A chemical method has been devised for the quantitative determination of plasma antithrombin. A concentrate of antithrombin has been prepared from plasma. It also contains heparin cofactor activity. The two distinct activities could not be separated and may quite possibly be the properties of a single substance. When the concentrate is combined with heparin the capacity of the concentmte to destroy thrombin is not increitsed but there is interference with the thrombin fibrinogen interaction. In a mixture of the concentrate and thrombin there is simultaneous destruction of antithrombin, and thrombin activities. Small amounts of the concentrate significantly prolong the clotting time of blood.
X concentrate of antithroinbin was made from bovine plasma. When examined with an analytical ultracentrifuge it was found to contain a main component and a sinall ail~ount of more rapidly sediinentating material. For the main component S j , , was 4.01 S. The antithroinbin concentrate destroyed the activity of purified autoprothrombin C, and most likely antithrombin and antiautoprothrombin C are one and the same substance. The antithrombin preparation was limited in its capacity to destroy autoprothrombin C activity, and equilibriunl conditions were reached in 3 to 4 hours. At equilibrium the quantity of autoprothrombin C destroyed \\;as proportional to the antithrombin added to an "excess" of autoprothroinbin C. 'The last traces of the latter activity could only be neutralized with difficulty. 1;nder optimum conditions 1 ml of plasma can neutralize the autoprothrombin C that inight generate froill 27 ml of plasma. About 0.83 ml of oxalated bovine plasma can neutralize the activity in 1 mg of purified autoprothrombin C.
Antithrombin levels were measured following the addition of varying amounts of thrombin to defibrinated plasma and following the intravenous injection of thrombin into rats. In the in vitro studies the decrease in antithrombin was proportional to the thrombin added. The addition of 30–40 u of thrombin to 1 ml of plasma caused a 50% decrease in antithrombin. Similar results were obtained in the in vivo studies. Six-hundred units of thrombin injected into heparinized rats caused a 40% reduction in plasma antithrombin. Thrombin generation in samples of recalcified plasma was measured according to the method of Pitney and Dacey ( J. Clin. Path. 6:9, 1953) and the values compared with changes in antithrombin. The amount of thrombin generated when full strength plasma was recalcified was only a fraction of the amount potentially available according to estimations by the two-stage method. When plasma was diluted with one to two volumes of saline before activation the amount of thrombin generated was increased two- to threefold. The antithrombin level of the serum from the diluted samples was correspondingly decreased.
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