Plasmin is the principal endogenous protease in bovine milk. Distribution of overall proteolytic potential among milk fractions was determined using Coumarin Substrate as a synthetic substrate. Casein containing fractions had a higher amidolytic potential. However, preparation of casein by acid treatment produced increased dissociation of plasminogen and plasmin from casein. The variable results obtained from milks of different cows could be due in part to the influence of inhibitors and activators of the fibrinolytic system present in milk.

We have shown, for the first time, the occurrence of α₂-M in bovine skimmilk (using SRID) at a level (using ELISA) of 1.54 +/- 0.91 mg/ml. This inhibitor appeared to primarily associate with the acid whey fraction. A high level (< 1 mg/ml) of α₂-M was also detected in human skimmilk. The other major fibrinolytic inhibitor, α₂-AP, as well as the complex formed between this inhibitor and plasmin were also shown to occur in human milk.

We used a coupled colorimetric assay to demonstrate the occurrence of a fibrin-independent plasminogen activator (similar to u-PA) in bovine skimmilk. The occurrence of a u-PA like activator in bovine milk was further confirmed in co-polymerized gel electrophoresis. Moreover, u-PA could also be detected in a sample of human skimmilk. However, the electrophoretic gel patterns also contained additional zones of clearing which may be due to the occurrence of other activators in bovine milk. These plasminogen activators may be fragments of u-PA, or t-PA (shown to occur in sow milk) which retain catalytic activity.

The occurrence of such high levels of α₂-M (~ 4% of the total protein) and plasminogen activators may be of tremendous significance to the dairy industry, as they may not only influence plasmin-mediated proteolysis of milk proteins, but may also interfere with the action of milk clotting enzymes.