Effects of Dextran Sulfate on Aprotinin Activity

Effects

of Dextran

Sulfate Tadahiro

Department

on Aprotinin SHIKIMI

of Pharmacology,

Accepted

Activity

Shimane

June

Medical

University,

7, 1985

Abstract-Dextran sulfate has an action on aprotinin activity similar to that heparin: preincubation of dextran sulfate with aprotinin enhanced the effect of aprotinin on the esterolytic activity of trypsin, but did not change on the proteolytic activity of trypsin or on the esterolytic and proteolytic of chymotrypsin. The enhancing effects of heparin and dextran sulfate due to increase in the active form of aprotinin which reacts with trypsin, due to a change in the aprotinin molecule which would lead to an increase tinin activity.

Aprotinin, a basic polypeptide with an isoelectric point close to pH 10.5 and with a molecular weight of about 6,500, is obtained from bovine organs, and it is pre scribed for patients with acute pancreatitis and shock due to acute peritonitis, owing to its inhibitory effects on trypsin, chymotrypsin, plasmin and kallikrein (1). In bovine tissue, aprotinin is localized in the mast cells (2-4). Preincubation of aprotinin with heparin enhanced the inhibitory effect of aprotinin on the esterolytic activity of trypsin (5). Dextran sulfate is a sulfated polysaccharide like heparin and has effects similar to heparin. For example, dextran sulfate has an anti coagulant action (6, 7), and its dependence on factor XII has been reported (8). On the other hand, the paradoxical platelet ag gregating activity of heparin (9) has also been reported, and in an in vitro study, both heparin and dextran sulfate inhibited the antiaggregating effect of prostacyc!in, pro staglandin D2 and prostaglandin El on platelets (10, 11). The ability of heparin and dextran sulfate to modulate the activity of hormone-sensitive adenylate cyclase derived from several tissues has been observed. In these cases, both compounds have similar action, except on the dopamine-sensitive adenylate cyclase from rat caudate nucleus (12). This report shows that dextran sulfate also has an action similar to that seen with

heparin enhancing aprotinin

seen with inhibitory the effect activities were not but rather in apro

on

aprotinin activity, effect of dextran activity is investigated.

Materials

and sulfate

the on

and Methods

Materials: A commercial sample of Trasylol (Bayer, AG, FRG), which cor responds to 1.4 mg of crystalline aprotinin, was used as the aprotinin preparation, and the following reagents were used: tosyl-L arginine methyl ester hydrochloride (TAME) and benzoly-L-tyrosine ethyl ester (BTEE) (Peptide Institute, Inc., Japan), casein ac cording to Hammarsten (Wako Pure Chemicals, Japan); trypsin (EC 3.4. 21.4) and a-chymotrypsin (EC 3.4. 21.1), which were both 3 times recrystallized (Miles Laboratories, U.S.A.); diphenylcarbamyl chloride (DPCC)-treated trypsin, type XI, and tosyl-L-lysine chloromethyl ketone (TLCK)-treated a-chymotrypsin, type VII (Sigma, U.S.A.); heparin [1,000 IU per ml (Novo Industry, Denmark)' 1 60 IU per mg (Nakarai Chemicals, Ltd., Japan)]; dextran sulfate, molecular weight of about 500,000 (Nakarai Chemicals, Ltd., Japan). Determination of aprotinin activity: Aprotinin was preincubated with various amounts of dextran sulfate at 37'C for 6, 24 and 48 hr. For determination of the inhibitory effects of aprotinin on the esterolytic activities of trypsin and a-chymotrypsin, the preincu

bation mixture was incubated with trypsin or a-chymotrypsin at 37°C for 5 min, and then the substrate (TAME or BTEE, respectively) was added. The enzymic activity of trypsin or a-chymotrypsin was measured by follow ing the rate of change in absorbance at 395 nm of the indicator, m-nitrophenol, as described by Simlot and Feeney (13). For determination of the inhibitory effects of aprotinin on the caseinolytic activities of trypsin and a-chymotrypsin, the pre incubation mixture of dextran sulfate and aprotinin was incubated with DPCC-treated trypsin or TLCK-treated a-chymotrypsin and casein, prepared by the method of Kunitz (14), at 37°C for 10 min, and then the amount of amino groups liberated from casein was determined by the previously described method (5). Difference spectrum of aprotinin: For the difference spectroscopy of aprotinin, com ponent 1, separated from the commercial product of aprotinin (Trasylol) on Sephadex G-50 as described previously (3), and dextran sulfate or heparin in crystalline form (160 IU/mg) were used. The difference spectrum of aprotinin in the ultraviolet region was measured using a Hitachi 220 A spectro photometer. Results Effects of dextran sulfate on aprotinin activity: Figure 1 shows the effects of dextran sulfate on aprotinin activity. The concentration of dextran sulfate added did not per se have any effect on the esterolytic or caseinolytic activity of trypsin or a chymotrypsi n. Preincubation of dextran sulfate with aprotinin enhanced the in hibitory effect of aprotinin on the esterolytic activity of trypsin, but did not change the effect on the caseinolytic activity of trypsin or on the esterolytic and caseinolytic activities of chymotrypsin. The aprotinin used in this study con tained at least 2 components with molecular weights of about 6,500 (component 1) and about 900 (component 2) (3). The former component showed the inhibitory activity of aprotinin on trypsin (3). Figure 2 shows that the preincubation of dextran sulfate with component 1 for 24 hr enhanced the in

hibitory effect of component 1 on the esterolytic activity of trypsin, whereas com ponent 2 did not have any effect on the esterolytic activity of trypsin. Figure 2 also shows that the change in the inhibitory activity of aprotinin on the esterolytic activity of trypsin after preincubation with dextran sulfate is probably due to component 1 per se, not to component 2 or a combination of component 1 with component 2. Trypsin titration of aprotinin activity: The inhibitory activity of aprotinin on the esterolytic activity of trypsin was titrated by trypsin. With this method, increasing amounts of trypsin are added to a fixed amount of aprotinin. As more trypsin is added, it over comes the aprotinin activity. Therefore, the equivalence point is identified as the point at which trypsin activity is first detected in the reaction mixture. In Fig. 3 (I), increasing amounts of trypsin were added to a fixed amount of aprotinin preincubated with or without dextran sulfate. The equivalence point of trypsin was identical in both groups. The same result was obtained with heparin (Fig. 3 (II)). Figure 3 also shows that dextran sulfate or heparin enhanced the inhibitory effect of aprotinin on the esterolytic activity of trypsin when comparing DS(+) and DS(-) or H(+) and H(-) in the inclination of the titration curves. Effect of dextran sulfate or heparin on the difference spectrum of aprotinin: The upper panel of Fig. 4 shows the difference spectra of component 1 dissolved in saline vs. saline (A) and of component 1 with dextran sulfate vs. dextran sulfate (B). The spectrum of aprotinin was identical with (A) and (B) just after the addition of dextran sulfate. However, at 48 hr after the addition of dextran sulfate, a change was observed at 200-240 nm between (A) and (B). The lower panel of Fig. 4 also shows the difference spectra of component 1 vs. saline (C) and of component 1 with heparin vs. heparin (D). Remarkable alteration in the spectrum of aprotinin was observed at 200-240 nm immediately after the addition of heparin. The change in the spectrum of aprotinin caused by heparin did not vary with incubation time.

Fig. 1. Effect of dextran sulfate on aprotinin activity. Various concentrations of dextran sulfate were preincubated with or without aprotinin at 37'C for the times shown. Then the enzyme and substrate (2 mM TAME, 0.5 mM BTEE or 0.3% w/v casein) were added. When esterolytic activities of trypsin and a-chymotrypsin were examined, 0.42 ug/ml of aprotinin for trypsin (5 fig/ml) and 0.7 ug/ml of aprotinin for a-chymotrypsin (15 ,pg/ml) were added; and when the caseinolytic activities of DPCC-treated trypsin (5 fig/ml) and TLCK-treated a-chymotrypsin (5 ,ug/ml) were examined, 0.28 ug/ml of aprotinin was added. The percentage inhibition was determined from the difference in enzymatic activities with aprotinin and without aprotinin. Columns show mean values±S.E. for 4 experiments. Significant differences from the control value (dextran sulfate concentration, 0) were determined by Student's t-test: *P<0 .05, **P<0.01.

Discussion In the present study, dextran sulfate enhanced the inhibitory effect of aprotinin on the esterolytic activity of trypsin. The similar action has also been observed with heparin in a somewhat different way (5). With extented preincubation with aprotinin, heparin further enhanced the aprotinin activity, whereas dextran sulfate did not further enhance the aprotinin activity. The

possibility that other sulfated polysaccharides such as heparan sulfate, dermatan sulfate and chondroitin sulfate also have an action similar to that seen with dextran sulfate or heparin on aprotinin activity awaits further investigation. In the solution, aprotinin exists in two states such as monomer and dimer (15-17), and the monomer form is regarded as an active form of aprotinin (18-20). Gelatin, a hydrophobic protein, which is used in

Fig. 2.

Effect of dextran

sulfate

on aprotinin

activity.

The commercial

product

study was separated into components 1 and 2 on a Sephadex G-50 column. 1 with the same inhibitory activity as the original aprotinin on the esterolysis

of aprotinin

used

in this

An amount of component of TAME with trypsin was

used. Component 2 was adjusted to the same volume in 0.9% NaCI solution as component 1. Various concentrations of dextran sulfate were preincubated with or without these components at 37 °C for 24 hr, and then Fig. 1 .

the

solutions

were

incubated

with

trypsin

and TAME

(2

mM).

Other

explanations,

as for

Fig. 3. Trypsin titration of aprotinin activity. Aprotinin (0.42 fig/ml) was preincubated with or without dextran sulfate (0.6 fiM) [or heparin (24 IU/ml)] at 37°C for 48 hr, and then the solution was incubated with various amounts of trypsin and 2 mM TAME. In the presence or absence of dextran sulfate [or heparin], the esterolytic activity of trypsin (8 ,ug/ml) without aprotinin was taken as 100%, and the relative percentages of the activity of trypsin were calculated from the corresponding trypsin activity with or without aprotinin. (I): Titration curve of trypsin to aprotinin preincubated with [DS(+)] or without [DS(-)] dextran sulfate. (II): Titration curve of trypsin to aprotinin preincubated with [H(+)] or without [H(-)] heparin.

Fig. 4. Difference spectrum of aprotinin. Component 1, separated from a commercial product of aprotinin on Sephadex G-50, and dextran sulfate or heparin in crystalline form were dissolved in 0.9% NaCI solution. Component 1 with or without dextran sulfate (or heparin) was incubated at 37'C for 48 hr, and the difference spectra of aprotinin were measured. Upper panel: A, component 1 (5.6 jig/ml) vs. 0.9% NaCl solution. B, component 1 (5.6 pg/ml) with dextran sulfate (8 pM) vs. dextran sulfate (8 IiM). Lower panel: C, component 1 (14 pig/ml) vs. 0.9% NaCI solution. D, component 1 (14 fig/ml) with heparin (800 IU/ml) vs. heparn (800 IU/ml).

complement assays as a stabilizing agent (21 ) and in the enzyme immunoassay of aprotinin as an eliminator of the influence of serum (22), also enhanced the inhibitory effect of aprotinin on the esterolytic activity of trypsin (23). The enhancing effect of gelatin was due to an increase in the active form of aprotinin in solution (23). In the present work, the equivalence point of trypsin to aprotinin preincubated with or without dextran sulfate overlapped each other. The

same result was obtained with heparin. These findings suggest that the enhancing effects of dextran sulfate and heparin are not due to increase in the active form of aprotinin which reacts with trypsin. In the difference spectrum of aprotinin, the degree of the change caused by dextran sulfate or heparin might be due to the differ ence in reactivity of aprotinin between dextran sulfate and heparin; i.e., heparin easily binds to aprotinin, whereas dextran

sulfate does not (24). No direct correlation between the change in the spectrum of aprotinin and the change of aprotinin activity, caused by dextran sulfate or heparin, was found in this study; however, the observation of the difference spectrum of aprotinin sug gests that both compounds affect the spectrum of aprotinin in the 200-240 nm region in which the absorbance of the protein peptide bond is observed (25). Among the amino acids residues which constitute aprotinin, lysyl residue 15 of aprotinin is essential for maintaining the activity of aprotinin and is involved in the interaction between aprotinin and trypsin (26). Lysyl residues of antithrombin and platelet factor IV serve as a specific binding site for heparin (27, 28). These findings are of interest in view of the enhancing effect of dextran sulfate or heparin on aprotinin activity. The interaction of dextran sulfate or heparin with the lysyl residue located in the reactive site of aprotinin requires study. In a preliminary study, dextran sulfate or heparin did not per se have any effect on the K,„ value of trypsin for TAME in the presence or absence of aprotinin; however, the dissociations of the trypsin-aprotinin complex and the trypsin TAME-aprotinin complex were suppressed by dextran sulfate or heparin. These findings also suggest that some qualitative change in the aprotinin molecule is induced by dextran sulfate or heparin. The combined use of heparin and aprotinin in the blood collection system is not recom mended, owing to the neutralization of heparin by aprotinin (29). The present results indicate that precaution is necessary in the combined use of dextran sulfate and aprotinin, for at least dextran sulfate has an effect on aprotinin activity. References 1 Vogel, R. and Zickgraf-Rudel, G.: Evaluation of the role of kinins in experimental, pathological, and clinical conditions; the therapeutic use of kallikrein inhibitor. In Bradykinin, Kallidin and allikrein, Edited by Erdos, E.G., p. 550-578, K Springer-Verlag, Berlin (1970) 2 Fritz, H., Kruck, J., Russe, I. and Liebich, H.G.: Immunofluorescence studies indicate that the basic trypsin-kallikrein-inhibitor of bovine organs (Trasylol) originates from mast cells. Hoppe

Seylers Z. Physiol. Chem. 360, 437-444 (1979) 3 Shikimi, T. and Kobayashi, T.: Production of antibody to aprotinin and location of this com pound in bovine tissue. J. Pharmacobiodyn. 3, 400-406 (1980) 4 Kobayashi, T. and Shikimi, T.: Histological studies of aprotinin-containing cells. Japan. J. Pharmacol. 30, Supp. 87P (1980) 5 Shikimi, T.: Enhancing effect of heparin on aprotinin activity. Experientia 37, 1179-1180 (1980) 6 Ricketts, C.R., Walton, K.W ; Van Leuven, B.D., Birbeck, A., Brown, A., Kennedy, A.C. and Burt, C.C.: Therapeutic trial of the synthetic heparin analogue, dextran sulfate. Lancet 2, 1004-1011 (1953) 7 Astrup, T. and Rosa, A.T.: A plasminogen proactivator-activator system in human blood effective in absence of hageman factor. Thromb. Res. 4, 609-613 (1974) 8 Miles, L.A., Rothschild, Z. and Griffin, J.H.: Dextran sulfate-dependent fibrinolysis in whole human plasma. J. Lab. Clin. Med. 101, 214-225 (1983) 9 Thomson, C., Forbes, C.D. and Prentice, C.R.M.: The potentiation of platelet aggregation and adhesion by heparin in vitro and in vivo. Clin. Sc. Mol. Med. 45, 485-494 (1973) 10 Eldor, A. and Weksler, B.B.: Heparin and dextran sulfate antagonize PGI2 inhibition of platelet aggregation. Thromb. Res. 16, 617-628 (1979) 11 Reches, A., Eldor, A. and Salomon, Y.: The effects of dextran sulfate, heparin and PGE1 on adenylate cyclase activity and aggregation of human platelets. Thromb. Res. 16, 107-116 (1979) 12 Amsterdam, A., Reches, A., Amir, Y., Minitz, Y. and Salomon, Y.: Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues. Biochim. Biophys. Acta 544, 273-283 (1978) 13 Simlot, M.M. and Feeney, R.E.: Relative reac tivities of chemically modified turkey ovomucoids. Arch. Biochem. Biophys. 113, 64-71 (1966) 14 Kunitz, M.: Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30, 291 310 (1946) 15 Anderer, F.A. and Hornle, S.: I. Molekularge wicht, Endgruppenanalyse und Aminosaure Zusammensetzung. Z. Naturforsch. - 20b, 457 462 (1965) 16 Burger, K., Zay, I., Gaizer, F. and Noszal, B.: Protonation and dimerization equilibria of the basic trypsin inhibitor (Kunitz base). Inorg.

Chim. Acta 34, L239-L241 (1979) 17 Wills, P.R. and Georgalis, Y.: Concentration dependence of the diffusion coefficient of a dimerizing protein: Bovine pancreatic trypsin inhibitor. J. Phys. Chem. 85, 3978-3984 (1981) 18 Kunitz, M. and Northrop, J.H.: Isolation from beef pancreas of crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound. J. Gen. Physiol. 19, 991-1007 (1936) 19 Fritz, H., Trautschold, I. and Werle, E.: Bestim mung der Molekulargewichte von neuen Trypsin

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