³H-Heparin と Antithrombin

抄録

Heparin itself has little or no antithrombin activity and it requires some of plasma protein fractions to neutralize the thrombin activities in its inhibitory mechanism (Antithrombin II, Heparin Cofactor). Thrombin activity was progressively inhibited by Antithrombin III without existence of heparin. Antithrombin II or Heparin Cof actor has an immediate type of antithrombin activity with the existence of Heparin.
Identity of Heparin Cofactor and Antithrombin III, however, is still questionable and there is some discussions on this point, and the assay methods of both Heparin Cofactor (Antithrombin II) and Antithrombin III are not established yet.
Authors established these assay methods according to their definition of both Antithrombins. Heat defibrinated plasma was fractionated by Sephadex G-200 column, and both activities were checked with these assay methods. The highest activities of Heparin Cofactor and Antithrombin III were observed on the third protein O. D. peak. Another peaks were also seen in the first protein peak of the plasma fraction, although both activities in this fraction were lower than those observed in the third protein peak, and each peaks of Heparin Cofactor and Progressive Antithrombin activities were observed in the different fractions.
³H-Heparin was utilized on Gel-filtration with Sephadex G-200. The ³H-Heparin treated plasma or heat defibrinated plasma showed two peaks of radioactivities. The first one is the heparin-protein complex and the other is noncomplexed free-heparin radioactivity. The first radioactivity peak is approximately corresponded with the peak of the first Heparin Cofactor activity which is apart from the first Progressive Antithrombin Activity peak, while this first radioactivity peak was not detected in the elution of ³H-Heparin alone. The Heparin-protein complex in the third protein fraction where the highest Heparin Cofactor and Antithrombin III activity was detectable, was not definite.
According to our experimental results, Heparin Cofactor and Progressive Antithrombin activities appeared in the fractions of late first protein peak. Recently proposed affinity chromatographic technique for the purification of Heparin Cofactor seems to yield the principally different plasma protein fraction from the fraction by gel-filtration and regular chromatographic techniques. However the binding affinity of heparin to plasma protein varies depending on the different heparins, and it is speculated that the Heparin Cofactor or Antithrombin II separation from plasma by affinity chromatography is related to their affinity of the used heparins as a ligand.