On the presence of a heat-stable, macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin.
By concentrating sonicates from rat liver microsomes containing prothrombin, the activity as measured by the one-stage prothrombin assay (Hjort, P., Rapaport, S. J. and Owren, P. A.(1965), J. Lab. Clin. Med. 46, 89-97) gradually decreased. Nearly a complete loss of prothrombin activity was found in sonicates being concentrated 10-fold. By adsorption of prothrombin on barium citrate and dissolving the precipitate in a solution of sodium citrate. NaCl and EDTA followed by gel filtration on a Sephadex G-50 column, the inhibitory effect on the bioassay of prothrombin disappeared. An inhibitor of the thrombin-fibrinogen reaction could be isolated from the supernatant after adsorption of prothrombin on barium citrate. The inhibitor was excluded from a Sephadex G-50 column equilibrated with 4 M NaCl in veronal buffer (pH 7.4) and was heat stable (70 degrees C for 10 min). No proteolytic or antitrypsin activity could be detected in the inhibitor preparation. The importance of removing the inhibitor of the microsomal sonicates prior to any bioassay of coagulation factors based on the thrombin-fibrinogen reaction is emphasized.
D000818:Animals; D001780:Blood Coagulation Tests; D002850:Chromatography, Gel; D004355:Drug Stability; D006358:Hot Temperature; D008862:Microsomes, Liver / Q000032:analysis*; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D011516:Prothrombin / Q000302:isolation & purification*; D051381:Rats; D013010:Sonication; D013917:Thrombin / Q000378:metabolism*
D011506:Proteins; D011516:Prothrombin; D013917:Thrombin