Modus Operandi of Commercial Heparin

Abstract

The full explanation of the clot-inhibiting action of heparin (Howell and Holt's 1 anticoagulant liver extract) has awaited the reconciliation of the conflicting results of various investigators. The present experiments were designed to assist in the search for the experimental variables involved. Three technical points received special attention: (1) adequate controls 2 ; (2) the time factor in certain reactions; (3) the effective concentration zones of the chief reagents as measured by a dilution method.

0.5 cc. of a moderately weak thrombin mixture [prothrombin soln. (10) + 0.1 % cephalin (1) + N/10 CaCl₂(1)] gave solid clots in 1.0 cc. of prothrombin-free dog fibrinogen in 15-60 sec. 2 Successive dilutions of a 1% stock solution of commercial (H. W. & D.'s) heparin were used in the following tests (all performed at 38°C).

A. A true antithrombic action could be demonstrated by adding 1-3 mg. heparin (1:1000 soln.) per one cc. of ‘ripe’ (i. e., maximally activated) thrombin and subsequently testing its clotting power. As long as the reagents were added simultaneously it was immaterial whether the heparin was added to the thrombin or to the fibrinogen first. In other words the inhibition was ‘immediate’. Nevertheless, if the heparin was incubated with the thrombin before the clotting test, the neutralization was definitely enhanced. The rapid and solid clots with control thrombins substantiate HoweH's (Cekada 3 ) claim that “antithrombin is absent from the solutions of prothrombin prepared by the acetone method.” There is nothing to suggest that this antithrombic action of heparin is other than direct. A flocculation of fibrinogen by the heparin proved troublesome in some of the tests but it was found possible to prevent this by controlled alkalinization with N/10 KOH.