Complex formation of human Val354-plasminogen with streptokinase

THROMBOSIS RESEARCH 30; 377-382, 0049-3848/83/100377-06$03.00/O Copyright

(c)

1983

COMPLEX

Pergamon

1983 Printed

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in

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Ltd.

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VAL354-PLASMINOGEN

WITH STREPTOKINASE

James Department

(Received

21.1.1983;

R. Powell

and Francis

3. Castellino

of Chemistry, University of Notre Notre Dame, Indiana 46556 U.S.A.

Accepted

in revised

form

21.2.1983

Dame,

by Editor

K.M. Mann)

ABSTRACT A controlled digestion of native human plasminogen (GluI-plasminogen) from either carbohydrate variant 1 or 2 with pancreatic elastase yields, among others, an activatable fragment, designated Va1354-plasminogen (Pgc). This species represents the smallest human plasminogen fragment (ca., 50,000 Mw) known to bind to Sepharose-lysine. Pgc forms a tight equimolar complex with streptokinase, as determined by sucrose density ultracentrifugation, whether formed from Va1354-plasminogen or Va1354plasmin. The Val 354-plasmin-streptokinase complex readily activates ovine plasminogen, which is normally insensitive to streptokinase, and this complex is essentially inactive toward casein, similar to the equimolar complexes formed with Lys 77-plasmin and the elastase fragment, Val 442-plasmin.

INTRODUCTION Streptokinase (SK) is a catabolic by-product of B-hemolytic streptococcus, which is known to activate several species of plasminogen (11, including human plasminogen, by an indirect mechanism, involving formation of the plasminogen activator, identified as the SK-human plasmin equimolar complex (for a review see reference 2). Various activatable low molecular weight forms of human plasminogen have been generated by limited proteolytic digestion of the zymogen. One form, termed Val 442-plasminogen, has been isolated, which lacks amino terminal residues 1-441 of native plasminogen (GluI-plasminogen) (3). Another form, Va1354plasminogen, has been purified, which lacks amino terminal residues l-353 of C&Iplasminogen (4). The availability of these two forms of plasminogen allows the assessment of the role of the amino terminal regions of plasminogen in the formation of the SK-plasmin equimolar complex and the ability of the complex, if formed, to serve as a plasminogen activator. Key words: Human plasminogen fragments, activation, streptokinase, plasmin formation,

streptokinase complexes, plasmin activity.

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PIasminogen

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PLASMINOCEN ACTIVATION

MATERIALS

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AND METHODS

Proteins Human Clul-plasminogen was prepared by Sepharose-lysine affinity chromatography with gradient elution to resolve the carbohydrate variants I and 2 (5). The human plasminogen fragments, Va1442-plasminogen and Val 354-plasminogen were prepared by controlled elastase-digestion of Clul-plasminogen, as previously outlined (3, 4), from carbohydrate variant 2. Ovine plasminogen was purified from whole titrated sheep plasma (6). Native streptokinase was purified from kabikinase (AB Kabi) as detailed by Castellino et al. (7), and pancreatic trypsin inhibitor (Trasylol) was obtained from FBA Pharmaceuticals. Sucrose

Density

Ultracentrifugation

Linear gradients of 5-20% sucrose (w/v) in 50 mM L-lysine, 0.02% sodium azide pH 8.0 (Tris/lysine buffer), at room temperature, were formed in 5.0 ml nitrocellulose tubes by a gradient apparatus previously described (8), and cooled to 4O. Six gradients were layered with 100 ~.rl each of the following incubations, all in Tris/lysine buffer: 4-plasminogen (52 /J M); cell 2, 100 yl SK (52 cc M); cell 3, 50 ~1 Zlll3&‘p”,9 ‘;d V,flSR, 37” for 2 minutes, then incubated with 10 ~1 Trasylol (520 PM) for 2 minutes; cell 4, 50 p 1 Val354-plasminogen, 10 11 Trasylol, then incubated with 50 1.r1SK at 37” for 2 minutes; cell 5, 100 ~1 Val354-plasminogen, 2 ~1 SK, 37” for 30 minutes, then incubated with 20 1.11Trasylol for 2 minutes; cell 6, 50 p I Va135 -Pg, I /Al SK 37” for 30 minutes, then incubated with 50 ~1 SK for 2 minutes, followe 2 by 10 1.r1Trasylol for 2 minutes. All generated species were cooled to 4O. The six gradients were subjected simultaneously to centrifugation in a Beckman LS-65 ultracentrifuge, employing a 50.1 SW swinging bucket rotor at 4O and 38,000 rpm for 12 hours. After sedimentation, each tube was drained from the bottom and about thirty fractions (ca., 0.15 ml) were collected. Each fraction was diluted with 0.5 ml Tris/lysine buffer and the absorbance obtained at 280 nm. Protein pools were dialyzed against 10 mM benzamidine hydrochloride at 4” , lyophilized and subjected to electrophoresis on reduced sodium dodecylsulfate (SDS) gels. Activator

Activity

and Caseinolysis

Ovine plasminogen variant 2 was dissolved in 50 mM TrisHCl/lOO mM NaCl, pH 7.4, at 37”, at a concentration of 14 PM. Urokinase-activated and glycerol-stabilized human plasmin stock solutions, prepared an outlined previously (9), for Lys77-plasmin, Val 54-plasmin and Vall14 -p lasmin, at concentrations of 3.0 PM, or plasmm-SK equi2M were incubated with ovine plasmin at 37” in a ratio of ma far complexes at 1.5 Cc The activation was followed by removal 1 l.c1:250 ~1 and 2 /~I:250 yl, respectively. of 10 ~1 of the incubation mixture at various times and addition to 0.79 ml of 0.75 mM S-2251 plasmin substrate (AB Kabi), in 50 mM Tris*HCI/IOO mM NaCl, pH 7.4 at 37O. The absorbance change at 410 nm was recorded and converted to units of substrate hydrolyzed per unit time. complexes was monitored Casein hydrolysis by the same plasmin and SK-plasmin by incubating 0.35 ml of 2% (w/v) azocasein (Miles Laboratories) in 50 .mM Tris.HCI/ 100 mM NaCl, pH 7.4, at 37”, with 0.15 ml of the various plasmins (3.0 MM), alone, and to which was added 0.025 ml of SK (20 PM) or protein buffer. At 20 minutes and 40 minutes, 0.25 ml of the digest was precipitated in 0.5 ml of cold 5% trichloroacetic acid and subjected to low-speed centrifugation to remove the precipitate. A volume of 0.5 ml of the supernatant was neutralized with 0.5 ml of 225 mM sodium hydroxide, and the absorbance at 410 nm was determined against a blank of azocasein carried through the assay.

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RESULTS Ultracentrifugation patterns of the various SK complexes with Val3 4-plasminogen are shown in Figure 1. Cells I and 2, respectively, s l?ow sedimentaand Val 354-plasmin tion behavior for Pgc (ca., 50,000 MW) and native streptokinase (ca., 45,000 MW) for comparison. Cell 3 illustrates the results of allowing a I:1 molecular complex to develop for 2 minutes at 37”, followed by a two-fold molar excess of Trasylol to prevent further autolysis. The more rapid sedimentation of peak 3 clearly demonstrates complex formation. The SDS-gel inset shows the presence of the slightly degraded SK species (designated SK*), as well as the plasmin light chain (LC) and remaining 4 plasmin (HC). These data demonstrate that the complex repreZZy~~~o%v~~Va1354-plasmin . In cell 4, the equimolar complex was allowed to form in the presence of a two-fold molar excess of Trasylol to prevent any peptide bond cleavages. The rather broad sedimentation profile of peak 4 indicated that some degree of aggregation occurred for this species, which may exist in a monomeroligomer equilibrium. The gel inset demonstrates the presence of intact Va1354plasminogen and native SK, indicating the complex to be SK-Va1354-plasminogen. Cell 5 represents the sedimentation of Val354-plasmin alone. Again, the plasmin light chain and the remaining Val plasmin heavy chain are shown by the gel inset. Finally, 354in cell 6, a molar equiva ent of SK was added to preactivated Val354-plasmin. The complex as in cell 3. material exists as the same equimolar Figure 2A depicts the activation of ovine plasminogen, normally insensitive to activation by streptokinase (lo), by a catalytic level of a preformed SK*Lys77-human plasmin complex, a preformed SKmVal254- human plasmin complex, and a preformed SK+Val 442-human plasmin complex. t a 14 PM concentration of ovine plasminogen the activator activity of the human activator complexes towards generation of ovine plasmin activity to the substrate, S-2251, proceed nearly at the same rate, in each case. By comparison the same plasmins not complexed to SK are essentially inactive to ovine plasminogen activation. In the case of hydrolytic activity towards a move generalized protein substrate, shown in Figure 2B, for the substrate azocasein, the streptokinase-complexed plasmins are, in contrast to ovine plasminogen activator activity, nearly totally inactive against this protein substrate. However, the uncomplexed plasmins, lacking in specific activator properties to ovine plasminogen, readily hydrolyze the azocasein substrate. DISCUSSION Previous studies from this and other laboratories have shown that SK will form equimolar complexes with human Glul-, Lys77-, and Val 42 plasminogen, as well as Clul-, Lys77-, and Val442-plasmin. We currently show t‘hat - these same complexes form with Val 354-plasminogen and Va1354-plasmin. This latter low molecular weight plasminogen represents molecules lacking the strong and most of the weak lysine binding sites, that are characteristic of Glul-plasminogen and Lys77-plasminogen (3, 4). The only obvious differences noted in this study and previous studies employing higher molecular weight forms of human plasminogen are that the SK*Va1354-plasminogen equimolar complex undergoes association to higher molecular weight forms. Considering all studies in the literature, it would appear as though the latent or actual heavy chain of human plasmin(ogen), and its attendant lysine binding sites, plays a minor rolein the ability of human plasminogen to generate stable equimolar complexes with SK. The ovine plasminogen activator activity and casineolytic activity of the various human plasmins and SK-plasmin complexes display no significant differences among members of each respective class. The uncomplexed plasmins will not serve as plasminogen activators, although they possess general proteolytic activity, and the plasmins

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PLASMINOGEN ACTIVATION

FRACTION

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FIG. I Sucrose density ultracentrifugation of Val -plasminogen (Pgc) and Va1354plasmin (Pmc) and their equimolar strepto %j4 mase (SK) complexes. The contents of each cell have been listed in Materials and Methods. The numbered peaks correspond to the SDS gel insets. The direction of sedimentation is to the left and the dotted line represents the sucrose concentration.

5

IO t

15 ( minutes

20

40

1

FIG. 2 A. Time course activation of ovine plasminogen variant 2 by equimolar SK complexes of Lys-,l-plasmin (Pmb), ~al~~4-~lasmin (Pmc) pnd Va144J-plasmin of azoc sem by (Pmd) (-), and u complexed plasmms --- . B. Hydrolysis the above enzymes, in the presence (--) of equimolar SK and in the absence (---) of SK.

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in complex with SK will yield plasminogen activator activity, but will not serve as general proteases. This conclusion has been revealed earlier upon study of the activity of the equimolar SKmLys77-plasmin complex (II). From the current data, it can be further concluded that the plasmin heavy chain is not essential for expression of activator activity in the SK-plasmin equimolar complex, at least in the in vitro systems analyzed. This observation is consistent with the finding of Summaria and Robbins (IZ), who showed that the isolated plasmin light chain could serve as plasminogen activator in equimolar complex with SK. Further, it can be concluded that the human plasminogen degraded in vivo to the low molecular weight forms can still be potentially activated to plasmiT aleast in the fluid phase of plasma. ACKNOWLEDGEMENTS These of Health.

studies

were

supported

by grant

HL-13423

from

the National

Institutes

REFERENCES 1.

WULF, R. J. and MERTZ, E. T. Studies on plasminogen. VIII. Species city of streptokinase. Can. J. Biochem.,s, 927-932, 1969.

2.

CASTELLINO, F. J. plasmin/streptokinase

3.

SOTTRUP-JENSEN, L., CLAEYS, H., ZAJDEL, M., PETERSEN, T. E. and MAGNUSSON, S. The primary structure of human plasminogen: isolation of two lysine-binding fragments and one “mini”-plasminogen (MW, 38,000) by eiastase-catalyzed-specif ic limited proteolysis. In: Progress in Chemical J. F. Davidson, EM. Rowan, M. M. Samama Fibrinolysis and Thrombolysis. and P. C. Desnoyers, eds. New York: Raven Press, pp. 191-209, 1978.

4.

POWELL, 3. R. and CASTELLINO, F. J. Isolation of human Val354-plasminogen as an elastolytic fragment of human GluI-plasminogen. Biochem. Biophys. Res. Commun., 102, 46-52, 1981.

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BROCKWAY, antifibrinolytic 151, 192-199,

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PAONI, N. F., VIOLAND, B. N. and CASTELLINO, F. J. Isolation and characterization of native and lower molecular weight forms of sheep plasminogen. -J. Biol. Chem., 252, 7725-7732, 1977. CASTELLINO, Streptokinase.

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F. J. Measurement of the binding of plasminogens. Arch. Biochem. Biophys.,

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POWELL, J. R. and CASTELLINO, F. J. Activation of human neoplasminogenVal442by urokinase and streptokinase and a kinetic characterization of neoplasmin-Va1442. J. Biol. Chem., 255, 5329-5335, 1980. MORRIS, J. P., BLATT, S., POWELL, J. R., STRICKLAND, D. K. and CASTELLINO, F. J. Role of Iysine binding regions in the kinetic properties of human plasmin. Biochemistry, 20, 4811-4816, 1981.

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10. PAONI, M. F. and CASTELLINO, F. J. A comparison of the urokinase and streptokinase activation properties of the native and lower molecular weight forms of sheep plasminogen. 3. Biol. Chem., 254, 2064-2070, 1979. 11. WERKHEISER, plasminogen. 12.

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of streptokinase

with

SUMMARIA, L. and ROBBINS, K. C. Isolation of a human plasmin-derived functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activating ability. 3. Biol. Chem., 25J, 5810-5813 (1976).