[32] Human α2-antiplasmin

[32]

HUMAN a2-ANTIPLASMIN

[32]

Human

395

a2-Antiplasmin ~

By BJORN WIMAN Introduction For many years it was believed that a2-macroglobulin and al-protease inhibitor were the major inhibitors of plasmin in plasma, z H o w e v e r , a few years ago a new and very efficient plasmin inhibitor in plasma was identified by several groups independently of each other. :~-~ Mfillertz 7 and also Collen and co-workers s suggested the existence of plasmin in complex with an unknown inhibitor in urokinase-activated plasma. This complex was later isolated and their suggestions verified. 3'4 Aoki and von Kaulla 9 studied an inhibitor of plasminogen activation, which later turned out to be a very efficient inhibitor of plasmin. 5 They showed that this inhibitor has affinity for plasminogen and this was used as step 4 in their five-step purification p r o c e d u r e 2 They were also the first to show that the inhibitor forms a very stable stoichiometric 1 : 1 complex with plasmin that is devoid of enzymatic activity. 5 Saldeen and co-workers ~ also demonstrated a fibrinolysis inhibitor with affinity for plasminogen that also turned out to be identical with o~z-antiplasmin. Name Different names have been used to designate this plasmin inhibitor in plasma, for example: primary plasmin inhibitor, '~ o~z-plasmin inhibitor, 5 antiplasmin, 4 primary fibrinolysis inhibitor, 0 and a2-antiplasmin. I° The international committee on thrombosis and hemostasis, subgroup on inhibitors of fibrinolysis, proposed the name a2-antiplasmin for the fastacting plasmin inhibitor in plasma, l° In accordance with this proposal we will use the name az-antiplasmin throughout this communication. In part supported by the Swedish Medical Research Council (Project No. 05193). A. Rimon, Y. Shamash, and B. Shapiro, J. Biol. Chem. 241, 5102 (1966). :~ S. Miillertz and I. Clemmensen, Biochem. J. 159, 545 (1976). 4 D. Collen, Eur. J. Biochem. 69, 209 (1976). 5 M. Moroi and N. Aoki, J. Biol. Chem. 251, 5956 (1976). " L. Bagge, I. BjTrk, T. Saldeen, and R. Wallin, Forensic Sci. 7, 83 (1976). r S. Miillertz, Biochem. J. 143, 273 (1974). D. Collen, F. DeCock, and M. Verstraete, Thromb. Res. 7, 245 (1975). "qN. Aoki and K. N. von Kaulla, Am. J. Physiol. 220, 1137 (1971). 10 Subgroup on Inhibitors, International Committee on Thrombosis and Haemostasis, Thromb. Haemostasis 39, 524 (1978).

METHODS IN ENZYMOLOGY,VOL. 80

Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181980-9

396

THE PLASMIN SYSTEM

[32]

Assay Different methods have been described for the specific determination of a2-antiplasmin, including immunochemical methods and procedures for measuring a2-antiplasmin activity. Immunochemical Assay Electroimmunoassay is the most frequently used immunochemical method for the determination of the a2-antiplasmin concentration, in which monospecific antisera produced in rabbits or in goats is used. ~'v' The method works well in purified samples as well as in plasma or serum samples. It has to be kept in mind, however, that the method also measures inactive forms of a2-antiplasmin (e.g., when complexed to plasmin). This is no problem under normal physiological conditions when no or very small amounts of plasmin-a2-antiplasmin complex has formed. However, in patients with an extensive activation of the fibrinolytic system (e.g., patients undergoing thrombolytic treatment or patients with intravascular coagulation and secondary fibrinolysis), falsely high values will be obtained. Functional Assay Several procedures for measuring a2-antiplasmin activity have been described. They all measure a decrease in plasmin activity after addition of an a2-antiplasmin sample to a specified amount of plasmin, using either synthetic substrates or clot-lysis methods? '~'6'1~ 13 Procedures using the synthetic-peptide substrate D-Val-Leu-Lys-Nan (S-2251) are most frequently and conveniently used. ~1-~3 Reagents Bt(ffer. O. 1 M sodium phosphate buffer, pH 7.3 Substrate Solution. D-Valine-L-Leucine-L-lysine-p-nitroanilide (DVal-Leu-Lys-Nan, S-2251, Kabi Diagnostica, Stockholm, Sweden), 6 mM dissolved in the phosphate buffer Plasrnin. A stable and soluble plasmin preparation is absolutely essential for a reliable result. Plasminogen is dissolved in 0.1 M sodium phos'~ B. Wiman and D. Collen, Eur. J. Biochem. 78, 19 (1977). v-, D. Collen, J. Edy, and B. Wiman, ill "Chromogenic Peptide Substrates: Chemistry and Clinical Usage" (M. F. Scully and V. V. Kakkar, eds.), p. 238. Churchill-Livingstone, Edinburgh and London, 1980. v.~A.-C. Teger-Nilsson, P. Friberger, and E. Gyzander, Stand. J. C/in. Lab. Invest. 37, 403 (1977).

[32]

HUMAN~2-ANTIPLASMIN

397

phate buffer (pH 7.3) containing 1 mM 6-aminohexanoic acid and 25% glycerol, to a concentration of about 20 mg/ml. Activation is performed by addition of streptokinase to a final concentration of 10.000 units/ml, and the sample is subsequently incubated for 2 hr at 0°C. The exact plasmin concentration is determined by active-site titration with p-nitrophenylp'-guanidinobenzoate, as described by Chase and Shaw, 1~ with the exception that the veronal buffer preferably should also contain 10 mM 6-aminohexanoic acid. The plasmin stock solution is stored frozen at -80°C in aliquots, under which conditions it is stable for many years. T M Prior to use it is diluted to a concentration of 10.0 /xM with ice-cold 0.1 M sodium phosphate buffer (pH 7.3) containing 25% glycerol and kept in an ice bath throughout the experiments. Procedm'e

Ten microliters of 10/zM plasmin solution is mixed in a cuvette with 50 txl az-antiplasmin solution (diluted to a concentration of about 1 /zM, which equals the concentration in normal plasma) and immediately diluted with 890/zl of 0.1 M phosphate buffer (pH 7.3) at 25°C, followed by the addition of 50/zl of 6 mM substrate (D-Val-Leu-Lys-Nan, final concentration 0.3 mM). The absorbance at 410 nm (or 405 nm) is spectrophotometrically recorded at 25°C for about 1 min. Blanks are run with buffer instead of o~2-antiplasmin solution. Plasmin of a final concentration 100 nM is expected to give an increase of A410 at 25°C of 0.39 A / m i n . lr If pure or partially purified o~2-antiplasmin samples are assayed, a straight relationship is obtained between the decrease in AA410 and the concentration of ~2-antiplasmin (provided that plasmin is still in excess). The ~2antiplasmin concentration (CAp) in nmol/liter can thus be calculated from the formula:

(z~kAblank -- ~l.sample ) X | 0 0 X f/0.39

where AA is the change in absorbance at 410 nm/min without o~2antiplasmin (blank) and with a2-antiplasmin (sample) and f is the dilution factor in the cuvette. When testing plasma samples, the best results are obtained when less than 50% of the plasmin is inactivated. Samples containing high ~2-antiplasmin concentration should thus be diluted to fulfill these conditions. Alternatively, a standard curve can be constructed by ~4 T. Chase, Jr., and E. Shaw, this series, Vol. 19, p. 20. ~:' B. Wiman, Eur. J. Biochem. 76, 129 (1977). t" B. Wiman and D. Collen, Eur. J. Biochem. 84, 573 (1978). ,7 B. Wiman, Thromb. Res. 17, 143 (1980).

398

THE PLASMIN SYSTEM

[32]

using different amounts (varying between 0 and 75 ~1) of citrated pooled plasma (at least 10 healthy donors). The unknown samples can thereafter be expressed as percentage of the plasma pool taking the value with 50 pJ as 100%. At normal and high concentrations of az-antiplasmin the accuracy of the method is good, but at low concentrations an overestimation is likely to occur, since other inhibitors will then be relatively more important.IS Furthermore plasma samples containing heparin should not be used, since a small but significant amount of the plasmin in this case will bind to antithrombin III. Such antifibrinolytic substances as 6-aminohexanoic acid and tranexamic acid strongly influence the reaction between plasmin and a2-antiplasmin and should therefore be avoided. 13'1~'~ Purification az-Antiplasmin has been shown to interact weakly with the lysinebinding sites in plasminogen? "la'"~ This can be used for afffinitychromatographic purification of the inhibitor, and if followed by D E A E Sephadex and concanavalin A - S e p h a r o s e chromatography, a pure a2antiplasmin preparation is obtained in good yield (30- 40%).11 Alternatively, affinity chromatography can be carried out on a fragment from plasminogen containing the three NH2-terminal triple-loop structures (LBS I) coupled to Sepharose. "° The material obtained from this step is already 80-90% pure, and the major contaminant, fibrinogen or highmolecular-weight (HMW) fibrinogen-degradation products, are easily removed by gel filtration on Ultrogel AcA 44. Procedure A ~J Reagents B t ~ e r . 0.04 M sodium phosphate buffer, pH 7.0 P l a s m i n o g e n - S e p h a r o s e . Human plasminogen (free from substances

containing amino groups) is dissolved in 0.1 M sodium phosphate buffer (pH 7.3) to a final concentration of 5-10 mg/ml and subsequently added to CNBr-activated Sepharose 4B 2l (1 ml of settled gel/ml of plasminogen solution). The coupling is usually completed in a few hours at 5°C with gentle stirring, but if left at 5°C overnight all reactive groups are destroyed 1~D. Collen, N. Semeraro, P. Telesforo, and M. Verstraete, Br. J. Haematol. 39, 101 (1978). "~ U. Christensen and I. Clemmensen, Biochem. J. 163, 389 (1977). ~(' B. Wiman, Biochem. J. 191, 229 (1980). 2t R. Ax6n, J. Porath, and S. Ernback, Nature (London) 214, 1302(1967).

[32]

HUMAN 0t2-ANTIPLASMIN

399

and treatment with NH2OH is then unnecessary. The gel is washed with 0.1 M sodium phosphate buffer (pH 7.3) containing 0.04% (w/w) sodium azide and stored in this solution at 5°C. DEAE-Sephadex A-50. The ion exchanger (Pharmacia, Uppsala, Sweden) is swollen in 0.04 M phosphate buffer containing 0.04% (w/w) sodium azide and stored in this way at 5°C. Prior to use it is washed on a Biichner funnel with the phosphate buffer without sodium azide. Concanavalin A-Sepharose. Concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden) is prepared according to the procedure described by the manufacturers. Plasminogen-depleted plasma is obtained by stirring outdated human citrated plasma with 100 ml lysine-Sepharose per liter of plasma at 5°C for 30 min. The slurry is subsequently filtered through a Biichner funnel.

Purification Procedure Affinity Chromatography on Plasminogen-Sepharose. Cohn fraction I is precipitated from 1 liter of plasminogen-depleted plasma by the addition of 70% ethanol to a final concentration of 10% while simultaneously lowering the temperature from 0°C to -3°C. After gentle stirring for 1 hr and centrifugation, the supernatant is mixed with 250 ml of plasminogenSepharose gel. The suspension is stirred gently at 0°C for 1 hr and the plasminogen-Sepharose is collected on a Biichner funnel. Washing is carried out with about 5 liters of cold 0.04 M sodium phosphate buffer (pH 7.0), and the gel is subsequently packed in a column (20 cm 2 × 12 cm) and washing is continued until the absorbance at 280 nm is below 0.05. Elution is then performed with the phosphate buffer containing 10 mM 6-aminohexanoic acid. A 200- to 400-fold purification of c~2-antiplasmin with a yield of about 50% is typically obtained in this step. DEAE-Sephadex Chromatography. The protein peak obtained from the plasminogen-Sepharose column is applied to a DEAE-Sephadex A-50 column (5 × 5 × 10 cm) equilibrated with 0.04 M sodium phosphate buffer, pH 7.0. Elution is performed with a linear gradient to 0.4 M NaC1 in this buffer. ~2-Antiplasmin is eluted between 0.15 and 0.2 M NaC1 in the second protein peak. The material from this step is about 70% pure and the yield from the starting material is about 40%. Chromatography on Concanavalin A-Sepharose. The c~2-antiplasmin peak from the DEAE-Sephadex column is applied without further treatment to a concanavalin A-Sepharose column (5 cm" × 7 cm) equilibrated with 0.04 M phosphate buffer, pH 7.0. Washing with phosphate buffer is performed until A280 has returned to the baseline and elution of ~2antiplasmin is subsequently obtained by 0.02 M ~-methylmannoside in

400

THE PLASMIN SYSTEM

[32]

phosphate buffer. The purified a2-antiplasmin is dialyzed in the cold against several changes of distilled water and lyophilized. Procedure

B 2°

Reagents Buffer. 0.04 M sodium phosphate buffer, pH 7.0 LBSI-Sepharose. Plasminogen is dissolved in 0. I M NH4HCO3 to a final concentration of about 10 mg/ml and aprotinin is added to a final concentration of 10 #M (60 me/liter). Elastase-Sepharose containing about 5 mg of porcine pancreatic elastase (Sigma, St Louis, Missouri) per milliliter of gel is added to give an enzyme/substrate ratio of 1/100 (w/w) and digestion is carried out at 25°C overnight with gentle stirring. The mixture is filtered through a Biichner funnel and the filtrate is passed through a lysine-Sepharose column (200 ml bed volume for 500 mg of digested material), and washing is continued with the equilibration buffer. The bound fragments are eluted with 50 mM 6-aminohexanoic acid in 0.1 M NH4HCO3 and subsequently chromatographed on a Sephadex G-75 column (800 ml bed volume for a 500-me digest) equilibrated with 0.1 M NH4HCO3. Two peaks are obtained, the first of which represents the three NH2-terminal triple-loop structures of plasminogen (LBSI), The material is dissolved after lyophilization in 0.1 M sodium phosphate buffer (pH 7.3) at a concentration of about 5 mg/ml and coupled to CNBr-activated Sepharose 4B in a similar way as described for plasminogen-Sepharose. Uhrogel AcA 44. Ultrogel AcA 44 is obtained from LKB (Stockholm, Sweden) and used according to the procedure given by the manufacturers. Starting Material. Plasminogen-depleted plasma can be used without further treatment, although fibrinogen interferes slightly with the purification procedure. However, since we normally use the plasma for purification of several other proteins, we have frequently used plasminogen-depleted supernatant Cohn fraction I or plasminogen-depleted supernatant after polyethylene glycol precipitation (final concentration 6%) of the plasma. Purification Procedure Affinity Chromatography oll LBSI-Sepharose. About 1 liter of plasminogen-depleted plasma is stirred with 50-100 ml of LBSISepharose at 0°C for 30 min and subsequently filtered through a BSchner funnel. Alternatively, the plasminogen-depleted plasma or supernatant after ethanol or polyethylene glycol precipitation is slowly passed (about 1 liter/hr) through the LBSI-Sepharose packed in an ordinary Bfchner fun-

[32]

HUMAN O~2-ANTIPLASMIN

401

nel. In this way the yield seems to be somewhat increased. The L B S I Sepharose is washed on the Biichner funnel with about 2-3 liters of 0.04 M sodium phosphate buffer (pH 7.3) and then transferred to a column; washing is continued with the phosphate buffer containing 0.5 M NaC1 until A280 is below 0.05. Elution of c~2-antiplasmin is performed with 10 mM 6-aminohexanoic acid in phosphate buffer and the protein peak is subsequently dialyzed at 0°C against several changes of distilled water. A small precipitate of fibrinogen is occasionally removed by centrifugation and the clear supernatant is thereafter lyophilized. This material is about 80-90% pure c~2-antiplasmin obtained in a yield of about 60%. Gel Filtration on Ultrogel AcA 44. The major contaminant (fibrinogen or H M W fibrinogen degradation products) can easily be r e m o v e d by gel filtration on Ultrogel AcA 44. The c~2-antiplasmin is eluted in the major second peak, which is dialyzed against distilled water in the cold and lyophilized. The obtained preparation is a homogeneous protein according to several chromatographic and electrophoretic criteria and fully active when titrated against plasmin.

Different Forms of ~z-Antiplasmin az-Antiplasmin is a glycoprotein that migrates as an a2-globulin 3-5 cently reported to contain a form of the inhibitor that does not bind to plasminogen-Sepharose. ~'' Using L B S I - S e p h a r o s e , which is much more efficient in binding ~2-antiplasmin than plasminogen-Sepharose, we have been able to confirm that a form of ~2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist even in unfractionated plasma. `-'() This form of the inhibitor, constituting 25-40% of the c~2antiplasmin antigen in plasma, has not yet been purified. It still is an active antiplasmin because it forms a complex with plasmin under highly competitive conditions, as evidenced by crossed immunoelectrophoresis (using an antiserum against ~2-antiplasmin) on a L B S I - S e p h a r o s e treated plasma sample after addition of plasmin to a final concentration of 1 #M. ~0 Properties

Physicochemical Properties c~2-Antiplasmin is a glycoprotein that migrates as an ~2-globulin 3-'~ on electrophoresis. Its molecular weight (MW) has been determined as 65,000-70,000 by both sedimentation equilibrium analysis and sodium e~ I. Clemmensen, in *'PhysiologicalInhibitors of Blood Coagulation and Fibrinolysis" (D. Collen, B. Wiman, and M. Verstraete, eds.), p. 131. Elsevier/North-Holland Publ., Amsterdam and New York, 1979.

402

THE PLASMIN SYSTEM

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TABLE I AMINO ACID COMPOSITION OF DIFFERENT O~2-ANTIPLASMIN PREPARATIONS Amino acid"

A t'

B~

C '~

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Lysine Histidine Arginine Tryptophan

40.9 25.3 41.1 79.3 41.2 30.3 33.5 5.2 21.4 13.2 9.7 73.0 5.2 34.8 23.0 10.3 21.6 7.4

41.3 23.4 43.3 80.3 40.6 31.6 32.2 5.5 23.8 12.6 11.8 75.1 4.9 32.7 18.2 10.1 22.2 7.6

38.6 24.8 39.1 75.9 38.0 29.8 29.9 5.6 30.0 19.0 10.1 75.7 6.8 30.7 21.7 14.0 21.3 8.1

o Figures are given as micromoles of amino acid per 70 mg of protein. Material obtained by procedure A (from Ref. 11). Material obtained by procedure B. ~ Adapted from the result reported by Moroi and Aoki. ~

dodecyl sulfate-polyacrylamide gel electrophoresis on reduced as well as nonreduced samples. T M The sedimentation constant was found to be 3.45 S, T M and the partial specific volume was calculated from the amino acid and c a r b o h y d r a t e compositions as 0.718 ml/g. 11 The Stokes radius and the frictional ratios were calculated as 5.0 nm and 1.8 respectively, in'dicating a very asymmetric or highly hydrated molecule. 11 This is in agreement with gelfiltration data, which have shown M W ~ 90,000. 6,23 Circular dichroism studies have demonstrated a molecule with about 16% a-helix, 19% fl-structure, and 65% r a n d o m coil. 24 The amino acid composition reported by Moroi and Aoki ~ is in excellent agreement with that obtained for our preparations ll'2° (Table I). ~:~ L. Bagge, I. BjSrk, T. Saldeen, and R. Wallin, Thromb. Haemostasis 39, 97 (1978). z~ B. Wiman, T. Nilsson, and I. Sj6holm, Prog. Chem. Fibrinolysis Thrombolysis 5, 302 (1981).

[32]

HUMAN Ot2-ANTIPLASMIN

403

TABLE II NH2-TERMINAE AMINO ACID SEQUENCES OBTAINED FROM INTACT Ot2-ANTIPLASMIN (a), DEGRADED Ot2-ANTIPLASMIN (b), THE 8000-MW PEPTIDE (C), AND

6 PURIFIEDCNBr FRAGMENTS(d-i) a. b. c. d. e. f. g. h. i.

Asn-Gln-Glu-Gln-ValLeu-Val-AsmGln-Glu-Val-Gly-Gly-GlnMet-Ser-Leu-Ser-Gly-PheLeu-Gly-Asn-Gln-X-ProSer-Phe-Val-Val-Leu-ValGln-Ala-Phe-Val-Tyr-Val-Leu Leu-Ala-X-Arg-TyrGlu-Glu-Asp-Tyr-Pro-Gln-Phe-Gly Tyr-Leu-Gln-Lys-Gly-Phe-Pro-Leu-Lys-Glu-Asp-Phe-

E d m a n degradation yielded 1 mol of NH2-terminal asparagine per mole of protein, and c a r b o x y p e p t i d a s e Y digestion indicated leucine as C O O H terminal amino acid. ~l'z~ C a r b o h y d r a t e analysis revealed about 11-14% c a r b o h y d r a t e ? 'll 10 mol sialic acid, 30 tool hexose, and 7 mol glucosamine per mole of c~2-antiplasmin. 11 The single-chain protein seems to be stabilized by 3 disulfide bridges, 24 2 of which can be very easily reduced and S - c a r b o x y - m e t h y l a t e d under nondenaturing conditions. 24 This results in a fully active molecule, as m e a s u r e d both by activity m e a s u r e m e n t and by kinetic analysis. The absorption coefficient, Al~Clcm, has been determined at 6.7011 or 7.05? c~2-Antiplasmin prepared according to the method of Moroi and A o k ? is reported to be very unstable. Thus it quite rapidly loses its activity in solution and lyophilization results in c o m p l e t e inactivation. H o w ever, our preparations 1~'2° are very stable provided that the p H is kept a b o v e 6.0. A solution of c~2-antiplasmin kept at 20°C in 0.1 M sodium phosphate buffer (pH 7.3) containing 0.04% (w/w') sodium azide retained o v e r 90% of the activity after 14 days. l~ F u r t h e r m o r e , no activity is lost on lyophilization. Indeed, lyophilization seems to be the best way of storing c~2-antiplasmin, since repetitive freezing and thawing of pure preparations in solution result in a significant decrease in activity. A m i n o A c i d Sequence

So far only minor parts of the c~2-antiplasmin molecule have been sequenced, including direct sequences of intact and partially degraded inhibitor, the 8000-MW peptide cleaved during the reaction with plasmin "5 (see later), and 6 purified C N B r fragments zG (Table II). A seventh C N B r ::' B. Wiman and D. Collen. J. Biol. Chem. 254, 9291 (1979). ~'; R. Lijnen. D. Collen. and B. Wiman, unpublished results.

404

THE PLASMINSYSTEM

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T A B L E III NH2-TERMINAL SEQUENCES OF THE TRYPTIC PEPTIDES ISOLATED FROM A C N B r FRAGMENT VqlTH A PRESUMABLY BLOCKED NH2-TERMINUS 1. (Set2, Thr, Pro, lie, Leu, Phe) -Leu-Phe-Glu-Asp(Tbr, Pro, Gly) -Phe-Val-Asn-Ser-Val-Arg 2. Glu-Gln-Gln-Asp-Ser-Pro-Gly-Asn-Lys 3. S e r - P h e - L e u - G l n - A s n - L e u - L y s 4. Gly-Phe-Pro-Arg 5. G l y - A s p - L y s 6. Leu-Phe-Gly-Pro-Asp- L e u - L y s 7. Leu-Ala-Arg 8. G l u - L e u - L y s 9. Asn-Pro-Asn-Pro-Ser-Ala-Pro-Arg 10. Leu-Val-(Leu, Pro~) HSer

fragment has been purified, but due to unknown reasons no NH~-terminal amino acid could be determined in this peptide. Nevertheless, all its tryptic peptides were purified and almost completely sequenced '6 (Table III). Peptide CB7-T1 could not be sequenced directly, but the data were obtained from chymotryptic peptides of this particular tryptic peptide. So far, only one small stretch has been found that shows clear homology with antithrombin III and ch-antitrypsin (Table IV). "~6 Reaction with Enzymes In purified systems, c~2-antiplasmin is capable of reacting with and forming stable enzymatically inactive complexes with many enzymes such as plasmin ";'~:' (very fast), trypsin ~;'''~ (fast), chymotrypsin 29 (intermediate), kallikrein 3° (slow), factor X, 3° (slow), urokinase ~ (very slow), and tissue-plasminogen activator ~ (very slow). Nevertheless, inhibition of plasmin is presumably its only physiologically important reaction. 32 Kinetics. The reaction between plasmin and c~z-antiplasmin can be divided into two steps, a very fast reversible second-order reaction followed ~7 T. E. Petersen, G. Dudek-Wojciechowska, L. Sottrup-Jensen, and S. Magnusson, in " T h e

Physiologic Inhibitors of Blood Coagulation and Fibrinolysis'" (D. Collen, B. Wiman, and M. Verstraete, eds.), p. 43. Elsevier/North-Holland Publ., Amsterdam and New York, 1979. ~ M. C. Owen, M. Lovier, and R. W. Carrell, FEBS Lett. 88, 234 (1978). ~ T. Nilsson and B. Wiman, unpublished results. :~" M. Moroi and N. Aoki, J. Biochem. (Tokyo) 82, 969 (1977). :" B. Wiman and P. Wall6n, unpublished results. :~ J. Edy and D. Collen, Biochim. Biophys. Acta 484, 423 (1977).

[32]

HUMAN Ot2-ANTIPLASMIN

405

T A B L E IV H O M O L O G I E S B E T W E E N A C B F R A G M E N T FROM C~2-ANTIPLASMIN ° A N D S T R E T C H E S IN A N T I T H R O M B I N III ~ A N D ~ I - A N T I T R Y P S I N c

C~z-Antiplasmin

L

A n t i t h r o m b i n III T

cq-Antitrypsin

G -

-

-D-

-Y

" Sequence i in Table II. Fro m Ref. 27. From Ref. 28.

by a slower irreversible first-order transition, and schematically represented as: p+A

K-~' pA ~ K-I

pA '

where P is plasmin, A is c~2-antiplasmin, and PA is the complex. 16'19Some of the kinetic constants for the reactions between c~2-antiplasmin and some enzymes are summarized in Table V. ~(~''a'33 The very high rate constant in its reaction with plasmin is dependent on an interaction between one of the so-called lysine-binding sites in the plasmin A chain and a complementary site in a2-antiplasmin. ~'29''~4 Thus 6-aminohexanoic acid in concentrations enough to block all the lysine-binding sites in plasmin decreases the rate of the plasmin-a2-antiplasmin reaction about 100-fold.l~ A similar rate constant is also obtained with low-molecular-weight (LMW) plasmin, which lacks lysine-binding sites? 3 Plasmin substrates, such as o-Val-LeuLys-Nan, also slow down the plasmin-a2-antiplasmin reaction, and a 50% reduction is obtained at a substrate concentration (0.38 mM) that is similar to the Km with plasmin. "; This shows that the active center of plasmin also plays a role in the fast formation of the reversible complex. Struct,ral Changes during Its Reaction with Plasmin. The reaction between plasmin and c~2-antiplasmin leads to the formation of a very stable enzyme-inhibitor complex that cannot be dissociated by reducing or denaturing agents. ~'x"''5':~ The complex is very easily degraded proteolytically.";'"~ Therefore, it is absolutely essential to ascertain inhibitor excess in mechanistic studies of the complex formation. We have shown that a peptide with MW ~ 8000, originating from the COOH-terminal portion of ~z2-antiplasmin, can be isolated from the complex in the presence of :~:~B. Wiman, L. Boman, and D. Collen, Fur. J. Biochem. 87, 143 (1978). :~ U. Christensen and I. Clemmensen, Biochem. J. 175, 635 (1978). :~:' M. Moroi and N. Aoki, Biochim. Biophys. Acta 482, 412 (1977).

406

THE PLASMIN SYSTEM

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TABLE V RATE CONSTANTS FOR THE INACTIVATION OF DIFFERENT PROTEOLYTIC ENZYMES BY O~2-ANTIPLASMIN Enzyme Plasmin I Plasmin II LMW plasmin Trypsin Chymotrypsin

Rate constant (M ~sec ~) 3.8 × 1.8 × 6.5 x 1.8 x 1.0 x

107 107 10~ 10'~ 10~

Ref. 16 16 33 16 29

sodium dodecyl sulfate. 25 A new NH2-terminal amino acid (methionine), in addition to those in the parent molecules, has been demonstrated in intact c c m p l e x e s between plasmin and a2-antiplasmin? 6 Methionine is also the NH2-terminal amino acid of the 8000-MW peptide (Table II), indicating that a peptide bond in the C O O H - t e r m i n a l portion of the inhibitor is cleaved as a result of its reaction with plasmin. "~'3" F u r t h e r m o r e , the plasmin-a2-antiplasmin complex is dissociated in 1.5 M N H 4 O H yielding up to 0.2 mol of active plasmin per mole of complex and a modified form of the inhibitor, which has lost a peptide from its COOH-terminal end. C a r b o x y p e p t i d a s e Y digestion of this modified form of the inhibitor indicated the new C O O H - t e r m i n a l dipeptide, Ala-Leu. 25 The reactive site in a~-antiplasmin may thus be a Leu-Met peptide bond, which is somewhat surprising in view of the specificity of plasmin. On the other hand, if c h y m o t r y p s i n reacts with the same site as plasmin, it m a y still be correct. The data obtained until now indicate that the stabilizing bond in the plasmin-a2-antiplasmin complex is an ester bond between the hydroxyl group of the active-site seryl residue in plasmin and the specific carbonyl group of the leucyl residue in the reactive site of a~-antiplasmin. 36

Preparation of Plasmin-o~2-Antiplasmin Complex Plasmin-a2-antiplasmin complex can be prepared either from purified reagents or from plasma after activation of the plasminogen or after addition of plasmin. Starting from purified reagents, a suitable procedure is as follows: Plasminogen and a2-antiplasmin are both dissolved to final concentrations of 10 mg/ml in 0.! M phosphate buffer, p H 7.3. This gives approximately 25% molar excess of a2-antiplasmin as c o m p a r e d to plasminogen. Urokinase is added to a final concentration of 5000 CTA units/ml and the mixture is incubated at 25°C for 2 hr to obtain complete activation of the a6 B. Wiman, T. Nilsson, and D. Collen, Protides Biol. Fluids 28, 379 (1980).

[32]

HUMAN Ct2-ANTIPLASMIN

407

plasminogen. The mixture is thereafter subjected to gel filtration on UItrogel AcA 44 equilibrated with 0.1 M NH4HCO3, containing 0.25 /xM aprotinin. The major first peak consisting of plasmin-a2-antiplasmin complex is lyophilized. If Glu-plasminogen has been used a complex containing Glu-plasmin is obtained. Preparation of the plasmin-a2-antiplasmin complex from plasma can be performed after activation of the plasma by urokinase (500 IU/ml) for 30 rain at 25°C, or preferably after addition of plasmin (final concentration 1 ~M) to plasminogen-depleted plasma at 5°C and incubation for several minutes. After addition of aprotinin to a final concentration of 1 ~zM, adsorption of the plasmin complexes is performed on lysine-Sepharose (100 ml/liter plasma) in a batch procedure. The lysine-Sepharose is collected on a Bfichner funnel, washed with 0.1 M NH4HCO3 containing 0.25 gM aprotinin, and subsequently packed in a column. Washing is continued until A280is less than 0. I, and elution is then performed with 0.1 M NH4HCO3 containing 0.25 ~M aprotinin and 50 mM 6-aminohexanoic acid. The protein peak, after concentration by ultrafiltration, is passed through an Ultrogel AcA 44 column, equilibrated with 0.1 M NH4HCOa, containing 0.25 tzM aprotinin. Three peaks are obtained, the second of which is the plasmin-a2-antiplasmin complex. The first and third peaks are plasmin in complex with a2-macroglobulin and aprotinin, respectively. :~,24

Physiological Role Turnover studies with radiolabeled a2-antiplasmin in humans indicated a half-life of 2.6 days and a synthetic rate of 1.4 mg/kg/day. The half-life of plasmin-a2-antiplasmin complex was found to be about 0.5 day. 37 The plasma concentration of a2-antiplasmin normally is 1.0 - 0.2 ~M (70 mg/liter). It seems to be a weak acute-phase reactant. Decreased values have been reported in patients with liver disease and with severe intravascular coagulation? s In patients undergoing thrombolytic therapy, the inhibitor may be immediately but temporarily exhausted, resulting in a severe fibrinogenolysis.39 A few patients with a2-antiplasmin deficiency have been described. They seem to suffer from severe lifelong bleeding tendencies. 4° a2-Antiplasmin plays an important role in the regulation of fi:~7D. Collen and B. Wiman, Blood 53, 313 (1979). ~ D. Collen and B. Wiman, Blood 51, 563 (1978). :~'~D. Collen and M. Verstraete, Thromb. Res. 14, 631 (1979). ~ N. Aoki, H. Saito, T. Kamiya, K. Koie, Y. Sakata, and M. Kobakura,J. 877 (1979).

Clin. Invest.

63,

408

THE PLASMIN SYSTEM

[33]

brinolysis. 4~-44 The very high reaction rate of the plasmin-a2-antiplasmin reaction is highly dependent on free active-site and lysine-binding sites in the plasmin molecules. 1~':~3This is not the case with the fibrinolytically active plasmin molecules 45'4~ formed at the fibrin surface by activation of fibrin-bound plasminogen by the tissue-plasminogen activator? 7 These plasmin molecules seem thus to be protected from the action of a2antiplasmin. This hypothesis explains in a plausible way the in vivo selective degradation of fibrin by plasmin. 4~ B. Wiman, in "Fibrinolysis: Current Fundamental and Clinical C o n c e p t s " (P. J. Gaffney and S. Balkov-Ulutin, eds.), p. 47. Academic Press, N e w York, 1978. 42 B. W i m a n and D. Collen, Nature (London) 272, 549 (19781. 4:~ B. W i m a n and D. Collen, in " T h e Physiological Inhibitors of Blood Coagulation and Fibrinolysis" (D. Collen, B. Wiman, and M. Verstraete, eds.), p. 177. Elsevier/NorthHolland Publ., A m s t e r d a m and New York, 1979. 44 D. Collen and B. Wiman, Prog. Chem. Fibrinolysis Thronlbolysis 4, 11 (1979). 4~ B. Wiman and P. Wall6n, Thromb. Res. 10, 213 (19771. 4" B. Wiman, H. R. Lijnen, and D. Collen, Biochim. Biophys. Acta 579, 142 (1979). 47 p. Wall~n, P. Kok, and M. RS.nby, in "Regulatory Proteolytic E n z y m e s and Their Control" (S. Magnusson, M. Ottesen, B. Foltman, K. Dan0, and H. Neurath, eds.), p. 127. Pergamon, Oxford, 1978.

[33] A C o u p l e d P h o t o m e t r i c A s s a y for P l a s m i n o g e n A c t i v a t o r By PATRICK L. COLEMAN and GEORGE D. J. GREEN Introduction Plasminogen activators are important in fibrinolysis, cell transformation, metastasis, and inflammatory responses, 1 and in some systems are under hormonal control. ",3 The great interest in understanding the many roles of plasminogen activator has led to the evolution of several different types of assays for the enzyme designed to meet the particular objectives of the investigators. Among those designed for assay with living cells are the 1'~5I-labeled fibrin-coated tissue culture plates, 4'5 the fibrin-agar overJ. K. Christman, S. C. Silverstein, and G. Acs, in " P r o t e i n a s e s in M a m m a l i a n Cells and Tissues" (A. J. Barrett, ed.), p. 91. Elsevier/North-Holland Publ., A m s t e r d a m and N e w York, 1977. 2 W. H. Beers, S. Strickland, and E. Reich, Cell 6, 387 (1975). 3 S. C. Seifert and T. D. Gelehrter, Proc. Natl. Aead. Sei. U.S.A. 75, 6130 (1978). 4 j. C. Unkeless, A. Tobia, L. Ossowski, J. P. Quigley, D. B. Rifkin, and E. Reich, J. Exp. Med. 137, 85 (1973). :' J. C. Taylor, D. W. Hill, and M. Rogolsky, Exp. Cell Res. 73, 422 (1972).

METHODS IN ENZYMOLOGY,VOL. 8(1

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