66
Biochimica et Biophysica Acta, 535 ( 1 9 7 8 ) 6 6 - - 7 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 37943
A CATALYTIC ROLE FOR HEPARIN EVIDENCE FOR A TERNARY COMPLEX OF HEPARIN COFACTOR THROMBIN AND HEPARIN
M A R K W. P O M E R A N T Z a n d W H Y T E G. OWEN
Department o f Pathology, University o f Iowa, Iowa City, Iowa 52242 (U.S.A.) (Received F e b r u a r y 2nd, 1 9 7 8 )
Summary The interaction of heparin with chemically modified thrombin and heparin cofactor is studied. Amidinated heparin cofactor does not bind to heparinagarose and the reaction rate of the amidinated inhibitor with unmodified thrombin is not affected by heparin. Likewise, thrombin modified with 1,2-cyclohexanedione does not bind to heparin agarose and the reaction rate of the modified enzyme with unmodified inhibitor is not affected by heparin. In the absence of heparin, the modified and unmodified proteins react at the same rate in all possible combinations. Affinity chromatography of diisopropylphosphoryl thrombin on heparin cofactor coupled to Sephadex G-50 is used to study the binding of heparin cofactor and thrombin to heparin. The thrombin for all experiments is tritium-labeled and then inactivated with diispropylfluorophosphate. Thrombin is not bound to heparin cofactor-Sephadex columns. However, after treatment of the columns with a heparin solution, thrombin binds tightly, and is eluted at high ionic strength. Bound thrombin can also be eluted with either excess non-radioactive thrombin or excess free heparin. Heparin-dependent binding of thrombin does not occur if the heparin cofactor-Sephadex is heat-denatured. The ability of heparin to couple solutionphase thrombin to solid-phase heparin cofactor indicates that a ternary complex is formed. Analysis of the binding of the proteins to heparin by a dye displacement method suggests that at least one site on heparin binds to thrombin but not to heparin cofactor. Further support for a catalytic role for heparin derives from the ability of catalytic concentrations of heparin to enhance the rate of hydrolysis of prothrombin by thrombin, another protein pair which bind mutually to heparin.
Abbreviations used are: iPr2PF, diisopropylfluorophosphate; iPr2P-, diisoprolylphosphoryl.
67 Introduction Thrombin in plasma or serum gradually loses its enzymatic activity. This inactivation, as well as that of other proteinases involved in the coagulation cascade is due in part to the presence in plasma of heparin cofactor (antithrombin III, factor Xa inhibitor), a glycoprotein with a molecular weight of about 62 000 [1,2]. The interaction of heparin cofactor and thrombin yields, with 1 : 1 stoichiometry, an enzymatically inactive complex which is stable in the presence of denaturing and reducing agents [1--3]. Thrombin with its active center blocked cannot interact with the inhibitor to form the complex
[2]. Heparin, a highly sulfated mucopolysaccharide anticoagulant, in catalytic concentrations accelerates the rate of inhibition of thrombin by heparin cofactor [4,5] but does not modify the 1 : 1 stoichiometry of the reaction [2,4,5]. The effect of heparin on this reaction distinguishes heparin cofactor from other plasma thrombin inhibitors. Both heparin cofactor and thrombin bind tightly to heparin [6--8]. By chemically modifying heparin cofactor, Rosenberg and Damus [2] have shown that lysyl residues on the inhibitor are required for binding to the highly negative heparin molecule. Blocking these lysines reduced heparin cofactor activity significantly, whilz not affecting the activity of the inhibitor in the absence of heparin. This behavior was interpreted as suggesting that heparin functions as an allosteric effector of the inhibitor, making it more reactive with thrombin. However other studies have shown that when arginyl residues of thrombin are blocked, the rate of inhibition of the modified thrombin by heparin cofactor is likewise unaffected by heparin [9]. On this basis it was suggested that heparin is an allosteric modifier of thrombin. However the latter study employed a crude, poorly characterized.thrombin preparation, and the experimental conditions, especially protein concentrations, were significantly different from those employed by Rosenberg and Damus [2]. Yin and Wessler [10] and Gitel [8] proposed a model for heparin activity in which heparin acts as a catalyst to which thrombin and the inhibitor, a specialized thrombin substrate, mutually bind. Such binding could increase the stability of the putative Michaelis complex, thereby enhancing the rate of formation of the covalent complex. This model is based on the assumption that thrombin and the inhibitor are bound simultaneously to a single heparin molecule. However, since available heparin preparations are highly heterogeneous [11], direct binding studies [8] do not distinguish between two proteins binding mutually to a single heparin molecule as opposed to each protein binding independently to different species of heparin. In the present study, both thrombin and heparin cofactor are modified chemically to abolish their affinity for heparin, without affecting their heparinindependent reactivity. In addition, the binding of thrombin and heparin cofactor is analyzed by affinity chromatography. Evidence is presented that thrombin and heparin cofactor bind to independent sites on a single heparin molecule and that the resultant catalysis of their reaction requires that both proteins bind to heparin.
68 Materials and Methods Heparin (porcine intestinal mucosa), sodium dodecyl sulfate, Sephadex G-50, and proflavine hemi-sulfate were obtained from Sigma Chemical Co., St. Louis, Mo. Proflavine was recrystalized from ethanol before use. Biogel A-15m was purchased from Biorad Laboratories, Richmond, Calif. Cyanogen bromide and diisopropylfluorophosphate (iPr2PF) were obtained from Aldrich Chemical Co., Milwaukee, Wis. iPr2PF was used as a 1 M solution in redistilled dimethylformamide. Methyl acetimidate, 1,2-cyclohexanedione, and 2,4-dinitrofluorobenzene were obtained from Pierce Chemical Co., Rockford, Ill. NaB3H4, spec. act. 2.0 Ci/mmol, was obtained from ICN, Cleveland, Ohio. Prothrombin, thrombin and heparin cofactor were prepared and assayed as described previously [3]. Thrombin was tritium-labeled by introducing 3H into sialic acid residues of prothrombin [ 12,13 ]. After activation and purification [ 3], the [ 3H] thrombin was inactivated with 10 -3 M iPrzPF. The product had a specific radioactivity of 106 dpm/mg protein. Lysyl residues of thrombin and heparin cofactor were amidinated as described previously [14]. Reactions were carried out for 2 0 h at 0°C in 0.25 M sodium borate/NaOH buffer, pH 9.0. The modified proteins were dialyzed in 0.2 M N-ethylmorpholine acetate buffer, pH 7.5. Unmodified lysine was determined by reaction with 2,4-dinitrofluorobenzene. Arginine residues were modified by reaction with 1,2-cyclohexanedione [15]. Modification was carried out for 6 min at 35°C, and the protein was separated from reagents by chromatography on biogel P-10 (Bio-rad Laboratories, Richmond, Calif.). Unmodified arginine was determined by the Sakaguchi reaction [16]. Heparin cofactor was coupled to Sephadex G-50 by the cyanogen bromide method [17]. Fifteen grams CNBr were reacted with 20 ml (packed volume) Sephadex G-50 to couple 5--10 mg heparin cofactor. Coupling was carried out at pH 8.3, and remaining reactive Sephadex was blocked with excess ethanolamine. Coupling yields was 200 pg protein/ml of packed Sephadex G-50, as estimated by absorbance at 280 nm after digestion with trypsin. For affinity chromtography, 1.0 ml (packed volume) of heparin cofactor Sephadex was poured into a silicone (Clay-Adams SilicladR)-coated Pasteur pipette (0.6 cm internal diameter) with silicone-coated glass wool as a bed support. All eluants were buffered with 0.02 M Tris • HC1 (pH 7.5), and contained 0.1% 7-globulin to minimize adsorption of thrombin to glass. Omission of carrier protein reduced recoveries of thrombin by about 50%, in both controls and heparin-treated columns, as well as columns of plain Sephadex G-50. Albumin was slightly less protective than 7-globulin, and was avoided also because of possible contamination of albumin with small amounts of native or degraded heparin cofactor. Samples of 10 drops each (~ 0.1 ml) were collected into scintillation vials, to which 10 ml scintillation cocktail were added. Each experiment reported was performed with an unused bed, although re-use of columns produced no differences in the results. Although heparin cofactor couples to agarose beads has a higher binding capacity and was used in preliminary experiments (not reported), the use of Sephadex G-50 for the support
69 medium gave substantially higher yields (see results) and was used for all studies reported below. Binding in solution phase of thrombin and heparin cofactor to heparin was studied by the dye displacement m e t h o d described by Li et al. [18,19]. Difference spectra were obtained at ambient temperature with a Beckman ACTA VI spectrophotomer. The binding of native and modified proteins to heparin-agarose was determined by applying native and modified thrombin (200 pg) or heparin cofactor (400 pg) to columns (0.6 X 4 cm) of heparin-agarose equilibrated in 0.15 M NaC1, 0.02 M Tris • HC1, pH 7.5. The columns were washed and eluted, and protein determined as absorbance at 280 nm. Gel electrophoresis in dodecyl sulfate was performed as described previously [20]. Proteins were quantitated by microdensitometry after staining with Coomassie Blue. Results
Reaction of thrombin for 6 min with 1,2-cyclohexanedione produced no change in specific activity, but reduced the affinity of thrombin for heparin agarose. The modified enzyme was eluted in the breakthrough fractions of a heparin-agarose column equilibrated with 0.15 M NaC1, whereas the unmodified enzyme bound to the column and required 0.6 M NaC1 for elution. Analysis of the modified thrombin by the Sakaguchi reaction indicated that 15% (3/20) of arginines were modified. When the reaction time was extended from 6 to 12 min, thrombin was inactivated completely, and even with a 6 min reaction, the fractions which were eluted last from the desalting column (those longest in contact with the reagents) had a 50% reduction in specific activity. Reaction of thrombin overnight with 1,2-cyclohexanedione modified about 50% (10/20) of arginine residues as assayed by the Sakaguchi reaction, and likewise inactivated the enzyme. Complete amidination of thrombin had no effect on its specific activity nor on its affinity for heparin-agarose. Complete amidination of heparin cofactor reduced its affinity for heparinagarose. The modified protein was eluted in the breakthrough fractions of a heparin-agarose column equilibrated with 0.15 M NaC1, while the unmodified protein required 1.0 M NaC1 for elution. The completely modified protein had no free amino groups detectable by reaction with 2,4-dinitrofluorobenzene. In the absence of heparin, inhibition of unmodified thrombin with amidinated heparin cofactor was identical to that with ummodified heparin cofactor (Fig. 1, closed and open circles). With unmodified inhibitor, addition of heparin increased the apparent second order rate constant about 15 fold (Fig. 1, triangles). However, the rate of inhibition of unmodified thrombin by amidinated inhibitor was n o t affected by heparin (Fig. 1, squares.). Analogous results were obtained for inhibition of modified thrombin with unmodified heparin cofactor (Fig. 2). In the absence of heparin, the rate of inhibition of modified thrombin by unmodified inhibitor (Fig. 2, open circles) was that of the control (Fig. 2, closed circles), and heparin had no effect on the rate of inhibition of the modified thrombin (Fig. 2, squares). The rate of inhibiton of modified thrombin by modified inhibitor was
70
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Fig. 1. I n h i b i t i o n of u n m o d i f i e d t h r o m b i n with modified heparin cofaetor. Reaction mixtuxes contained 50#g/ml (150 NIH units/ml) of t h r o m b i n , 100 # g / m l heparin cofactor, 0.2 M N - e t h y l m o r p h o l i n e acetate, pH 7.5. The results are presented as a second-order rate plot where a = t h r o m b i n c onc e nt ra t i on at t i me t, and a 0 = initial t h r o m b i n concentration. The reactions are: u n m o d i f i e d inhibitor, no heparin (o e); u n m o d i f i e d inhibitor, I #gfrnl h e p ~ (~ ~): modii~ied ]ni~ibitor, no h e p a r i n (o o); modified inhibitor, 1 # g / m l heparin (D ~).
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Fig. 2. Inhibition of modified t h r o m b i n by unmodified inhibitor. Reaction conditions are the same as for Fig. 1. The reactions are: u n m o d i f i e d thrombin, no heparin ( / l); unmodified thrombin, 1 #g/mi heparin (~ "-~); modified thrombin, no heparin (o o); modified t h r o m b i n , 1 # g / m l heparin
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71 identical to that of the unmodified proteins, and the reaction rate was not sensitive to heparin. Affinity chromatography was used to investigate the non-covalent interactions among heparin cofactor, thrombin, and heparin. It was reasoned that if thrombin and the inhibitor bind mutually to single heparin molecules, then heparin should act as a tether, and interlink fluid-phase thrombin noncovalently to solid-phase heparin cofactor. Initial experiments determined that iPr2P-thrombin has little, if any, affinity for either Sephadex G-50 or Sephadex G-50 to which heparin cofactor was coupled (Fig. 3). In both chromatograms, greater than 80% of the applied iPr2P-thrombin was recovered in the breakthrough fractions, and no iPr2Pthrombin was eluted with either 1.5 M NaC1 or 1% dodecyl sulfate (Fig. 3, arrow). Thrombin was bound tightly to the heparin cofactor-Sephadex column only when the column first was treated with a heparin solution (Fig. 4). Chromatography of iPr2P-thrombin on a column untreated with heparin resulted in the recovery of more than 95% of the thrombin in the breakthrough fractions (Fig. 4, closed circles), while less than 5% of the applied sample was eluted with 1.5 M NaCl (arrow). The same column was re-equilibrated with 0.1 M NaC1, treated with heparin, and then washed with 0.1 M NaC1 to remove excess (unbound) heparin. When a sample of iPr2P-thrombin was then applied to this heparin-treated column, less than 1% of the counts were recovered in the break-
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F i g . 3. C h r o m a t o g r a p h y of iPr2P-[3H]thrombin on Sephadex G-50 (e e) and on heparin cofactorS e p h a d e x (© c)). A s a m p l e c o n t a i n i n g 1 5 # g o f i P r 2 P - [ 3 H ] t h r o m b i n in 50 pl of 0.1 M NaCl was applied to the column. The column was washed with one bed volume of 0.1 M NaCl and then was eluted with 1.5 M NaCl (arrow). Other details arc described under "Methods." F i g . 4. E f f e c t o f h e p a r i n o n c h r o m a t o g r a p h y of iPr2P-[3H]thromhin on heparin cofaetor-Sephadex. A 1 5 ttg s a m p l e i f i P r 2 P - [ 3 H ] t h r o m b i n was chromatographed on heparin cofactor-Sephadex as described in Fig. 3.(e -'). A f t e r e l u t i o n w i t h 1 . 5 M N a C l ( a x r o w ) , t h e b e d w a s r e - e q u i l i b r a t e d w i t h 0 . 1 M N a C 1 , a n d t h e n 1 0 0 # g o f h e p a r i n i n 1 0 0 ~tl o f 0 . 1 M N a C l w a s a p p l i e d t o t h e c o l u m n . T h e c o l u m n w a s w a s h e d with two bed volumes of 0.1 M NaCl to remove excess unbound heparin, a second 15 pg sample of iPr2P[3H]thrombin w a s a p p l i e d , t h e c o l u m n w a s w a s h e d w i t h o n e b e d v o l u m e o f 0 . 1 M NaC1, a n d t h e n e l u t e d w i t h 1 . 5 M N a C l (© o). In separate experiments (not shown), increasing by threefold the volume of wash with 0.1 M NaCI neither eluted significant 3H from the column nor altered the recovery after elution with 1.5 M NaCl.
72
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Fig. 5. A f f i n i t y c h r o m a t o g r a p h y o f heparin o n heparin c o f a c t o r - S e p h a d e x (e o) and heparin c o f a c t o r S e p h a d e x t r e a t e d a t 1 0 0 ° C f o r 10 m i n (o "3). H e p a r i n ( 5 0 u n i t s , 0 . 3 r a g ) in 1 m l o f 0.1 M N a C I , 0 . 0 2 M T r i s • HC1, P h 7.5, w a s applied t o a c o l u m n ( 0 . 6 X 4 c m ) o f heparin c o f a c t o r - S e p h a d e x equilib r a t e d in the s a m e b u f f e r . T h e c o l u m n w a s w a s h e d w i t h t w o b e d v o l u m e s o f starting b u f f e r and t h e n w a s e l u t e d ( a r r o w ) w i t h 1.2 M N a C I . H e p a r i n w a s a s s a y e d b y its ability t o increase the c l o t t i n g t i m e o b t a i n e d o n a d d i t i o n o f t h r o m b i n t o n o r m a l c i t r a t e d p l a s m a . D a t a p o i n t s are c a l c u l a t e d f r o m a s t a n d a r d c u r v e c o n s t r u c t e d w i t h the crude heparin.
through fractions (Fig. 4, open circles). The bound thrombin was eluted quantitatively with 1.5 M NaC1 (arrow). The correlation of the intact tertiary structure of heparin cofactor with the heparin-dependent binding of iPr2P-thrombin was tested with an experiment identical to that shown in Fig. 4, except that the heparin cofactor-Sephadex was first treated at 100°C for 10 min. After heat denaturation, the Sephadexbound inhibitor lost more than 90% of its capacity to bind heparin (Fig. 5); treatment of the heat-inactivated column with heparin did not induce an affinity for iPr2P-thrombin (Fig. 6). In addition, heparin did not induce an affinity of iPr2P-thrombin for plain Sephadex G-50 (not shown). The specificity and reversibility of the binding of iPr2P-thrombin to heparin
~0
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15
FRACTIONS Fig. 6. C h r o m a t o g r a p h y o f i P r 2 P - [ 3 H ] t h r o m b i n o n h e a t - t r e a t e d heparin c o f a e t o r - S e p h a d e x . C h r o m a t o g r a p h y w a s p e r f o r m e d e x a c t l y as d e s c r i b e d in Fig. 4, e x c e p t t h a t the c o l u m n had b e e n h e a t e d in a boiling w a t e r b a t h f o r 10 rain. C h r o m a t o g r a p h y b e f o r e heparin t r e a t m e n t (c --); a f t e r heparin t r e a t m e n t (o o).
73
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Fig. 7. E l u t i o n of i P r 2 P - [ 3 H ] t h r o m b i n f r o m h e p a r i n - t r e a t e d h e p a r i n c o f a c t o r - S e p h a d e x w i t h excess free ligand, i P r 2 P - [ 3 H ] t h r o m b i n w a s a d s o r b e d t o h e p a r i n - t r e a t e d h e p a r i n c o f a c t o r - S e p h a d e x as d e s c r i b e d in Fig. 4. C o l u m n s w e r e e l u t e d ( a r r o w ) w i t h 1 m l of: h e p a r i n , 1 0 0 p g / m l ( e -~), cold i P r 2 P - t h r o m b i n , 1 0 0 # g / m l (o o), or cold i P r 2 P - t h r o m b i n , 10 /~g/ml (z~ A). E a c h c h r o m a t o g r a m r e g r e s e n t s a separate experiment.
cofactor-Sephadex was studied by competition experiments, iPr2P-thrombin was adsorbed to heparin-treated columns as described in Figs. 3 and 4. The columns then were eluted with either excess heparin or excess cold iPr2Pthrombin (Fig. 7). The bound iPr2P-thrombin was eluted quantitatively by 1 ml of either a 1 mg/ml heparin solution (Fig. 7, closed circles) or by a 100 pg/ml cold iPr2P-thrombin solution Fig. 7, open circles). Bound thrombin was eluted
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Fig. 8. E f f e c t s of t h r o m h i n a n d h e p a r i n c o f a c t o r o n d i f f e r e n c e s p e c t r a of p r o f l a v i n e - h e p a r i n vs. p r o f l a v i n e s o l u t i o n s . B o t h c u v e t t e s c o n t a i n e d 3.0 • 1 0 -S M p r o f l a v i n e in 0 . 0 2 M NaC1, 0.01 M Tris • HC1, p H 7.5, a n d t h e s a m p l e c u v e t t e c o n t a i n e d 2 0 0 /~g/ml ( 1 . 8 • 10 -5 M) h e p a r i n . ( A ) A d d i t i o n of t h r o m b i n t o s a m p l e ( h e p a r i n - c o n t a i n i n g ) c u v e t t e : a, n o t h ~ o m b i n ; b, 1 2 . 5 /~g/ml; c, 25 /~g/ml, d, 50 ~ g / m l ( 1 . 4 • 1 0 -6 M). C u r v e e is t h e b a s e l i n e o b t a i n e d w i t h p r o f l a v i n e a l o n e in s a m p l e c u v e t t e . (B) A d d i t i o n o f h e p a r i n c o f a c t o r t o s a m p l e c u v e t t e : a, n o h e p a r i n c o f a e t o r ; b , 2 0 0 /~g/ml, ( 3 . 2 • 1 0 -6 M). I n c u r v e c, t h e r u m p l e e u v e t t e c o n t a i n e d 2 0 0 / ~ g / m l h e p a r i n c o f a c t o r , a n d n o h e p a r i n . C u r v e d is t h e baseline.
74
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Fig. 9. E n h a n c e m e n t b y h e p a r i n of the r a t e of p r o t e o l y s i s of p r o t h r o m b i n b y t h r o m b i n . R e a c t i o n m i x t u r e s contained 1 m g / m l of p r o t h r o m b i n , the concentratio ns of heparin indicated below, and 25 p g / m l t h r o m b i n . R e a c t i o n s w e r e c a r r i e d o u t in 0.1 M NaCI, 0 . 0 2 M Tris • HC1, p H 7.5, 35 ° -+ I ° C . A t 4,8, a n d 20 rain a f t e r a d d i t i o n o f t h r o m b i n , s a m p l e s w e r e r e m o v e d , a d d e d t o 1 / 1 0 v o l u m e of 10% s o d i u m d o d e c y l sulfate, h e a t e d at 1 0 0 ° C f o r 2 m i n a n d t h e n a n a l y z e d b y gel e l e c t r o p h o r e s i s in d o d e c y l sulfate. All gels were stained and destained simultaneously, and scanned with a microdensitometer. Percent p r o t h r o m b i n is 1 0 0 times the a r e a of the prothrombin peak d i v i d e d b y t h e t o t a l area of t h e e l e c t r o p h e r o g r a m . T h e mixtures contained: no heparin (e -~), 1 / ~ g / m l h e p a r i n (o o), a n d 10 /~g/ml h e p a r i n
(~
A).
more slowly with a less concentrated (10 ug/ml) cold thrombin solution (Fig. 7, triangles). A dye displacement technique [18,19] was used to explore the possibility that thrombin and heparin cofactor bind to nonidentical sites on the heparin molecule. In agreement with the results of Li et al. [18], difference spectra of proflavine/heparin/thrombin mixtures demonstrate that thrombin and proflavine compete for a binding site on heparin (Fig. 8A). Addition of thrombin to a proflavine-heparin mixture causes a shift (Fig. 8A, curves b--d, ~ma~ = 447 nm) in the difference spectrum of heparin/proflavine vs. proflavine (Fig. 8A, curve a) toward the proflavine vs. proflavine blank (Fig. 8A, curve e). A smaller shift with ~max -~ 470 nm is due to the binding of proflavine to the active site of thrombin [18]. Unlike thrombin, heparin cofactor, at a molar concentration twice that of thrombin in Fig. 8A, produced no spectral shifts in heparinproflavine mixtures (Fig. 8B, curve b). Evidence that proflavine does not bind to heparin cofactor is shown by curve c in Fig. 8B, which is the difference spectrum of heparin cofactor-proflavine vs. proflavine. If the effect of heparin on the thrombin-heparin cofactor reaction is a consequence of the formation of a ternary complex of the two proteins and heparin, then appropriately oriented ternary complexes of other reactive protein pairs and heparin might undergo comparable rate enhancement. Such a reactive pair is represented by prothrombin and thrombin. Prothrombin is cleaved by thrombin to yield Fragment 1 and Prethrombin 1 [21], and both prothrombin and thrombin bind to heparin [8]. The effect of catalytic concentrations of heparin on the rate of hydrolysis of prothrombin by thrombin is shown in Fig. 9. At heparin concentrations which inhibit up to 80% of the
75 hydrolysis by thrombin of 0.4 mM benzoyl-Gly-Pro-Arg-p-nitroanilide [22] the hydrolysis of prothrombin is enhanced at least 2--3 fold. Inhibition with the tripeptide substrate is n o t due to adsorption of the substrate to heparin, since, with a m o n o m e r molecular weight of heparin of about 200, the inhibition is seen at a 10--100 fold excess of substrate over heparin monomer. Discussion
If catalysis by heparin of the thrombin-heparin cofactor reaction is the result of formation of a ternary complex of heparin, the enzyme, and its inhibitor, then both proteins must bind simultaneously to heparin. Such a complex is n o t demonstrated unambiguously by the binding of both thrombin and heparin cofactor to insolubilized heparin [8], since the heterogeneity of heparin [11] might allow the binding of each protein to dissimilar species of heparin. However, the ability of solution-phase heparin to interlink thrombin non-covaler;tly with solid-phase heparin cofactor requires that a single heparin molecule binds to both proteins. However, binding studies alone cannot demonstrate unambiguously that a ternary complex is required for catalysis. The dependence of catalysis on the binding of both proteins to heparin is shown by the chemical modification studies. The effects of amidination of the inhibitor are comparable to those reported by Rosenberg and Damus [2]. The rate of reaction of the modified inhibitor with thrombin is insensitive to heparin. This finding, together with the finding that the modified inhibitor does n o t bind :to heparin-agarose, verifies that catalysis requires the binding of the inhibitor to heparin. However, in agreement with the results of Machovich [9], reaction of arginine-modified thrombin with unmodified inhibitor is likewise unaffected by heparin, despite binding of the unmodified inhibitor to heparin. Modification of the highly specific protease, thrombin, did not affect its specific activity toward the protein substrates fibrinogen and heparin cofactor, which would be highly sensitive to perturbation of the specificity of the enzyme. Therefore, insensitivity to heparin of the modified enzyme-unmodified inhibitor reaction implies that catalysis is not simply a consequence of an allosteric effect of heparin on the inhibitor, b u t must depend absolutely on the binding of thrombin as well as the inhibitor to heparin. Such a model for the activity of heparin was suggested by Yin and Wessler [10] and Gitel [8] as analogous to the prothrombin activation complex, where a phospholipid bilayer can enhance the rate of prothrombin activation by providing a binding surface for prothrombin and activated factor X [23--25]. The binding surface model is supported further by the finding that the rate of hydrolysis of prothrombin by thrombin, another protein pair which binds mutually to heparin [8], is enhanced by that binding. Furthermore, enhancement of proteolysis by heparin concentrations which inhibit the activity of thrombin with substrates other than prothrombin (or heparin cofactor) implies that heparin is not a positive allosteric effector of thrombin, and the inhibition of thrombin by heparin, whether steric or all~)steric, must be small compared to the catalytic effect of heparin. Our data do not exclude conformational effects of heparin on either throm-
76
bin or heparin cofactor. However, the effect of blocking the binding of either protein to heparin is the same: the enzyme-inhibitor reaction rate becomes insensitive to heparin. Therefore if heparin is an allosteric effector, then complimentary changes in the conformations of both proteins must occur simultaneously, and the change in conformation of only one protein of the pair must have neither a positive nor negative effect on their reaction rate. Li et al. [18,19] have determined that the binding of proflavin to heparin produces the spectral shift with ~ m a x = 447 nm, and that binding of thrombin to heparin reverses the shift by displacing proflavine from heparin. With experimental conditions identical to those under which thrombin was examined, heparin cofactor exerted no effect on the difference spectrum between proflavine and proflavine bound to heparin. In addition, there was no evidence of the spectral shift which Li et al. [19] found to result from binding of proflavine to heparin cofactor and which partially obscured a comparison of the spectra. Whether the result of minor changes in heparin or proflavine preparations or in ionic environment, the absence of this spectral shift permits the comparison of heparin-proflavine spectra obtained with and without heparin cofactor. The virtual identity of the two spectra suggests that at least one site on heparin binds tightly to thrombin but not to heparin cofactor. An alternate but unlikely possibility is that thrombin binds more tightly and heparin cofactor much less tightly than proflavine to heparin. However affinity chromatographic data [8] indicate that heparin cofactor binds more tightly than thrombin to heparin. The data in the present study are consistent with the hypothesis t h a t heparin acts as a mutual binding substrate for thrombin (and presumably other proteinases) and heparin cofactor. Since binding and chemical modification data alone cannot determine the mechanism by which the ternary complex is reactive, the extent to which this mutual binding accounts for the catalytic activity of heparin can best be evaluated by detailed kinetic analyses. However such studies must await a more thorough understanding and resolution from one another of the various molecular species which comprise available h e p ~ i n preparations.
Acknowledgements This work was supported by National Institutes of Health, Grant No. 1423005S1 (Specialized Center of Research in Atherosclerosis). M.P. was a trainee of the National Science Foundation, Secondary School Training Program.
References 1 A b i l d g a a r d , U. ( 1 9 6 9 ) S c a n d . J . Clin. L a b . I n v e s t . 2 4 , 2 3 - - 2 7 2 R o s e n b e r g , R . D . a n d D a m u s , P.S. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 6 4 9 0 - - 6 5 0 5 30wen. W.G. (1975) Biochim. Biophys. Acta 405, 380--387 4 A b i l d g a a r d , U. ( 1 9 6 8 ) S c a n d . J . Clin. L a b . I n v e s t . 2 1 , 8 9 - - 9 1 5 Biggs, R . , D e n s o n , K . W . E . , A k m a n , N., B o r r e t t , R . a n d H a d d e n , M. ( 1 9 7 0 ) B r i t . J . H a e m a t o l . 1 9 , 283--305 6 Porter, P., Porter,M.C. and Shanberg, J.N. (1967) Biochemistry 6, 1854--1863 7 Wilson-Gentry, P. and Alexander, B. (1973) Fed. Proc. 32, 290 8 Gitel, S. (1975) in Heparin: Structure, Function, and Clinical Implications (Bradshaw, R.A. and Wessler, S., eds.), p p . 2 4 3 - - 2 4 7 , P l e n u m Press. N e w Y o r k
77 9 Machovich, R. (1975) Biochim. Biophys. Acta 412, 13--17 10 Yln, E.T. and Wessler, S. (1970) Biochim. Biophys. Acta 2 0 1 , 3 8 7 - - 3 9 0 11 Cifonelli, J.A. (1975) in Heparin: Structure, F u n c t i o n , and Clinical I m p l i c a t i o n s (Bradshaw, R.A. and Wessler, S. eds.), pp. 95--103, Plenum Press, New York 12 Van Lenten, L. and Ashwell, G. (1971) J. Biol, Chem. 246, 1 8 8 9 - - 1 8 9 4 13 Owen, W.G., Esmon, C.T. and Jackson, C.M. (1974) J. Biol. Chem. 249, 594--605 14 Hunter, M~I. and Ludwig, M.L. (1972) in Methods in E n z y m o l o g y (Hits, C.H.W. and Timasheff, S.N., eds.), VoL 25, pp. 585--596, E n z y m e Structure Part B 15 P a t t h y , L. and Smith, L.E. (1975) J. Biol. Chem. 250, 557--564 16 Izumi, Y. (1965) Anal. Biochem. 12, 1--7 17 Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3 0 5 9 - - 3 0 6 5 18 Li, E.H.H., Orton, C. and Feinman, R.D. (1974) Biochemistry 13, 5012--5017 19 Li, E.H.H., Fen ton, J.W. and Feinman, R.D. (1970) Arch. Biochem. Biophys. 175, 153--159 20 Laemmli, U.K. (1970) Nature 2 2 7 , 6 8 0 - - 6 8 5 21 Mann, K.G. Heldebrant, C.M. and Fass, D.N. (1971) J. Biol. Chem. 246, 6 1 0 6 - - 6 1 1 4 22 Owen, W.G. (1977) Biochim. Biophys. Acta 494, 182--190 23 Papahadjopoulos, D. and Hanahan, D~I. (1964) Biochim. Biophys. Acta 90, 436--439 24 Barton, P.G. and Hanahan, D.J. (1969) Biochim. Biophys. Acta 1 8 7 , 3 1 9 - - 3 2 7 25 Jackson, C.M., Owen, W.G., Gitel, S.N. and Esmon, C.T. (1974) Thromb. Diath. Haemorrh. Suppl. 57, 273--293
Biochimica et Biophysica Acta, 535 ( 1 9 7 8 ) 6 6 - - 7 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 37943
A CATALYTIC ROLE FOR HEPARIN EVIDENCE FOR A TERNARY COMPLEX OF HEPARIN COFACTOR THROMBIN AND HEPARIN
M A R K W. P O M E R A N T Z a n d W H Y T E G. OWEN
Department o f Pathology, University o f Iowa, Iowa City, Iowa 52242 (U.S.A.) (Received F e b r u a r y 2nd, 1 9 7 8 )
Summary The interaction of heparin with chemically modified thrombin and heparin cofactor is studied. Amidinated heparin cofactor does not bind to heparinagarose and the reaction rate of the amidinated inhibitor with unmodified thrombin is not affected by heparin. Likewise, thrombin modified with 1,2-cyclohexanedione does not bind to heparin agarose and the reaction rate of the modified enzyme with unmodified inhibitor is not affected by heparin. In the absence of heparin, the modified and unmodified proteins react at the same rate in all possible combinations. Affinity chromatography of diisopropylphosphoryl thrombin on heparin cofactor coupled to Sephadex G-50 is used to study the binding of heparin cofactor and thrombin to heparin. The thrombin for all experiments is tritium-labeled and then inactivated with diispropylfluorophosphate. Thrombin is not bound to heparin cofactor-Sephadex columns. However, after treatment of the columns with a heparin solution, thrombin binds tightly, and is eluted at high ionic strength. Bound thrombin can also be eluted with either excess non-radioactive thrombin or excess free heparin. Heparin-dependent binding of thrombin does not occur if the heparin cofactor-Sephadex is heat-denatured. The ability of heparin to couple solutionphase thrombin to solid-phase heparin cofactor indicates that a ternary complex is formed. Analysis of the binding of the proteins to heparin by a dye displacement method suggests that at least one site on heparin binds to thrombin but not to heparin cofactor. Further support for a catalytic role for heparin derives from the ability of catalytic concentrations of heparin to enhance the rate of hydrolysis of prothrombin by thrombin, another protein pair which bind mutually to heparin.
Abbreviations used are: iPr2PF, diisopropylfluorophosphate; iPr2P-, diisoprolylphosphoryl.
67 Introduction Thrombin in plasma or serum gradually loses its enzymatic activity. This inactivation, as well as that of other proteinases involved in the coagulation cascade is due in part to the presence in plasma of heparin cofactor (antithrombin III, factor Xa inhibitor), a glycoprotein with a molecular weight of about 62 000 [1,2]. The interaction of heparin cofactor and thrombin yields, with 1 : 1 stoichiometry, an enzymatically inactive complex which is stable in the presence of denaturing and reducing agents [1--3]. Thrombin with its active center blocked cannot interact with the inhibitor to form the complex
[2]. Heparin, a highly sulfated mucopolysaccharide anticoagulant, in catalytic concentrations accelerates the rate of inhibition of thrombin by heparin cofactor [4,5] but does not modify the 1 : 1 stoichiometry of the reaction [2,4,5]. The effect of heparin on this reaction distinguishes heparin cofactor from other plasma thrombin inhibitors. Both heparin cofactor and thrombin bind tightly to heparin [6--8]. By chemically modifying heparin cofactor, Rosenberg and Damus [2] have shown that lysyl residues on the inhibitor are required for binding to the highly negative heparin molecule. Blocking these lysines reduced heparin cofactor activity significantly, whilz not affecting the activity of the inhibitor in the absence of heparin. This behavior was interpreted as suggesting that heparin functions as an allosteric effector of the inhibitor, making it more reactive with thrombin. However other studies have shown that when arginyl residues of thrombin are blocked, the rate of inhibition of the modified thrombin by heparin cofactor is likewise unaffected by heparin [9]. On this basis it was suggested that heparin is an allosteric modifier of thrombin. However the latter study employed a crude, poorly characterized.thrombin preparation, and the experimental conditions, especially protein concentrations, were significantly different from those employed by Rosenberg and Damus [2]. Yin and Wessler [10] and Gitel [8] proposed a model for heparin activity in which heparin acts as a catalyst to which thrombin and the inhibitor, a specialized thrombin substrate, mutually bind. Such binding could increase the stability of the putative Michaelis complex, thereby enhancing the rate of formation of the covalent complex. This model is based on the assumption that thrombin and the inhibitor are bound simultaneously to a single heparin molecule. However, since available heparin preparations are highly heterogeneous [11], direct binding studies [8] do not distinguish between two proteins binding mutually to a single heparin molecule as opposed to each protein binding independently to different species of heparin. In the present study, both thrombin and heparin cofactor are modified chemically to abolish their affinity for heparin, without affecting their heparinindependent reactivity. In addition, the binding of thrombin and heparin cofactor is analyzed by affinity chromatography. Evidence is presented that thrombin and heparin cofactor bind to independent sites on a single heparin molecule and that the resultant catalysis of their reaction requires that both proteins bind to heparin.
68 Materials and Methods Heparin (porcine intestinal mucosa), sodium dodecyl sulfate, Sephadex G-50, and proflavine hemi-sulfate were obtained from Sigma Chemical Co., St. Louis, Mo. Proflavine was recrystalized from ethanol before use. Biogel A-15m was purchased from Biorad Laboratories, Richmond, Calif. Cyanogen bromide and diisopropylfluorophosphate (iPr2PF) were obtained from Aldrich Chemical Co., Milwaukee, Wis. iPr2PF was used as a 1 M solution in redistilled dimethylformamide. Methyl acetimidate, 1,2-cyclohexanedione, and 2,4-dinitrofluorobenzene were obtained from Pierce Chemical Co., Rockford, Ill. NaB3H4, spec. act. 2.0 Ci/mmol, was obtained from ICN, Cleveland, Ohio. Prothrombin, thrombin and heparin cofactor were prepared and assayed as described previously [3]. Thrombin was tritium-labeled by introducing 3H into sialic acid residues of prothrombin [ 12,13 ]. After activation and purification [ 3], the [ 3H] thrombin was inactivated with 10 -3 M iPrzPF. The product had a specific radioactivity of 106 dpm/mg protein. Lysyl residues of thrombin and heparin cofactor were amidinated as described previously [14]. Reactions were carried out for 2 0 h at 0°C in 0.25 M sodium borate/NaOH buffer, pH 9.0. The modified proteins were dialyzed in 0.2 M N-ethylmorpholine acetate buffer, pH 7.5. Unmodified lysine was determined by reaction with 2,4-dinitrofluorobenzene. Arginine residues were modified by reaction with 1,2-cyclohexanedione [15]. Modification was carried out for 6 min at 35°C, and the protein was separated from reagents by chromatography on biogel P-10 (Bio-rad Laboratories, Richmond, Calif.). Unmodified arginine was determined by the Sakaguchi reaction [16]. Heparin cofactor was coupled to Sephadex G-50 by the cyanogen bromide method [17]. Fifteen grams CNBr were reacted with 20 ml (packed volume) Sephadex G-50 to couple 5--10 mg heparin cofactor. Coupling was carried out at pH 8.3, and remaining reactive Sephadex was blocked with excess ethanolamine. Coupling yields was 200 pg protein/ml of packed Sephadex G-50, as estimated by absorbance at 280 nm after digestion with trypsin. For affinity chromtography, 1.0 ml (packed volume) of heparin cofactor Sephadex was poured into a silicone (Clay-Adams SilicladR)-coated Pasteur pipette (0.6 cm internal diameter) with silicone-coated glass wool as a bed support. All eluants were buffered with 0.02 M Tris • HC1 (pH 7.5), and contained 0.1% 7-globulin to minimize adsorption of thrombin to glass. Omission of carrier protein reduced recoveries of thrombin by about 50%, in both controls and heparin-treated columns, as well as columns of plain Sephadex G-50. Albumin was slightly less protective than 7-globulin, and was avoided also because of possible contamination of albumin with small amounts of native or degraded heparin cofactor. Samples of 10 drops each (~ 0.1 ml) were collected into scintillation vials, to which 10 ml scintillation cocktail were added. Each experiment reported was performed with an unused bed, although re-use of columns produced no differences in the results. Although heparin cofactor couples to agarose beads has a higher binding capacity and was used in preliminary experiments (not reported), the use of Sephadex G-50 for the support
69 medium gave substantially higher yields (see results) and was used for all studies reported below. Binding in solution phase of thrombin and heparin cofactor to heparin was studied by the dye displacement m e t h o d described by Li et al. [18,19]. Difference spectra were obtained at ambient temperature with a Beckman ACTA VI spectrophotomer. The binding of native and modified proteins to heparin-agarose was determined by applying native and modified thrombin (200 pg) or heparin cofactor (400 pg) to columns (0.6 X 4 cm) of heparin-agarose equilibrated in 0.15 M NaC1, 0.02 M Tris • HC1, pH 7.5. The columns were washed and eluted, and protein determined as absorbance at 280 nm. Gel electrophoresis in dodecyl sulfate was performed as described previously [20]. Proteins were quantitated by microdensitometry after staining with Coomassie Blue. Results
Reaction of thrombin for 6 min with 1,2-cyclohexanedione produced no change in specific activity, but reduced the affinity of thrombin for heparin agarose. The modified enzyme was eluted in the breakthrough fractions of a heparin-agarose column equilibrated with 0.15 M NaC1, whereas the unmodified enzyme bound to the column and required 0.6 M NaC1 for elution. Analysis of the modified thrombin by the Sakaguchi reaction indicated that 15% (3/20) of arginines were modified. When the reaction time was extended from 6 to 12 min, thrombin was inactivated completely, and even with a 6 min reaction, the fractions which were eluted last from the desalting column (those longest in contact with the reagents) had a 50% reduction in specific activity. Reaction of thrombin overnight with 1,2-cyclohexanedione modified about 50% (10/20) of arginine residues as assayed by the Sakaguchi reaction, and likewise inactivated the enzyme. Complete amidination of thrombin had no effect on its specific activity nor on its affinity for heparin-agarose. Complete amidination of heparin cofactor reduced its affinity for heparinagarose. The modified protein was eluted in the breakthrough fractions of a heparin-agarose column equilibrated with 0.15 M NaC1, while the unmodified protein required 1.0 M NaC1 for elution. The completely modified protein had no free amino groups detectable by reaction with 2,4-dinitrofluorobenzene. In the absence of heparin, inhibition of unmodified thrombin with amidinated heparin cofactor was identical to that with ummodified heparin cofactor (Fig. 1, closed and open circles). With unmodified inhibitor, addition of heparin increased the apparent second order rate constant about 15 fold (Fig. 1, triangles). However, the rate of inhibition of unmodified thrombin by amidinated inhibitor was n o t affected by heparin (Fig. 1, squares.). Analogous results were obtained for inhibition of modified thrombin with unmodified heparin cofactor (Fig. 2). In the absence of heparin, the rate of inhibition of modified thrombin by unmodified inhibitor (Fig. 2, open circles) was that of the control (Fig. 2, closed circles), and heparin had no effect on the rate of inhibition of the modified thrombin (Fig. 2, squares). The rate of inhibiton of modified thrombin by modified inhibitor was
70
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Fig. 1. I n h i b i t i o n of u n m o d i f i e d t h r o m b i n with modified heparin cofaetor. Reaction mixtuxes contained 50#g/ml (150 NIH units/ml) of t h r o m b i n , 100 # g / m l heparin cofactor, 0.2 M N - e t h y l m o r p h o l i n e acetate, pH 7.5. The results are presented as a second-order rate plot where a = t h r o m b i n c onc e nt ra t i on at t i me t, and a 0 = initial t h r o m b i n concentration. The reactions are: u n m o d i f i e d inhibitor, no heparin (o e); u n m o d i f i e d inhibitor, I #gfrnl h e p ~ (~ ~): modii~ied ]ni~ibitor, no h e p a r i n (o o); modified inhibitor, 1 # g / m l heparin (D ~).
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Fig. 2. Inhibition of modified t h r o m b i n by unmodified inhibitor. Reaction conditions are the same as for Fig. 1. The reactions are: u n m o d i f i e d thrombin, no heparin ( / l); unmodified thrombin, 1 #g/mi heparin (~ "-~); modified thrombin, no heparin (o o); modified t h r o m b i n , 1 # g / m l heparin
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71 identical to that of the unmodified proteins, and the reaction rate was not sensitive to heparin. Affinity chromatography was used to investigate the non-covalent interactions among heparin cofactor, thrombin, and heparin. It was reasoned that if thrombin and the inhibitor bind mutually to single heparin molecules, then heparin should act as a tether, and interlink fluid-phase thrombin noncovalently to solid-phase heparin cofactor. Initial experiments determined that iPr2P-thrombin has little, if any, affinity for either Sephadex G-50 or Sephadex G-50 to which heparin cofactor was coupled (Fig. 3). In both chromatograms, greater than 80% of the applied iPr2P-thrombin was recovered in the breakthrough fractions, and no iPr2Pthrombin was eluted with either 1.5 M NaC1 or 1% dodecyl sulfate (Fig. 3, arrow). Thrombin was bound tightly to the heparin cofactor-Sephadex column only when the column first was treated with a heparin solution (Fig. 4). Chromatography of iPr2P-thrombin on a column untreated with heparin resulted in the recovery of more than 95% of the thrombin in the breakthrough fractions (Fig. 4, closed circles), while less than 5% of the applied sample was eluted with 1.5 M NaCl (arrow). The same column was re-equilibrated with 0.1 M NaC1, treated with heparin, and then washed with 0.1 M NaC1 to remove excess (unbound) heparin. When a sample of iPr2P-thrombin was then applied to this heparin-treated column, less than 1% of the counts were recovered in the break-
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F i g . 3. C h r o m a t o g r a p h y of iPr2P-[3H]thrombin on Sephadex G-50 (e e) and on heparin cofactorS e p h a d e x (© c)). A s a m p l e c o n t a i n i n g 1 5 # g o f i P r 2 P - [ 3 H ] t h r o m b i n in 50 pl of 0.1 M NaCl was applied to the column. The column was washed with one bed volume of 0.1 M NaCl and then was eluted with 1.5 M NaCl (arrow). Other details arc described under "Methods." F i g . 4. E f f e c t o f h e p a r i n o n c h r o m a t o g r a p h y of iPr2P-[3H]thromhin on heparin cofaetor-Sephadex. A 1 5 ttg s a m p l e i f i P r 2 P - [ 3 H ] t h r o m b i n was chromatographed on heparin cofactor-Sephadex as described in Fig. 3.(e -'). A f t e r e l u t i o n w i t h 1 . 5 M N a C l ( a x r o w ) , t h e b e d w a s r e - e q u i l i b r a t e d w i t h 0 . 1 M N a C 1 , a n d t h e n 1 0 0 # g o f h e p a r i n i n 1 0 0 ~tl o f 0 . 1 M N a C l w a s a p p l i e d t o t h e c o l u m n . T h e c o l u m n w a s w a s h e d with two bed volumes of 0.1 M NaCl to remove excess unbound heparin, a second 15 pg sample of iPr2P[3H]thrombin w a s a p p l i e d , t h e c o l u m n w a s w a s h e d w i t h o n e b e d v o l u m e o f 0 . 1 M NaC1, a n d t h e n e l u t e d w i t h 1 . 5 M N a C l (© o). In separate experiments (not shown), increasing by threefold the volume of wash with 0.1 M NaCI neither eluted significant 3H from the column nor altered the recovery after elution with 1.5 M NaCl.
72
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Fig. 5. A f f i n i t y c h r o m a t o g r a p h y o f heparin o n heparin c o f a c t o r - S e p h a d e x (e o) and heparin c o f a c t o r S e p h a d e x t r e a t e d a t 1 0 0 ° C f o r 10 m i n (o "3). H e p a r i n ( 5 0 u n i t s , 0 . 3 r a g ) in 1 m l o f 0.1 M N a C I , 0 . 0 2 M T r i s • HC1, P h 7.5, w a s applied t o a c o l u m n ( 0 . 6 X 4 c m ) o f heparin c o f a c t o r - S e p h a d e x equilib r a t e d in the s a m e b u f f e r . T h e c o l u m n w a s w a s h e d w i t h t w o b e d v o l u m e s o f starting b u f f e r and t h e n w a s e l u t e d ( a r r o w ) w i t h 1.2 M N a C I . H e p a r i n w a s a s s a y e d b y its ability t o increase the c l o t t i n g t i m e o b t a i n e d o n a d d i t i o n o f t h r o m b i n t o n o r m a l c i t r a t e d p l a s m a . D a t a p o i n t s are c a l c u l a t e d f r o m a s t a n d a r d c u r v e c o n s t r u c t e d w i t h the crude heparin.
through fractions (Fig. 4, open circles). The bound thrombin was eluted quantitatively with 1.5 M NaC1 (arrow). The correlation of the intact tertiary structure of heparin cofactor with the heparin-dependent binding of iPr2P-thrombin was tested with an experiment identical to that shown in Fig. 4, except that the heparin cofactor-Sephadex was first treated at 100°C for 10 min. After heat denaturation, the Sephadexbound inhibitor lost more than 90% of its capacity to bind heparin (Fig. 5); treatment of the heat-inactivated column with heparin did not induce an affinity for iPr2P-thrombin (Fig. 6). In addition, heparin did not induce an affinity of iPr2P-thrombin for plain Sephadex G-50 (not shown). The specificity and reversibility of the binding of iPr2P-thrombin to heparin
~0
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FRACTIONS Fig. 6. C h r o m a t o g r a p h y o f i P r 2 P - [ 3 H ] t h r o m b i n o n h e a t - t r e a t e d heparin c o f a e t o r - S e p h a d e x . C h r o m a t o g r a p h y w a s p e r f o r m e d e x a c t l y as d e s c r i b e d in Fig. 4, e x c e p t t h a t the c o l u m n had b e e n h e a t e d in a boiling w a t e r b a t h f o r 10 rain. C h r o m a t o g r a p h y b e f o r e heparin t r e a t m e n t (c --); a f t e r heparin t r e a t m e n t (o o).
73
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Fig. 7. E l u t i o n of i P r 2 P - [ 3 H ] t h r o m b i n f r o m h e p a r i n - t r e a t e d h e p a r i n c o f a c t o r - S e p h a d e x w i t h excess free ligand, i P r 2 P - [ 3 H ] t h r o m b i n w a s a d s o r b e d t o h e p a r i n - t r e a t e d h e p a r i n c o f a c t o r - S e p h a d e x as d e s c r i b e d in Fig. 4. C o l u m n s w e r e e l u t e d ( a r r o w ) w i t h 1 m l of: h e p a r i n , 1 0 0 p g / m l ( e -~), cold i P r 2 P - t h r o m b i n , 1 0 0 # g / m l (o o), or cold i P r 2 P - t h r o m b i n , 10 /~g/ml (z~ A). E a c h c h r o m a t o g r a m r e g r e s e n t s a separate experiment.
cofactor-Sephadex was studied by competition experiments, iPr2P-thrombin was adsorbed to heparin-treated columns as described in Figs. 3 and 4. The columns then were eluted with either excess heparin or excess cold iPr2Pthrombin (Fig. 7). The bound iPr2P-thrombin was eluted quantitatively by 1 ml of either a 1 mg/ml heparin solution (Fig. 7, closed circles) or by a 100 pg/ml cold iPr2P-thrombin solution Fig. 7, open circles). Bound thrombin was eluted
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Fig. 8. E f f e c t s of t h r o m h i n a n d h e p a r i n c o f a c t o r o n d i f f e r e n c e s p e c t r a of p r o f l a v i n e - h e p a r i n vs. p r o f l a v i n e s o l u t i o n s . B o t h c u v e t t e s c o n t a i n e d 3.0 • 1 0 -S M p r o f l a v i n e in 0 . 0 2 M NaC1, 0.01 M Tris • HC1, p H 7.5, a n d t h e s a m p l e c u v e t t e c o n t a i n e d 2 0 0 /~g/ml ( 1 . 8 • 10 -5 M) h e p a r i n . ( A ) A d d i t i o n of t h r o m b i n t o s a m p l e ( h e p a r i n - c o n t a i n i n g ) c u v e t t e : a, n o t h ~ o m b i n ; b, 1 2 . 5 /~g/ml; c, 25 /~g/ml, d, 50 ~ g / m l ( 1 . 4 • 1 0 -6 M). C u r v e e is t h e b a s e l i n e o b t a i n e d w i t h p r o f l a v i n e a l o n e in s a m p l e c u v e t t e . (B) A d d i t i o n o f h e p a r i n c o f a c t o r t o s a m p l e c u v e t t e : a, n o h e p a r i n c o f a e t o r ; b , 2 0 0 /~g/ml, ( 3 . 2 • 1 0 -6 M). I n c u r v e c, t h e r u m p l e e u v e t t e c o n t a i n e d 2 0 0 / ~ g / m l h e p a r i n c o f a c t o r , a n d n o h e p a r i n . C u r v e d is t h e baseline.
74
i
J '\\
\
e.
\\
4
_
8
12 MINUTES
16
20
Fig. 9. E n h a n c e m e n t b y h e p a r i n of the r a t e of p r o t e o l y s i s of p r o t h r o m b i n b y t h r o m b i n . R e a c t i o n m i x t u r e s contained 1 m g / m l of p r o t h r o m b i n , the concentratio ns of heparin indicated below, and 25 p g / m l t h r o m b i n . R e a c t i o n s w e r e c a r r i e d o u t in 0.1 M NaCI, 0 . 0 2 M Tris • HC1, p H 7.5, 35 ° -+ I ° C . A t 4,8, a n d 20 rain a f t e r a d d i t i o n o f t h r o m b i n , s a m p l e s w e r e r e m o v e d , a d d e d t o 1 / 1 0 v o l u m e of 10% s o d i u m d o d e c y l sulfate, h e a t e d at 1 0 0 ° C f o r 2 m i n a n d t h e n a n a l y z e d b y gel e l e c t r o p h o r e s i s in d o d e c y l sulfate. All gels were stained and destained simultaneously, and scanned with a microdensitometer. Percent p r o t h r o m b i n is 1 0 0 times the a r e a of the prothrombin peak d i v i d e d b y t h e t o t a l area of t h e e l e c t r o p h e r o g r a m . T h e mixtures contained: no heparin (e -~), 1 / ~ g / m l h e p a r i n (o o), a n d 10 /~g/ml h e p a r i n
(~
A).
more slowly with a less concentrated (10 ug/ml) cold thrombin solution (Fig. 7, triangles). A dye displacement technique [18,19] was used to explore the possibility that thrombin and heparin cofactor bind to nonidentical sites on the heparin molecule. In agreement with the results of Li et al. [18], difference spectra of proflavine/heparin/thrombin mixtures demonstrate that thrombin and proflavine compete for a binding site on heparin (Fig. 8A). Addition of thrombin to a proflavine-heparin mixture causes a shift (Fig. 8A, curves b--d, ~ma~ = 447 nm) in the difference spectrum of heparin/proflavine vs. proflavine (Fig. 8A, curve a) toward the proflavine vs. proflavine blank (Fig. 8A, curve e). A smaller shift with ~max -~ 470 nm is due to the binding of proflavine to the active site of thrombin [18]. Unlike thrombin, heparin cofactor, at a molar concentration twice that of thrombin in Fig. 8A, produced no spectral shifts in heparinproflavine mixtures (Fig. 8B, curve b). Evidence that proflavine does not bind to heparin cofactor is shown by curve c in Fig. 8B, which is the difference spectrum of heparin cofactor-proflavine vs. proflavine. If the effect of heparin on the thrombin-heparin cofactor reaction is a consequence of the formation of a ternary complex of the two proteins and heparin, then appropriately oriented ternary complexes of other reactive protein pairs and heparin might undergo comparable rate enhancement. Such a reactive pair is represented by prothrombin and thrombin. Prothrombin is cleaved by thrombin to yield Fragment 1 and Prethrombin 1 [21], and both prothrombin and thrombin bind to heparin [8]. The effect of catalytic concentrations of heparin on the rate of hydrolysis of prothrombin by thrombin is shown in Fig. 9. At heparin concentrations which inhibit up to 80% of the
75 hydrolysis by thrombin of 0.4 mM benzoyl-Gly-Pro-Arg-p-nitroanilide [22] the hydrolysis of prothrombin is enhanced at least 2--3 fold. Inhibition with the tripeptide substrate is n o t due to adsorption of the substrate to heparin, since, with a m o n o m e r molecular weight of heparin of about 200, the inhibition is seen at a 10--100 fold excess of substrate over heparin monomer. Discussion
If catalysis by heparin of the thrombin-heparin cofactor reaction is the result of formation of a ternary complex of heparin, the enzyme, and its inhibitor, then both proteins must bind simultaneously to heparin. Such a complex is n o t demonstrated unambiguously by the binding of both thrombin and heparin cofactor to insolubilized heparin [8], since the heterogeneity of heparin [11] might allow the binding of each protein to dissimilar species of heparin. However, the ability of solution-phase heparin to interlink thrombin non-covaler;tly with solid-phase heparin cofactor requires that a single heparin molecule binds to both proteins. However, binding studies alone cannot demonstrate unambiguously that a ternary complex is required for catalysis. The dependence of catalysis on the binding of both proteins to heparin is shown by the chemical modification studies. The effects of amidination of the inhibitor are comparable to those reported by Rosenberg and Damus [2]. The rate of reaction of the modified inhibitor with thrombin is insensitive to heparin. This finding, together with the finding that the modified inhibitor does n o t bind :to heparin-agarose, verifies that catalysis requires the binding of the inhibitor to heparin. However, in agreement with the results of Machovich [9], reaction of arginine-modified thrombin with unmodified inhibitor is likewise unaffected by heparin, despite binding of the unmodified inhibitor to heparin. Modification of the highly specific protease, thrombin, did not affect its specific activity toward the protein substrates fibrinogen and heparin cofactor, which would be highly sensitive to perturbation of the specificity of the enzyme. Therefore, insensitivity to heparin of the modified enzyme-unmodified inhibitor reaction implies that catalysis is not simply a consequence of an allosteric effect of heparin on the inhibitor, b u t must depend absolutely on the binding of thrombin as well as the inhibitor to heparin. Such a model for the activity of heparin was suggested by Yin and Wessler [10] and Gitel [8] as analogous to the prothrombin activation complex, where a phospholipid bilayer can enhance the rate of prothrombin activation by providing a binding surface for prothrombin and activated factor X [23--25]. The binding surface model is supported further by the finding that the rate of hydrolysis of prothrombin by thrombin, another protein pair which binds mutually to heparin [8], is enhanced by that binding. Furthermore, enhancement of proteolysis by heparin concentrations which inhibit the activity of thrombin with substrates other than prothrombin (or heparin cofactor) implies that heparin is not a positive allosteric effector of thrombin, and the inhibition of thrombin by heparin, whether steric or all~)steric, must be small compared to the catalytic effect of heparin. Our data do not exclude conformational effects of heparin on either throm-
76
bin or heparin cofactor. However, the effect of blocking the binding of either protein to heparin is the same: the enzyme-inhibitor reaction rate becomes insensitive to heparin. Therefore if heparin is an allosteric effector, then complimentary changes in the conformations of both proteins must occur simultaneously, and the change in conformation of only one protein of the pair must have neither a positive nor negative effect on their reaction rate. Li et al. [18,19] have determined that the binding of proflavin to heparin produces the spectral shift with ~ m a x = 447 nm, and that binding of thrombin to heparin reverses the shift by displacing proflavine from heparin. With experimental conditions identical to those under which thrombin was examined, heparin cofactor exerted no effect on the difference spectrum between proflavine and proflavine bound to heparin. In addition, there was no evidence of the spectral shift which Li et al. [19] found to result from binding of proflavine to heparin cofactor and which partially obscured a comparison of the spectra. Whether the result of minor changes in heparin or proflavine preparations or in ionic environment, the absence of this spectral shift permits the comparison of heparin-proflavine spectra obtained with and without heparin cofactor. The virtual identity of the two spectra suggests that at least one site on heparin binds tightly to thrombin but not to heparin cofactor. An alternate but unlikely possibility is that thrombin binds more tightly and heparin cofactor much less tightly than proflavine to heparin. However affinity chromatographic data [8] indicate that heparin cofactor binds more tightly than thrombin to heparin. The data in the present study are consistent with the hypothesis t h a t heparin acts as a mutual binding substrate for thrombin (and presumably other proteinases) and heparin cofactor. Since binding and chemical modification data alone cannot determine the mechanism by which the ternary complex is reactive, the extent to which this mutual binding accounts for the catalytic activity of heparin can best be evaluated by detailed kinetic analyses. However such studies must await a more thorough understanding and resolution from one another of the various molecular species which comprise available h e p ~ i n preparations.
Acknowledgements This work was supported by National Institutes of Health, Grant No. 1423005S1 (Specialized Center of Research in Atherosclerosis). M.P. was a trainee of the National Science Foundation, Secondary School Training Program.
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77 9 Machovich, R. (1975) Biochim. Biophys. Acta 412, 13--17 10 Yln, E.T. and Wessler, S. (1970) Biochim. Biophys. Acta 2 0 1 , 3 8 7 - - 3 9 0 11 Cifonelli, J.A. (1975) in Heparin: Structure, F u n c t i o n , and Clinical I m p l i c a t i o n s (Bradshaw, R.A. and Wessler, S. eds.), pp. 95--103, Plenum Press, New York 12 Van Lenten, L. and Ashwell, G. (1971) J. Biol, Chem. 246, 1 8 8 9 - - 1 8 9 4 13 Owen, W.G., Esmon, C.T. and Jackson, C.M. (1974) J. Biol. Chem. 249, 594--605 14 Hunter, M~I. and Ludwig, M.L. (1972) in Methods in E n z y m o l o g y (Hits, C.H.W. and Timasheff, S.N., eds.), VoL 25, pp. 585--596, E n z y m e Structure Part B 15 P a t t h y , L. and Smith, L.E. (1975) J. Biol. Chem. 250, 557--564 16 Izumi, Y. (1965) Anal. Biochem. 12, 1--7 17 Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3 0 5 9 - - 3 0 6 5 18 Li, E.H.H., Orton, C. and Feinman, R.D. (1974) Biochemistry 13, 5012--5017 19 Li, E.H.H., Fen ton, J.W. and Feinman, R.D. (1970) Arch. Biochem. Biophys. 175, 153--159 20 Laemmli, U.K. (1970) Nature 2 2 7 , 6 8 0 - - 6 8 5 21 Mann, K.G. Heldebrant, C.M. and Fass, D.N. (1971) J. Biol. Chem. 246, 6 1 0 6 - - 6 1 1 4 22 Owen, W.G. (1977) Biochim. Biophys. Acta 494, 182--190 23 Papahadjopoulos, D. and Hanahan, D~I. (1964) Biochim. Biophys. Acta 90, 436--439 24 Barton, P.G. and Hanahan, D.J. (1969) Biochim. Biophys. Acta 1 8 7 , 3 1 9 - - 3 2 7 25 Jackson, C.M., Owen, W.G., Gitel, S.N. and Esmon, C.T. (1974) Thromb. Diath. Haemorrh. Suppl. 57, 273--293
