Кoличecтвeннoe oпpeдeлeниe cпeцифичecкич ингибитopoв пoлимepизaции фибpинa. Eдиницы тopмoзящeй aктивнocти

Abstract

Fibrinogen and its degradation products are to be regarded as specific inhibitors of fibrin polymerization because of the protein-protein recognizing manner of their action. Some effects of specific inhibitors, e.g. prolongation of clotting time are easily measurable, yet, a proper quantitative presentation of the inhibitory activity is a separate problem requiring consideration. No series of direct measurements of inhibitory effects can adequately reflect the relative activities of inhibitors in question as the inhibitor concentration - effect dependence is non-linear. In Figure 1 curves for fibrinogen and fragment D are shown. Ordinate - relative prolongation of pure fibrin monomer clotting, abscissa - inhibitor concentration. The curves suggest some cooperativity in fibrin-inhibitor interactions. Dotted lines indicate a striking change in relative activity of the two inhibitors due to altering the concentration chosen for the activity-comparison test. Thus, the mere values of the inhibitory effects can give hardly more than semi-quantitative hints on the intrinsic inhibitory activities. To gain true values we made resort to the estimated weight quantities of inhibitors producing the same effect. As these quantities are obviously inversely proportional to the specific activity values of the respective inhibitors they immediately provide the information wanted. An arbitrary standard was chosen: 10-fold retardation of fibrin monomer clotting at definite medium conditions. So it became possible to express activities of specific inhibitors in terms of special units. In mixtures of inhibitors the overall activity (the total amount of activity units) was found equal or near to the sum of unit contents of all the components present. Consequently, the behaviour of components is virtually additive (Tables 2 and 3). This additivity means that the effects of individual components enhance each other in the same co-operative manner as the effectiveness of a single inhibitor is raised by its concentration increase (Figure 1). The arithmetical sum of separately exerted effects of components generally proved far below the actual effect of the corresponding mixture. This difference, as expected theoretically, was maximal if the components were represented in equivalent amounts, and it was less for a two-component system than for a three-component one. These results warrant the adequacy of the new method. Following determinations were carried out with this method. In fibrinogen solutions subjected to tryptic hydrolysis the activity unit content increased by about 300 per cent under optimal conditions, i.e. in the presence of Ca 2+ and at 1:2250 trypsin-fibrinogen weight ratio. Specific activities of fibrinogen and purified fragment D were found to amount to 1.4 and 10.9 units per mg respectively. Throughout our work on fragment D isolation from tryptic fibrinogen digests activity yields were determined to check the procedure under trial.