High Molecular Weight Forms of Antithrombin III Complexes in Blood

Summary

A double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 ± 21 μg/ml. Complexes of AT III with thrombin or factor X a crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT III complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

Keywords

Antithrombin III - Radioimmunoassay - Enzyme - inhibitor complex - Thrombin - Factor Xa

• References

• 1 Downing MR, Bloom JW, Mann KG. Comparison of the inhibition of thrombin by three plasma protease inhibitors. Biochemistry 1978; 17: 2649-2653
• 2 Shapiro SA, Anderson DB. Thrombin inhibition in normal plasma. In: Chemistry and Biology of Thrombin. Lundblad RL, Fenton JW, Mann KG. (Eds) pp 361-374 Ann Arbor Science Publishers Inc; Ann Arbor, Mich: 1977
• 3 Binder B. On the complex formation of antithrombin III with thrombin. Gelfiltration studies on human plasma and serum. Thrombos Diathes Haemorrh 1973; 30: 280-283
• 4 Pepper DS, Banhegyi D, Cash JD. The different forms of antithrombin III in serum. Thromb Haemostas 1977; 38: 494-503
• 5 Marciniak E. Thrombin-induced proteolysis of human antithrombin III: an outstanding contribution of heparin. Br J Haematol 1981; 48: 325-336
• 6 Thaler E, Schmer G. A simple two-step isolation procedure for human and bovine antithrombin II/III (heparin cofactor): a comparison of two methods. Br J Haematol 1975; 31: 233-243
• 7 Marciniak E. Antithrombin III and heparin inactivation in thrombin involving reactions. Thromb Haemostas 1977; 38: 486-493
• 8 Fenton JW, Fasco JJ, Stackrow AB, Aronson DL, Young AM, Finlayson JS. Human thrombins. Production, evaluation and properties of a-thrombin. J Biol Chem 1977; 252: 3587-3598
• 9 Marciniak E. Factor-Xa inaktivation by antithrombin III: evidence for biological stabilization of factor Xa by factor-V-phospholipid-complex. Br J Haematol 1973; 24: 391-400
• 10 Furie BC, Furie BF. Coagulant protein of Russell’s viper venom. Methods Enzymol 1976; 45: 191-205
• 11 Hunter MW. Radioimmunoassay. In: Handbook of Experimental Immunology. Weir DM. (Ed) pp 17.1-17.36 Blackwell Scientific Publications; Oxford: 1967
• 12 Marciniak E. Differential role of fractionated heparin in antithrombin-III proteolysis. Blood 1982; 59: 576-581
• 13 Fish WW, Orre K, Bjork I. The production of an inactive form of antithrombin through limited proteolysis by thrombin. FEBS Lett 1979; 98: 103-106
• 14 Chandra S, Bang NU, Marks C. Radiolabeled AT III as a probe for the detection of activation of blood coagulation in vivo. Thromb Res 1976; 09: 09-23
• 15 Chan V, Chan TK, Wong W, Tso SC, Todd D. The determination of antithrombin III by radioimmunoassay and its clinical application. Br J Haematol 1979; 41: 563-572
• 16 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
• 17 Ohlsson K. Interactions in vitro and in vivo between dog trypsin and dog plasma proteinase inhibitors. Scand J Clin Lab Invest 1971; 28: 219-223
• 18 Ohlsson K, Olsson I. Neutral proteases of human granulocytes III. Interaction between human granulocyte elastase and plasma proteinase inhibitors. Scan J Clin Lab Invest 1974; 34: 349-355
• 19 Schapira M, Scott CF, Colman RW. Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma. J Clin Invest 1982; 69: 462-468
• 20 Machovich R. Mechanism of action of heparin through thrombin on blood coagulation. Biochim Biophys Acta 1975; 379: 193-200
• 21 Carlstrom AS, Lieden K, Bjork I. Decreased binding of heparin to antithrombin following the interaction between antithrombin and thrombin. Thromb Res 1977; 11: 785-797
• 22 Villanueva G, Danishefsky I. Conformational changes accompanying the binding of antithrombin III to thrombin. Biochemistry 1979; 18: 810-817
• 23 Marciniak E. Adverse effect of heparin in thrombin-antithrombin III interaction. Thrombos Diathes Haemorrh 1975; 34: 748-762
• 24 Bocci V. Catabolism of plasma proteins. In: Structure and Function of Plasma Proteins. Allison AC. (Ed) pp 168-188 Plenum Press; New York and London: 1976
• 25 Putnam FW. Haptoglobin. In: The Plasma Proteins. Structure, Function and Genetic Control. Putnam FW. (Ed) Vol 02. pp 02-50 Academic Press; New York, London: 1975
• 26 Shifman MA, Pizzo SV. The in vivo metabolism of antithrombin IE and antithrombin III complexes. J Biol Chem 1982; 257: 3243-3248
• 27 Lam LS L, Regoeczi E, Hatton MW C. In vivo behaviour of some antithrombin-III-protease complexes. Br J Exp Pathol 1979; 60: 151-160
• 28 Collen D, Schetz J, De CockF, Holmer E, VeTStraete M. Metabolism of antithrombin III (heparin cofactor) in man: effects of venous thrombosis and of heparin administration. Eur J Clin Invest 1977; 07: 27-35
• 29 Teitel JM, Bauer KA, Lau HK, Rosenberg RD. Studies on the prothrombin activation pathway utilizing radioimmunoassays for the F₂/F₁+ 2 fragment and thrombin-antithrombin complex. Blood 1982; 59: 1086-1097