Although there is almost a 75% homology between bovine and human PA, extrapolation of results from human to bovine milk is not justified due to the difference in the composition of the milk between the species. Therefore results from research on human milk can be utilised as a guideline but should not be accepted for different models.
2.4.1. Human plasminogen and plasmin
During the first few days after birth high levels of PA and plasmin activity occur in human milk. After a few weeks the PA activity gradually decreases [37]. Comparison of the molecular weights of different PA's in human milk correlates well with the PA in blood, suggesting that the PA in milk originates from the blood, rather than being produced by the tissue of the mammary gland itself [38- 41].
Human plasminogen is a single peptide chain with the N-terminal glutamine and the C-terminal asparagine [42,43]. Native plasminogen can be divided into two subgroups, namely Glu-plasminogen 1 (Mr 91,500 Da) and Glu-plasminogen 2 (Mr 89,300 Da) [40]. Glu-plasminogen 1 has four subforms with different pI values, namely 6.2, 6.3, 6.4 and 6.6. Glu-plasminogen 2 has 2 different subforms with different pI values, namely 6.4 and 6.6 [44]. Activation of
plasminogen to plasmin by t-PA or u-PA is as a result of the proteolyitic cleavage of a single peptide bond at Arg561-Val562 in human plasminogen [46]. The N-
terminal peptide is released, forming Lys-plasminogen 1 and Lys-plasminogen 2. Lys-plasminogen 1 has three subforms with different isoelectric (pI) points, namely 6.7, 7.2 and 7.5. Lys-plasminogen 2 has three different subforms with different pI values, namely 7.5, 7.8 and 8.1 [40,41]. A three dimensional model of plasminogen reveals triple disulfide loops or the so-called kringles, whereas plasmin contains five kringle domains on the N-terminal A chain [44].
Human plasmin (Mr approximately 90,000 Da) is capable of hydrolysing a broader spectrum of proteins than any other blood protease, but is highly specific for the peptide bonds containing lysine residues [38,39]. Plasmin catalyses the hydrolysis of the peptide bonds on the C-terminal side of the Lys and Arg residues [40]. The active site on the plasmin molecule is located at the serine residue on the C-terminal B chain and is connected to the N-terminal A chain two by a disulfide bridge [41,42].
Plasmin digests fibrin to form soluble degradation products, thus solubilising the fibrin-clot. Both plasmin and PA is then released into the fluid phase of the blood; subsequent inactivation by their natural inhibitors, PAI and PI, renders these molecules inactive [38,43].
2.4.2. Human plasminogen activators
Plasminogen activator activity is present in human milk and human mammary gland tissue [37]. The sources of PA are the mammary epithelial cells, endothelial cells or the leakage of PA from the mammary epithelial tissue via the blood into the milk. PA is native to human milk and can be divided into two distinct groups, namely: u-PA and t-PA [38-46]. Plasmin and plasminogen is functionally the same in milk and blood. PA activates plasminogen to plasmin;
PA is inhibited by PAI. The activation of plasmin results in an increase in proteolysis in blood and milk, in the latter instance casein is converted into smaller peptides. Plasmin inhibitors (PI) also inhibit the conversion of plasmin activity [21,23].
2.4.2.1. Urokinase plasminogen activators
Urokinase-PA is a serine proteinase and is probably the best-characterised PA with a molecular weight of 54,000 Da that can be divided into two subunits, a C- terminal B chain (containing the serine proteinase domain) and a N-terminal A chain, with molecular weights of 27,000 Da each. These two subunits are linked with a disulfide bond. The N-terminal A-chain contains a growth factor domain (amino acids 1-49), a kringle domain (amino acids 50-131), and an inter-domain linker region (amino acids 132-158) [40]. The one chain zymogen precursor of u-PA, pro-u-PA, has an activity of 250-fold less than that of the two chained u- PA. The activation of the pro-u-PA occurs by proteolytic cleavage of Lys158-Ile159
in human u-PA [47]. U-PAs with molecular weight of 93, 57, 42, 35 and 27 kDa have been isolated from bovine and human milk and are associated with the somatic cells (leukocytes) in milk. The u-PA is synthesised by all cell types such as macrophages, fibroblasts and epithelial cells. The main function of u-PA is the degradation of extracellular matrix.
Glu-plasminogen 1 and 2 are converted to plasmin due to activation by urokinase. There are two proteolytic cleavages that firstly release a pre- activation peptide from the N-terminal with a molecular weight of 7,000. The cleavage takes place between Lys76 and Lys77 to yield either Lys-plasminogen 1
or 2 [40,41,43]. The second takes place between Arg560 and Val561 to yield Glu-
plasminogen [45]. If the pre-activation peptide has already been released Glu- plasminogen will be converted to Lys-plasminogen. The release of the pre- activation peptide induces conformational changes in the Glu-plasminogen that enhance the activation of Lys-plasminogen. The dissociation of non-covalent
bonds at residues 45-51 and unidentified residues elsewhere on the molecule result in the Arg561-Val562 bond being more accessible for proteolytic cleavage
[40,44,45].
2.4.2.2. Tissue type plasminogen activator
Tissue type PA is a serine-protease (Mr approximately 90,000 Da) which is released from the vascular endothelium tissue in a single-chain form. The single chain t-PA can be proteolytically converted to a two-chained form, by a cleavage of a single polypeptide bond at Arg275-Ile276 [45]. The two chains are held
together by a disulfide bond, The N-terminal A chain (starting from the N- terminal) contains a fibronectin type II domain, a growth factor domain, and two kringle domains. The C-terminal B chains contains the serine proteinase domain [48,49]. T-PA is released into the blood stream where it binds to fibrin, and is biologically inactive when it is not bound to fibrin. Upon binding with fibrin, the t- PA cleaves the inactive precursor zymogen, plasminogen, between the Arg561-
Val562 bond to render the two-chain active protease plasmin [45,48,49]. The
plasminogen activity of the single-chain t-PA is 10 to 50-fold lower than that of the two-chained form [48,49].