A simple method to measure dermatan sulfate at sub-microgram concentrations in plasma.
A simple method for biological assay of dermatan sulfate (DS) in plasma is described. DS accelerates thrombin inhibition by heparin cofactor II (HC II). The principle of the assay is to measure the residual amidolytic thrombin activity after a short period of incubation with HC II in defibrinated plasma at low ionic strength. For this method we take advantage of two observations. Firstly, at fixed concentrations of DS and of HC II, the rate of thrombin inhibition increases when the ionic strength of the medium decreases. Secondly, defibrination by bentonite absorption also removes antithrombin III, HC II and for a large part alpha-2 macroglobulin from the plasma, so that no other thrombin inhibitor competes with HC II added as a reagent in a second step. In the conditions described, there is a linear relationship between DS concentrations in plasma from 0 to 2 micrograms/ml and the log of residual thrombin activity. The limit of sensitivity is 0.1 micrograms/ml. The assay displays an acceptable reproducibility in intra-assay, inter-assay and inter-individual experiments. It can be used to measure DS in human, rabbit and rat plasmas. The assay is also sensitive to other HC II activators such as heparin and pentosan polysulfate. DS is effective in experimental thrombosis without any detectable anticoagulant effect ex vivo. Pharmacological concentrations of DS in plasma fall into the range of sensitivity of this assay, which would be helpful in experimental or clinical studies of DS and related glycosaminoglycans.
Dupouy
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Laboratoire d'Hémostase, Centre de Transfusion Sanguine, Hôpital Purpan, Toulouse, France.
Sié
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Dol
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Boneu
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B
D000990:Antithrombin III / Q000032:analysis; D000991:Antithrombins / Q000032:analysis; D001774:Blood Chemical Analysis / Q000379:methods; D002807:Chondroitin / Q000031:analogs & derivatives*; D003871:Dermatan Sulfate / Q000097:blood*; D005337:Fibrin / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D015844:Heparin Cofactor II; D006801:Humans; D007477:Ions; D015203:Reproducibility of Results
D000991:Antithrombins; D006023:Glycoproteins; D007477:Ions; D003871:Dermatan Sulfate; D015844:Heparin Cofactor II; D000990:Antithrombin III; D005337:Fibrin; D002807:Chondroitin