Technical datasheet
Description
The monoclonal antibody RCR-252 recognizes human endothelial protein C receptor (EPCR), a highly glycosylated type I transmembrane protein of 221-amino-acids. These amino acids comprise an extracellular domain, a 25-aa transmembrane domain, and a short (3 aa) intracytoplasmic sequence coding for an ~46 kDa protein. Deglycosylation will reduce the protein mass to 25 kDa. EPCR is expressed strongly on the endothelial cells of arteries and veins in heart and lung, less intensely in capillaries in the lung and skin, and not at all in the endothelium of small vessels of the liver and kidney.
EPCR is the receptor for protein C, a key player in the anticoagulation pathway. The protein C anticoagulant pathway serves as a major system for controling thrombosis, limiting inflammatory responses, and potentially decreasing endothelial cell apoptosis in response to inflammatory cytokines and ischemia. The essential components of the pathway include thrombin, thrombomodulin, the endothelial cell protein C receptor (EPCR), protein C and protein S. The pathway is initiated when thrombin binds to thrombomodulin on the surface of endothelium. EPCR augments protein C activation by binding protein C and presenting it to the thrombin-thrombomodulin activation complex. Activated protein C (aPC) retains its ability to bind EPCR, and this complex appears to be involved in some of the cellular signaling mechanisms that down-regulate inflammatory cytokine formation (TNF, IL-6). EPCR is shed from the vasculature by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1. Furthermore, EPCR can undergo translocation from the plasma membrane to the nucleus.
EPCR can be cleaved to release a soluble form (sEPCR) in the circulation. This sEPCR is detected as a single species of 43 kDa, resulting from shedding of membrane EPCR by the action of a metalloprotease, which is stimulated by thrombin and by some inflammatory mediators. Soluble EPCR binds PC and aPC with similar affinity, but its binding to aPC inhibits the anticoagulant activity of aPC by blocking its binding to phospholipids and by abrogating its ability to inactivate factor Va. sEPCR can be detected in plasma. In normal persons, sEPCR is present in levels of 83.6 +/- 17.2 ng/ml. Elevated levels of sEPCR are positively correlated to a higher risk for thrombosis. Furthermore, a haplotype (A3 allele) has been linked to elevated levels of sEPCR (264 +/-174 ng/ml).
Immunogen
Human EPCR-positive RE-1 cells
Formulation
1 ml (100 µg/ml) 0.2 µm filtered antibody solution in PBS, containing 1% bovine serum.
Species
Rat IgG1
Application
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F
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FC1,2
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FS1-4
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IA
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IF
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IP5
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P
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W5
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Yes
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No
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N.D.
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N.D.= Not Determined; F = Frozen sections; FC = Flow Cytometry; FS = Functional Studies; IA = Immuno Assays; IF = Immuno Fluorescence; IP = Immuno Precipitation; P = Paraffin sections; W = Western blot
Application notes
FC: Antibody RCR-252 stains the extracellular domain of human EPCR. As positive control RE-1 cells were used and as negative control N1 cells. (Ref.1)
W: A non-reduced sample treatment was used. The observed band size is/ 50 kDa (Ref.5).
FS: Antibody RCR-252 functions as blocking antbody. It ablates APC binding to EPCR and blocks APC-mediated signaling. The antibody was functionally tested by pretreatment of cells with 10-25 µg/ml RCR-252 (Ref.1-4).
Use
For flow cytometry and Western blot dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50. For functional studies, in vitro dilutions have to be optimized in user’s experimental setting.
Aliases
Activated protein C receptor, CD201, Endothelial cell protein C receptor
Positive control
RE-1 (rat 3Y1 fibroblasts stably transfected with human EPCR.
Negative control
N1-cells
Storage and stability
Product should be stored at 4°C. Under recommended storage conditions, product is stable for at least one year. The exact expiry date is indicated on the label.
References
1. Ye, X et al; The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries. Biochem Biophys Res Commun 1999, 259: 671
2. Dömötör, E et al; Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. Blood 2003, 101: 4797
3. Sturn, D et al; Expression and function of the endothelial protein C receptor in human neutrophils. Blood 2003, 102: 1499
4. Finigan, J et al; Activated protein C mediates novel lung endothelial barriere enhancement. J Biol Chem 2005, 280: 17286
5. Bae J et al; Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. PNAS 2006, 104: 2867
Precautions
For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result from the use or derivation of this product.
References
1. Ye, X et al; The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries. Biochem Biophys Res Commun 1999, 259: 671
2. Dömötör, E et al; Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. Blood 2003, 101: 4797
3. Sturn, D et al; Expression and function of the endothelial protein C receptor in human neutrophils. Blood 2003, 102: 1499
4. Finigan, J et al; Activated protein C mediates novel lung endothelial barriere enhancement. J Biol Chem 2005, 280: 17286
5. Bae J et al; Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. PNAS 2006, 104: 2867