Investigations on antithrombin III in normal plasma and serum.
Studies on antithrombin III (AT-III) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-III from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-III displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin III1 : IAT-III1) dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-III2 and IAT-III3) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the AT-III concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasma and serum and were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-III concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-III2 and IAT-III3) had higher molecular size than IAT-III1, that the concentration of IAT-III2 and IAT-III3 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-III and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quantitation can be achieved using the heparin/agarose crossed immunoelectrophoresis system.