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Effects of some amino acids on the inhibition of plasmin by antiplasmin
BIOCHIMICA ET BIOPHYSICA ACTA
34 2
EFFECTS OF SOME AMINO ACIDS ON THE INHIBITION OF PLASMIN BY ANTIPLASMIN
RICHARD E. MAXWELL, VERA LEWANDOWSKI AND VIOLET S. NICKEL Research Laboratories, Parke, Davis and Company, Ann Arbor .Mich, (U.S.A.j (Received November 24th, 1964)
SUMMARY
Variations in inhibition of plasmin (EC 3-4+14) and potentiation of antiplasmin by s-arninocaproic acid were studied as functions of relative concentrations, reaction times, and type of plasmin preparation. tS-Aminovaleric acid and y-aminobutyric acid were also potentiators of antiplasmin inhibition. Lysine and ornithine, as well as s-aminocaproic acid, could be shown either to enhance plasmin activity, or act as potentiators of antiplasmin at higher concentrations. Arginine and citrulline did not appear to act as potentiators. Lysine methyl ester and p-toluenesulfonylarginine methyl ester were effective antagonists of antiplasmin at appropriate concentrations.
INTRODUCTION
Certain reversible inhibitors of plasmin (EC 3.4-4.14) protected the latter from irreversible inactivation by antiplasmin'. However, it was found that s-aminocaproic acid, while being an effective and easily reversible inhibitor, potentiated the action of antiplasmin at the concentrations studied. In view of the theoretical implications of these observations, and the proposed clinical applications of plasmin and s-aminocaproic acid, additional experiments with s-aminocaproic acid and related structures were indicated. MATERIALS AND METHODS
Plasmin preparations Kline-type plasmin was obtained as previously described", Euglobulin-type plasmin was isolated from human serum, all steps being carried out at 0-5°. Euglobulins were precipitated by I :10 dilution with water and adjustment to pH 5.4 'with acetic acid, collected by centrifugation and frozen until needed. About 25 g of the Abbreviations: TAME, p-toluenesulfonylarginine methyl ester; LME, lysine methyl ester.
Biochim. Biophys, Acta, 99 (19 65) 34-2-35 1
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
343
frozen paste was dispersed with a mechanical blendor into 300 ml 0.05 M acetate buffer (pH 5-4). After centrifuging for IS min at 10 000 X g, the extraction was repeated with ISO ml acetate buffer. The sediment was dispersed into 300 ml 0.1 satd. (NH4)2S04, and the pH was raised to 8.0 with drop-wise addition of 1.0 N NaOH and rapid mechanical stirring. After further stirring for 30 min, the mixture was centrifuged for 30 min at 10 000 X g, the supernatant was filtered through glass wool, and the filtrate was frozen overnight. After thawing in cold tap water, insoluble material was removed by centrifuging as before, and a fraction precipitating between 0.25 and 0.3 satd. (NH4)2S04 was collected and dissolved in 50 ml of TrisNaCl buffer 2. After dialysis for 48 h vs. 5 2-1 changes of the latter buffer, any remaining solids were removed by centrifuging at 20 000 X g for 30 min. This procedure yielded about I g of protein from 5 g in the original paste, with a 4- to 5-fold increase in specific activity and apparently complete recovery of total activity, a result probably due to the presence of inhibitory material in the original euglobulin, since some discarded fractions had activity. These preparations had very low spontaneous activity, were stable for months stored as frozen solutions, and served as convenient starting material for further purification. For conversion to plasmin, the plasminogen was diluted to a concentration of 10 absorbance units per ml (i.e., that concentration which would have an absorbance of 10.0 cm ? at 280 mf.l) in Tris-NaCI buffer containing 500 Ploug units" of urokinase per ml. After IS min at 37°, the euglobulin-type plasmin was frozen in aliquots. The specific activity was about the same as that of Kline-type plasmin, or 4-5 RPMI units- per mg of protein, if one assumes E~~~m = 14.0 at 280 mf.l. The urokinase used was obtained from Leo Pharmaceutical Products, Copenhagen, and had an activity of 5000 units/mg. A ntiplasmin Antiplasmin was isolated from bovine serum", Bovine antiplasmin was used, since similar fractionation procedures applied to human serum consistently failed to yield material of high potency. In several experiments, unfractionated human serum was used as an effective source of human antiplasmin. Chemicals Chemicals were of the highest purity obtainable from various commercial sources. All amino acids and derivatives with asymmetric centers were of the Lconfiguration. Fibrinogen and thrombin Human fibrinogen and bovine thrombin were obtained as previously described 6 , except that the fibrinogen was prepared from Human Fibrinogen Grade L, supplied by A. B. Kabi, Stockholm. This product was suspended in Tris-NaCl buffer and dialyzed against a large volume of the latter for 8 h at 37°. The Darco G-60 treatment was then applied, and was consistently effective in removing plasminogen contamination. These preparations were 95-98% clottable by thrombin, and clots showed no lysis at 37° for 24 h in the presence of 400 units of urokinase per ml. Fluorescein-labeled fibrinogen was prepared by modification of the method of RINDERKNECHT 7. Fibrinogen at 10 mg/ml in 0,175 M Na 2CO a buffer containing Biochim, Biophys. Acta, 99 (19 65) 34 2-35 1
344
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
0.9% NaCl (pH 8.8) was shaken at 25° for 30 min with 4 mg/ml 10% fluorescein isothiocyanate on celite. After removal of the celite by centrifugation, excess fluorescein was removed and the labeled fibrinogen transferred into Tris-NaCI buffer with a column of Sephadex G-2S. The fibrinogen was quantitatively recovered with no significant change in clottability or resistance to urokinase, but the clots were somewhat more susceptible to plasmin action.
Fibrinolytic measurements A z-phase system with I ml clots and I ml fluid in vials of 2S mm diameter was employed and results were expressed as per cent lysis of the clotl,2. In experiments involving serum or TAME, which interfered with the usual measurements in the ultraviolet region, fluorescein-labeled clots were used and the lysed material was determined at the activation wavelength of 500 m/-, and fluorescence wavelength of 530 mp,. Protein levels of plasmin preparations are expressed in terms of absorbance, one absorbance unit being that amount with an absorbance of I ' 10-8 cm- l at 280 mfJJ. The value for the extinction coefficient of plasmin is controversial", Absorbance values were corrected by readings at 320 ID/-' and 360 m/-'9.
40
30 .~
z-"' 15 u
....c::
s
~ 20
10
~==~==
30
/
__
. l - _... _.l
60
LySIS
90
J
120
period (min)
Fig. 1. Kinetics of inhibition of euglobulin-type plasmin (EP) by s-aminocaproic acid (EAC). /::,.-l::" 100 absorbance units EP; A-A, 100 absorbance units EP + 0.,5 flmole EAC; 0-0, 50 absorbance units EP; e-e, 50 absorbance units EP + 0·5 flmole EAC; 0-0,25 absorbance units EP; . - . , 25 absorbance units EP + c.y zzrnole EAC.
Biochim. Biopbys, Acta, 99 (19 65) 342-351
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
345
RESULTS
Studies with e-aminocaproic acid Fig. I serves to illustrate the general course of the lysis of the fibrin substrate in the experimental arrangement used, at different levels of plasmin over a z-h period. Inhibition by e-aminocaproic acid was reversible, and it is evident that the degree of inhibition was a function of the ratio of plasmin to e-aminocaproic acid, and of the time of observation at a given plasmin level. As seen in Fig. 2, similar fibrinolytic levels of euglobulin-type plasmin and Kline-type plasmin varied signi20
n
Euglobulin type plasmin
10
o Vl
~ .... .Q u
....c:
Kline type
OJ
l:
~ 20
10
o
~
o
3
~
l 5
10
g-Amlnocaprolc (IJ-nnoles)
Fig. 2. Effects of mixtures of s-arninocaproic acid and antiplasmin (AP) on 300 absorbance units euglobulin-type plasmin and 333 absorbance units Kline-type plasmin. Plasmin and plasmin plus inhibitor mixtures were preincubated for 2 h at 37° and then placed over the clots for a 30-min period of lysis. Left bar, no AP; center bar, 25 pg AP; right bar, 50 fig AP.
ficantly in their sensitivity to s-aminocaproic acid, and to mixtures of s-aminocaproin acid and antiplasmin. Inhibition of euglobulin-type plasmin by antiplasmin was potentiated by the lowest level of s-aminocaproic acid tested in this particular experiment, while potentiation of Kline-type plasmin inhibition was not found at these inhibitor levels and at this period of interaction, In fact, as also reported by others10,1.l, enhancement of activity was noted at lower s-aminocaproic acid levels with both Biochim: Biophys, Acta, 99 (19 65) 34~-351
R . E. MAXWELL, V. LEWANDOWSKI , V. S. NICKEL
TABLE I IR REVERS I B LE INHIBITION OF I(LINE-TYPE PLASulIN
Mixtures with K line-type p lasmin (333 absorbance u nits) were preincubated w ith fibrin.
2
Inh ibitors
Per cent clot lysis after 22 II
None s-aminocaproic acid (10 }lmoles) AntipJasmin (100 Itg ) s-aminocaproic acid ( 1 0 p,molcs)
100
h before contact
95
+ antiplasrnin
20
( 100
Ilg)
3
Kline-typ e pla smin and euglobulin-t y pe plasmin ; as reversal of initial inhibition pro ceede d (Fig. r), t he ra te of lysis in the presence of s-aminocaproic acid becam e greater than in its absence. At higher levels of antiplasmin , potentiat ion by s-aminocaproic acid an d irreversible inact ivation of Kline-type plasmin was observe d.'. This is further demonstrat ed in Table I, the data in dicat ing insig nificant reversal of anti plasmin or antiplasmin + s-aminocaproic acid inhibition even after 22 h of contact with the fibrin substrate. Fig. 3 shows earlier stages of the interaction and serves to illustrate the marked differences in the kinetics of progressive irreversible inhibition by antipl asmin , and t h e reversal of initial inhibiti on by s-aminocaproic acid . It is also apparent tha t t he combination of s-ami nocaproic acid and antiplasmin broug ht about almost im mediat e inhib ition. 80 r
'0; ~
60
..... 0 u ..... c 40 ~
L
n,
20
Lysis period
(mi n)
Fig. 3. Kinetics of inhibition of 333 absor bance units Kline-type plasmin (KP) by r o urnoles s-aminocaproic acid (EAC) and 200 f.tg antiplasmin (AP) . 0 -0, KP; 0-0, KP AP; 6.-'/::,., KP EAC ; V - V , KP AP + EAC.
+
+
+
Other amino acids Lysine, like s-aminocaproic acid, has also been found to stabilize or enhance p lasmin activity at lower concentrations or produce inhibition at higher concentrations- ", Therefore, a range of levels of lysine was examined in the presence of two con centrations of anti plasmin; ornithine, citrulline, and arginine were included for comparison. The results (Fig. 4) indicated t hat ornithine, lysine , and arginine had a Biocbim, B iophys, Acta , 99 (19 65) 34 2 -35 1
347
THE INHIBITION OF PLASMIN BY ANrIPLASMIN Ornithine
Lysine
....S2 \J
Citrulline
I~!f~~~ 5
10 15 20 Amino acid level (I-l. moles)
Fig. 4. Effects of amino acids on plasmin and on inhibition by antiplasmin (AP). Mixtures were preincubated for 2 h at 37° and then placed over the clots for a 3D-min period of lysis. Bars represent results with 333 absorbance units Kline-type plasmin: left bar, no AP; center bar, 25 ~Ig AP; right bar, 50 fig AP. The lines similarly represent results with 300 absorbance units euglobulin-type plasmin.
stabilizing effect in the absence of antiplasmin, ornithine and lysine being the most effective. In the presence of antiplasmin, however, potentiated inhibition was evident at higher levels of ornithine and lysine. As in the case of s-aminocaproic acid interaction, euglobulin-type plasmin was more sensitive than Kline-type plasmin to this potentiation. A kinetic study of the euglobulin-type plasmin (50 flg antiplasmin and 10 flmoles lysine) system over a period of 150 min gave no indication of reversal in this period of time. The above experiments involved a preincubation period in the absence of fibrin, in order to assess the stabilizing effects of the amino acids and their interaction with lower levels of antiplasmin. It was also of interest to determine the effects of various compounds initially in the presence of fibrin and a higher antiplasmin level, and over a sufficiently long time to allow for reversal of initial inhibition by reversible inhibitors. Table II summarizes results with several amino acids, and it is evident that the potentiated inhibitions with lysine and ornithine were still significant after 5 h of contact with the fibrin. s-Aminocaproic acid and two other structures with activity in this test, Cl-aminovaleric and y-aminobutyric acids, are included in the table for comparison. Biochim, Biophys, Acta, 99 (1965) 34 2-35 1
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
TABLE II POTENTIATION OF ANTI PLASMIN INHIBITION OF KLINE-TYPE PLASMIN
Mixtures containing 333 absorbance units Kline-type plasmin with or without 200 tLg antiplasmin were added over the clots without preincubation and incubated for 5 h. At this time, all of the mixtures without antiplasmin had given 100% lysis of the clots. ---_._------.---~---------
Compound added
Level
PC1'
cent clot
( umoles ) lysis with aniiplasmin present
None s-Aminocaproic acid o-Aminovaleric acid y-Aminobutyric acid Lysine Ornithine Arginine Citrulline
10 10
22
SO
30 30
50 50 50 50
42 58 61
Lysine and arginine esters Lysine and arginine esters are plasmin substrates and stabilizers, and competitive inhibitors of plasmin proteolytic activity13,l4. It was postulated, therefore, that they might be able to antagonize the action of antiplasmin and still allow fibrinolysis to take place, as previously found for some other reversible inhibitors", This could be readily shown (Fig. 5), provided proper account was taken of time and concentration parameters. The dissociation of plasmin-LME in the presence of fibrin was relatively rapid, so that stabilization and antiplasmin antagonism became evident in 30min oflysis. Plasmin-TAME dissociatec1less readily, so that the effective antagonism of antiplasmin by TAME could be overlooked by limiting observations to an initial 30-min period of lysis, but was apparent after I h of lysis. These experiments are representative of numerous others which led to the not unexpected conclusion that only within limits was it possible to balance higher levels of reversible inhibitors against higher levels of antiplasmin, and to increase the effectiveness of the stratagem by use of reversible inhibitors with lower K, values (e.g., TAME .!!l
50
'"
~ 40
+'
a
"0 +'
30
c
u
20
10
L
0
25
0 LME ( f-io moles)
20 TAME
(t: moles)
Fig. 5. Antagonism of antiplasmin (AP) by LME and TAME. Mixtures containing 333 absorbance units Kline-type plasmin were preincubated for :
h.
Bioohim, Biophys, Acta. 99 (19 65) 34:
349
THE INHIBITION 011 PLASMIN BY ANTIPLASMIN
TABLE III EFFECT OF AMINO ACIDS ON HUMAN SERUM INHIBITIOH
Systems containing 300 absorbance units euglobulin-type plasmin (EP) or 333 absorbance units Kline-type plasmin (KP) were preincubated 2 h at 37° and then placed over the clots for a 30min lysis period.
Enzyme
Amino acid added
Level Clot lysis (%) at serum level of (flmoles) 0 0.025 ml 0.05 ml
-----~-
EP EP EP EP EP EP EP KP KP KP KP
None Ornithine Ornithine Lysine Lysine Citrulline Arginine None Lysine Lysine Lysine
25 100 10 50 10 50 5 50 100
24 32 26 31 20 30 29 23 30 36 29
16 19 10 18 6 17 20 12 23 21 12
8 10 2 10 2 10 14 9 17 8 5
cf. LME) together with longer lysis periods. As time and inhibitor concentrations were increased, larger fractions of the plasmin were inevitably destroyed before becoming effectively associated with fibrin.
Experiments with serum Since the above experiments were done with isolated bovine antiplasmin of fairly high potency, it was desirable to compare unfractionated human Serum as the source of antiplasmin activity. Table III shows representative results with a selection of levels of amino acids. Qualitatively, the findings were similar to those with bovine antiplasmin : stabilization by all four compounds at lower concentrations and lower 100
80
~
..... 60
~
..... c: <1J
u
L
40
<1J
0..
20
~'-----:!::------=l:---_-L--
30
60
90
__.J
120
Lysis period (min)
Fig, 6. Potentiation of serum inhibition of 333 absorbance units Kline-type plasmin (KP) by s-arninocaproic acid (EAC). Mixtures were placed over clots without preincubation. 0-0, KP; 6.-/:::" KP + 0.1 ml serum; 0-0, KP + 5flmoles EAC; V-V'. KP + 0.1 ml serum and 5 p,moles EAC.
Biochim. Biophys, Acta, 99 (1965) 342-351
350
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
serum levels, but a decrease in favorable effect or even potentiation of inhibition at higher ornithine and lysine concentrations. Again, quantitative variations were found in comparing the two plasmin preparations, as illustrated with lysine. Fig. 6 is an example of s-aminocaproic acid potentiation of serum inhibition in the initial presence of fibrin. DISCUSSION
The present model of the fibrinolytic system, including reversible, irreversible, antagonistic and potentiated inhibitions, as well as the phase separation of the enzyme solution from the insoluble substrate, is not designed for facile analysis of kinetics and mechanisms. Conventional Michaelis-Menten kinetics do not necessarily apply, and rates of reactions are determined by adsorptive factors at the fibrin surface as well as by time-dependencies of interactions in the fluid phase as compared to those at the interface. Possible quantitative approaches to some of the kinetic problems in less involved heterogeneous or multiple-inhibitor systems have been discussed by McLAREN 15 and WEBB 16 . Qualitatively, it is apparent that even in the relatively simple case of a plasmin-reversible inhibitor mixture (or of plasmin + slowly acting irreversible inhibitor with which the substrate competes kineticallyw) the degree of inhibition can be a complex function of concentrations of inhibitor, substrate and enzyme, as well as of the particular time of measurement of the reaction. Conventional kinetic analyses, if based on a single reaction time, may be of little significance. It is presumed that these complexities, along with many others that could be added, exist in vivo, and it is hoped that examination of some of the variables may aid in the interpretation of other experiments in which similar considerations could apply in part. It has been reported that e-aminocaproic acid was a specific inhibitor of plasminogen activation 10, that s-arninocaproic acid was equally inhibitory after activation had taken place-t, that s-aminocaproic acid was a fairly potent inhibitor of plasmin fibrinolysis 18, or that s-aminocaproic acid did not inhibit plasmin fibrinolysis at all l D. Additional contradictory results could be cited, but these are adequate to illustrate the situation. The quoted studies cover a remarkable range of time factors, enzyme to inhibitor ratios, and assay systems, often within the same investigation. At least some of the inconsistencies might be rationalized on the basis of their being different aspects of the reversible inhibition of plasmin or of plasminogen activation. In the case of heterogeneous systems 17 ,19, as demonstrated in the present study also, the effective substrate concentration, being limited as a function of the surface area in contact with the fluid phase, is far less than in comparatively homogeneous caseinolytic or esterolytic assays 10, and it is possible to obtain the impression that a given reversible inhibitor is either potent or impotent, depending on time of observation and initial enzyme and inhibitor concentrations. In the study of activation systems in which the end point of the assay depends on plasmin activity in the presence of the added s-aminocaproic acid-", the inhibitor to plasmin ratio is obviously very high initially, and it is hardly surprising that the inhibition can be greater in comparison to that obtained with some relatively large, arbitrarily selected level of preformed plasmin. It is also now apparent that s-aminocaproic acid inhibition in blood or plasma systems can be markedly affected by the Biocbim. Biophys. Acta, 99 (19 65) 34 2-35 I
351
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
levels of antiplasmin or perhaps other inhibitors which may be potentiated. This potentiation may help explain the apparently greater activity of s-aminocaproic acid in vivo as compared to some in vitro tests with purified componentsw, and it may be hazardous to conclude that s-aminocaproic acid inhibition per se can be evidence for distinction between activator activity and low levels of preformed plasmin. It has been frequently noted that low concentrations of s-aminocaproic acid or lysine enhance rather than inhibit plasmin activity l O,12 . It is apparent from the present and previously reported results 1 that other related and unrelated reversible inhibitors may produce similar enhancement; the simplest interpretation would be on the basis of stabilization of the plasmin or its protection from irreversible inhibitors that might be present, although more complicated mechanisms can not be excluded. An explanation of concentration effects and structural variations that lead to potentiation or antagonism of antiplasmin is of course not available at this time. Intellectually, some satisfaction may be obtained by visualizing interactions at or near a flexible active site which may result either in interference between inhibitors or enhancement of irreversible attachments-. Conceivably, additional correlations of structure with potentiating or antagonistic effects might eventually contribute to a further understanding of the characteristics of such a mobile active site. Proposals have been made for the use of e-aminocaproic acid, lysine and other amino acids, or lysyl and arginyl esters in the formulation offibrinolytic products 12 , 14 , 22 . While the relevance of the present investigations to events in vivo is naturally speculative, the possible influence of substances which may either enhance plasmin activity or potentiate its inhibition, depending on time and concentration parameters, should be kept in mind. REFERENCES I R. E. 2 R. E.
MAXWELL, V. LEWANDOWSKI AND V. S. NICKEL, Life Sciences, 4 (1965) MAXWELL AND V. LEWAI-lDOWSKI, Anal. Biochem., 4 (1962) 407. 3 PLOUG AI-lD N. O. KJELDGAARD, Biochim, Biophys, Acta. 24 (1957) 278. 4 W. BAUMGARTEI-l, C. M. AMBRUS, K. B. MCCALL AI-lD R. B. PEI-lNELL, Am.
45.
J.
]. Cardiol., 6 (1960) 447. 5 E. C. LOOMIS, A. RYDER AI-lD C. GEORGE, Arch, Biochem., 20 (1949) 4446 R. E. MAXWELL, V. S. NICKEL AI-lD V. LEWANDOWSKI, Biochem, Biophys. Res. Commun., 7 (19 62) 5°· 7 H. RINDERKI-lECHT, Nature, 193 (1962) 167. 8 K. C. ROBBII-lS AI-lD L. SUMMARIA, ]. Bioi. Chem., 238 (1963) 952. 9 G. H. BEAVEN AI-lD E. R. HOLIDAY, in M. L. ANSOI-l, K. BAILEY AI-lD J. T. EDSALL, Advances in Protein Chemistry, Vol. 7, Academic Press, N.Y., 1952, p. 376. 10 N. ALKJAERSIG, A. P. FLETCHER AI-lD S. SHERRY, J. Bioi. Chem., 234 (1959) 832. II K. EGEBLAD AND T. ASTRUP, Proc. Soc. Exptl. Bioi. Med., II2 (1963) 1020. 12 M. N. RICHARD AI-lD B. E. SAI-lDERS, Can. ]. Biochem. Phvsiol.. 41 (1963) 211. 13 W. TROLL, S. SHERRY AND J. WACHMAI-l, j. Bioi. Chem., 208 (1954) 85· 14 D. L. KLINE, Yale J. Bioi. Med., 26 (1954) 365. 15 A. D. McLAREN, Enzymologia, 26 (1963) 238. 16 ]. L. WEBB, Enzyme and Metabolic Inhibitors, Vol. I, Academic Press, N.Y., 196 3. I7 S. Br.tx, Acta Med. Scand., 172 (1962) Suppl. 386. 18 F. B. ABLOI-lDI, J. J. HAGAN, M. PHILIPS AI-lD E. C. DERENZO, Arch. Biochem. Biophys., 82 (1959) 153· 19 A. SJOERDSMA AND 1. M. NILSSON, Proc, Soc. Exptl. Bioi. Med., ra3 (1960) 533· 20 W. DOLESCHEL, W. AUERSWALD AND A. VON LUTZOW, Thromb. Diath. Haemorrhg., 8 (1962) rOI. 21 D. E. KOSHLAND, JR., Federation Proc., 23 (1964) 719. 22 British Patent 897,539. April 29, 1960.
Biochim, Biophys, Acta, 99 (19 6 5) 34 2-35 1
34 2
EFFECTS OF SOME AMINO ACIDS ON THE INHIBITION OF PLASMIN BY ANTIPLASMIN
RICHARD E. MAXWELL, VERA LEWANDOWSKI AND VIOLET S. NICKEL Research Laboratories, Parke, Davis and Company, Ann Arbor .Mich, (U.S.A.j (Received November 24th, 1964)
SUMMARY
Variations in inhibition of plasmin (EC 3-4+14) and potentiation of antiplasmin by s-arninocaproic acid were studied as functions of relative concentrations, reaction times, and type of plasmin preparation. tS-Aminovaleric acid and y-aminobutyric acid were also potentiators of antiplasmin inhibition. Lysine and ornithine, as well as s-aminocaproic acid, could be shown either to enhance plasmin activity, or act as potentiators of antiplasmin at higher concentrations. Arginine and citrulline did not appear to act as potentiators. Lysine methyl ester and p-toluenesulfonylarginine methyl ester were effective antagonists of antiplasmin at appropriate concentrations.
INTRODUCTION
Certain reversible inhibitors of plasmin (EC 3.4-4.14) protected the latter from irreversible inactivation by antiplasmin'. However, it was found that s-aminocaproic acid, while being an effective and easily reversible inhibitor, potentiated the action of antiplasmin at the concentrations studied. In view of the theoretical implications of these observations, and the proposed clinical applications of plasmin and s-aminocaproic acid, additional experiments with s-aminocaproic acid and related structures were indicated. MATERIALS AND METHODS
Plasmin preparations Kline-type plasmin was obtained as previously described", Euglobulin-type plasmin was isolated from human serum, all steps being carried out at 0-5°. Euglobulins were precipitated by I :10 dilution with water and adjustment to pH 5.4 'with acetic acid, collected by centrifugation and frozen until needed. About 25 g of the Abbreviations: TAME, p-toluenesulfonylarginine methyl ester; LME, lysine methyl ester.
Biochim. Biophys, Acta, 99 (19 65) 34-2-35 1
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
343
frozen paste was dispersed with a mechanical blendor into 300 ml 0.05 M acetate buffer (pH 5-4). After centrifuging for IS min at 10 000 X g, the extraction was repeated with ISO ml acetate buffer. The sediment was dispersed into 300 ml 0.1 satd. (NH4)2S04, and the pH was raised to 8.0 with drop-wise addition of 1.0 N NaOH and rapid mechanical stirring. After further stirring for 30 min, the mixture was centrifuged for 30 min at 10 000 X g, the supernatant was filtered through glass wool, and the filtrate was frozen overnight. After thawing in cold tap water, insoluble material was removed by centrifuging as before, and a fraction precipitating between 0.25 and 0.3 satd. (NH4)2S04 was collected and dissolved in 50 ml of TrisNaCl buffer 2. After dialysis for 48 h vs. 5 2-1 changes of the latter buffer, any remaining solids were removed by centrifuging at 20 000 X g for 30 min. This procedure yielded about I g of protein from 5 g in the original paste, with a 4- to 5-fold increase in specific activity and apparently complete recovery of total activity, a result probably due to the presence of inhibitory material in the original euglobulin, since some discarded fractions had activity. These preparations had very low spontaneous activity, were stable for months stored as frozen solutions, and served as convenient starting material for further purification. For conversion to plasmin, the plasminogen was diluted to a concentration of 10 absorbance units per ml (i.e., that concentration which would have an absorbance of 10.0 cm ? at 280 mf.l) in Tris-NaCI buffer containing 500 Ploug units" of urokinase per ml. After IS min at 37°, the euglobulin-type plasmin was frozen in aliquots. The specific activity was about the same as that of Kline-type plasmin, or 4-5 RPMI units- per mg of protein, if one assumes E~~~m = 14.0 at 280 mf.l. The urokinase used was obtained from Leo Pharmaceutical Products, Copenhagen, and had an activity of 5000 units/mg. A ntiplasmin Antiplasmin was isolated from bovine serum", Bovine antiplasmin was used, since similar fractionation procedures applied to human serum consistently failed to yield material of high potency. In several experiments, unfractionated human serum was used as an effective source of human antiplasmin. Chemicals Chemicals were of the highest purity obtainable from various commercial sources. All amino acids and derivatives with asymmetric centers were of the Lconfiguration. Fibrinogen and thrombin Human fibrinogen and bovine thrombin were obtained as previously described 6 , except that the fibrinogen was prepared from Human Fibrinogen Grade L, supplied by A. B. Kabi, Stockholm. This product was suspended in Tris-NaCl buffer and dialyzed against a large volume of the latter for 8 h at 37°. The Darco G-60 treatment was then applied, and was consistently effective in removing plasminogen contamination. These preparations were 95-98% clottable by thrombin, and clots showed no lysis at 37° for 24 h in the presence of 400 units of urokinase per ml. Fluorescein-labeled fibrinogen was prepared by modification of the method of RINDERKNECHT 7. Fibrinogen at 10 mg/ml in 0,175 M Na 2CO a buffer containing Biochim, Biophys. Acta, 99 (19 65) 34 2-35 1
344
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
0.9% NaCl (pH 8.8) was shaken at 25° for 30 min with 4 mg/ml 10% fluorescein isothiocyanate on celite. After removal of the celite by centrifugation, excess fluorescein was removed and the labeled fibrinogen transferred into Tris-NaCI buffer with a column of Sephadex G-2S. The fibrinogen was quantitatively recovered with no significant change in clottability or resistance to urokinase, but the clots were somewhat more susceptible to plasmin action.
Fibrinolytic measurements A z-phase system with I ml clots and I ml fluid in vials of 2S mm diameter was employed and results were expressed as per cent lysis of the clotl,2. In experiments involving serum or TAME, which interfered with the usual measurements in the ultraviolet region, fluorescein-labeled clots were used and the lysed material was determined at the activation wavelength of 500 m/-, and fluorescence wavelength of 530 mp,. Protein levels of plasmin preparations are expressed in terms of absorbance, one absorbance unit being that amount with an absorbance of I ' 10-8 cm- l at 280 mfJJ. The value for the extinction coefficient of plasmin is controversial", Absorbance values were corrected by readings at 320 ID/-' and 360 m/-'9.
40
30 .~
z-"' 15 u
....c::
s
~ 20
10
~==~==
30
/
__
. l - _... _.l
60
LySIS
90
J
120
period (min)
Fig. 1. Kinetics of inhibition of euglobulin-type plasmin (EP) by s-aminocaproic acid (EAC). /::,.-l::" 100 absorbance units EP; A-A, 100 absorbance units EP + 0.,5 flmole EAC; 0-0, 50 absorbance units EP; e-e, 50 absorbance units EP + 0·5 flmole EAC; 0-0,25 absorbance units EP; . - . , 25 absorbance units EP + c.y zzrnole EAC.
Biochim. Biopbys, Acta, 99 (19 65) 342-351
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
345
RESULTS
Studies with e-aminocaproic acid Fig. I serves to illustrate the general course of the lysis of the fibrin substrate in the experimental arrangement used, at different levels of plasmin over a z-h period. Inhibition by e-aminocaproic acid was reversible, and it is evident that the degree of inhibition was a function of the ratio of plasmin to e-aminocaproic acid, and of the time of observation at a given plasmin level. As seen in Fig. 2, similar fibrinolytic levels of euglobulin-type plasmin and Kline-type plasmin varied signi20
n
Euglobulin type plasmin
10
o Vl
~ .... .Q u
....c:
Kline type
OJ
l:
~ 20
10
o
~
o
3
~
l 5
10
g-Amlnocaprolc (IJ-nnoles)
Fig. 2. Effects of mixtures of s-arninocaproic acid and antiplasmin (AP) on 300 absorbance units euglobulin-type plasmin and 333 absorbance units Kline-type plasmin. Plasmin and plasmin plus inhibitor mixtures were preincubated for 2 h at 37° and then placed over the clots for a 30-min period of lysis. Left bar, no AP; center bar, 25 pg AP; right bar, 50 fig AP.
ficantly in their sensitivity to s-aminocaproic acid, and to mixtures of s-aminocaproin acid and antiplasmin. Inhibition of euglobulin-type plasmin by antiplasmin was potentiated by the lowest level of s-aminocaproic acid tested in this particular experiment, while potentiation of Kline-type plasmin inhibition was not found at these inhibitor levels and at this period of interaction, In fact, as also reported by others10,1.l, enhancement of activity was noted at lower s-aminocaproic acid levels with both Biochim: Biophys, Acta, 99 (19 65) 34~-351
R . E. MAXWELL, V. LEWANDOWSKI , V. S. NICKEL
TABLE I IR REVERS I B LE INHIBITION OF I(LINE-TYPE PLASulIN
Mixtures with K line-type p lasmin (333 absorbance u nits) were preincubated w ith fibrin.
2
Inh ibitors
Per cent clot lysis after 22 II
None s-aminocaproic acid (10 }lmoles) AntipJasmin (100 Itg ) s-aminocaproic acid ( 1 0 p,molcs)
100
h before contact
95
+ antiplasrnin
20
( 100
Ilg)
3
Kline-typ e pla smin and euglobulin-t y pe plasmin ; as reversal of initial inhibition pro ceede d (Fig. r), t he ra te of lysis in the presence of s-aminocaproic acid becam e greater than in its absence. At higher levels of antiplasmin , potentiat ion by s-aminocaproic acid an d irreversible inact ivation of Kline-type plasmin was observe d.'. This is further demonstrat ed in Table I, the data in dicat ing insig nificant reversal of anti plasmin or antiplasmin + s-aminocaproic acid inhibition even after 22 h of contact with the fibrin substrate. Fig. 3 shows earlier stages of the interaction and serves to illustrate the marked differences in the kinetics of progressive irreversible inhibition by antipl asmin , and t h e reversal of initial inhibiti on by s-aminocaproic acid . It is also apparent tha t t he combination of s-ami nocaproic acid and antiplasmin broug ht about almost im mediat e inhib ition. 80 r
'0; ~
60
..... 0 u ..... c 40 ~
L
n,
20
Lysis period
(mi n)
Fig. 3. Kinetics of inhibition of 333 absor bance units Kline-type plasmin (KP) by r o urnoles s-aminocaproic acid (EAC) and 200 f.tg antiplasmin (AP) . 0 -0, KP; 0-0, KP AP; 6.-'/::,., KP EAC ; V - V , KP AP + EAC.
+
+
+
Other amino acids Lysine, like s-aminocaproic acid, has also been found to stabilize or enhance p lasmin activity at lower concentrations or produce inhibition at higher concentrations- ", Therefore, a range of levels of lysine was examined in the presence of two con centrations of anti plasmin; ornithine, citrulline, and arginine were included for comparison. The results (Fig. 4) indicated t hat ornithine, lysine , and arginine had a Biocbim, B iophys, Acta , 99 (19 65) 34 2 -35 1
347
THE INHIBITION OF PLASMIN BY ANrIPLASMIN Ornithine
Lysine
....S2 \J
Citrulline
I~!f~~~ 5
10 15 20 Amino acid level (I-l. moles)
Fig. 4. Effects of amino acids on plasmin and on inhibition by antiplasmin (AP). Mixtures were preincubated for 2 h at 37° and then placed over the clots for a 3D-min period of lysis. Bars represent results with 333 absorbance units Kline-type plasmin: left bar, no AP; center bar, 25 ~Ig AP; right bar, 50 fig AP. The lines similarly represent results with 300 absorbance units euglobulin-type plasmin.
stabilizing effect in the absence of antiplasmin, ornithine and lysine being the most effective. In the presence of antiplasmin, however, potentiated inhibition was evident at higher levels of ornithine and lysine. As in the case of s-aminocaproic acid interaction, euglobulin-type plasmin was more sensitive than Kline-type plasmin to this potentiation. A kinetic study of the euglobulin-type plasmin (50 flg antiplasmin and 10 flmoles lysine) system over a period of 150 min gave no indication of reversal in this period of time. The above experiments involved a preincubation period in the absence of fibrin, in order to assess the stabilizing effects of the amino acids and their interaction with lower levels of antiplasmin. It was also of interest to determine the effects of various compounds initially in the presence of fibrin and a higher antiplasmin level, and over a sufficiently long time to allow for reversal of initial inhibition by reversible inhibitors. Table II summarizes results with several amino acids, and it is evident that the potentiated inhibitions with lysine and ornithine were still significant after 5 h of contact with the fibrin. s-Aminocaproic acid and two other structures with activity in this test, Cl-aminovaleric and y-aminobutyric acids, are included in the table for comparison. Biochim, Biophys, Acta, 99 (1965) 34 2-35 1
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
TABLE II POTENTIATION OF ANTI PLASMIN INHIBITION OF KLINE-TYPE PLASMIN
Mixtures containing 333 absorbance units Kline-type plasmin with or without 200 tLg antiplasmin were added over the clots without preincubation and incubated for 5 h. At this time, all of the mixtures without antiplasmin had given 100% lysis of the clots. ---_._------.---~---------
Compound added
Level
PC1'
cent clot
( umoles ) lysis with aniiplasmin present
None s-Aminocaproic acid o-Aminovaleric acid y-Aminobutyric acid Lysine Ornithine Arginine Citrulline
10 10
22
SO
30 30
50 50 50 50
42 58 61
Lysine and arginine esters Lysine and arginine esters are plasmin substrates and stabilizers, and competitive inhibitors of plasmin proteolytic activity13,l4. It was postulated, therefore, that they might be able to antagonize the action of antiplasmin and still allow fibrinolysis to take place, as previously found for some other reversible inhibitors", This could be readily shown (Fig. 5), provided proper account was taken of time and concentration parameters. The dissociation of plasmin-LME in the presence of fibrin was relatively rapid, so that stabilization and antiplasmin antagonism became evident in 30min oflysis. Plasmin-TAME dissociatec1less readily, so that the effective antagonism of antiplasmin by TAME could be overlooked by limiting observations to an initial 30-min period of lysis, but was apparent after I h of lysis. These experiments are representative of numerous others which led to the not unexpected conclusion that only within limits was it possible to balance higher levels of reversible inhibitors against higher levels of antiplasmin, and to increase the effectiveness of the stratagem by use of reversible inhibitors with lower K, values (e.g., TAME .!!l
50
'"
~ 40
+'
a
"0 +'
30
c
u
20
10
L
0
25
0 LME ( f-io moles)
20 TAME
(t: moles)
Fig. 5. Antagonism of antiplasmin (AP) by LME and TAME. Mixtures containing 333 absorbance units Kline-type plasmin were preincubated for :
h.
Bioohim, Biophys, Acta. 99 (19 65) 34:
349
THE INHIBITION 011 PLASMIN BY ANTIPLASMIN
TABLE III EFFECT OF AMINO ACIDS ON HUMAN SERUM INHIBITIOH
Systems containing 300 absorbance units euglobulin-type plasmin (EP) or 333 absorbance units Kline-type plasmin (KP) were preincubated 2 h at 37° and then placed over the clots for a 30min lysis period.
Enzyme
Amino acid added
Level Clot lysis (%) at serum level of (flmoles) 0 0.025 ml 0.05 ml
-----~-
EP EP EP EP EP EP EP KP KP KP KP
None Ornithine Ornithine Lysine Lysine Citrulline Arginine None Lysine Lysine Lysine
25 100 10 50 10 50 5 50 100
24 32 26 31 20 30 29 23 30 36 29
16 19 10 18 6 17 20 12 23 21 12
8 10 2 10 2 10 14 9 17 8 5
cf. LME) together with longer lysis periods. As time and inhibitor concentrations were increased, larger fractions of the plasmin were inevitably destroyed before becoming effectively associated with fibrin.
Experiments with serum Since the above experiments were done with isolated bovine antiplasmin of fairly high potency, it was desirable to compare unfractionated human Serum as the source of antiplasmin activity. Table III shows representative results with a selection of levels of amino acids. Qualitatively, the findings were similar to those with bovine antiplasmin : stabilization by all four compounds at lower concentrations and lower 100
80
~
..... 60
~
..... c: <1J
u
L
40
<1J
0..
20
~'-----:!::------=l:---_-L--
30
60
90
__.J
120
Lysis period (min)
Fig, 6. Potentiation of serum inhibition of 333 absorbance units Kline-type plasmin (KP) by s-arninocaproic acid (EAC). Mixtures were placed over clots without preincubation. 0-0, KP; 6.-/:::" KP + 0.1 ml serum; 0-0, KP + 5flmoles EAC; V-V'. KP + 0.1 ml serum and 5 p,moles EAC.
Biochim. Biophys, Acta, 99 (1965) 342-351
350
R. E. MAXWELL, V. LEWANDOWSKI, V. S. NICKEL
serum levels, but a decrease in favorable effect or even potentiation of inhibition at higher ornithine and lysine concentrations. Again, quantitative variations were found in comparing the two plasmin preparations, as illustrated with lysine. Fig. 6 is an example of s-aminocaproic acid potentiation of serum inhibition in the initial presence of fibrin. DISCUSSION
The present model of the fibrinolytic system, including reversible, irreversible, antagonistic and potentiated inhibitions, as well as the phase separation of the enzyme solution from the insoluble substrate, is not designed for facile analysis of kinetics and mechanisms. Conventional Michaelis-Menten kinetics do not necessarily apply, and rates of reactions are determined by adsorptive factors at the fibrin surface as well as by time-dependencies of interactions in the fluid phase as compared to those at the interface. Possible quantitative approaches to some of the kinetic problems in less involved heterogeneous or multiple-inhibitor systems have been discussed by McLAREN 15 and WEBB 16 . Qualitatively, it is apparent that even in the relatively simple case of a plasmin-reversible inhibitor mixture (or of plasmin + slowly acting irreversible inhibitor with which the substrate competes kineticallyw) the degree of inhibition can be a complex function of concentrations of inhibitor, substrate and enzyme, as well as of the particular time of measurement of the reaction. Conventional kinetic analyses, if based on a single reaction time, may be of little significance. It is presumed that these complexities, along with many others that could be added, exist in vivo, and it is hoped that examination of some of the variables may aid in the interpretation of other experiments in which similar considerations could apply in part. It has been reported that e-aminocaproic acid was a specific inhibitor of plasminogen activation 10, that s-arninocaproic acid was equally inhibitory after activation had taken place-t, that s-aminocaproic acid was a fairly potent inhibitor of plasmin fibrinolysis 18, or that s-aminocaproic acid did not inhibit plasmin fibrinolysis at all l D. Additional contradictory results could be cited, but these are adequate to illustrate the situation. The quoted studies cover a remarkable range of time factors, enzyme to inhibitor ratios, and assay systems, often within the same investigation. At least some of the inconsistencies might be rationalized on the basis of their being different aspects of the reversible inhibition of plasmin or of plasminogen activation. In the case of heterogeneous systems 17 ,19, as demonstrated in the present study also, the effective substrate concentration, being limited as a function of the surface area in contact with the fluid phase, is far less than in comparatively homogeneous caseinolytic or esterolytic assays 10, and it is possible to obtain the impression that a given reversible inhibitor is either potent or impotent, depending on time of observation and initial enzyme and inhibitor concentrations. In the study of activation systems in which the end point of the assay depends on plasmin activity in the presence of the added s-aminocaproic acid-", the inhibitor to plasmin ratio is obviously very high initially, and it is hardly surprising that the inhibition can be greater in comparison to that obtained with some relatively large, arbitrarily selected level of preformed plasmin. It is also now apparent that s-aminocaproic acid inhibition in blood or plasma systems can be markedly affected by the Biocbim. Biophys. Acta, 99 (19 65) 34 2-35 I
351
THE INHIBITION OF PLASMIN BY ANTIPLASMIN
levels of antiplasmin or perhaps other inhibitors which may be potentiated. This potentiation may help explain the apparently greater activity of s-aminocaproic acid in vivo as compared to some in vitro tests with purified componentsw, and it may be hazardous to conclude that s-aminocaproic acid inhibition per se can be evidence for distinction between activator activity and low levels of preformed plasmin. It has been frequently noted that low concentrations of s-aminocaproic acid or lysine enhance rather than inhibit plasmin activity l O,12 . It is apparent from the present and previously reported results 1 that other related and unrelated reversible inhibitors may produce similar enhancement; the simplest interpretation would be on the basis of stabilization of the plasmin or its protection from irreversible inhibitors that might be present, although more complicated mechanisms can not be excluded. An explanation of concentration effects and structural variations that lead to potentiation or antagonism of antiplasmin is of course not available at this time. Intellectually, some satisfaction may be obtained by visualizing interactions at or near a flexible active site which may result either in interference between inhibitors or enhancement of irreversible attachments-. Conceivably, additional correlations of structure with potentiating or antagonistic effects might eventually contribute to a further understanding of the characteristics of such a mobile active site. Proposals have been made for the use of e-aminocaproic acid, lysine and other amino acids, or lysyl and arginyl esters in the formulation offibrinolytic products 12 , 14 , 22 . While the relevance of the present investigations to events in vivo is naturally speculative, the possible influence of substances which may either enhance plasmin activity or potentiate its inhibition, depending on time and concentration parameters, should be kept in mind. REFERENCES I R. E. 2 R. E.
MAXWELL, V. LEWANDOWSKI AND V. S. NICKEL, Life Sciences, 4 (1965) MAXWELL AND V. LEWAI-lDOWSKI, Anal. Biochem., 4 (1962) 407. 3 PLOUG AI-lD N. O. KJELDGAARD, Biochim, Biophys, Acta. 24 (1957) 278. 4 W. BAUMGARTEI-l, C. M. AMBRUS, K. B. MCCALL AI-lD R. B. PEI-lNELL, Am.
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]. Cardiol., 6 (1960) 447. 5 E. C. LOOMIS, A. RYDER AI-lD C. GEORGE, Arch, Biochem., 20 (1949) 4446 R. E. MAXWELL, V. S. NICKEL AI-lD V. LEWANDOWSKI, Biochem, Biophys. Res. Commun., 7 (19 62) 5°· 7 H. RINDERKI-lECHT, Nature, 193 (1962) 167. 8 K. C. ROBBII-lS AI-lD L. SUMMARIA, ]. Bioi. Chem., 238 (1963) 952. 9 G. H. BEAVEN AI-lD E. R. HOLIDAY, in M. L. ANSOI-l, K. BAILEY AI-lD J. T. EDSALL, Advances in Protein Chemistry, Vol. 7, Academic Press, N.Y., 1952, p. 376. 10 N. ALKJAERSIG, A. P. FLETCHER AI-lD S. SHERRY, J. Bioi. Chem., 234 (1959) 832. II K. EGEBLAD AND T. ASTRUP, Proc. Soc. Exptl. Bioi. Med., II2 (1963) 1020. 12 M. N. RICHARD AI-lD B. E. SAI-lDERS, Can. ]. Biochem. Phvsiol.. 41 (1963) 211. 13 W. TROLL, S. SHERRY AND J. WACHMAI-l, j. Bioi. Chem., 208 (1954) 85· 14 D. L. KLINE, Yale J. Bioi. Med., 26 (1954) 365. 15 A. D. McLAREN, Enzymologia, 26 (1963) 238. 16 ]. L. WEBB, Enzyme and Metabolic Inhibitors, Vol. I, Academic Press, N.Y., 196 3. I7 S. Br.tx, Acta Med. Scand., 172 (1962) Suppl. 386. 18 F. B. ABLOI-lDI, J. J. HAGAN, M. PHILIPS AI-lD E. C. DERENZO, Arch. Biochem. Biophys., 82 (1959) 153· 19 A. SJOERDSMA AND 1. M. NILSSON, Proc, Soc. Exptl. Bioi. Med., ra3 (1960) 533· 20 W. DOLESCHEL, W. AUERSWALD AND A. VON LUTZOW, Thromb. Diath. Haemorrhg., 8 (1962) rOI. 21 D. E. KOSHLAND, JR., Federation Proc., 23 (1964) 719. 22 British Patent 897,539. April 29, 1960.
Biochim, Biophys, Acta, 99 (19 6 5) 34 2-35 1