A comparison of the urokinase and streptokinase activation properties of the native and lower molecular weight forms of sheep plasminogen.

Native sheep plasminogen (SPg-a), of molecular weight 86,000 to 90,000, in the presence of sheep plasmin (SPm), is rapidly and specifically degraded to a plasminogen (SPg-b), of molecular weight 80,000 to 82,000, by loss of a peptide from the NH2 terminus of SPg-a. More extensive treatment of SPg-b with SPm results in further loss of a single peptide (P) of molecular weight 29,000 to 32,000 from the NH2 terminus of SPg-b, yielding a much lower molecular weight (50,000 to 52,000) plasminogen (SPg-c) which is fully activatable to SPm. The two affinity chromatography forms of SPg-a (Paoni, N., Violand, B. N., and Castellino, F. J. (1977) J. Biol. Chem. 252, 7725-7732) are activated to SPm by urokinase at approximately the same rate and to the same extent. Although SPg-b is activated to SPm in a manner similar to that of SPg-a, SPg-c free of P appears to be activated significantly more rapidly by urokinase when compared to SPg-b and SPg-a. Addition of P to SPg-c restores the SPg-b and SPg-a activation rates to SPg-c. We show SPg-a, SPg-b, and SPg-c to be insensitive to activation to SPm by streptokinase. However, all sheep plasminogen forms are fully activated by catalytic levels of a 1:1 molar complex of streptokinase and human plasmin. A major reason for the insensitivity of SPg-a, SPg-b, and SPg-c to streptokinase activation results from the rapid degradation of streptokinase to inactive fragments by small amounts of SPm initially formed in the activation.

252, 7725-7732) are activated to SPm by urokinase at approximately the same rate and to the same extent. Although SPg-b is activated to SPm in a manner similar to that of SPg-a, SPg-c free of P appears to be activated significantly more rapidly by urokinase when compared to SPg-b and SPg-a. Addition of P to SPg-c restores the SPg-b and SPg-a activation rates to SPg-c. We show SPg-a, SPg-b, and SPg-c to be insensitive to activation to SPm by streptokinase. However, all sheep plasminogen forms are fully activated by catalytic levels of a 1:1 molar complex of streptokinase and human plasmin.
A major reason for the insensitivity of SPg-a, SPg-b, and SPg-c to streptokinase activation results from the rapid degradation of streptokinase to inactive fragments by small amounts of SPm initially formed in the activation.

Plasminogen
is the inactive form of the proteolytic enzyme plasmin and has been found in the plasma of all mammalian species tested to date. This protein can be readily purified from any species by affinity chromatography (1,2) and detailed analyses of the properties of plasminogen have been forwarded for the rabbit (3)(4)(5)(6)(7)(8), human (2, 8-U), and sheep (14) systems. There are many similarities in the plasminogens derived from several species. It has been established that a high degree of multiplicity exists in human, rabbit, and sheep plasminogen. At least two forms of this protein can be resolved by affinity chromatography (2,14) and each major form consists of several subforms (3,13,14). In addition, human, rabbit, and sheep plasminogen undergo dramatic alterations in conformation as a consequence of their binding small mol-* 'I'his work was supported by Grant HL-13423 from the National ecules of the 6-Ahx' class (2,8,14,15). Notable differences also exist between the various plasminogens. The ability of the bacterial protein streptokinase to activate plasminogen differs from species to species. Human plasminogen is highly sensitive, while rabbit plasminogen is only weakly sensitive to activation by this agent (16)(17)(18). The observation that sheep euglobulin, when treated with crude streptokinase, did not develop further proteolytic activity suggested that sheep plasminogen was not activated by streptokinase (16). The above species of plasminogen also differ in the nature of the products obtained as a result of plasminolysis. It has been previously shown that plasminolysis of human (19) and rabbit (20)(21)(22) plasminogen leads to loss of an M, = 6,000 to 8,000 peptide from the NH:! terminus of the native plasminogen molecule. The remaining plasminogen is an altered, lower molecular weight form of the native molecule. We have previously shown that an analogous reaction takes place in the sheep system (14). Treatment of native sheep plasminogen (SPg-a) with sheep plasmin (SPm) results in rapid loss of a small peptide of M, = 6,000 to 8,000, yielding sheep plasminogen b (SPg-b). In addition, however, protracted treatment of SPg-b with SPm results in the loss of a second, and much larger peptide (P) of M, of approximately 30,000 to 32,000. The portion of the molecule that remains is a fully activatable plasminogen (SPg-c) of M,. = 50,000 to 52,000. Sheep plasminogen is pertinent to our studies on the mechanism of activation of plasminogen, since it is apparently insensitive to action of streptokinase alone. Further, the plasminolysis reaction of sheep plasminogen provides convenient natural cleavage products with which the structure-function relationships of the molecule can be examined. We have previously described the purification and physical characterization of the native and altered, lower molecular weight forms of sheep plasminogen, as well as the large peptide released from SPg-b by SPm. In this paper, we extend our studies to comparison of the urokinase and streptokinase sensitivities of the various forms of the sheep plasminogen molecule.

EXPERIMENTAL PROCEDURES
Proteins-Wg-a, SPg-b, SI'g-c, and P were prepared as previously described (14). The only exception was that the Sl'g-c and P used in these studies was prepared by incubating SPg-a with urokinase-free SPm for 2.5 h, rather than 4 h. Only slightly lower yields of SPg-c and P were obtained with this modification. Urokinase was obtained from G. H. Barlow of Abbott Laboratories, and further purified as previously described (19 with urokinase-free SPm in a molar ratio of streptokinase: plasmin equal to 251 (Fig. 5). The streptokinase was rapidly

RESULTS
The urokinase activation of the two major affinity chromatography forms of SPg-a is shown in Fig. 1 was followed by DodS04-gel electrophoresis in the presence and absence of reducing agent (Fig. 2, A and B, respectively). ,s The results shown for affinity form 2 SPg-a indicate that the final plasmin formed has a much lower molecular weight than that reported for human and rabbit plasmin. The heavy and light chains of human and rabbit plasmin have molecular weights of approximately 60,000 and 24,000, respectively. The molecular weights of the heavy and light chains of SPm have been determined by calibrated DodS04-gel electrophoresis. The SPm heavy chain has a molecular weight of 29,000 to 32,000 and the light chain has a molecular weight of 23,000 to 26,000. The molecular weights of the SPm-component chains A are similar to those previously reported for bovine plasmin (heavy chain, M, = 35,000; light chain, M, = 23,500 (30)).
SPg-a affinity chromatography form 2 was incubated with 1 2 3 4 various amounts of streptokinase and the results are shown in Fig. 3. Little or no plasmin activity was detected when SPg-a SPg-a was incubated with streptokinase at molar ratios of SPg-a: streptokinase equal to 8.6:1,5:1,2.6:1, or l:l. However, plasmin activity was rapidly generated when SPg-a was incubated  degraded to fragments electrophoretically similar to those produced during the incubation of SPg-a with streptokinase. The nature of the streptokinase fragmentation is qualitatively consistent with previous studies employing human and rabbit plasmin (18,31,32) for this purpose, although the stability of the various fragments is different in all three systems.
The NHz-terminal amino acid\sequences of SPg-a, SPg-b, SPg-c, and P derived from affinity chromatography form 2 are shown in Table I. SPg-b was found to bind the solvent used during the automatic Edman degradation of the protein. As a result, the phenylthiohydantoin derivatives obtained from SPg-b could not be resolved by routine gas chromatographic analysis. Instead, the samples were hydrolyzed as described under "Experimental Procedures," and the residues were identified by amino acid analysis on a Beckman model 121 amino acid analyzer. Clearly, the NH1 terminus of the native sheep plasminogen molecule is lost during the formation of SPg-b. Furthermore, the same NHs-terminal amino acid sequence found on SPg-b is also present on the large peptide released during extended treatment of SPg-a with plasmin. This indicates that P originates as the NH, terminus of SPg-b. The urokinase-mediated activation rates of the native and lower molecular weight forms of sheep plasminogen were compared utilizing potentiometric techniques and the synthetic substrate, TosArgOMe.
In all cases, urokinase activations were performed at 30°C in 0.05 M Tris-HCl, 0.1 M Llysine, pH 8.0. The activation rates of SPg-a and SPg-b from affinity chromatography forms 1 and 2 are shown in Fig. 6, A and B. Clearly, SPg-a and SPg-b are activated at the same rate and to the same extent by urokinase. The activation of SPg-c and P (affinity chromatography form 2) were compared to SPg-a under identical conditions with those employed for SPg-b (Fig. 7). There is approximately a 33% difference in the initial activation rate of SPg-c over SPg-a. This difference is consistently noted and appears significant. When urokinase was added to the large peptide produced by plasminolysis of SPg-a, no plasmin activity resulted.
To test the effect of the large peptide on the activation of SPg-c, a 2-fold molar excess of P was added to SPg-c, and the activation rate of this mixture was compared to SPg-c alone, and to SPg-a (Fig. 7). The mixture of SPg-c and P activated at approximately 67% of the initial activation rate of SPg-c alone. Similar results were obtained when SPg-c and P were mixed in a molar ratio of SPg-c:P equal to 1:5 (SPg-c + P activated at an initial rate equal to 60% of SPg-c alone, data not shown). As also shown in Fig. 7, the addition of the large peptide to SPg-c produces an activation rate which is very similar to that of SPg-a.
The observed decrease in activation rate when P is added to SPg-c is not due to an inhibition of plasmin activity by the   plus urokinase, as described in the text. Activity is expressed as micromoles of TosArgOMe cleaved per min per nmol of protein.
B, relative rate of activation of affinity form 2 SPga and SPg-b to plasmin by urokinase.
As described in A, except that affinity chromatography form 2 plasminogens were used. IO   While SPg-c is insensitive to activation by streptokinase alone, it is rapidly activated by small amounts of a mixture of human plasmin and streptokinase ( Fig. 9). In this regard, SPgc closely resembles SPg-a (Fig. 3). DISCUSSION

Sheep plasminogen
has previously been reported to be insensitive to activation by the bacterial protein streptokinase. Wulf and Mertz (16), using crude sheep plasminogen preparations, reported that this zymogen could not be activated by crude streptokinase at any level of streptokinase tested. The streptokinase sensitivity of sheep plasminogen was also examined in this present study utilizing highly purified sheep plasminogen and streptokinase preparations. Streptokinase was incubated with SPg-a, at molar ratios of streptokinase: SPg-a up to and including 1:l (Fig. 3). In each case, little or no plasmin activity could be detected in the activation mix- sheep plasminogen activation (32). It is seen, however, in Fig.  4, upon examination of the longer incubation times, that significant amounts of SPg-c and P are formed long after the streptokinase has disappeared from the gels. Since SPg-c and P are readily produced from SPg-b, by small amounts of SPm, these results indicate that some small amount of SPm is likely produced during the reaction. Fig. 5 shows the effect of urokinase-free SPm on streptokinase.
Here, low levels of SPm rapidly degrade the streptokinase to fragments electrophoretically similar to those produced upon incubation of SPg-a, SPg-b, and SPg-c with streptokinase.
Thus, the inability of streptokinase to activate sheep plasminogen is contributed to by the extreme instability of streptokinase in the presence of initial small quantities of SPm formed. Concomitant with the small amounts of SPm formed, rapid degradation of the streptokinase, to lower molecular weight fragments (SK-3 and SK-4), precludes further SPm formation (32). The instability of streptokinase in the presence of SPm is apparently not pronounced when formed in a complex with human plasmin. The results given in Fig. 3 show that low levels of a complex of streptokinase and human plasmin can rapidly activate sheep plasminogen.
A significant observation in the sheep plasminogen system deals with the existence of significantly smaller plasminogen intermediates, produced by plasminolysis of SPg-a. Sheep plasmin rapidly cleaves a small peptide, M, = 6,000 to 8,000, from SPg-a, forming an altered and somewhat lower molecular weight form of the molecule, SPg-b. Continued exposure of SPg-b to SPm results in the loss of a second and much larger peptide (P), M, = approximately 30,000, from SPg-b. The portion of the molecule that remains, SPg-c, possesses an approximate Mr = 51,000. In the conversion from SPg-a to SPg-c, more than 40% of the molecular weight of the SPg-a molecule is lost.
Urokinase activations of the various forms of the sheep plasminogen molecule were performed to assess what affect the loss of a large portion of the NH2 terminus had on the urokinase-mediated activation of sheep plasminogen. While SPg-c possesses a substantially smaller molecular weight than SPg-a, it remains a fully activatable form of the sheep plasminogen molecule. The large M, = 30,000 peptide P, cleaved during the formation of SPg-c, appears to be significant in describing the mechanism of activation of sheep plasminogen by urokinase. SPg-c activates significantly faster than SPg-a or SPg-b. The addition of P to the SPg-c activation mixture, however, restores the SPg-a and SPg-b activation rate. The exact process by which the large peptide modifies the activation rate of SPg-c is uncertain. One possible mechanism would be through a noncovalent interaction with SPg-c, which may restore the SPg-a and SPg-b conformation.
It should be pointed out that the urokinase activations of the native and lower molecular weight forms of sheep plasminogen were performed in the presence of 0.1 M L-lysine.
This was necessary since the SPm formed was extremely insoluble in all solvents which did not contain L-lysine or 6 AHx. Lysine and 6-AHx are known to cause a gross conformational change in SPg-a, a process which greatly facilitates its activation by urokinase (12,15,33,34). In comparison, SPg-b and SPg-c do not exhibit the dramatic decrease in .&, in the presence of 6-AHx which is characteristic of the gross conformational alteration of SPg-a in the presence of this agent (14). While the studies described above represent a necessary comparison of the activation rates of the various forms of sheep plasminogen in 0.1 M L-lysine, similar results may not be obtained in the absence of this amino acid.
DodS04-gel electrophoretic analysis of the plasmins produced by urokinase activation of SPg-a, SPg-b, and SPg-c (Fig. 8) reveals that all of the plasmins are electrophoretically similar in nature. We have previously shown that the plasmin derived from SPg-c possesses fibrinolytic activity (35). Thus, it appears that a large portion of the NH2 terminus of SPg-a is not necessary for the cleavage of fibrin.
The streptokinase sensitivities of SPg-b and SPg-c were also tested by separately incubating each altered form of the plasminogen molecule with an equimolar amount of streptokinase. In each case, the streptokinase was rapidly cleaved to fragments electrophoretically similar to those produced by the incubation of SPg-a with streptokinase, while no appreciable amount of plasmin was formed (data not shown). SPgc was tested to determine whether it retained the ability to be activated by human activator (Fig. 9). As with SPg-a, human activator rapidly activated the SPg-c to plasmin. Thus, it appears as though the streptokinase sensitivity of sheep plasminogen is not dramatically altered by the loss of a substantial portion of the NH2 terminus of the molecule.
While the in vitro studies reported here on the sheep plasminogen system serve to compare some of the properties of the various forms of the plasminogen molecule in the purified state, little is known of the function of the lower molecular weight forms of sheep plasminogen in plasma. It is not unreasonable to suspect that plasminolysis of SPg-a occurs in uiuo when there is an accumulation of excess plasmin. Observations contained in the text (Fig. 8), and previously reported (14), indicate that P is extremely stable in the presence of SPm or urokinase. The size and stability of P may suit it to a regulatory role in the mechanism of fibrinolysis, blood coagulation, or other biological pathways. While such a role is totally speculative, it is not without precedent for peptides released in other humoral systems. Peptides released during the activation of the third and fifth components of the complement system have dramatic biological activities. Each causes contraction of smooth muscle; release of histamine from mast cells; and directed, chemotactic migration of polymorphonuclear leukocytes (36). Finally, the comparison of the activation properties of the native and lower molecular weight forms of sheep plasminogen may be particularly valuable in light of recent preliminary observations by Yecies and Kaplan (37), suggesting that lower molecular weight forms of the plasminogen molecule exist in human plasma.