TIXE JOURNAL OF BIOLOGICAI, CHEMISTRY Vol. 235, No. 6, June 1960

Printed in U.S. A.

On the Formation of Different Types of Plasmin

by Streptokinase Activation*?

GABOR MARKUS$ AND CLARA M. AMBRUS

From the Roswell Park Memorial Institute and the University of Bu$alo Medical and Graduate Schools,

Bufalo 3, New York

(Received for publication, August 24, 1959)

Plasminogen, a normal constituent of human blood plasma, can be activated in various ways to yield the proteolytic enzyme plasmin capable of dissolving fibrin clots and of hydrolyzing a variety of other proteins and synthetic amino acid esters. The activation by the bacterial product streptokinase has been widely studied in the past and appears to proceed through a two-stage reaction: the interaction of streptokinase with plasminogen (1) (or a hypothetical substance called proactivator) (2) yielding “activator,” which in turn converts plasminogen to plasmin. The conversion of plasminogen to plasmin by streptokinase has been reported to result in a decrease in molecular weight from 143,000 to 120,000 (3). The fibrinolytic and caseinolytic activi- ties of streptokinase-activated plasminogen have been invariably attributed to the end product of this reaction.

The studies reported here were designed to test the functional homogeneity of the product resulting from the addition of strep- tokinase to human plasminogen. The possibility that more than one enzyme is produced by streptokinase activation was suggested by our earlier studies (reported in abstracts, 4-6) which indicated that maximal fibrinolytic activities develop within one minute after the addition of streptokinase, before measurable amounts of peptides are released. This finding sug- gested that at least part of the hydrolytic activity of plasmin may be attributed to a molecular species which appears earlier in the course of activation than the one characterized by the loss of a peptide moiety.

The existence of several active components was demonstrated by the measurement of the rates at which activities toward different substrates develop and by the use of inhibitors. Ex- periments with physical separation methods are in progress.

MATERIALS AND METHODS

Human plasminogen was prepared from Cohn Fraction III by the method of Kline (7). After the final dialysis step the pH of the solution was adjusted to 5.5 and centrifuged at 9,000 r.p.m. in an SS-4 Servall centrifuge for 30 minutes. The super- natant fluid was found to contain about 50% of the total pro- tein. This fraction had no plasminogen or proactivator activity and most of it migrated toward the cathode during electrophore-

* Supported by grants from the National Instit.utes of Health, United States Public Health Service, the American Heart Associ- at,ion, Parke Davis and Company, and Merck-Sharpe and Dohme Company.

t A preliminary report was presented at the 1959 Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, New Jersey (6).

t: Established investigator of the American Heart Association.

sis on paper. A 5% solution at pH 4.0 was prepared by dissolv- ing the precipitate in dilute acid. This stock solution was kept frozen at -20” for several months without loss of activity. When examined as a 1% solution in 0.1 M Tris buffer in the ultracentrifuge (see below) it showed three peaks, with sedimen- tation constants of 1, 4, and 7. The fraction with the constant of 4 comprised 75% of the total material. Fresh solutions were prepared for each experiment at a final concentration of 1% protein in 0.1 M Tris buffer, pH 8.5. These solutions showed no turbidity. This preparation had some spontaneous activity as shown in the zero time activities on Figs. 1 and 2.

Xtreptokinase (Varidase-Lederle) was dissolved in 0.1 M Tris buffer to give the desired concentration. Some of the experi- ments were duplicated with purified streptokinase recently ob- tained from Merck, Sharp and Dohme Company.

Case& (Nutritional Biochemical Corporation) was purified according to the method of Mtillertz (8) and made to 3% in 0.1 M phosphate buffer, pH 7.4.

p-Toluene sulfonyl-z-arginine methyl ester HCl (Mann Research Laboratories, Inc.) was prepared for each experiment to give 0.03 M TAMeI in 0.36 M Tris buffer, pH 9.0.

Human fibrinogen was prepared from Cohn Fraction I by the method of Laki (9). In order to achieve the required purity with respect to the plasminogen contaminant (see below) the extraction procedure was repeated three times.

Thrombin was purified from bovine topical thrombin (Parke, Davis and Company) by stepwise alcohol precipitation. (A detailed account of this method will be published elsewhere.) The purity of both fibrinogen and thrombin with respect to plasminogen was estimated by forming clots and incorporating increasing amounts of streptokinase. The amount of strepto- kinase necessary to achieve the shortest lysis time depends on the amount of the plasminogen contamination in the clot. A clot prepared from the purified components used in this study lysed in 3 hours when incubated with 10 units of streptokinase. Lower or higher amounts of streptokinase gave either longer lysis times or did not affect the stability of the clot. A a-hour lysis time corresponds to a plasminogen concentration of less than 0.01 unit and is therefore negligible in comparison with the values (about 1 unit) encountered in this study.

Diisopropylphosphorojluoridate was obtained from K and K Laboratories, Inc.

Fibrinolysis-A modified version of the Loomis method was used (10). It consisted in the determination of the lysis time

1 The abbreviations used are: TAMe, p-toluene sulfonyl-n- arginine methyl ester; DFP, diisopropylphosphorofluoridate.

1673

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167-1 Different Types of Plasmin by Streptokinasc Activation Vol. 235, so. G

pair of samples was equivalent to the amount of TA\AIe hydro- lyzed in 5 minutes.

80

% OF MAX. ACT.

60

Streptokinase activation :in(l cawin and ‘l’,~~In hydrolysis wre performed in a water bath at 24“.

i 0.D 050

PEPTIDE RELEASE

0 . 4

.*

5 IO 15 20 25 30

TIME IN MINUTES AFTER SK ACT.

025

300

LZtraccntrifugal Anal&s-This was carried out with a Spinco model E ultraccntrifugc at 59,780 r.p.m. at 20” with the USC of the synthetic boundary ~11. Pictures wrc taken at S-minute intervals with a wire angle of GO”. The samples to be :malyzt?d rrere obtained in the following ~:ty: a so!ution of lo/o plasmino- gen in 0.1 M Tris buffer, pII 8.5, KBS activntcld nith 100 L of strcptokinasc per mg of plasminogcn. At specified timc,s l-ml samples were n-ithdran-n and mix-cd with 0.15 ml of 1 IV HCl which lowcrcd the p1-I to 2.5. A sxmple tak(,n before activation w-as trcatcd similarly. That this proccdurc stopped the actiya- tion process was shown by the fact that pcptidc material IT-hi& regularly accompanies activation was not rclrascd when aciclifi- cation was carried out immc~tliately after activation.

FIG. 1. Time course of development of protcolyt,ic and TAXle- ester&se activities after streptokinase (SK) activation. 1% plas- minogen in 0.1 M Tris buffer, pH 8.5, 21”. SK, 100 units per mg of plasminogen.

in a 45” water bath of a clot prepared by mixing 0.1 ml of puri- fied thrombin solution with 0.2 ml of the test solution and 0.3 ml of a O.GO10 purified fibrinogen solution. The lysis times indi- catcd by rise of the air bubbles trapped in the clot, wcrc converted to units of fibrinolytic activity on the basis of a calibration curve made by assigning 1 unit to a lysis time of 2 minutes.

Casein Assa!~-A modification of the method of 1\Iiillertz (8) was used. Aftcxr the addition of strcptokinase to a plasminogcn pool, 0.5-ml portions of this mixture were pip&ted at varying time intervals into two centrifuge tubes containing 0.5 ml of a 3y0 casein solution. Two milliliters of 1.7 31 perchloric acid Twre immcdiatcly added to one tube and 5 minutes later to the other enc. The precipitated solutions wrc centrifuged at 9,000 r.p.m. for 40 minutes, after \T+-hich the supcrnates wcrc pip&ted off and their optical dcnsitics measured in a Beckman model DU spectrophotometer at 275 rnp. The difference in optical density between each pair of san~ples indicated the breakdown of cnscin during the 5-minute digestion period. The series of samples v&i& lucre precipitated immcdintcly after the addition of plas- min also scrvcd as the basis for calculating the amount of pcptidc released from the plnsminogcn preparation during activation. Since prior interaction of cnsein and plasmin (or plasminogcn) seems ncccssnry for complctc precipitation of the enzyme in thcsc samples, pcrchloric acid was added last. The slight casein digestion occurring during the 5 seconds between the addition of plasmin and perchloric acid ITas corrected for by linear extrapolation to zero time of the line connecting the 5 second to the 5 minute optical density.

TdMel Assay-The proccdurc of Troll et al. (11) was modi- fied for this purpose. Into t\vo bcakcrs containing 1.5 ml of T-Me-Tris solution, (0.03 RI in T,K&, 0.36 M in Tris, pH 9.0) 0.5.ml samples of the nctivatcd plnsmin solution wcrc pipetted at varying times after activation. One of thcsc bcakcrs also contained 2 ml of 38y0 forn~nldel~ydc. To the other beaker 2 ml of t)his formaldehyde solution wrc added after a 5-minute digestion period. The solutions were then titrated to pH 8.0 \yith 0.1 K KaOII from a syringe-microburette with a Beckman pH meter. Tllc diffcrcncc in the amount of alkali used for ench

RESULTS

The cxpcrimcnts to be tltscribccl in this section wcrc designed to compare the rates at which hydrolytic activity toward three difftrent substrates dcvclops after the addition of strcptokinasc to plasminogcn.

Pibrinol~/sis-Fig. 1 show the rate of dcvclopmcnt of fibrino- lytic activity after the addition of 100 units of streptokinsse per mg of plasminogcn which ~vas found to be the optimal ratio for maximal plasminogen activation with the batch used in this study. It can be seen that the sample taken 30 seconds after the addition of strcptokinnse already showed maximal activity. The lysis time of all the samples taken from the activated plas- minogen pool was npprosimatcly 2 minutes. Since the samples taken at 1, 3, and 5 minutes showed no increase in activity, it is unlikely that the 2 minutes required for the assay influenced t.hcsc results; full activation must have taken place already at 30 seconds. The dcgrw of activity dcpcnded on the amount of enzyme and was not limited by the particular mak(x-up of the clot since increased cnz~-me concentrations rcsultcd in a shorter lysis time.

Case& 11~clroZ~~sis-The ability to digest cascin (Fig. 1) de- veloped as fast as the fibrinolytic activity when plasminogen KUS activated with 100 units of strcptokinasc per mg of plasminogcn at pH 8.5. Hcrc, a sample taken 15 seconds after the addition of strcptokinase already showd maximum activity. Again, the constamy of protcolytic activity cannot bc due to a limitation imposed by the substrate since the reaction was found to be first order in plasmin up to a concentration of 3c/,, using the same substrate concentrations. To test whcthcr the hydrolytic activities reflected the state of activation at the time the samples were taken, or activation continued during cascin digestion, the time course of the hydrolysis itself was dctcrmincd for several samples at different times after the addition of strcptokinasc (Fig. 2). Scvcral plasminogcn samples were activated with streptokinase, and at varying times after activation substrate lvas added to each sample. .\liquots of thcsc enzyme-substrate mixtures were withdrawn at l-minute intervals for a period of 5 minutes, immcdintcly prccil)itatctl, and analyzed for break- down products as dcscribcd above. Thcrc is no departure from linearity in any of the curves so obtnincd, and this indicates that the 5-minute values used in these cxpcrimcnts indcccl rc- fleeted the state of activation at the time of sampling. The slopes of the cascin curves do not differ significantly in the I-,

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June 1960 G. Markus and C. M. Ambrus 1675

ACTIVATION TIME

CASEIN TAMe

.400

.300

A 0.0 275 mlr

.200

.I00

0’

l

/ t’ --i-ST

5’

p

60

A#\-S 3.1 N NoOH

60

I’

0

;p

5’

0

ii

7

15’

/

DIGESTION TIME IN MINUTES FIG. 2. Rates of digestion of casein and TAMe in samples taken at indicated time intervals after activation with 100 U

streptokinase per mg of plasminogen. 1% plasminogen in 0.1 M Tris buffer, pH 8.5, 24”.

5-, and 15-minute samples as could be expected from Fig. 1. TABLE I

In plasmin samples with a slower rate of development of caseino- lytic activity (suboptimal streptokinase concentrations) an up-

Time course of development of streptokinase-induced caseinolytic and p-toluene sulfonyl-L-arginine methyl ester-esterase

ward curvature in the slopes of the casein digestion curves of activities

samples taken shortly after activation was observed. The absence of such an effect in the casein curves of Fig. 2 further

1% plasminogen in 0.1 M Tris buffer, pH 8.5. Activation at

supports the conclusion that the development of caseinolytic zero time with 100 units streptokinase per mg of plasminogen at 240.

activity has reached its maximum within 1 minute after the addition of streptokinase.

TAMe Hydrolysis-In contrast to these two proteolytic ac- tivities the ability to hydrolyze TAMe develops gradually, achieving its maximum level 10 to 15 minutes after the addition of streptokinase. Again, the individual hydrolysis rates are linear (Fig. 2). The relatively slow development of TAMe hydrolytic activity is expressed by the differences in the slopes of the subsequent lines. The constancy of the slopes of the individual lines also indicates that the addition of substrate in this case stops the process of activation.

The difference in the rate of development of caseinolytic and

Time

nzinb 0 -0.5 0.5-l 1 -3 3 -5 5 -8

Caseha I

TAMP

Experiment

1 2 3 4 5 6 1 8 9 10 --

@W an&T 94 92 64 77 89 32 31 20 30 19 26

100 98 76 91 61 46 60 61 46 55 98 97 92 100 97 78 72 77 88 65 76 90 100 100 97 87 81 77 81 92 93 98 97 100 94 94 96 91 95

TAMe-hydrolytic activities in 10 separate experiments done o Expressed as percentage of maximum streptokinase-induced under identical conditions is summarized in Table I. activity achieved during each 30-minute experiment.

The difference in the rate at which the proteolytic and TAMe- b Following addition of streptokinase.

hydrolytic activities develop after the addition of optimal strep- tokinase concentration suggests that two different enzymes, or streptokinase concentrations (Fig. 3), it became apparent that at least two different sites may develop under the influence of at lower than optimum streptokinase concentrations the devel- streptokinase: a proteolytic enzyme with a very high rate of opment of the caseinolytic activity itself consists of two distinct formation capable of hydrolyzing both fibrin and casein, and a phases: an initial rapid phase, such as has been described earlier, slowly developing esterase enzyme with the ability to hydrolyze followed by a much slower, linearly rising activity. An in- TAMe. The observation that the proteolytic activity develops teresting feature of this second phase was the decrease in slope faster than the esterase activity suggests that the first kind of associated with increasing streptokinase concentration (see “Dis- enzyme does not cleave TAMe, whereas the absence of an addi- cussion”). The development of TAMe-hydrolytic activity (Fig. tional rise in the proteolytic activity concomitant with the de- 3) did not resemble the second phase of the cas&nolytic activity, velopment of TAMe-hydrolytic ability suggests that the second since even the lowest streptokinase concentration resulted in enzyme may not have proteolytic activity. a plateau in 10 to 20 minutes, whereas the caseinolytic activity

On closer examination, however, even this interpretation was still rising at this time. Moreover, 10 units of streptokinase appears to be oversimplified. When an activation experiment, per mg of plasminogen produces essentially the same final ac- such as the one described above, was carried out at a series of tivity as 100 units. This again suggests that the ability to hy-

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1676 Diferent Types of Plasmin by Xtreptolcinase Activation Vol. 235, No. 6

a) CASEIN

100 U SK

50 U SK 25 ll SK

IO USK

TIME AFTER SK ACTIVATION

IN MINUTES FIG. 3. Time course of development of hydrolytic activities

toward casein and TAMe at different streptokinase (SK) concen- trations. 1% plasminogen in 0.1 M Tris buffer, pH 9.2, 24”.

ioo-

so-

PER CENT OF *‘- MAX. ACT.

70-

I MIN.

FIG. 4. pH dependence of caseinolytic activity at 1 minute and 10 minutes after activation in two separate experiments. 100 units streptokinase per mg of plasminogen, in 0.1 M Tris buffer, 24”. Circles refer to one set of data, triangles to the other.

drolyze TAMe is not due to the same site as the caseinolytic activity.

The two phases of the caseinolytic activity can further be demonstrated by carrying out the activation at different pH values. Fig. 4 shows the results of such an experiment. Sam- ples of 1% plasminogen were adjusted to pH values ranging

TABLE II

Effect of p-toluene suljonyl-L-arginine methyl ester concentration on e&erase activity of plasmin at two streptokinase concentrations

Streptokinase TAMe

units/mg +?2016T/l

100 2.86 X 1O-2 100 2.86 X 1O-3 100 2.86 X lo+

1000 2.86 X 1OF 1000 2.86 X lo-* 1000 2.86 X lo-’

Maximal activity

%

100 15 17 97 23 23

-

from 7.9 to 9.2 and were activated with 100 units of strepto- kinase per mg of plasminogen. Samples were tested for caseino- lytic activity at 1 and at 10 minutes after activation. A plot of these values against pH shows that the early activity proceeds optimally at pH 8.2, whereas the late activity has its optimum at pH 8.6.

Peptide Release-Fig. 1 shows the optical density of peptide material soluble in 1.13 M perchloric acid that has been released from plasminogen in the course of activation. Since the plas- minogen preparation is not pure it cannot be ascertained whether this material has been released from plasminogen itself as part of the activation process or is the hydrolytic product of a con- taminating protein broken down by plasmin. The amount of peptide material can be calculated to be about 1% of the total protein. This finding is in contrast to the large decrease in molecular weight reported by Shulman et al. (3) as quoted ear- lier. The possibility that larger fragments had also been liber- ated but were precipitated by the perchloric acid is made im- probable by the finding that the sedimentation patterns obtained at different stages during activation do not show changes com- patible with the postulated transformation2 (Fig. 5). Large changes in sedimentation patterns have been noted in this laboratory also, but these were found to be associated with decline of enzymatic activity rather than with its acquisition.

More significant for the process of activation, however, is the finding that the development of maximal fibrinolytic and casein- olytic activity occurs before any peptide material has been re- leased. This indicates that the enzyme formed first in the course of activation differs little in its molecular dimensions from its precursor, plasminogen. If the small amount of pep- tide released stems indeed from plasminogen, it is possible that this release accompanies the development of esterase activity and may mark the appearance of a second enzyme.

High Xtreptokinase Concentrations-Streptokinase at increas- ingly high concentrations becomes progressively less effective as an activator of the fibrinolytic property of plasmin (Fig. 6). This loss of effectiveness can be observed for the caseinolytic activity as well. The behavior of the TAMe hydrolytic activity, however, is quite different both at low and at high streptokinase concentrations. Maximal activity develops at lower strepto- kinase concentrations than does proteolytic activity and no sig- nificant decrease can be observed even at concentrations as high as 4,000 units of streptokinase per mg of protein. The fact that full esterase activity develops at high streptokinase levels strongly suggests that it is not due to the same enzymatic site

2 G. Markus, C. M. Ambrus, F. Wissler, and D. Woernley, in preparation.

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June 1960 G. Markus and C. M. Ambrus 1677

FIG. 5. Sedimentation runs on 8 different samples of plasminogen taken from the same pool at different times after activation with 100 units streptokinase per mg of plasminogen. (Times are indicated on each picture.) Pictures taken 43 minutes after at- tainment of full speed. For details see “Materials.”

as the proteolytic activity.3 It should be pointed out that the question whether the inhibitory substance is streptokinase itself or a contaminant in the Varidase preparation is irrelevant to this conclusion.

An alternative explanation favoring the existence of a single enzyme for both activities would be that TAMe has a much greater affinity for plasmin than does streptokinase and conse- quently displaces the inhibitor from plasminogen. This expla- nation is made improbable by the following observations: (a) At a constant TAMe concentration of 0.02 M, a 400-fold increase in streptokinase concentration failed to produce any decrease in the esterase activity of plasmin (Fig. 6). (b) A lo-fold and a loo-fold reduction in the TAMe concentration at a strepto- kinase level (1000 units per mg) which is strongly inhibitory for caseinolysis and fibrinolysis, did not produce any inhibition of the esterase activity. Table I shows the effect of three sub- strate concentrations at the optimum and at an inhibitory strep- tokinase concentration. The data show considerable decrease in activity at low substrate levels for both streptokinase con- centrations as was observed by Troll et al. (11) but this decrease was not greater in the high streptokinase group.

Finally, the possibility has to be considered that the inhibitory action of streptokinase may be due to shielding of the active site of plasmin by adsorbed streptokinase molecules. The re- sulting steric hindrance could interfere with the approach of the large protein substrates but might still permit access of the small TAMe molecule to the active site. Although such a steric hindrance could explain the effect of high streptokinase concen- trations, it cannot be invoked to account for the differences between esterase and proteolytic activities at low streptokinase concentrations thus necessitating a separate explanation for the latter effect.

Effect of DFP-DFP has been shown to be inhibitory for a number of enzymes. Its effect on plasmin has been studied by Mounter and Shipley (12) who obtained complete inhibition of casein hydrolysis 30 minutes after addition of 0.01 M DFP to streptokinase-activated plasmin (see Fig. 2 in (12); plasmin con-

3 Dr. Philip Norman, The Johns Hopkins University, informs us that he obtained a similar discrepancy in the effect of high streptokinase concentrations on the csseinolytic and TAMe hy- drolytic activities of plasmin.

PER CENT

o-1 IO 100 1000 l0,000

SK UNITS/mg PLASMINOGEN

FIG. 6. Proteolytic and esterase activities as a function of streptolrinase (SK) concentration measured 20 minutes after ac- tivation. (The fibrin curve was obtained with a plasminogen preparation of smaller specific activity than the one used in the other experiment,s.) 1% plasminogen in 0.1 M Tris buffer, pH 8.5, 24”.

DF.P ,n MOLES/L

FIG. 7. The inhibitory effect of DFP on casein and TAMe hydrolysis. 100 units streptokinase per mg of plasminogen in 0.1 M Tris buffer, pH 8.5, 24’.

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1678 Di’erent Types of Plasmin by Streptokinase Activation Vol. 235, h-o. 6

centration is not given). We were unable to obtain complete inhibition even with 0.02 M DFP. Fig. 7 shows the effect of increasing DFP concentrations, assayed after 30 minutes of incubation with streptokinase-activated plasmin, on casein and TAMe hydrolysis. The inhibition of casein hydrolysis obtained with 0.02 M DFP was 86% whereas that of TAMe hydrolysis was only 677,. Although this difference is not very large, it is significant and is indicative of heterogeneity in the substrate specificities of the enzymatic sites involved. Since the effect of DFP on other enzymes has been extremely difficult to reverse, the likelihood of different degrees of competition by the two substrates for the same active site is small.

DISCUSSION

The results reported in this paper provide a description of the complex phenomena encountered in the activation of plas- minogen by streptokinase. Proteolytic activity appears very rapidly following the addition of streptokinase to plasminogen. The development of this activity occurs before measurable amounts of peptide material had been released from the pre- cursor. At the same time a slower process is initiated giving rise to esterase activity. This process may be accompanied by the release of a small amount of pcptide material. It has been known for some time (2) that when human plasminogen is acti- vated with streptokinase there arises besides fibrinolytic activity also “activator activity,” i.e. the ability to convert further amounts of plasminogcn to plasmin. As mentioned earlier it was assumed that this activator activity is responsible for the formation of plasmin. The activator itself was considered to be a stoichiometric complex between either streptokinase and plasminogen (1) or streptokinase and a hypothetical substance called proactivator (2). It stems reasonable to suppose that the enzyme responsible for the rapidly appearing proteolytic activity found in our experiments might be identical with the activator. This supposition is strengthened by the observation of Troll and Sherry (13) who found that although plasmin can hydrolyze TAMe, activator-activity is not associated with TAMe-hydrolytic ability. This observation has been confirmed by Ablondi and Hagan (14). The early appearance of a pro- tcolytic factor, inactive toward TAMe, seems to be in agreement with these observations. The rapidity with which this compo- nent is formed in addition to the lack of concomitant peptide rclcase suggests that this component might well be the result of complex formation between strcptokinase and plasminogen. In carlicr studies with streptokinase and with urokinase (unpub- lishcd) it was found that lowering the temperature of activation considerably slowed the activation by urokinasc, almost certainly a protcolytic enzyme (15), whereas the activation by strepto- kinase was not measurably slomcd. The slowing encountered in the cast of urokinase is characteristic for enzymatic reactions while the lack of such an effect for the streptokinase activation suggests that this reaction may be a direct interaction between streptokinase and plasminogen. If such is the case, the mecha- nism of formation of the early component would be identical with that postulated for the formation of activator. This component characterized by its early appearance, its apparent proteolytic specificity and its optimal pH of formation at 8.2 will now be referred to as a-plasmin, bearing in mind its possible identity with activator.

The next events to bc considered in the course of activation arc the slow phase of the development of caseinolytic activity

and the development of estcrase activity. As shown in Fig. 3 the slowly developing caseinolytic activity is apparent only at suboptimal streptokinase concentrations, and its most remark- able feature is the decrease in its rate of development with in- increasing streptokinase concentrations. The component re- sponsible for this activity will be referred to as P-plasmin.

If ol-plasmin is indeed the activator then an increase in its concentration, as streptokinase is increased, should result in an increase, rather than a decrease, in the rate of P-plasmin forma- tion. Further studies on this complex phenomenon are in prog- ress.

Esterase activity develops considerably more slowly than ar-plasmin activity but its rate of development does not parallel that of fl-plasmin. The release of a small amount of peptide material seems to accompany this process. It is possible that the development of esteratic activity reflects the appearance of still another enzyme y-plasmin. The finding that high strep- tokinase concentrations selectively inhibit the development of proteolytic activity but not that of esterase activity also argues for the existence of the esterase enzyme as a separate entity.

Our results concerning the rates of development of proteolytic and esterase activity as well as the dissociability of these two functions by the use of inhibitors appears to be in disagreement with those obtained by Troll and Sherry (13) who observed parallel development of caseinolytic and ThMc-hydrolytic ac- tivities. Part of this disagreement can be accounted for by the differences in the treatment of the samples used for measure- ments of enzymatic activity. In the work quoted, the authors stopped the activation at several time intervals by lowering the pH to 2, reprecipitated the resulting sediment with 1 M NaCl and after solubilization carried out the assays on this material. This procedure is stated to remove both streptokinase and “activator” and is said to be essential for obtaining first order kinetics (13, 16). It seems reasonable to suppose that this procedure, although yielding a more homogeneous preparation, resulted in the removal of c-r-plasmin from the activation mix- ture and thus made impossible the observation of the early ap- pearing proteolytic activity. I f such was the case, the parallel rise of the two activities would argue for the identity of p-plas- min with the esterase enzyme. These contradictory results, however, are not strictly comparable, since activation was car- ried out in most of our studies at pH 8.5 and at 24”, whereas in the work referred to it was done at pH 7.6 at 37”. (These values are not stated in (13) and are only inferred from (16).) It is conceivable that at different pH values and temperatures streptokinase activation may yield different end products. An indication of this is indeed present in our study (Fig. 4).

Finally, it should be pointed out that the distinctions between the separate enzyme forms tentativclg established in this study do not necessarily imply that each form is associated with a different molecular species. It is possible that some of these activities are due to different enzymatic sites on the same mole- cule or, that they reflect subsequent stages in the evolution of a single molecule, as has been described for chymotrypsin by Neurath et al. (17).

In previous studies from this laboratory, the thcrapcutic use- fulncss of various preparations of plasmin was tested on dogs with IWabeled fibrin clots and patients suffering from various thrombo-embolic disorders (18, 19). When strcptokinase was added to human plasminogen, apparently several enzymes were formed and during the treatment period a constantly varying

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enzyme population was infused. Experiments are in progress to isolate the various types of plasmin and, if successful, to test them for therapeutic activity.

SUMMARY

The time course of activation of human plasminogen by strep- tokinase at pH 8.5, 24” has been studied by measurement of the rates at which activities toward different substrates develop. At optimal streptokinase concentrations proteolytic activity toward fibrin and casein develops to its final value within 1 minute after the addition of streptokinase. Esterase activity toward p-toluene sulfonyl-L-arginine methyl ester develops con- siderably more slowly, reaching its peak at 5 to 15 minutes after activation. The latter process is accompanied by the release of a small amount of peptide material. By the use of suboptimal streptokinase concentrations it can be shown that the caseinolytic activity itself consists of two distinct phases: a fast process al- ready mentioned and a subsequent linearly rising slower process. The development of esterase activity does not parallel the course of either one of the proteolytic activities. The two phases of the proteolytic activity can further be distinguished by their different pH optima. These two phases are attributed to the action of two distinct enzyme forms (or enzymatic sites) develop- ing at different rates which are referred to as CZ- and @-plasmin, respectively. The esterase activity appears to be associated with a third enzyme form, y-plasmin. The use of inhibitors permits further differentiation between the proteolytic and the esterase activities. Increasing the streptokinase concentration above the optimum progressively inhibits the development of pro- teolytic but does not affect that of esterase activity. Diisopro- pylphosphofluoridate inhibits the two activities to different extents.

The relationship of a-plasmin to “activator” is considered.

Acknowledgments-The authors are indebted to Rudolf Hans& MS., James Freeman, B.A., and Rosalie Benson, B.S., for tech- nical assistance, and to Drs. W. C. Werkheiser and A. Nisonoff for many stimulating discussions.

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REFERENCES

KLINE, D. L., AND FISHMAN, J. B., Ann. N. Y. Acad. Sci., 68, 25 (1957).

M~LLERTZ, S., AND LASSEN, M., Proc. Sot. Exptl. Biol. Med., 82, 265 (1953).

SHULMAN, S., ALKJAERSIG, N., AND SI~ERRY, S., J. Biol. Chem., 233, 86 (1958).

MARKUS, G.. AMBRUS. C. M., WISSLER, F., ARTD WOERNLEY, D. L.,‘Federation Pioc., 17, 104 (1958).

MARKUS. G.. AMBRUS. C. M.. WISSLER. F.. AND WOERNLEY. D. L.,‘Ab&acts of ihe IVth Internatronai congress of Bio: chemistry, Pergamon Press, London, 1958. p. 164.

MARKUS, G., AND AMBRUS, C. M., Federation Proc., 18, 98 (1959).

KLINE, D. L., J. Biol. Chem., 204,949 (1953). M~~LLERTZ, S., Biochem. J., 61, 424 (1955). LAKI, K., Arch. Biochem., 32, 317 (1951). LOOMIS, E. C., GEORGE, C., AXD RYDER, A.? Arch Biochem.,

12, 1.(1947).. TROLL. W.. SHERRY. S.. AND WACHMAN. J., J. Biol. Chem.,

208, 85 (i954). ’ ’ MOUNTER, L. A., AND SHIPLEY, B. A., J. Biol. Chem., 231, 855

(1958). TROLL, W., AND SHERRY, S., J. Biol. Chem., 213, 881 (1955). ABI,ONDI, F. B., AND HAGAN, J., Proc. Sot. Exptl. Biol. Med.,

96, 195 (1957). KJELDGAARD, N. O., AND PLOUG, J., Biochim. et Biophys. Acta,

24, 283 (1957). 16. ALKJAERSIG. N.. FLETCHER. A. P.. AND SHERRY. S.. J. Biol.

Chem., 23$, 8i (1958). ’ ’ I I

17. NEURATH, H., AND DIXON, G. H., Federation Proc., 16, 791 (1957).

18. AMBRUS, J. L., AMBRUS, C. M., BACK, N., SOKAL, J. E., COL- LINS, G. L., Ann. Ai. Y. Acad. Sci., 68, 97 (1957).

19. SOKAL, J. E., AMBRUS, J. L., AMBRUS, C. M., J. Am. Med. It is suggested that they may be identical. Assoc., 168, 1314 (1958).

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Gabor Markus and Clara M. AmbrusOn the Formation of Different Types of Plasmin by Streptokinase Activation

1960, 235:1673-1679.J. Biol. Chem. 

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