Following the principles described by Wohl, Summaria and Robbins we have studied the kinetics of human plasminogen activation by streptokinase. Plasma plasminogen activation kinetic parameters were measured in the following way: Citrated plasma was mixed with a Tris/NaCl buffer, pH 7.4, and increasing amounts of streptokinase were added. After incubation at 37°C for 5 min, synthetic substrate, H-D-val- -L-leu-L-lys-p-nitroanilide was added and initial plasmin rates were recorded. We also determined the Michaelis-Man- ten parameters for the plasmin of the plasma samples following maximal activation of the plasminogen with streptokinase. This was done by incubating citrated plasma (diluted 1:200) with streptokinase (5000 U/ml) for 10 min at 37°C.In the normal individuals the zymogen.streptokinase complex gave a mean catalytic efficiency, kcat/KM, of 0.15 μM-1S-1. For the streptokinase activator species the average catalytic efficiency, kplg/Kplg, was 26 μM-1min-1, Iμ(95%) = {8.4; 44.5} μM-lmin-1.So far we have studied eight patients with a history of thrombosis. With the exception of one patient, all showed values within the range mentioned above. The deviating patient had a significantly lower concentration of plasminogen, 6.7 mg/dl, as measured by immunodiffusion, and a significantly higher kplg/Kplg-value, 54 μM-1min-1, but a similar kcat/KM-value, 0.2 μM-1S-1. The lower plasminogen concentration is thus matched by an apparent increase of streptokinase activation. We do not know if the observed effect is caused by a mutation in the enzyme. An explanation for the low concentration of plasminogen could be chronic fibrinolysis of thrombi.We will investigate this further and also screen more patients with a history of thrombosis for possible variants with lowered catalytic efficiency of zymogen activation.
The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity.
