Vol. 254, Nr, 6, Issue of March 25, pp. 2064-2070, 197’1 Prmted m CJ S.A.

A Comparison of the Urokinase and Streptokinase Activation Properties of the Native and Lower Molecular Weight Forms of Sheep Plasminogen”

(Received for publication, July 7, 1978, and in revised form, October 5, 1978)

Nicholas F. Paoni and Francis J. CastellinoS

From the Department of Chemistry, The Uniwrsity of Notre Dame, Notre Dame, Indiana 46556

Native sheep plasminogen (SPg-a), of molecular weight 86,000 to 90,000, in the presence of sheep plas- min (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 molec- ular 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 activata- ble 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 insen- sitive 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 insensitiv- ity of SPg-a, SPg-b, and SPg-c to streptokinase activa- tion 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 de- tailed analyses of the properties of plasminogen have been forwarded for the rabbit (3-8), 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 Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “ud~vrtisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4: Recipient of a Research Career Development Award (HL-70717) from the National Institutes of Health.

ecules of the 6-Ahx’ class (2, 8, 14, 15). Notable differences also exist between the various plasmin-

ogens. 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 (16-18). The observation that sheep euglobulin, when treated with crude streptokinase, did not develop further proteolytic activity suggested that sheep plasminogen was not activated by strep- tokinase (16). The above species of plasminogen also differ in the nature of the products obtained as a result of plasminol- ysis. It has been previously shown that plasminolysis of human (19) and rabbit (20-22) 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 al- tered, 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 plasmin- ogen (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 treat- ment 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 mech- anism of activation of plasminogen, since it is apparently insensitive to action of streptokinase alone. Further, the plas- minolysis 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 character- ization 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 previ- ously described (19). The starting material used for purification of

’ The abbreviations used are: &Ahx, 6-aminohexanoic acid; Tos- ArgOMe, N-a-tosyl-1,.arginine methyl ester; DodSOi, sodium dodecvl sulfate; iI’rJ’F, diisopropyl phosphorofluoridate; Sl’g-a, native sheep plasminogen; SPm, sheep plasmin; SPg-b, sheep plasminogen b; P, beptide; SI’g-c, sheep plasminogen c; ETA, Committee on Throm- bolytic Agents.

2064

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Sheep Plasminogen Activation 2065

streptokinase was Kabikinase, obtained from AB Kabi. The material had as its major impurity plasma albumin, added by the manufac- turers as a stabilizer. The albumin was removed as previously pub- lished (23). Urokinase-free SPm was prepared by treatment of SPg-a with Sepharose-urokinase (14, 21).

Quantitative NH2-terminal Amino Acid Analysis-The method used was essentially that of Stark (24). The concentration of protein taken for analysis was determined by amino acid analysis of an aliquot of the mixture obtained at the cyclization step.

NHZ-terminal Amino Acid Sequence Analyses-These were per- formed in collaboration with K. G. Mann, of the Mayo Clinic, and T. C. Vanaman, of Duke University. Automatic Edman degradation of the protein samples was performed with a Beckman model 890B Sequencer. The program was structured to the basic Quadrol modi- fications recommended by Hermodson (25). For the SPg-a, SPg-c, and P samples, the phenylthiohydantoins were identified with the aid of a Beckman GC 45 gas chromatograph on a glass column (2 mm x

4 feet) packed with SP-400 (10% on Supelcoport, lOO/ZOO mesh, Supelco, Inc.). Silica thin layer chromatography, employing Solvent XM (26), was used for discriminations between Thr and Gly, and between Thr and Pro derivatives.

SPg-b was found to strongly bind Quadrol, thus making gas chro- matographic analysis of the phenylthiohydantoins difficult to accom- plish. Instead, the samples obtained after each cycle were hydrolyzed for 24 h at 140°C in 5.7 N HCl, 0.01% 2-mercaptoethanol, and analyzed on a Beckman model 121 amino acid analyzer. Ser and Thr were destroyed by this process, and blanks appear in the sequence of SPg- b in place of these residues.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis- The methodology employed was essentially as described by Weber and Osborn (27), except that 6 M deionized urea was substituted for Hz0 in the preparation of all DodS04 gel solutions except for the tray buffer. The gels used in this study were 5% in polyacrylamide.

Protein Concentration Determinations-Protein concentrations were determined spectrophotometrically using the following&, val- ues at 280 nm: human plasminogen, 17.0 (28); streptokinase, 9.49 (29); SPg-a, 20.0; SPg-b, 20.0; SPg-c, 21.0; and P, 23.0.

Plasmin Assays. Potentiometric Method-The equipment used was purchased from Radiometer and consisted of a PHM 26 pH meter, type TTT 11 Titrator, TTA-31 titration assembly, ABU-11 Auto Burette, and a REC-51-S Servograph Recorder. Constant tem- perature was maintained with a Haake model FE circulating water bath. The syringe was filled with carefully standardized NaOH at concentrations of approximately 0.02 M. The temperature of the pH- stat cell was maintained at 30°C and the samples were stirred contin- uously. The synthetic substrate utilized for plasmin assays was 0.15 M TosArgOMe, which was adjusted to approximately pH 8.0 with 0.05 N NaOH. One milliliter of substrate was placed in the pH-stat vessel and allowed to reach temperature. The pH was then adjusted to pH 8 by automatic addition of base. Plasmin was added in O.OlO- to 0.050- ml aliquots such that approximately 45 to 60 pg of active plasmin was present per ml in the reaction vessel. The reaction rate was followed for at least 2 min on the recorder. The results were converte( to micromoles of TosArgOMe hydrolyzed min-’ nmoll’ of protein .dm- ple added to the reaction vessel.

I

Urokinase Activations of Sheep Plasminogens-a, -b, -c, and the Large Peptide Derived from Plasminolysis of Sheep Plasminogen- a-Prior to the start of these studies, various levels of urokinase were tested, and a range of urokinase concentration was determined at which the initial activation rate of plasminogen varied in a linear fashion with urokinase concentration. The concentration of urokinase used in these experiments was within this range. The rate of formation of plasmin activity was measured using the synthetic substrate TosArgOMe and potentiometric techniques, as described above. In separate studies, using SPg-a, plasmin activity, measured utilizing initial concentrations of 0.1 M TosArgOMe, was found to be 95% of that observed when 0.15 M TosArgOMe was utilized. Thus, 0.15 M TosArgOMe concentration will be considered saturating for the pur- pose of these experiments. In all cases, activations were performed at 30°C in 0.05 M Tris-HCl, 0.1 M L-lysine, pH 8.0. The final concentra- tion of urokinase in the activation mixtures was 562 CTA units/ml. The final concentrations of the native and lower molecular weight forms of sheep plasminogen were all approximately 54 nmol/ml, or expressed as milligrams/ml; SPg-a, 4.67 mg/ml; SPg-b, 4.36 mg/ml; SPg-c, 2.71 mg/ml. The large peptide obtained from protracted treat- ment of SPg-b with sheep plasmin (SPm) was also tested for plasmin activity upon the addition of urokinase. The final concentration of the large peptide in the activation mixture was equal to 98.2 nmol/ml

(3.04 mg/ml), and the concentration of urokinase was the same as that used for the various forms of sheep plasminogen. The activation rate of SPg-c was examined in the presence of the large peptide derived from plasminolysis of SPg-b. The final SPg-c and urokinase concentrations were as described above, and the large peptide was added to form a final concentration of 98.9 nmol/ml (3.07 mg/ml), or approximately a 2-fold molar excess of peptide over SPg-c.

To determine whether the large peptide (P) has an effect on the TosArgOMe activity of the plasmin produced from SPg-c, the follow- ing study was conducted. Samples of the urokinase activation mixture of SPg-c were removed after maximum plasmin activity was attained. The samples were mixed with equal volumes of either 0.05 M Tris- HCl, 0.1 M L-lysine, pH 8.0, or peptide, dissolved in the same solvent, such that the final molar ratio of SPg-c to peptide was equal to 1:3. The time course of the reaction was then analyzed by the esterase assay described above.

Samples of the various activation mixtures, containing maximum plasmin activity, were removed and analyzed by reduced DodSOr-gel electrophoresis.

Reduced and nonreduced DodSOd-gel electrophoretograms of the time course of urokinase activation of SPg-a are shown in the text. This activation was performed at 30°C in 0.05 M Tris-HCl, 0.1 M L-

lysine, pH 8.0. The final concentration of SPg-a was 3.07 mg/ml, and the final concentration of urokinase was 649 CTA units/ml. At the times indicated, 0.01 ml of the activation mixture was added to 0.05 ml of 0.1% DodSO+ 0.01 M phosphate, 6 M urea, pH 7.0, for nonreduced samples, and to 0.05 ml of the same solvent adjusted to 5% in 2- mercaptoethanol for reduced samples. The samples were analyzed by DodSO,-gel electrophoresis, 5% in polyacrylamide and 6 M in urea.

Streptokinase Activation of the Native and Lower Molecular Weight Forms of Sheep Plasminogen-The streptokinase sensitivity of SPg-a (affinity chromatography form 2) was tested under a variety of molar ratios of streptokinase to SPg-a, with similar results obtained in each case. A representative selection of experiments were chosen for presentation in this text, and the conditions used in those studies were as follows: (a) 8.6:1 molar ratio of SPg-a:streptokinase; final SPg-a concentration = 2.84 mg/ml; final streptokinase concentration = 0.17 mg/ml. The solvent used was 0.05 M Tris-HCl, 0.05 M z-lysine, pH 8.0. (b) 5:l molar ratio of SPg-a:streptokinase; final SPg-a concen- tration = 2.98 mg/ml; final streptokinase concentration = 0.31 mg/ ml. The SPg-a and streptokinase were dissolved in 0.05 M Tris-HCl, pH 8.0, and the mixture was made 0.02 M in L-lysine 20 min after the start of the reaction. (c) 2.6:1 molar ratio of SPg-a:streptokinase; final SPg-a concentration = 6.12 mg/ml; final streptokinase concentration = 1.2 mg/ml. The solvent used was 0.05 M Tris-HCl, 0.02 M L-lysine, pH 8.0. (d) 1:l molar ratio of SPg-a:streptokinase; final SPg-a concen- tration = 3.43 mg/ml; final streptokinase concentration = 2.0 mg/ml. The solvent used was 0.05 M Tris-HCl, 0.02 M L-lysine, pH 8.0.

The activations were performed at 3O”C, and the time course of the reactions was monitored by potentiometric techniques utilizing the synthetic substrate TosArgOMe.

The streptokinase sensitivities of affinity chromatography form 2 SPg-a, SPg-b, and SPg-c were examined by DodS04-gel electropho- resis under reducing conditions. The various forms of plasminogen were pretreated with iPrpPF in order to remove traces of active plasmin. This was accomplished by incubating the samples, dissolved in 0.05 M Tris-HCl, 0.1 M L-lysine, pH 8.0, with a final concentration of IO-’ M iPrsPF overnight at 5°C. The samples were then dialyzed for a total of 4 h against 2 x 4 liters of 0.05 M Tris-HCl, pH 8.0, in the cold, to remove excess iPrZPF. The solutions were removed from the dialysis sacks and adjusted to 0.02 M in L-lysine by the addition of 0.05 M Tris-HCl, 0.1 M L-lysine, pH 8.0. The activations with strep- tokinase were in each case performed at 30°C in 0.05 M Tris-HCl, 0.02 M L-lysine, pH 8.0. The molar ratio of the various plasminogens: streptokinase was equal to 1:l. In each case, the concentration of plasminogen was equal to approximately 22.3 nmol/ml, which was in terms of milligrams per ml equal to: SPg-a, 1.93 mg/ml; SPg-b, 1.79 mg/ml; SPg-c, 1.15 mg/ml. The concentration of streptokinase in the activation mixtures was 1.01 mg/ml (22.4 nmol/ml). At the times indicated, 0.02 ml of the incubation mixtures were removed and added to 0.02 ml of 0.2% DodSOI, 0.01 M phosphate, 3 M urea, pH 7.0, which was 10% in 2-mercaptoethanol. The time course of the reaction was analyzed by DodSO1-gel electrophoresis, 5% in polyacrylamide and 6 M in urea.

Human Activator Activation of Sheep Plasminogen-a and Plas- minogen-c-Human plasminogen activator was prepared by mixing 0.015 ml of human plasminogen (5.95 mg/ml in 0.05 M Tris-HCl, 0.1 M L-lysine, pH 8.0) with 0.005 ml of streptokinase (6.47 mg/ml in 0.05

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2066 Sheep Plasminogen Activation

M Tris-HCl, pH 8.0). The molar ratio of human plasminogenstrep- with a human plasminogen . streptokinase activator complex tokinase was equal to 1.4:1, and the final lysine concentration was 0.08 M. The mixture was incubated at 22°C for 5 min, after which 0.01

at a molar ratio of SPg-a to activator equal to 17.4:1.

ml was withdrawn and added to 0.538 mg of SPg-a (affinity chroma- SPg-a and streptokinase were mixed in equal molar quan-

tography form 2) dissolved in 0.14 ml of 0.05 M Tris-HCl, 0.1 M L- tities and the time course of the reaction was analyzed under

lysine, pH 8.0. The activation was performed at 30°C in 0.05 M Tris- reducing conditions by DodSO,-gel electrophoresis (Fig. 4). HCl, 0.1 M L-lysine, pH 8.0. The final SPg-a concentration was equal The gels show that the streptokinase in the activation mixture to 41.3 nmol/ml (3.59 mg/ml), and the molar ratio of SPg-a:human was rapidly degraded. No change in the migration of the activator was equal to 17.4:1. The time course of the activation was monitored by potentiometric techniques using the synthetic substrate

SPg-a or streptokinase bands were observed when the active

TosArgOMe. Controls for the experiment included a human activator site titrantp-nitrophenyl-p’guanidinobenzoate was present in

blank, to determine the amount of plasmin activity contributed by the incubation mixture (data not shown). Examination of the the activator, and individual mixtures of human plasminogen with 120-min sample of Fig. 4 reveals significant amounts of SPg-c SPg-a, and streptokinase with SPg-a. and P formed during the incubation, at times beyond the

The human activator activation of SPg-c (affinity chromatography stability limit of streptokinase, or streptokinase fragments, as form 2) was performed as described above, except that the human plasminogen and streptokinase mixture was incubated at 22°C for 10

evidenced by their disappearance from the gel profiles. This

min, and the final concentration of SPg-c in the activation mixture indicates that a small amount of plasmin was likely produced

was 50.7 nmol/ml (2.59 mg/ml). by the reaction, subsequently degrading SPg-a to these pep-

Plasminolysis of Streptokinase-Streptokinase, at a final concen- tides (14). tration of 2.9 mg/ml in 0.05 M Tris-HCl, 0.02 M L-lysine, pH 8.0, was The time course of stability of streptokinase in the presence incubated with urokinase-free SPm at 30°C. The molar ratio of of SPm was next determined. Streptokinase was incubated streptokinase to SPm was 25:l. The time course of the reaction was analyzed by DodS04-gel electrophoresis under reducing conditions.

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 chro- matography forms of SPg-a is shown in Fig. 1. Both form 1

I 2 3:4

and form 2 SPg-a are activated at the same rate, and to the spg-a I, same extent, by urokinase. The time course of the reaction * I / w

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

5 Spg-b

s Pg-c

H,P

L

“g-b

H+L,SPgmc

1.2 / -. 1.0 A’ /’

z 0.8 / / 5 / i= 0.6 0 /’ a

;,,/I/- I’

0.4 1’ 6 0.2

P

FIG. 1. Relative

1 FIG. 2. A, DodSOd-gel electrophoretic analysis under reducing con- ditions of the time course of activation of SPg-a to plasmin by

IO 20 30 40 50 60 urokinase. Gel 1, SPg-a control. SPg-a and urokinase were incubated

TIME (min.) for times of: Gel 2, 20 min; Gel 3, 50 min; Gel 4, 120 min; and Gel 5, 240 min. H and L refer to the heavy and light chains of SPm,

rate of activation of form 1 and form 2 SPg-a to respectively. All other abbreviations are as previously noted in the plasmin by urokinase. O--O, affinity chromatography form 1; and text. The conditions used were 3.07 mg/ml of SPg-a and 649 CTA l - - -0, affinity chromatography form 2 SPg-a plus urokinase, as units of urokinase/ml. B, DodS04-gel electrophoretic analysis of the described in the text. Activity is expressed as micromoles of Tos- time course of urokinase activation of SPg-a to plasmin by urokinase. ArgOMe cleaved per min per nmol of SPg-a originally added to the As described in A, except that the samples were not reduced prior to pa-stat vessel. electrophoresis.

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Sheep Plasminogen Activation 2067

IO 20 30 60 60 100

TIME (min.)

FIG. 3. Sensitivity of SPg-a to activation by various levels of streptokinase, and by a mixture of human plasminogen and strepto- kinase (human activator complex). The streptokinase sensitivity of SPg-a was tested by incubating SPg-a with streptokinase at molar ratios of SPg-a:streptokinase equal to 8.&l (A), 5:l (El), 2.6:1 (+), and I:1 (0). SPg-a was also incubated with a mixture of human plasmin- ogen atid streptokinase (human activator complex) at a molar ratio of SPg-a:activator complex equal to 17.4:1 (M). The time coarse of formation of plasmin activity was monitored by potentiometric tech- niques utilizing the synthetic substrate TosArgOMe. Plasmin activity is expressed as micromoles of TosArgOMe cleaved per min per nmol of SPg-a originally present. All incubations were performed at 30°C.

12 345678

FIG. 4. DodS04-gel analysis under reducing conditions of the in- cubation of SPg-a with streptokinase (SK). The time course of the reaction of SPg-a with streptokinase, at a molar ratio of SPg-a: streptokinase of 1:l (m/m) was analyzed by DodS04-gel electropho- resis. Gel 1, SPg-a control; Gel 2, streptokinase control. Incubation of the two components was carried out for times of: Gel 3, 1 min; Gel 4, 2 min; Gel 5, 5 min; Gel 6, 15 min; Gel 7, 30 min; and Gel 8, 120 min. The reaction was performed at 30°C. The nature of the streptokinase fragments (SK-I, SK-2, SK-3, SK-4, and SK-5) has been described in previous publications (18, 31, 32). Other abbreviations have been described in the text.

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 iden- tified 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 indi- cates 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 syn- thetic substrate, TosArgOMe. In all cases, urokinase activa- tions were performed at 30°C in 0.05 M Tris-HCl, 0.1 M L- lysine, 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

1 234567

El

SK-3

SK-4

FIG. 5. Plasminolysis of streptokinase (SK). The time course of reaction of streptokinase with urokinase-free SPm, at a molar ratio of streptokinase:SPm of 25:1, at 3O”C, was analyzed by DodSO1-gel electrophoresis under reducing conditions. Gel 1,60 min streptokinase control. The proteins were incubated for times of: Gel 2, 1 min; Gel 3, 2 min; Gel 4, 4 min; Gel 5, 8 min; Gel 6, 15 min; and Gel 7, 30 min. The nature of the streptolinase fragments is described in Fig. 4.

TABLE I

NHZ-terminal amino acid sequence analysis of form-2 spg-a, SPg- b, SPg-c, and P

Amino acid present in sequence no.

SPg-a SPg-b P SPg-c

1 ASP Ser Met 2 Leu Ile Ile ASP 3 Leu Tyr Tyr Ala 4 Asp Leu Leu Ser 5 Asp Ser Val 6 Tyr Glu Glu Pro 7 Val Ser 8 Asn LYS LYS Glu 9 Ile Ile Thr

10 GUY GUY GUY Pro

Quantitative NHS-termi- Asp 0.80 n.d.” n.d. Met 0.79 nal analysis (mol/mol sample)

a n.d., not determined.

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2068

FIG. 6. A, relative rate of activation of affinity form 1 SPg-a and SPg-b to plasmin by urokinase. Affinity chroma- tography form 1 SPg-a (O- - -0) and SPg-b (M) plus urokinase, as de- scribed 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 SPg- a and SPg-b to plasmin by urokinase. As described in A, except that affinity chro- matography form 2 plasminogens were used.

Sheep Plasminogen Activation

IO 20 30 40 50 60 70 SO

= 08 5 = 06 a

0.4

IO 20 30 40 50 60 70 60

TIME (min.) TIME (min.)

= 0.6 4

0.4

0.2

t AI IPI I IPI1

IO 20 30 40 50 60 70 60

TIME (min.)

FIG. 7. Relative rate of activation of SPg-c, P, and SPg-c + P to plasmin by urokinase. Shown are the urokinase-mediated activation rates of affinity chromatography form 2 SPg-c (H), P (A), and SPg-c + P (A.). Activities are expressed as micromoles of TosArgOMe cleaved per min per nmol of protein added to the pH-stat vessel. For comparison, the activation rate of SPg-a, under similar conditions (O- - -O), is also indicated.

large peptide. SPg-c was activated with urokinase until max- imum plasmin activity was reached. The plasmin produced was then separately mixed with a 3-fold molar excess of P and buffer alone. The results showed that little or no change in plasmin activity occurred.

DodSO1-gel electrophoresis under reducing conditions of the plasmins produced by partial activations of SPg-a, SPg-b, and SPg-c showed electrophoretically similar plasmins upon activation by urokinase, while urokinase does not affect the mobility of P.

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, SPg- c 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 prepa- rations, 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 ex- amined 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- tures. Similar results have been obtained for 1:l mixtures of SPg-b, and SPg-c with streptokinase. DodS04 electrophoretic analysis of this process reveals that in 1:l mixtures of SPg-a, SPg-b, and SPg-c with streptokinase, the streptokinase is rapidly degraded to fragments (SK-3 and SK-4) which are inactive, when formed in a complex with human plasmin, in

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 electrophoret- ically 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 plas- minogen 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 activa- tion 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 plas- minogen were performed in the presence of 0.1 M L-lysine.

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Sheep Plasminogen Activation

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 confor- mational 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 pro- duced 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 strepto- kinase. 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 appre- ciable amount of plasmin was formed (data not shown). SPg- c 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 plas- minogen 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 pres- ence 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

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FIG. 8. DodSOa-gel electronhoretograms produced under reducing - - _ conditions of the plasmins produced by the urokinase-mediated act; vation of SPe-a. Spa-b. and SPe-c. Gel 1. SPe-a: Gel 2. vartial activation of SPg-a; Gel 3, SPg-b;-Gel4, partial activation of SPg-b; Gel 5, SPg-c; Gel 6, partial activation of SPg-c. Also shown are P (Gel 7), abd P + urokinase (Gel 8). H and L refer to the heavy and light chains of SPm, respectively. Other abbreviations are as used previ- ously.

2.5

2.0

E LJ 1.5 i=

: 1.0

0.5

5 IO 15 TIME (min.)

FIG. 9. Activation of SPg-c by a mixture of streptokinase and human plasmin. Here, SPg-c was incubated with a 1:l m/m complex (preformed) of streptokinase and human plasmin. The molar ratio of SPg-c to the complex was 17.4:1. The time course of the formation of SPm activity was monitored by TosArgOMe hydrolysis, as described in the text. Activities are expressed as micromoles of TosArgOMe cleaved per min per nmol of SPg-c originally present.

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 com- plement system have dramatic biological activities. Each causes contraction of smooth muscle; release of histamine from mast cells; and directed, chemotactic migration of poly- morphonuclear 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.

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N F Paoni and F J Castellinonative and lower molecular weight forms of sheep plasminogen.

A comparison of the urokinase and streptokinase activation properties of the

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