Abstract
Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.
Highlights
When the decrease in clotting activity is plotted against the loss of sialic acid residues, the experimental points fall on a line with a slope of
The amountof protein recovered was essentially the same in the two preparations. These results provide no evidence for aggregation and/or precipitationof factor IX aas consequence of removal of the negatively charged sialic acid residues
The data we have presented show that cleavage of sialic acid results in a decrease in the activity of factor IX andof factor IX
Summary
Bean trypsin inhibitor (Type I-S), imidazole, Russell’s viper venom (Received for publication, August 24, 1983). Tions for theloss of factor IX activity: proteolytic degradation, inhibitoryeffects of free sialic acid, and nonspecific inhibition of thcelotting assays. There- Covalent coupling of Russell’s viper venom to Sepharose was done fore, we suggest that the loss of factor IX activity is according to Kohn and Wilchek (1982),with the following modificathe direct result of cleavage of sialic acidfromthe tions. In the case of many glycoproteins, the cleavage of 1-2 residues of sialic acid/molecule of protein results in rapid clearance of theprotein from circulation (McFarlane, 1983). Factor IX, a plasma glycoproteinconstituent of the clotting cascade, is a vitamin K-dependent serine protease which contains y-carboxyglutamic acid residues. We have investigated the effects of removal of terminal sialic acids on the clotting activity of factor IX.
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