The Effect of Enzymatic Removal of Sialic Acids on the Functional Properties of Protein C

Summary

The effect of enzymatic removal of sialic acid residues on the functional properties of human protein C was examined. The rates of activation of native and asialo protein C were identical using either the thrombin thrombomodulin complex or the snake venom activator, protac. Desialylated activated protein C (APC) was, two-three-fold more active than native protein C, both as an anticoagulant and in its ability to hydrolyze chromogenic substrates. The potentiating effect of the desialylation was confirmed by initially fully activating protein C followed by the desialylation process. In these studies, a two-three-fold enhancement in the activity of the protein was observed which paralleled the extent of the desialylation. The activity of asialo-protein C was completely abolished by monoclonal and polyclonal antiprotein C antibodies, confirming that the effect was mediated by APC. Our studies suggest that the sialic acid component of protein C is not essential for the expression of APC activity but may act to modulate the function of the protein.

Keywords

Protein C - Coagulation - Sialic acid

• References

• 1 Schauer R. In: Advances in Carbohydrate Chemistry and Biochemistry. Tipson RS, Horton D. (eds) 1982; 40: 132-232
• 2 Morell AG, Gregoriadis G, Scheinberg IH, Hickman J, Ashwell G. The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem 1971; 246: 1461-1467
• 3 Ashwell G, Morell A G. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol Relat Areas Mol Biol 1974; 41: 99-128
• 4 Sodetz JM, Pizzo SV, McKee P. Relationship of sialic acid to function and in vivo survival of human factor VUI/von Willebrand factor protein. J Biol Chem 1977; 252: 5538-5546
• 5 Chavin SI, Weidner SM. Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues. J Biol Chem 1984; 259: 3387-3390
• 6 Martinez J, MacDonald KA, Palascak J E. The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains. Blood 1983; 61: 1196-1202
• 7 De MarcoL, Shapiro SS. Properties of human asialo-factor VIII. A ristocetin-independent platelet aggregating factor. J Clin Invest 1981; 68: 321-328
• 8 Walker FJ, Sexton PW, Esmon C T. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochem Biophys Acta 1979; 571: 333-342
• 9 Kisiel W. Human plasma protein C. Isolation characterization and mechanism of activation by a-thrombin. J Clin Invest 1979; 64: 761-769
• 10 Salem HH, Esmon NL, Esmon CT, Majerus PW. Effect of thrombomodulin and factor Va-light chain on protein C activation in vitro. J Clin Invest 1984; 73: 968-972
• 11 Dahlback B. Purification of human Vitamin K-dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846
• 12 Salem HH, Maruyama I, Ishii H, Majerus PW. Isolation and characterization of thrombomodulin from human placenta. J Biol Chem 1984; 259: 12246-12251
• 13 Miletich JP, Jackson CM, Majerus PW. Properties of the factor Xa binding site on human platelets. J Biol Chem 1978; 253: 6908-6916
• 14 Miletich JP, Broze GJ, Majerus PW. The synthesis of sulphated dextran beads for the isolation of human coagulation factors II IX, and X. Anal Biochem 1980; 105: 304-310
• 15 Owen WG. Evidence for the formation of an ester between thrombin and heparin cofactor. Biochim Biophys Acta 1975; 405: 307-887
• 16 Warren L. The thiobarbituric acid assay of sialic acid. J Biol Chem 1959; 234: 1971-1975
• 17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 1970; 227: 680-685
• 18 Mitchell CA, Hau L, Salem HH. Control of thrombin mediated cleavage of protein S. Thromb Haemostas 1986; 56: 151-154
• 19 Mitchel CA, Salem HH. Cleavage of protein S by a platelet membrane protease. J Clin Invest 1987; 79: 374-379
• 20 Tishkoff GH, Pechet L, Alexander B. Some biochemical and electrophoretic studies on purified prothrombin factor VII (Proconvertin) and factor X (Stuart). Blood 1960; 15: 778-790
• 21 Nelsesten GL, Suttie JW. Properties of asialo and aglycoprothrombin. Biochem Biophys Res Commun 1971; 45: 198-203
• 22 Henriksen A, Christensen TB, Helgeland L. On the significance of the carbohydrate moieties of bovine protrombin for clotting activity. Biochim Biophys Acta 1976; 421: 348-352
• 23 Federici AB, Elder JH, De MarcoL, Ruggeri ZM, Zimmerman TS. Carbohydrate moiety of von Willebrand factor is not necessary for maintaining multimeric structure and ristocetin cofactor activity but protects from proteolytic degradation. J Clin Invest 1984; 74: 2049-2055