Anticoagulant activity assay heparins

Anticoagulant Activity Assay. The anticoagulant activities of IV-acetylated heparin and all further derivatized heparins were determined based on activated partial thromboplastin time (APTT) assay methods (15). Bovine blood was collected in 3.8% sodium-citrated (9 parts blood to 1 part citrate solution) and centrifuged at 5000 x g for 15 min. The supernatant plasma was collected and pooled for subsequent APTT testing. Prior to testing plasma was kept refrigerated no longer than 6 h after... 

The final yield and purity of a heparin preparation depend largely on the use of appropriate, analytical methods at different stages of extraction and purification. Heparin in tissue extracts is still most commonly determined biologically, by such assays as the U.S.P. assay for anticoagulant activity. It is now recognized10 that the anticoagulant activity does not measure the actual concentration of heparin (see also Sections XII and XIII). 

The oleosin fusion procedure was used for the purification of the commercially valuable plant-based blood anticoagulant hirudin in transgenic Brassica carinata and Brassica napus. Hirudin, a natural protein from the medicinal leech Hirudo medicinalis, is superior to other anticoagulants such as heparin. Recombinant hirudin was cleaved from oil-bodies using endoproteinase Factor Xa. Released hirudin was biologically active, as determined by a colorimetric thrombin inhibition assay. 

The variety of methods that have been used for assaying anticoagulant activity makes difficult a comparison of the results of different workers."-It is claimed that the most satisfactory, although not the most convenient, method involves animal tests. By the use of three groups of animals, for control and for injection of standard heparin and of synthetic products, the effect of the latter group is thought to be most accurately assessed. Subsequent references to the activity of sulfated polysaccharides will be limited to comparisons with that of the heparin preparations used by individual authors and not with that of a common standard. 

The assay of heparin has been discussed in detail by Jaques and Bell and been recently given in a book by Tocantins and Kazal . The possible properties of these compounds which have been used for assay, given in order of descending interest from the standpoint of pharmacology, are (1) prevention of thrombosis (2) anticoagulant activity on fresh whole blood (3) mcta-... 

We have just begun a series of experiments in which citrated rabbit blood, heparinized at a level of 10 units/mL (153 units/mg), was passed through a Sepharose-heparinase column (0.5mL) at a flow rate of 0.5 mL/min (Figure 8). After 5 min the blood leaving the bottom of the column was sampled and assayed for heparin by whole blood clotting time and Factor Xa heparin assays. In the active column, 50% of the heparin was removed. However, when the same heparinized blood was treated with a control column, less than a 5% decrease in anticoagulant activity was observed. 

Heparin fractions were chemically characterized as described (11) by measurements of uronate (UA), sulfaminohexose (SAH) and sulfate (SO ). Anticoagulant activities were determined on aqueous solutions of heparin fractions containing a known amount of uronate using the USP assay in recalcified sheep... 

Directly compared with International Standard Heparin. MA = metachromatic Ac= = anticoagulant activity on fresh whole blood AT=inhibition of clotting of blood by thrombin USP = U.S. pharmacopeia XV assay of inhibition of clotting of sheep plasma. Modified from [3]. 

Assay of the PABA standard individually in the presence of each plasma sample is necessary (Table 3.7.1 tubes 7 and 8) since some heparin-based anticoagulating agents sometimes used to prepare plasma may diminish the colour development, thus reducing the intensity of the colour. Falsely low activities would be measured in such a sample if the standard factor required to convert the absorbance units to nanomoles (see Calculation below), is determined in the absence of the plasma. 

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