The Preparation of Human Fibrinolysin (Plasmin)*

Abstract

The physiological role of the fibrinolytic system in maintaining the fluidity of the blood is now generally accepted [2]. The persistence of intravascular clots may be associated in part with some deficiency of the normal fibrinolytic mechanism. I n view of the frequency of thrombotic episodes the need for an effective, rational fibrinolytic therapy is very great, particularly in dangerous situations where clots form or lodge in the pulmonary, cerebral or coronary vascular systems. There are three fundamental approaches t o thrombolysis. The first is based on the induction of a fibrinolytic state b y adding amounts of fibrinolysin more than sufficient t o overcome the antifibrinolysin. The second approach is based on the ability of a n "activator" t o induce a fibrinolytic state. The third approach is to use a combination of a n activator and the fibrinolysin. A number of materials are being studied as activators to induce a fibrinolytic state: such as, streptokinase [7], urokinase [14], pyrogens [8], and chemicals [27].Our report deals with a study involving the preparation for clinical evaluation of human fibrinolysin with and without the activator, urokinase. The source of our profibrinolysin was the Fraction I11 paste usually discarded in the preparation of gamma globulin from outdated blood [S, 131. The source of the activator, urokinase, was stable a t neutral pH a t 25" C. These solutions are readily prepared in sterile condition for clinical use. All protein components have been subjected to a heat treatment period of 10 hours a t 60" C. during their purification. MaterialsBlood protein source. Human plasma Fraction I11 paste was kindly provided by the Blood Program of the American National Red Cross. This fraction was prepared b y Method 6 of Cohn, et al. [5] and Method 9 of Oncley, et al. [13] by E. R. Squibb & Sons, New Brunswick, New Jersey. Glycerol. Shell Chemical Corporation, 99.5 % U.S.P. synthetic glycerol. Phosphate-saline bufler. p H 7.4, 0.1 M sodium phosphate -0.9% NaCl buffer.Streptokinase. Lederle Varidase(R) was dissolved in phosphatesaline buffer a t 3,000 units per milliliter and stored a t 0-2" C.Casein solution. Devitaminized and high nitrogen caseins were kindly supplied by the Sheffield Company+. Stock solutions of 6% (w/v) casein were prepared by dissolving the material in 0.1 M phosphate-saline buffer and adjusting the p H to 7.4 with 1 N NaOH. The solution was heated in a boiling water bath for 15-30 minutes t o destroy any proteolytic activity present. The final solution was filtered, adjusted to p H 7.40 and stored in 50 ml. volumes in the frozen state (-30" C.).

Increased Plasminogen Activator (Urokinase) in Tissue Culture After Fibrin Deposition

“…Glycerol-activated human plasmin3 (11) was introduced into the culture medium in amounts similar to those detected in cultures after exposure of cells to plasminogen-rich fibrin clots (0.1-0.2 CTA U/ml of medium). of 0.01-1.0 mg/ml.…”

“…Activation of UK precursor by proteases, particularly plasmin and thrombin, also should be examined in light of factors such as presence of impurities and possible cross-contamination of the enzymes used in the experiments. Plasmin is obtained by spontaneous activation of purified plasminogen (11) which is free of known contaminants such as prothrombin, thrombin, and thromboplastic material (11). A minor contaminating component observed in purified preparations of both plasmin and plasminogen (27) is thought to represent an "inert" form of plasmin (25).…”

Studies on the structural abnormality of fibrinogen Paris I.

“…The degradative phases of plasmin hydrolysis of fibrinogen have been divided into stages 1, 2, and 3 (28 The final protein concentration (normal or Paris I fibrinogen) was 1 mg/ml. Plasmin (10.2 caseinolytic U/ml) had been prepared at the Michigan Department of Health (33). It was used at final concentrations varying from 0.06 to 1.0 U/ml.…”

Fibrinolysis during Extracorporeal Circulation

“…The plasminogen was estimated by the caseinolytic method of Sgouris et al (4). Euglobulin lysis time was measured by the method of Iatridis and Ferguson (5) in which purified fibrinogen is added to the euglobulin sample to eliminate shortening of the lysis time due to hypofibrinogenemia.…”