVX-208: A Small Molecule That Increases Apolipoprotein.
Figure 3.1. Chromatogram of L-ficolin elution from the CysNAc column. The relative L-ficolin concentration (closed circles, left Y-axis, arbitrary units)
CHROMATOGRAPHY 1Biochemistry of Medics. Chromatography 2Biochemistry of Medics.
One-Stage Quantitative
MLAB Coagulation Keri Brophy-Martinez
Elvira Maličev Blood Transfusion Centre of Slovenia
Pro-apoptotic low-density lipoprotein subfractions in type II diabetes
Adiponectin Correlation With Plasma Lipoprotein Subclasses Determined By NMR And With The Risk Of Venous Thrombosis. Fernández JA, Deguchi H, Pecheniuk.
Antiphospholipid Antibody Syndrome
Factor H inhibits complement activation induced by liposomal and micellar drugs and the therapeutic antibody rituximab in vitro  Tamás Mészáros, MSc,
Triglycerides (mmol/L)
General Approach of Haemostasis
Volume 63, Issue 2, Pages (February 2003)
General Approach in Investigation of Hemostasis
Regulation of serum-induced lipid accumulation in human monocyte-derived macrophages by interferon-γ. Correlations with apolipoprotein E production, lipoprotein.
Pro-apoptotic low-density lipoprotein subfractions in type II diabetes
Taste sensitivity to 6-n propylthiouracil (PROP) is associated with fungiform taste buds density and body fat mass in humans Anna Valenzano1, R. Viscecchia2,
WCC12-ABS-1469 P751 Influence of the concentration and molecular composition on the LDL and HDL functional characteristics in patients with the metabolic.
Evidence for an exclusive association of matrix metalloproteinase-9 with dysfunctional high-density lipoprotein: A novel finding  S. Sini, D. Deepa, S.
Mixing Studies-aPTT or PT 1:1 Mix
Adipose Tissue ATP Binding Cassette Transporter A1 Contributes to High-Density Lipoprotein Biogenesis In VivoClinical Perspective by Soonkyu Chung, Janet.
Human Apolipoprotein A-II Enrichment Displaces Paraoxonase From HDL and Impairs Its Antioxidant Properties by Vicent Ribas, José Luis Sánchez-Quesada,
Volume 6, Issue 3, Pages (March 1997)
Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression by Chang-jun Hu, Frank A. Baglia, David C.B.
Volume 132, Issue 5, Pages (May 2007)
Glycated hemoglobin (HbA1c)
John B. Massey, Henry J. Pownall  Biophysical Journal 
BRCA1 Is Associated with a Human SWI/SNF-Related Complex
by Laurent O. Mosnier, Andrew J
Evaluation of Candidate Standard XX (97/650)
Age-related variations in follicular apolipoproteins may influence human oocyte maturation and fertility potential  Tiffany Von Wald, M.D., Yevgeniya.
The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding.
by Monica Galli, Luisa Ruggeri, and Tiziano Barbui
by Xingwei Sui, Sanford B. Krantz, and Zhizhuang Zhao
Volume 94, Issue 1, Pages (July 1998)
by Andrew J. Gale, Mary J. Heeb, and John H. Griffin
Volume 140, Issue 5, Pages (May 2011)
by Subburaj Ilangumaran, Anne Briol, and Daniel C. Hoessli
Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of.
Volume 72, Issue 7, Pages (October 2007)
ImmunoWELL Zika Virus Serology.
Volume 94, Issue 1, Pages (July 1998)
Factor Va Increases the Affinity of Factor Xa for Prothrombin
Chlamydomonas Shortens Its Flagella by Activating Axonemal Disassembly, Stimulating IFT Particle Trafficking, and Blocking Anterograde Cargo Loading 
Transforming Growth Factor-Alpha: A Major Human Serum Factor that Promotes Human Keratinocyte Migration  Yong Li, Jianhua Fan, Mei Chen, Wei Li, David.
Volume 67, Issue 2, Pages (February 2005)
ClpX-Mediated Remodeling of Mu Transpososomes
Leslie J. Bannon, Betty L. Cabrera, Kathleen J. Green 
Antti Nykänen, Benjamin Haley, Phillip D. Zamore  Cell 
Volume 16, Issue 4, Pages (April 2009)
Volume 54, Issue 2, Pages (August 1998)
Richard A. Zager, Ali C.M. Johnson, Sherry Y. Hanson 
Purification of chromogranin B from over-expressing insect sf9 cells.
Victor Faúndez, Jim-Tong Horng, Regis B Kelly  Cell 
The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin  Shusuke Tada, James P.J. Chong,
Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes by Edward N. van den Brink, Ellen A. M. Turenhout,
Gaku Mizuguchi, Toshio Tsukiyama, Jan Wisniewski, Carl Wu 
Duality of statin action on lipoprotein subpopulations in the mixed dyslipidemia of metabolic syndrome: Quantity vs quality over time and implication.
Richard W. Deibler, Marc W. Kirschner  Molecular Cell 
Volume 116, Issue 2, Pages (February 1999)
Volume 23, Issue 2, Pages (July 2006)
John M. Lamar, Vandana Iyer, C. Michael DiPersio 
Acta Pathol Microbiol Scand and Clin Lab Med use in PPT for external audience verified via Rightsphere 25Jul
Molecular Therapy - Methods & Clinical Development
Activated protein C light chain provides an extended binding surface for its anticoagulant cofactor, protein S by José A. Fernández, Xiao Xu, Ranjeet K.
Volume 84, Issue 2, Pages (January 1996)
Presentation transcript:

Materials used in the different studies Superose 6 Chromatography Profile of HDL Prep’s #2249 The fractions were tested for their ability to enhance FVa inactivation and prothrombin activation. The inactivation of purified coagulation FVa by APC and its cofactor, protein S, was studied using a two-step procedure. First, FVa was incubated with HDL fractions or buffer, APC, and protein S to inactivate FVa. Second, residual FVa activity was quantitated using prothrombinase assays. The pool of fractions corresponding to apo AI peak markedly enhanced APC-dependent loss of FVa. The fractions on the void volume also show inhibition although less potent. Fractions in the void volume were also found to stimulate prothrombin activation, whereas the ApoAI containing fractions did not stimulated prothrombin activation. Thus, the anticoagulant effects that were observed in HDL preparations isolated by ultracentrifugation were associated with the apoAI fractions. Jose A. Fernandez1), Hiroshi Deguchi1), Natalie Pecheniuk1), Subramanian Yegneswaran1), Carole L. Banka2), John H. Griffin1) Dept of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, USA1), Dept of Medicine, UCSD, USA2) To define the active molecular species in HDL, immobilized antibodies against apoA-I, were tested for their ability to absorb the anticoagulant activity of superpose 6 fractions. Anti–apoA-I IgG adsorbed 86% of the anticoagulant activity, whereas no significant (<5%) adsorption was observed for the control Sepharose beads. Thus, HDL fractions containing apoA-I provided the anticoagulant activity observed. When the anticoagulant property of these HDL subtractions was studied, we found that the lightest HDL subfractions were more anticoagulantly active as APC-cofactors than other HDL fractions. These HDL subfractions with anticoagulant activity correspond to the peak of phospholipid, cholesterol and protein in HDL2 containing large HDL particles. Plasma High Density Lipoprotein and Anticoagulant Response to Activated Protein C and Protein S Reevaluation ? 1) Analyze and assay 4 different HDL preparations using Gel Filtration. 2) Remove HDL particles from HDL prep. using anti - ApoAI immunoadsorbtion . 3) Correlate APC / ProS anticoagulant response with HDL levels in plasmas. Previously we reported that plasma high density lipoprotein (HDL) enhances activated protein C (APC)/protein S (PS) anticoagulant action in plasma clotting and factor Va inactivation assays (APC/PS enhancement) and that lower HDL levels are found in male venous thrombosis patients or in patients with recurrent venous thrombosis versus controls, giving rise to our hypothesis that HDL helps protect against venous thrombosis. In this study, we sought (1) to identify which HDL particles enhance APC/PS activity, (2) to assess correlations between this activity and HDL particle size, and (3) to evaluate the recent challenge to our HDL anticoagulant activity hypothesis (Oslakovic et al, J Clin Invest, 2010). To identify HDL subfractions with APC/PS enhancing activity, we subfractionated HDL by sequential density gradient ultracentrifugation over the HDL density range of 1.063-1.21 mg/dl. When the anticoagulant property of these HDL subtractions was studied, we found that the less dense HDL subfractions corresponding to HDL2 particles enhanced APC/PS anticoagulant activity much more than other HDL fractions. Thus, we identified larger HDL particles as the key subfraction for this anticoagulant property of HDL. Hence, we hypothesized that plasma levels of large HDL particles would correlate with plasma sensitivity of APC/protein S. To test this hypothesis, we used proton NMR to quantify the levels of large, medium and small HDL particles and to determine the average size of HDL particles in plasmas from 39 normal adults.  We performed dilute tissue factor-induced clotting assays in the absence or presence of APC/protein S to define APC/PS sensitivity and assessed correlations.  There was a positive correlation between large HDL particle concentration and plasma sensitivity to APC/protein S (r=0.40, p=0.02). The size of HDL particles was also positively correlated with plasma sensitivity to APC/protein S (r=0.42, p=0.01). As previously reported, apoAI concentrations which is major apolipoprotein in HDL correlated with plasma sensitivity to APC/protein S (r=0.52, p=0.0007). Thus, as hypothesized, apoAI-containing large HDL particle concentrations in plasma correlate very well with the anticoagulant response to APC/PS.  Recently Oslakovic et al purported to show that APC/PS enhancement was not an intrinsic property of HDL and claimed that this activity was due to negatively charged phospholipid contaminants of HDL based on gel filtration (Superose 6) analysis of their frozen HDL preparation. Therefore, we applied Superose 6 gel filtration analyses to four different fresh, never-frozen HDL preparations coming either from commercial sources or from in-house preparations. Our studies showed that when each HDL prep was subjected to Superose 6 fractionation, the column fractions containing large HDL (apoAI positive fractions) enhanced APC/protein S anticoagulant activity in plasma clotting assays and also for the inactivation of factor Va in purified systems. Moreover, immobilized anti-apoAI-antibodies removed the APC/PS enhancing effect, further establishing the fact that apoAI-containing HDL particles enhance APC/PS activity. When we analyzed HDL preps stored at 4 °C over successive weeks, we found that HDL fractions lost the ability to enhance APC/PS, showing the relatively labile nature of this activity.  Freezing and thawing HDL was also deleterious for this activity. Thus, the APC/PS enhancing activity of fresh, never-frozen HDL preps is primarily due to HDL particles. All of our new findings confirm our previous conclusion that HDL enhances APC/PS anticoagulant actions. Our extensive data set strongly contradicts the conclusions from the recent HDL study by Oslakovic et al who unfortunately performed their bioassays using frozen HDL made from previously frozen lipidmic plasma.  In summary, freshly purified, non-frozen large HDL particles made from fresh plasma enhance APC/PS activity.  The sensitivity of plasma of healthy adults to APC/PS anticoagulant effects is significantly correlated with the plasma levels of large HDL particles, suggesting the physiological importance of large HDL particles as enhancing the anticoagulant APC/PS system and supporting our hypothesis that large HDL particles may be protective against venous thrombosis, at least in part, via this activity. Conclusions