Cephalin Content of Prepared Fibrinogen and Prothrombin Solutions

Abstract

Mills 1 prepared fibrinogen and prothrombin solutions from platelet-free plasma; the fibrinogen remained unclotted by both calcium and cephalin for 24 hours at 40° C.; the prothrombin needed cephalin as well as calcium for its activation. Few other investigators have secured preparations which could fulfil these rigorous tests, and Howell, 2 in particular, has held to the idea that calcium alone is sufficient to activate prothrombin, the more especially since he and Cekada 3 were able to obtain active thrombins that were lipoid-free. In view of Mills'demonstration that (a) the prothrombin prepared by Howell's acetone method contained cephalin which could be extracted with cold benzene, leaving a preparation which needed cephalin as well as calcium for its activation, and that (b) an appreciable amount of cephalin-like lipoid could be extracted by boiling ether from platelet-free goose and dog plasma, we wondered whether Howell's pure thrombins had been tested on fibrinogen solutions free from lipoids. The following experiment emphasizes the need for satisfying this inquiry.

A dog, not fasted, was anesthetized with sodium amytal. Blood was collected, via a paraffined cannula in the carotid artery, into cooled paraffined centrifuge tubes containing sodium citrate (to a final concentration of 0.5%). After immediate low speed centrifugalization to remove red and white cells, the platelets were separated by prolonged high speed centrifugation. The plasma was salted with 15% (solid) NaCl, followed by 18% (NH₄)₂SO₄, the respective fibrinogen and prothrombin fractions being purified by washing and reprecipitation, and tested for their coagulation reactions. The final solution of prothrombin was found to be activated by calcium alone, and formed a firm clot with fibrinogen solution in 4–5 minutes. After extraction for a few minutes with cold benzene, calcium was unable to activate the coagulant.