Abstract:

During our study of the action of water-soluble poly-a-amino acids on blood clotting (1), it was observed that the basic poly-amino acids: poly-lysine (2), poly-ornithine (3) and poly-arginine (3), retard fibrinolysis of human clotted blood. A more detailed analysis of this phenomenon was therefore undertaken. Fibrinolytic activity of oxalated human plasma was induced by mixing the plasma with a suspension of ,- hemolytic streptococci (4, 5), by treatment with a cellfree broth containing streptokinase (6), or by shaking the plasma with chloroform (6, 7). The activated plasma was then treated with the poly-amino acids (prepared in this laboratory), and a fibrin clot obtained by the addition of thrombin. The final mixtures were incubated at 370 C for 15-24 hr to determine -if lysis occurred. When the preparations used did not interfere with fibrinolysis, dissolution of the clot occurred. Inhibition of fibrinolysis was indicated by the maintenance of the fibrin clot, obtained as described *above, for 24 hr. A typical experiment with poly-L-lysine and a streptococei-activated plasma is described below. Oxalated human plasma (0.5 ml) was mixed with a suspension of 1-hemolytic streptococci (0.4 ml), and the mixture added to 1 ml saline solution containing 40y poly-L-lysine hydrochloride. Clotting was induced by adding 4 units of bovine thrombin (Upjohn Company) in 0.1 ml saline with vigorous shaking. The clot was incubated at 370 C for 24 hr. No visual change in the clot was observed. In the control experiment where the 1 ml poly-lysine solution was substituted by saline, a complete lysis of the clot was evident within 30 min. The fibrinolytic activity of plasma activated by Ihemolytic streptococci was not inhibited either by the neutral poly-DL-alanine (8) or by the acidic poly-Laspartic (9) and poly-D-glutamic (10) acids up to concentrations of 500y/ml final test mixture. The basic poly-a-amino acids, poly-DL-lysine hydrochloiide (average chain length n = 35) (2), poly-DL-ornithine hydrochloride (n = 30) (3), and poly-DL-arginine sulfate (n = 30) (3), on the other hand, prevented fibrinolysis at concentrations greater than 30y40y/ml test mixture. In the presence of the basic poly-amino acids, fibrinolysis Was inhibited equally well when the streptococcal culture suspension was replaced (in the test mixture) by a cell-free supernatant containing streptokinase. Furthermore, it has been demonstrated that the fibrinolytic activity of chloroform-treated plasma and of menstrual blood was also inhibited by relatively low concentrations of poly-DL-lysine and poly- DL-arginine. It thus seems justified to assume that the basic poly-amino acids are capable of inhibiting hydrolysis of fibrin by plasma fibrinolysin (plasmin) under the experimental conditions used. Preliminary experiments indicated that the average molecular weight of the basic poly-amino acids plays a profound role in ther determination of their antifibrinolytic properties. L-lysine monomer, as well as L-lysyl-L-lysine (11), did not show any antifibrinolytic activity up to a concentration of 750y/ml. A tetra-Llysine showed slight antifibrinolytic activity at 750y/ ml, whereas poly-L-lysine of average chain length n= 7, 35, and 100 showed distinct antifibrinolytic activity at concentrations of 500y, 40y, and 35y/ml test mixture, respectively. No great difference was observed in the antifibrinolytic activity of poly-L-, poly-D-, and poly-DL-lysine of similar average molecular weights. In our previous study on the action of water-soluble poly-amino acids on blood clotting (1), it was demonstrated that the acidic poly-amino acids, poly-D-glutamic acid and poly-L-aspartic acid, as well as heparin, are capable of neutralizing the anticoagulant activity of the basic poly-amino acids. A similar relationship was found to hold for the antifibrinolytic effect of the basic poly-amino acids. Heparin, as well as poly-Laspartic acid (n = 50), was found to obviate the antifibrinolytic activity of poly-lysine. The neutralization of the antifibrinolytic activity of the basic poly-amino acids occurred when approximately equivalent concentrations of the basie -and acidic poly-amino acids were applied. The ability of the basic synthetic peptides to inhibit rev,ersibly the. proteolytic activity of fibrinolysin resembles, the antiproteolytic properties of some natural peptides, such as the pepsin,inhibitor and the' trypsin inhibitors (12). The interaction of the natural as well as the synthetic peptides with the different proteolytic enzymes is probably determined in both cases by some specific groups present in the enzyme and the inhibitor, as well as by the electrostatic forces prevailing between the enzyme and the relatively high molecular weight inhibitor. Further studies with the 'synthetic amino acid polymers may contribute to our basic knowledge of the mode of actlon of naturally naturally occurring polypeptides on enzyme behavior.