Mechanism of Bovine Plasminogen Activation by Human Plasmin and Streptokinase

Abstract

It has been mown for some time that conversion of bovine plasminogen to plasmin can be effected by addition of streptokinase in presence of trace amounts of either human plasminogen 01' plasmin. This is in contrast to activation a f human plasminogen, elicited by addition ol f streptokinase alone. In (both systems, the formation of plasmin has been ascribed by some investigators ( 1,2,3) to conversion of a specific proactivator by streptokinase to a sinlylar activator which then acts on plasminogen to produce plasmin. Other investigators(4,5,6), however, have expressed the possibility that the proactivator of bovine plasminogen, .Nhen activation mixtures of streptokinase and human plasminogen or plasmin are used, may be identical with either human plasminog en or plasmin. The following studies were performed to examine the mechanism of bovine plasminogen conversion by human plasmin and streptokinase under various experimental conditions, and to determine whether this conversion is brought about by a specific activator principle, formed by action of streptokinase on a separate distinct proactivator, or whether streptokinase may form a complex with human plasmin which can directly convert bovine plasminogen to plasmin.Materials arid methods. Human plasmin. This enzyme was obtained from human plasma Fraction TTTt by a method previously described ( 7 1. frovine plasminogen. Twentyfour liters of Freshly collected bovine blood were permitted to clot a t room temperature; * Died Sept. 30, 1959. after 2 hours standing, the clots were dispersed and strained through gauze. Final serum clarification was obtained by centrifugation for 30 minutes at 2,000 rpm a t 0°C. The serum obtained (approximately 8 liters) was cooled to 0°C and brought to 257% ammonium sulfate saturation by addition of pre-cooled saturated ammonium sulfate solution, stirred for 30 minutes, then left for 1 to 2 hours at 0°C. The precipitate obtained following centrifugation was discarded and the supernatant solution adjusted to 33% saturated ammonium sulfate by further addition of pre-cooled saturated ammonium sulfate solution a t 0°C. After remaining overnight a t 4"C, the precipitate was collected by centrifugation, dispersed in distilled water, and dialyzed against running tap water for 18 hours at 4°C. The precipitate formed on dialysis was collected by centrifugation, dispersed in 100 to 150 ml of 0.9% sodium chloride and then diluted 1:8 with distilled water a t room temperature. To achieve maximum solubilization, p H was adjusted to 9.4 with 1 I\J sodium hydroxide, and the solution was then brought to p H 5.3 with 1% acetic acid. The precipitate that formed was allowed to settle overnight a t 4°C. After centrifugation, the precipitate was dissolved in 1 liter of phosphate buffer p H 7.0, ionic strength 0.15. The solution was cooled to 0°C and brought to 25% saturated ammonium sulfate by addition of pre-cooled satut Obtained through courtesy cf Am. Nat.

Purification of Streptokinase and Human Plasmin and Their Interaction

“…When SK was present in relative excess, residual inhibitory activity was noted. Previously reported studies on activation of bovine and of human plasmingen have demonstrated the loss of activator potency at low pH and restoration upon addition of SK at neutral pH (1,2). Consideration of the present study with these data support the following concept of plasmin-SK interaction.…”

Mechanism of Human Plasminogen Activation by Streptokinase and Human Plasmin

“…Our studies on bovine plasminogen activation (8) indicated formation of an activator moiety on interaction of human plasmin and SK. These studies suggest the same finding inasmuch as the activating potency of plasmin-SK mixtures on human plasminogen was destroyed by low pH treatment, the reactants were shown to be individually stable at pH 2.0 and no degradation of the SK by plasmin a t reduced p H could be demonstrated.…”