Abstract
It had been previously reported that platelet-clumping substance (PCS) appeared after acidification of heparinized plasma of rabbit. The pH in acidification was around 6.0 which was observed in ischemic or inflammatory lesions in vivo. As the chemical structure is still not determined, we tried to isolate it from rabbit plasma. Citrated rabbit plasma was mixed with 100U/ml of heparin and 0.1M CaCl2, and was centrifuged at 10, 000×g for 30 minutes to obtain clumping activity in the precipitate. The precipitate was soluble in 20mM Tris-HCl containing 1M KBr pH 7.4 (high salt buffer), while fine aggregates of plasma proteins were observed in 20mM Tris-HCl without 1M KBr (low salt buffer). The dissolved material was applied to the Sapharose 6B column, and the clumping activity was eluted between the void volume and albumin fractions. The molecular weight was estimated to be about 800, 000. The fractions contained 3-4 bands in the globulin fraction on 7.5% polyacrylamide electrophoresis. Heparin was not detected in these PCS fractions by metachromatic staining on 6% agarose electrophoresis. The clumping activity was adsorbed on the fibrin monomeraffinity column. After the immunization of the active fraction to guinea-pig, the antiserum prevented platelet aggregation induced by PCS. The antiserum showed an identical precipitation line against PCS fraction as anti-fibrinogen antiserum. These results suggest that platelet-clumping substance may be composed of aggregated plasma proteins which mainly contained fibrinogen-fibrin.
