THE JOUHNAL OF I~IOLOGIC~L Cmni~sr~~u Vol. 24R, So. 14, Issue of #July 25, pp. 4F41-4G-17, 1071

Printed in U.S.A.

The Mechanism of the Inhibition of Plasmin Activity

by E-Aminocaproic Acid*

(Received for publication, December 21, 1970)

WILLIAM J. BROCKWAY AND FRANCIS J. CASTELLINO~

From The Department of Chemistry, Program in Biochemistry and Biophysics, The University of Notre Dame, Notre Dame, Indiana 46556

SUMMARY

The streptokinase-induced conversion of human plasmino- gen to plasmin is inhibited by concentrations of e-amino- caproic acid which stimulates the esterolytic activity of plasmin on the synthetic substrate, tosyl-L-arginine methyl ester. The inhibitory effect of e-aminocaproic acid decreases as its concentration is decreased and is eliminated upon in- cubation of plasminogen with streptokinase, regardless of the presence of e-aminocaproic acid. At increased levels, e-aminocaproic acid further functions as a competitive in- hibitor of plasmin activity on tosyl-L-arginine methyl ester with a K1 of 0.32 M. In the presence of concentrations of c-aminocaproic acid (0.05 M) sufficient to nearly saturate its inhibitory effect on the plasminogen to plasmin conversion, the sedimentation coefficient (S%,u) of plasminogen decreases from a native value of 5.0 GX 0.1 S to 3.8 f 0.1 S without a decrease in molecular weight suggesting a gross conforma- tional change in plasminogen induced by c-aminocaproic acid. This conformational alteration is also evidenced in circular dichroism measurements. The effect of c-amino- caproic acid on the conformation of plasminogen is readily reversible and restoration of the native structure is apparent after dialysis.

A mechanism for the inhibition of the plasminogen to plasmin conversion by e-aminocaproic acid is postulated in- volving the formation of a plasminogen-e-aminocaproic acid complex, which due to the altered conformation of plasmino- gen, is not acted upon by streptokinase. This inactive com- plex is rapidly reversible, yielding a fully streptokinase re- active plasminogen.

Plasmin (EC 3.4.4.14), a proteolytic enzyme which hy- drolyzes fibrin clots is formed from llla.sminogen,l its inactive plasma protein precursor. Specific activators isolated from sev-

* These studies were supported by a grant-in-aid from the Indiana Heart Association and Grant HE-13423 from the Na- tional Institute of Health.

f To whom inquiries should be addressed. 1 In this report plasminogen refers to human plasminogen.

When plasminogens from other species are ment.ioned their sources will be specified.

era1 sources are known to induce plasmin formation from plas- minogen. These activators can be isolated from either bac- terial sources (streptokinase), or human origin (urokinase and plasma activator), and are also present in a variety of animal tissues (pig heart activator).

Robbins and coworkers have demonstrated that the activation of human plasminogen to plasmin by urokinase or trace amounts of streptokinase takes place by the urokinase- or streptokinase- induced cleavage of a single arginyl-valine bond in the plasmino- gen molecule (1, 2). Human plasminogen consists of a single ltolypeptide chain and activation of the molecule to l)lasmill results in a two chain structure stabilized by disulfide bridges (1, 2). The two chains have molecular weights of 25,700 and 57,200 daltons and are called the light, and heavy chains, respectively

(3). Several investigators have noted that compounds such as

c-aminocaproic acid and p-aminomethylbenzoic acid are potent antifibrinolytic agents. Although there are numerous papers published on these compounds, the mechanism of their anti- fibrinolytic activity is not clear. Alkjaersig, Fletcher, and Sherry (4) reported that eAcp2 acts as an inhibitor of the plas- minogen-plasmin conversion, thus manifesting its antifibrinolytic activity. Other theories have been presented such as ~Acp acting as an antiplasmin (5), and papers reporting that tAcp was antifibrinolytic due to its inducing a conformational altera- tion in the structure of the substrate, fibrin, have been published (6, 7). More recently, it has been demonstrated that ~Acp has no effect on the activation of human plasminogen (8).

Due to these apparent inconsistencies, we have undertaken a study of the mechanism of the inhibition of plasmin activity 1)~ ehcp. Our results conclusively demonstrate that this inhibitiol~ is complex and the mechanism proposed involves both inhibition at the level of the conversion of plasminogen to plasmin and the inhibition of the proteolytic activity of plasmin.

EXPERIMENTAL PROCEDURE

Materials

PurQkation of Plamlinogen-Human plasminogen was pre- pared in one step from Cohn III, prepared from age outdated c&rated human plasma, by an affinity chromatography technique utilizing L-lysine bound to Sepharose 4B (Pharmacia) to selec-

2 The abbreviations used are: ~Acp, c-aminocaproic acid; tosyl- AMe, N-c+tosyl-L-arginine methyl ester.

4641

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

4642 Inhibition of Plasmin Activity Vol. 246, No. 14

Volume (ml1

FIG. 1. Purification of human plasminogcn by affinity chroma- tography on Sepharose-L-lysine. The column (1.1 X 6 cm) was equilibrated with 0.3 M phosphate buffer, pH 7.5. Approximately 50 ml of Cohn III extract were applied and the column was eluted with the same buffer. When no further absorbance at 280 nm was obtained, 0.1 M phosphate buffer-O.2 M EACI). KIH 7..5. was added. The peak obtained after this addition &l poolii and found to contain human plasminogen. E-ACA, E-aminocaproic acid.

tively retard the plnsminogen (9). Our Sepharose-L-lysine

columns were prepared by suspending 100 ml of Scpharose 4B in water and adding IO g of cyanogen bromide (Eastman). The

p1-I was adjusted to and maintained at pR 11 by repeated

additions of 4 N sodium hydroxide. After approximately 20 mill of reaction the activated Scpharose 4B was washed with 2 liters of cold 0.1 M sodium bicarbonate on a Buchner funnel. The resin was then suspended in 0.1 M phosphate buffer, pH 9.0, for the coupling reaction.

In order to couple L-lysine to the activated Sepharose, 100 ml of a solution cont,aining 15 mmoles of L-lysine in 0.1 M 1)hosphate buffer, p1-I 9.0, were added to 100 ml of the cyanogen bromide-

activated Sepharose 4B. The solution was allowed to react overnight at 4” with gentle stirring. The solution was then filterrd and the gel suspended in a buffer of 0.1 RI phosphate, pH

7.5. 1Tnder these conditions approximately 25 pmoles of L-lysine were coupled per ml of gel.

In order to purify plasminogen from human plasma using this technique, 5 ml of Sepharose-L-lysine were packed into an ll-mm (diameter) column and equilibrated with 0.3 M phosphate buffer, pH 7.5. Approximately 50 ml of Cohn III extract were passed through the column and eluted with 0.3 M phosllhate buffer, pH 7.5, until a steady base-line, indicating no further absorbance, was obtained. At this point, a solution of 0.1 M

phosphate buffer-O.2 nr ~Acp, $1 7.5, was percolated through the column and a sharp peak was immediately obtained. The yield of plasminogen is 85% under these conditions. No de- t,ectable plasmin activity was found in this plasminogen prepa- ration. Fig. 1 shows a typical elution profile.

Other Proteins-Streptokinasc (Varidase) was obtained from Lederle Laboratories through a local drug outlet in vials contain- iug 20,000 units of activity.

Reagents--EAcp, was purchased from Calbiochem and tosyl- AlIe was purchased from Cycle Chemical Company. All other reagent)s were the best commercially available.

Methods

PZa,smin Assays-Since these assays were done under a variety of conditions they are described in appropriate sections of the manuscript. All assay components were prepared in 0.1 RI

Tris.hydrochloride, 1~11 8.0, and all assays were performed at 30”.

In general, the assays consisted of converting plasminogen to plasmin with streptokinase and following the action of plasmin

on tosyl-AMe. Analysis of the amounts of tosyl-AMe cleaved by plasmin were performed essentially as described by Hestrin (10) with the following minor modifications. ilfter a given time of reaction a 0.2.ml aliquot of the reaction mixture was added to a solution containing 0.2 ml of 4 N sodium hydroxide aud 0.2 ml of 2 N hydroxylamine hydrochloride. These conditions were suff- cient to immediately stop the enzymatic reaction. The reaction was allowed to proceed for 30 min, and 0.2 ml of a solution of 4 N hydrochloric acid containing 6 g of trichloroacetic acid was added followed by addition of 0.2 ml of water. Following t,his, 4 ml of a solution containing 0.11 JI ferric chloride in 0.004 M hydrochloric acid were added, and the absorbances of these solutions lvere determined on a Gilford model 240 spectrophotomct’er at 525 nm against an appropriate blank. This Iprocedure allowed ~1s to de- termine the final concentration of tosyl-AMe from a standard curve. Initial concentrations of tosyl-AMe were obtained in the

same fashion by preparing incubation mixtures in the absence of any enzymes. The rate of reaction of plasmin &h tosyl-AMe was shown to be linear at the times of incubat’ion used in these studies.

Ultracentrijuge Studies~ -Sedimentation coefficients of plas- minogen in 0.1 M l)hosph:rte, $1 7.5, and plasminogen in 0.1 M phosl)hate-0.05 M ~Acp, pII 7.5, were measured in a Spinco model E analytical ultraccntrifuge using absorption optics at 280 nm. Protein concentrations were approximately 0.2 mg per ml. Sedimentation coefficient’s were calculated in the usual manner and corrected to the density and viscosity of water at 20” (11).

Circular Dichroism &u&es-These were performed lvith a Cary 60 spectropolarimeter circular dichroism apparatus using I-, 5-, and lo-mm cells. These cells were interchanged during a run so that the optical density of the protein did not exceed 1.0 at any wave length. The ellipticity [0] values were recorded directly from the instrument and converted to molecular ellipticity [0] expressed in degree cm2 per dmole of amino acid according to the relationship

[el = g

where Ma is the mean residue weight of the protein, 1, the path length in the sample solution in centimeters, and C is the protein concentration in grams per ml.

Polyacrylamide Gel Electrophoresis--These experiments n-ere performed at $19.5 (12), pl-I 4.3 (13), pH 3.2 in 6.25 M urea (13), and in sodium dodecyl sulfate (14).

I1ESULTS

Characterization of Plasminogen-The plasminogen isolated b? the affinity chromatography technique indicated multiple bands when examined by polyacrylamide gel electrophoresis at pH 9.5, 4.3, or 3.2 in 6.25 M urea. However, all bands exhibited plasmin activity upon addition of streptokinase as demonstrated in gel slicing experiments. These results are consistent with the obser-

vations that human plasminogen consists of multiple molecular forms (4, 9, 15). In agreement with these facts, plasminogen gave only one band when examined by polyacrylamide gel elec- trophoresis in sodium dodecyl sulfate. The molecular weight of

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

Issue of July 25, 1971 W. J. Brockway and F. J. Castellino 4643

plasminogen calculated by this technique was 82,000, a value in

agreement with previously published values (I). Moreover,

pl~;sminogen exhibited a boundary indicating that a single 1)rotein

was present when examined by sedimentation velocity in the ul-

traceutrifuge. Thus, there is no evident heterogeneity in size of

the plusminogen preparations, and the heterogeneity in charge

obtained on gel electrophorcsis C:LIL be explained by the multiple

molecular form hypothesis.

Fig. 2 presents Lineweaver-Burke plots from which the I<,

and V,,, values for plasmin, obtained from streptokinase activa-

tion of plasminogen, on tosyl-A,lle were determined. The Zi,

was found to be 0.0065 M and t’he 1 ,,lax was 12.1 pmoles of tosyl-

AMe cleaved per min per mg. This is the highest specific activ-

ity reported for plasmin to this timo and reflects the high quality

of the plasminogen prepa&ion.

Inhibition of Conversion of Plaminogen to Plasmin by eAcp in Absence of Prior Incubation of Plasminogen with Streptokinase- Fig. 3 shows the inhibitory effect of &cp on the activation of

plasminogen to plasmin. In these experiments prior incubation

of plasminogen with streptokinasc n-as omitted. The data for

the curve in the absence of &cl) in Fig. 3 was obtained by incu-

bating the assay components’in the following order: 0.1 ml of

plasminogen (26 pg), 0.025 ml of tosy1-Alle (100 bmoles per ml),

0.115 ml of 0.1 M Tris,TICl, pH 8.0. The reaction was initiated

by the addition of 0.01 ml of strcptokinase containing 10 to 400

units/O.01 ml. The reaction was allowed to proceed for 10 min

and the concentration of tosyl-AMe in a 0.2-ml aliquot was detcr-

mined as described under “~Icthotls.” The data for the curve in

t,he presence of 0.02 M &cl3 in Fig. 3 was obtained by incubating

the assay components in the following order of addiCon: 0.1 ml of

plasminogen (26 /.g), 0.01 ml of 0.5 RI ~Acp, 0.025 ml of tosyl-AMe

(100 pmoles per ml), 0.105 ml of 0.1 nf Tris I-ICI, pH 8.0. The re-

action was initiated and the tosyl-AMe concentration determined

as described above.

E#ect ot ~Acp on Activity of Plasmin--Fig. 4 shows the direct

effect of ~Acp on the activit,y of plasmin. These experiments

were conducted as follows. Plasminogen, 0.1 ml, was incubated

with 0.010 ml (200 units) of streptokinase for 10 min. Under

these conditions plasminogen is completely converted to plasmin.

Following this 0.1 ml of ~Acp containing 0.005 to 0.1 mmole was

added followed by 0.015 ml of 0.1 nl Tris.HCl, pH 8.0. The re-

action was initiated by addition of 0.026 ml of tosyl-Ahle (100

pmoles per ml) and incubated for 10 min. The concentration of

tosyl-ARle was determined as described under “Rlethods.”

The results from Fig. 4 show that ~Acp has a complex cffcct

directly on the activity of plasmin. It is quite evident from Kg. 4

that at concentratjions of ~Acp to approximately 0.1 M a direct stimulation of plasmin occurs and at increased levels a direct

iuhibition of plasmin occurs.

Since, as Fig. 4 indicates, high concentrations of ~Acp appeared

t,o directly inhibit the activity of plasmin, it was of interest to

determine the type of inhibition exhibited by ~Acp on plasmin

esterolytic activity to tosyl-Ahle. These experiments were per-

formed as shown above escept that the eAcp concentration was

held constant at 0.2 M or 0.4 M and the concentration of tosyl-

Anle varied. Parallel experiments were performed in the ab-

sence of eAcp in order to determine K,, and Tr,,, for plasmin.

These results are shown in Fig. 2. Clearly, competitive inhibi-

tion is obtained and the KI for ~Acp on plasmin is 0.32 M. There

was no significant effect of the high ~Acp concentrations on the

calorimetric assay of tosyl-AMe and control experiments demon-

1 I I , / I

I t I 1 I I I too 200 300

I/S (M-l)

FIG. 2. Variation in initial velocity of plasmin on tosyl-Ahlc with the initial tosyl-AMe concentration. The initial velocities a.re expressed in micromoles of tosyl-AMe cleaved Der min Der mg of plasmin and the substra.te concentrations are expressed in molarity. Curve 1 (0): plasminogen was fully converted to plasmin by 800 units per ml of streptokinase arior to addition of tosyl-AMe; Curve 2 (O), after plagminogen &as fully converted to plasmin as in Curve 1, the solution was made 0.2 M in tarp prior to substrate addition; Cuurve 3 (A), as in Curve 2 except that the l Acp concentration was 0.4 BC. The experimental procetlllre is described in the text.

0.6

9 0.4

z

0.2

I I I I I I I

4 8 12 16 20

I/SK x to3(units”‘mt)

FIG. 3. Inhibitlion of the conversion of plasminogen to plasmin by ~Acp at various concentrations of streptokinase. Curve 1 (o), the effect of the concentration of streptokinase on the conversion of plasminogen to plasmin. In these cxpcriments there was no preliminary incubation of plasminogcn wit.h streptokinase. V. is the initial velocity of plasmin on 0.010 M tosyl-AMe expressed in micromoles of tosyl-AMc cleaved per min per mg of plasmin. C~IYW, 2 (A) is identical with Curve 1 except that the incubation mixture contained 0.02 M EACP, added as described in the text. Since this concentration of ~kcp had only a stimulatory effect directly on the activity of plasmin (Fig. 4), the inhibition ob- served must be due to an inhibition of the conversion of pla.smino- gen to plasmin.

strated that this inhibitory effect of ~Acp was specific and not due

to increased ionic strength.

Effect of Concentration of ~Acp on Inhibition of Conversion of

Plasminogen to Plasmin-Since t’he data of Fig. 4 indicated that

e&p had bot,h a stimulatory and an inhibitory effect directly on

plasmin, these effects required a consideration when attempting

to isolate the effects of EACI) on the conversion of plasminogen to

plasmiu. Therefore, we performed studies to measure the effect

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

Inhibition of Plasmin Activity Vol. 246, Xo. 14

50

8 .- 30 f .e f 20 c

‘t- IO A! % 0

-20

-40

i- I

L/! 0.005 0.010 0.110 0.210 0.310

c-ACA Concentration(M)

Fro. 4. The effect of eAcp (E-ACA) on the activity of plasmin. In these experiments plasminogen was fully converted to plasmin by 900 units per ml of streptokinase prior to addition of the indi- cated concentrations of eAcp. Therefore, the activation or inhibition observed is due to the direct effect of ~Acp on plasmin. The initial concentration of tosyl-AMe was 0.010 M.

TAHLE I Effect of prior incubation of plasminogen with streptokinase on

inhibitory properties of E-aminocaproic acid

Assay conditionsa Initial velocityb

Experiment 1 Ko ~Acp, 200 units of streptokinase per ml; 10 min

of preincubation. Experiment 2

0.02 M eAcp, 200 units of streptokinase per ml; 10 min of preincnbation.. _.

Experiment 3 No eAcp, 800 units of streptokinase per ml; 10 min

of preincubation. Experiment 4

0.02 M eAcp, 800 units of streptokinase per ml; 10 min of preincubntion

7.2

7.3

7.5

7.5

a The concentrations referred to are final assay concentrations. o Units of micromoles per min per mg of plasminogen initially

added. The initial tosgl-AMe concentrat,ion is 8.0 pmoles per nil and the velocity is not maximal imder these conditions.

60 E z 40 r f 20

-20

-40 W' 0.010

c-ACA Concentration(M)

FIG. 5. The inhibition of the conversion of plasminogen to plasmin by various concentrations of aAcp (E-ACA). A, the percentage of inhibition was obtained by dividing the initial velocity of plasmin on 0.010 M tosyl-AMe in the presence of a given amonnt of l Acp by the initial velocity obtained in the absence of l Acp on the same concentration of tosyl-AMe and subtracting the result from 100%. There was no preliminary incubation of plasminogen with streptokinase in these experiments. The order of addition of reagents is described in the text. l , the data of Fig. 4 were added to the experimental data presented in Fig. 5 (A). This curve now represents the inhibition of the conversion of plusminogen to plasmin by ~Acp after correcting for the st,imula- tion of eAcp on plasmin.

of e&p on the conversion of plasminogen to plasmin in the fol- lowing manner. Experiments were conducted identical with those in Fig. 3 except that the eRcp concentration was varied and thr data is plotted in Fig. 5. This data shows the percentage of inhibition of plasmin activity plotted against the concentration of ~Acp and includes an inhibition of the conversion of plas- minogen to plasmin and a direct stimulation of plasmin at these &cl) concentrations. The concentration of eAcp was not high enough to consider the inhibition of plasmin directly by e9cp.

Since the data in Fig. 4 was obtained at the same initial concen- tration of tosyl-AMe as the data in Fig. 5, we have added the clcrzje in Fig. 4 to that in Fig. 5 to obtain the inhibition data of ehcp on the conversion of plasminogen to plasmin. This cor- rected data is also presented in Fig. 5. The experiments in Fig. 5 were carried out with additions in the order given: 0.1 ml of plas-

minogeu (27 pg), 0.01 ml of eilcp containing 0.05 to 25 pmoles, 0.025 ml of tosyl-ARIe (100 pmoles per ml), 0.105 ml of 0.1 81 Tris.1.ICI, pII 8.0. The reaction was initiated with 0.01 ml of streptokinase (225 units) and allowed to incubate for 10 min. The concentration of tosyl-Ahfe in a 0.2-ml aliquot of the reaction mixture was determined as described under “Methods.” The results obtained are presented in Fig. 5. Concentrations of 0.05 IVI eAcp represent the minimum concentration at which maximum inhibition occurs.

Eflect of Prior Incubation of Plasminogen with Streptokinase on Inhibitory Properties of CAcp---The effect of prior incubation of plasminogen and streptokinase in the presence of 0.02 M ~Acp has been examined at two levels of streptokinase concentration. These experiments were carried out as follows in the indicated orders of addition: 0.1 ml of plasminogen (17 pg), 0.01 ml of 0.5 1\1 eAcp, and 0.11 ml of 0.1 M Tris IICl, $1 8.0, were mixed. To this was added 0.01 ml of streptokinase (50 or 200 units), and the mixture was incubated for 10 min. At this time 0.02 ml of tosyl- AMe (100 pmoles per ml) was added to initiate the plasmin re- action and allowed to proceed for 10 min. The amount of tosyl- AMe cleaved was analyzed as previously described. Parallel experiments were performed with 0.01 ml of Tris buffer added in

place of ~Acp at each streptokinase concentration. The results of these experiments are presented in Table I. Clearly, the prior incubation abolishes the inhibitory effect of ~Acp at the streptokinnse concentrations indicated.

E$ect of I’ime of Incubation of ~Acp with Plasminogen on Its Inhibitory Properties of Plasminogen to Plasmin Conversion- ~Acp was incubated with plasminogen for various times and the extent of inhibition of the conversion of plasminogen to plasmin was noted in order to determine whether the time factor was im- portant in e&p exhibiting its inhibitory effect. These experi- ments were carried out as follows in the indicated orders of addi- tion: 0.1 ml of l)lasminogen and 0.01 ml of 0.5 M ~hp were incu- bated for various times. Following this 0.110 ml of Tris buffer and 0.02 ml of tosyl-AMe (100 pmoles per ml) were added. The reaction was initiated by addition of 0.01 ml of streptokinase and tosyl-A?\ie cleaved in a 0.2.ml aliquot was determined as de-

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

Issue of July 25, 1971 W. J. Broclcway and F. J. Castellino 4645

Ei7~f.t of lime oj incubation of plasminogen with E-aminocaproic acid on inhibition of plasminogen to plasmin conversion

Time Initial velocitya

nrin

O* 7.2

1 2.9

2 2.8 5 3.0

10 2.9 20 2.8

a Units of micromoles per min per mg of plasminogen initially added. The initial tosyl-AMe concentration is 8.0 pmoles per ml

and the velocity is not maximal under these conditions. * Refers to no ~Acp.

T>~BLE III

s:o,~ values of plasminogen in presence and absence of e-aminocaproic acid

Conditions

1. PlasminogeninO.lMphosphate,pH7.5 5.0 s*

2. Plasminogen in 0.1 M phosphate, 0.05 M ~Acp, pH 7.5.................................... 3.8 S*

3. No. 2 dialyzed against 0.1 M phosphate, pH 7.5. 5.0 s*

a Although these values were not actually extrapolated to zero protein concentration, the low concentrat.ions (0.2 mg per ml)

of plasminogen required for these experiments make this extrap- olation unnecessary.

b Average of three determinations.

scribed under “Methods.” The results are presented in Table II. Clearly, the time of incubation of plasminogen with ~Acp is not an important factor and the inhibition observed is attained very rapidly.

Sedimentation Coejicient of Plasminogen in Presence and Ab- sence of &q-The effect of 0.05 M ~Acp on the sedimentation coefficient (s~O,w) of pl asminogen is given in Table III. There is a decrease in the sio,W of plasminogen upon addition of ~Acp and a return to the native value is evident upon dialysis against buffers containing no ~Acp. The decrease in the .s$, value of plas-

minogen upon eAcp addition can reflect either a dissociation of plasminogen into a smaller molecular weight component or a gross unfolding of the molecule. Since plasminogen consists of

only one polypeptide chain, there can be no dissociation into a smaller molecular weight component and the effect of ~Acp upon

the s~o,~ of plasminogen must be due to an CAcp-induced confor- mational change in plasminogen resulting in a more randomly coiled structure. This .s!& vnlue is not significantly decreased

at higher concentrations of eAcp. Circular Dichroism Analysis of Plasminogen in Presence and

Absence oj CAcp-Since the s$~ values of plasminogen indicated that a gross conformational change took place upon treatment with &cp, circular dichroic spectra were performed from 250 rnp to 190 rnp to confirm this conformational alteration. Plots of molecular ellipticity [0] versus wave length are given in Fig. 6. There are obvious differences in the two curves in both magnitude of ellipticity at any wave length and shape of the spectra. The

200 210 220 230 240 250

Wavelength (mq)

FIG. 6. Representative circldar dichroism spectra of plasmino- gen in the presence and absence- of 0.05 M eAcp. [e] is expressed in terms of mean residue weight = 114. Upper curve is a plot of molar ellipticity [e] versus wave length of plasminogen in 0.05 M eAcp. Lower c?Lrve is as upper except that no eAcp is present.

spectrum of plasmiuogen in &cl) appears almost devoid of any helical structure. These results support the inferences made from analysis of the .s$+ values of plasminogen in eAcp in that a gross conformational alteration takes place resulting in a more random polypeptide chain.

DISCUSSION

The studies presented here show that the mechanism of the inhibition of plasmin activity by ~Acp is complex and involves both inhibition of the conversion of plasminogen to plasmin and stimulation of plasmin at low eAcp concentrations and direct in- hibition of plasmin activity at high eAcp concentrations.

A schematic mechanism for these effects can be illustrated which is in accord with the data presented in this manuscript. This scheme is as follows:

SK Pg- Pm+S--+Pm+P

EACP i? M ~ACP Pg. CAcp Pm. CAcp

In this diagrammatic representation, under normal conditions, plasminogen (Pg) is activated by streptokinase (SK) to form plasmin (pm). The plasmin possesses esterase activity on tosyl- AMe (8). In Fig. 2, in the absence of ~Acp and without prior incubation of plasminogen with streptokinase, the enzymatic ac- tivity of plasmin increases as the streptokinase concentration is

increased. This effect can best be explained by considering plas- minogen as the substrate for streptokinase action. Then, as the streptokinaee concentration is increased there is a more rapid conversion of plasminogen into plasmin and thus a higher rate of reaction of plasmin with tosyl-AMe. Consistent with this hypothesis, when plasminogen is incubated with different concentrations of streptokinase for various times prior to addi- tion of tosyl-AMe, there is no dependence of plasmin activity on streptokinase concentration given a sufficient time of incuba- tion. This occurs since all the plasminogen present will even- tually be converted into plasmin. We have shown this to be the case during the course of these studies and others have published confirmatory data (16).

In considering the inhibition data in Fig. 3 at 0.02 M ~Acp and no prior incubation of plasminogen with streptokinase, all one

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

Inhibition of Plasmin Activity Vol. ,346, x0. 14

can conclude at this point is that plasmin activity is competi- tively inhibited by ~hcp. This inhibition can occur at the level of the conversion of plnsminogerl to plasmin or ~Xcp can be a direct inhibitor of plasmin. This question is resolved by cou- pling the datn of Fig. 3 with that of Fig. 4. In Fig. 4 we con- clusivcly demonrtratc that tAcp concentrations up to 0.1 M

have a stimulatory effect on the activity of plasmin. Thus, the inhibition of the plasminogen to plasmin conversion should in fact be greater than what is observed by performing experi- ments as in Fig. 3. We have made a rough correction for this stirnulatory effect as illustrated in Fig. 5. Clearly, the net in- hibition of plaemin activity which occurs at ~Acp concentrations to 0.1 IVY is due only to the inhibition of the plasrninogen to plas- min conversion by ~Acp. These effects are illustrated in the scheme prcPentcd above. It can be seen that plasminogen can react with tAcp and foErn a l)lasrninogell.Ehcp complex. This complex is refractive to activation by streptokinase. We feel that this complex can be rapidly converted to I&~sminogen and thus be activated by streptokinase for the following reason. As shown in Table I, the inhibitory effect of 0.02 M ~Acp on the conversion of plasminopen to plasmin can be abolishctl by incu- bation of plasminogen, 0.02 hf tAcp, and streptokinasc prior to addition of tosyl-AMe. What must bc happening in this case is that strcptokinase, in an irreversible manner, reacts with the free plasminogen in the equilibrium, plasminogen + ~Acp 4 &asminogen .&cl), to form plasmin. Thus, the equilibrium is pulled toward free plasminogen. Given enough time of incuba- tion before tosyl-AMc addition, the plasminogen ~Acp complex will completely dissociate into free plasminogen which will react with the streptokinase present to produce plasmin. Since, under these conditions all the plasrninogen present will be converted into plasmin, no inhibition occurs when tosyl-AMe is added, regardless of t,he presence of ~Acp. On the other hand, inhibition at 0.02 M ~Acp is only seen when all components of the assay mixture are added together in a definite order (Fig. 3-referred to as “no preincubatjon”). In this case the rate of plasmin reactivity with tosyl-AMe will be slower in the presence of .~Acp than in its absence. All the data collected in this study are con- sistent with these views.

;\t this point KC feel that we call perhaps explain some discrcp- :ulciea which appear in the literature concerning the activation of plasminofen b\- streptokiiiase. For example, Murarnatu et al. (16) proposed that t,hcrc wcrc two mechanisms of activation of plasrninogen by strcptokinasc. The first mechanism, the details of which are not important for discussion here, occurred at low streptokinase concentrations and was based on the fact that. when plasminogen mas activated ai low strel)tokinase concent,rations (10 units), the activity of plasmin incrcnsetl with time. In other words, incubation of streptokinase with plasminogcn before sub- strate addition was necessary to obtain full plasmin activity. The second mechanism of activation of plnsminogen by strepto- kinase, according to these authors, occurred at high strcptokinase concentrations and was based on the fact that at high strepto- kinase concentrations (900 units) a much shorter incubation time of st’reptokinase with plasminogen was necessary before substrate addition to obtain full plasminogcn activity. Although their mechanism is consistent with the data obtained, a rnore simple rate effect can also explain the data. According to our scheme, it is not necessary to propose two mechanisms of action. If one considers the action of streptokinase on plasminogen to be as stated above then at low streptokinaee concentrations the rate of

activation of plasminogeri Tao plasmin will be slower than nt higli streptokinase concentrations.

In addition, Muramatu et (11. feel that two mechanisms of RC- tion for streptokinase activation of plasminogen must exist since ~Acp did not inhibit plasmin activity at high concentrations of streptokinase (800 units), whereas inhibition did occur at low con- centrations of streptokinase (10 units). In these experiments incubation of plasminogen, ~Acp, and streptokinase before sul)- strate addition was performed. Bgain, we feel, based on our scheme, that it is not necessary to Ijropose a complicated esplana- tion. Clearly at 800 units of streptokinase, according to our scheme, all the plasminogcn I)rrsent is converted to plasnlin re- gardless of the formation of a plasminogen.tAcp inactive corn- plex. At these high streptokinase concentrations t,he rate of breakdown of this complex is very rapid. On the other hand, at 10 units of streptokinase, the rate of plasmirrogen. ~Acp complcs breakdown is suflicicnlly slow to require much larger I)rior illcan- bation times. We have lengthened the prior incubation times with 10 units of strcptokinasc in an effort to demonstrntr this point, but the rate of complex dissociation was so slow that in- activ&ioii of plasmin occurred, severc,l,v complicating our rr- sults. IIowever, wc have tlernonst~rated that at several strt,pto- kinase conccntrntions above 100 units the inhibition by low t.%cp can be abolished, supporting our contention.

Quite a different picture of the inhibition of plasmin nctivit> occurs at high ~hcp concentrations. Here, two effects are noted. (a) inhibition of plasmin artivit,y at the level of the conversion of plasminogen to plasmin still occurs and (b) at high concentrations &cp is a competitive inhibitor of plasmin activity with a K1 of 0.32 M. This effect is indicated in the scheme presented above. These results require no prolonged analysis since L&I) is a sub- strate analogue of L-lysine and L&sine methyl ester is a substrate of plasmin. Therefore, tL2cp probably binds at the substr:ltc binding site of plasmin thereby producing csompetitire illhibitiotl kinetics. The k’l of ~Acp toward plasmin is very high illtlicnting that ~Acp is not strongly bound to the enzyme. 1Ve art I)rcsently in the process of testing rnorc substrate analogues as inhibitors of plasmin and a report on these studies will shortly appear.

Although the inhibitory cffrc,t, of high collccntratiolls OC t.\cap on the enzymatic activit,y of plasmin appears to 1)~ rraao~rabl~ straightforward, the reason for the inhibitory effect of Ion- CO~I- centrations of .&cp on t’he c~onversion of 1)l:isminogen to I)l:lsmill st,ill requires explanation. Re f(lel that thi:: inhibition is tluc to ~Acp causing a freely reversible conformat ional change in the plasrninogen molecule. This conformational alteration ~“odnces a plasminogen which is not capable of being acted upon by strep tokinase. This conformational alteration is clearly evidenced 1)) analysis of the S&~ values of plasminogen in the presence alld ab- sence of 0.05 &CC ~Acp given in Table III. This conformational alteration is also evidenced in circular dichroism studies. X11:1ly-

sis of the data in Fig. 6 shows that plasminogen in the :Lbscll(~e of ~Acp possesses some, but’ not a great deal of, helical structure, as evidenced by the trough at 220 nm. This trough disappears upon addition of 0.05 M ~Acp and there is a decrease of the mo- lecular ellipticity. These conditions indicate that there is :I con- siderable loss of structure of plasminog~n upon addition OF ~.Zcp. The native structure reappears upon removing the .&p by tlinly- sis suggesting a freely reversible conformational transition.

With regard to the stimulatory effect of low concentrations of ~Acp on the activity of plasmin, this is due to a direct effect on plasmin and not on t’he plasminogen to plasmin conversion. Oul

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

Issue of July 25, 1971 W. J. Brockway and F. J. Caste&no 4647

data dots not allow us to malw a useful evaluation as to the mech- allism of this stimulation at this point.

In conclusion, many factors wql~irct control when the inhibit)ion of l)l:lsmin activity by thcp is studied. We feel that we have conclusively csplaincd the mechanism of the inhibition by ~Rcp as wvrlll ns pointed out the reaso~ls for some discrepawies which esist on this topic.

RlGFElUNCI?S

1. Rortnrns, K. C., SUMMULL, T,., I~SIEII, B., AND SHAN, R. J., J. Biol. Chem., 242, 2333 (1!)67).

2. S~MM~~RIA, L.. HSIEH, B., ‘ZXD ROIIBINS, K. C., J. Biol. Chem., 242, 4279 (1967).

3. SEMM~RI~I, L., HSIEH, B., GROSKOPF, W. R., ROBBINS, K. C., .~ND Baa~ow, G. H., J. Riol. Chem., 244, 359 (1969).

4. AIXJAEIISIG, N., FLIXCHEI~, A. P., AND SHERRY, S., J. Biol. Chem., 234, 832 (1959).

5. OIMMOTO, S., Keio J. Med., 8, 211 (1959). 6. EGEBLBD, K., Thromb. Diath. Haemorrh., 16, 137 (1966). 7. MASRELL, R. E., AND ALLJ~N, II., Nature, 209, 211 (1966). 8. iVIaxw~~~~+ II. E., Nawrtoc~~~, J. W., AND NICKEL, V. S.,

Thvomb. Diath. Haem.or,h., 19, 117 (1968). 9. DEUTSCH, D., AND MERTZ, E. T., Ped. Proc., 29, 2253 (1970).

10. HESTILIN, S., J. Biol. Chem., 180, 249 (1949). 11. SCHXHIVLIN, H. K., Ultvxentrifugation in biochemistry, Aca-

demic Press, New York, 1959, p. 82. 12. ORNSTISIN, L.,‘.~ND DAVIS,‘B. J.; bLsc electrophoresis, Dist.illa-

tion Products Industries. Rochester. New York. 1962. 13. PANYIM, S., IND CIIBLKLEY’, R., Arch. kochem. Bibphus., 130,

337 (1969). 14. WEBER, K., AR’D OSBORN, M. J., J. Uiol. Chem., 244, 4406

(1969). 15. SUMM.~RLI, I,., AND ROBI~INS, K. C., Fed. Proc., 29, 408 (1970). 16. MURGVIATU, M., HAYAKUMO, Y., ONISHI, T., SATO, T., AND

FGJII, S., J. Biochem. (Tokyo), 66, 329 (1969).

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from

William J. Brockway and Francis J. Castellino-Aminocaproic AcidεThe Mechanism of the Inhibition of Plasmin Activity by

1971, 246:4641-4647.J. Biol. Chem. 

  http://www.jbc.org/content/246/14/4641Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/246/14/4641.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Novem

ber 2, 2018http://w

ww

.jbc.org/D

ownloaded from