scholarly journals Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1255-1262 ◽  
Author(s):  
W Shi ◽  
BH Chong ◽  
CN Chesterman
Keyword(s):  
Cross Reactivity ◽  
Anticardiolipin Antibodies ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycoprotein I ◽  
Activated Platelets ◽  
Major Interest ◽  
Thrombin Activation ◽  
Beta 2

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

scholarly journals Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1255-1262 ◽  
Author(s):  
W Shi ◽  
BH Chong ◽  
CN Chesterman
Keyword(s):  
Cross Reactivity ◽  
Anticardiolipin Antibodies ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycoprotein I ◽  
Activated Platelets ◽  
Major Interest ◽  
Thrombin Activation ◽  
Beta 2

Abstract Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

Anticardiolipin Antibodies Block the Inhibition by β2-Glycoprotein I of the Factor Xa Generating Activity of Platelets

1993 ◽  
Vol 70 (02) ◽  
pp. 342-345 ◽  
Author(s):  
Wei Shi ◽  
Beng H Chong ◽  
Philip J Hogg ◽  
Colin N Chesterman
Keyword(s):  
Antiphospholipid Antibodies ◽  
Lupus Anticoagulant ◽  
Fetal Loss ◽  
Factor Xa ◽  
Anticardiolipin Antibodies ◽  
Recurrent Fetal Loss ◽  
Glycoprotein I ◽  
Activated Platelets ◽  
Inhibit Factor

SummaryAntiphospholipid antibodies, defined either by lupus anticoagulant (LA) activity or positive anticardiolipin immunoabsorbent assay (ACA) are associated with a predisposition to thromboses, recurrent fetal loss or thrombocytopenia. The mechanisms for these predispositions remain undefined. We have enriched immunoglobulin fractions from two patient plasmas to obtain antibodies with LA activity but no ACA, or conversely, with ACA positivity but no LA, in order to investigate in vitro characteristics which might explain a thrombotic propensity. β2-glycoprotein I (β2-GPI), the plasma cofactor required for ACA binding to negatively charged phospholipid, has previously been shown to inhibit prothrombinase generation in the presence of activated platelets (8). We now report that β2-GPI, at physiological concentrations, inhibits the generation of factor Xa in the presence of activated gel-filtered platelets. Further, ACA interferes with this inhibition, resulting in protracted, unopposed factor Xa generation. This interference with β2-GPI, a natural anticoagulant component of plasma, is potentially prothrombotic. LA immunoglobulins behave differently and inhibit factor Xa generation in a manner similar to β2-GPI. These findings provide the basis for a previously unsuspected mechanism for thrombosis in patients with aPL.

Antiphospholipid Syndrome: Diagnostic Aspects of Lupus Anticoagulants

2001 ◽  
Vol 86 (07) ◽  
pp. 83-91 ◽  
Author(s):  
J. Arnout
Keyword(s):  
Monoclonal Antibodies ◽  
Antiphospholipid Syndrome ◽  
Autoimmune Disorder ◽  
Coagulation Factors ◽  
Laboratory Diagnosis ◽  
Anticardiolipin Antibodies ◽  
Lupus Anticoagulants ◽  
Glycoprotein I ◽  
Antigen Antibody

SummaryAntiphospholipid syndrome (APS) is an autoimmune disorder in which antiphospholipid antibodies (aPL) are thought to be involved in the development of venous and/or arterial thrombosis. APL found in this syndrome are antibodies directed against a variety of phospholipid (PL) binding-proteins of which β2-glycoprotein I (β2GPI) and prothrombin are considered to be the major antigens. Some of these antibodies prolong PL-dependent clotting reactions and are termed lupus anticoagulants (LA). Autoimmune aPL which bind through β2GPI to cardiolipin are called anticardiolipin antibodies (aCL). Clinical studies indicate that LA is a stronger risk factor for thrombosis than aCL. The production of monoclonal antibodies against β2GPI and prothrombin has enabled us to understand the mechanism by which LA prolong coagulation in vitro. LA form bivalent antigen-antibody complexes with increased affinity for PL which compete with coagulation factors for the same catalytic surface. These LA positive monoclonal antibodies may be helpful in further improving the laboratory diagnosis of LA.

False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

1980 ◽  
Vol 26 (2) ◽  
pp. 345-347 ◽  
Author(s):  
G P James ◽  
M H DJang ◽  
H H Hamilton
Keyword(s):  
Ascorbic Acid ◽  
Ion Exchange ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
False Negative ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Negative Results ◽  
False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

1973 ◽  
Vol 58 (3) ◽  
pp. 405-419 ◽  
Author(s):  
M. JOAN REED ◽  
S. R. STITCH
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Supernatant Fraction ◽  
Low Molecular Weight ◽  
Molecular Weight Peak ◽  
Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

Intestinal Absorption of Dipeptides Containing Glycine, Phenylalanine, Proline, β-Alanine or Histidine in the Rat

1973 ◽  
Vol 45 (6) ◽  
pp. 849-858 ◽  
Author(s):  
D. J. Boullin ◽  
R. F. Crampton ◽  
Christine E. Heading ◽  
D. Pelling
Keyword(s):  
Intestinal Absorption ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Peptide Absorption ◽  
Physiological Conditions ◽  
Anaesthetized Rats ◽  
Tissue Homogenates ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

1. The intestinal absorption of carnosine, glycylglycine, glycyl-d-phenylalanine, glycyl-l-phenylalanine, glycyl-l-proline and l-prolylglycine were investigated after intraluminal injection of dipeptide into anaesthetized rats. 2. With all six dipeptides, the intact substance was detected by ion-exchange chromatography in blood samples taken from the superior mesenteric vein. 3. The rate of hydrolysis of the dipeptides in tissue homogenates was measured in vitro. 4. The relative rates of hydrolysis varied by a factor of 300; there was an apparent inverse relationship between rate of hydrolysis and detection of intact peptide. 5. Peptide absorption was accompanied by increases in venous concentrations of the component amino acids, which appeared in proportions appropriate to the view that peptide absorption preceded hydrolysis. 6. It is suggested that slowly hydrolysed dipeptides may pass intact through the intestine wall under physiological conditions.

scholarly journals Anticardiolipin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface.

1994 ◽  
Vol 179 (2) ◽  
pp. 457-462 ◽  
Author(s):  
E Matsuura ◽  
Y Igarashi ◽  
T Yasuda ◽  
D A Triplett ◽  
T Koike
Keyword(s):  
Photoelectron Spectroscopy ◽  
Gamma Ray ◽  
Solid Phase ◽  
Anticardiolipin Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polystyrene Surface ◽  
Glycoprotein I ◽  
Latex Beads ◽  
Beta 2 ◽  
Phase Surface

Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.

Isolation of Fasciola hepatica haemoglobin

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 209-215 ◽  
Author(s):  
S. McGonigle ◽  
J. P. Dalton
Keyword(s):  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Haemoglobin Molecule

SUMMARYA haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

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