The effect of arginine on coagulation and fibrinolysis in vitro

Fibrinolysrs 8 Profeolys~s ( 1999) 13 (1). 31-34 Q Harcourl Brace 8 Co. Lld 1999

The effect of arginine on coagulation and fibrinolysis in vitro A. S. Hovest, M. K. Horne III Hematology

SewiCe.

ChICal

Pathology

Department,

Warren

G. Magnuson

Clinical Center,

National

Institutes

of Health,

Bethesda,

USA

Arginine is added to commercially-available recombinant tissue plasminogen activator (rtPA) to promote its solubility. However, arginine is also known to inhibit certain proteases. To explore the possibility that arginine inhibits the highly concentrated rtPA in commercial formulations, we tested the amino acid in assays of rtPA function. Although arginine was found to inhibit rtPA cleavage of a chromogenic substrate, it did not interfere with rtPA-mediated fibrinolysis. However, whole-blood clotting time and thrombin clotting time of purified fibrinogen were prolonged by arginine. Therefore, the concentration of arginine in commercial preparations of rtPA appears to have an anticoagulant effect but does not retard fibrinolysis.

Summary

INTRODUCTION

MATERIALS

Recombinant tissue plasminogen activator @PA) is a serine protease that is given systemically by intravenous infusion to treat myocardial infarction and pulmonary emboli. Recently, we have shown that it is also very effective in lysing venous thrombi if the drug is injected directly into the clots.’ However, for this indication rtPA is administered in a highly concentrated form in a solution that contains 35 mg/ml L-arginine (Alteplase-r”, Genentech, Inc., San Franciso, CA). Although arginine is necessary to solubilize rtPA at this concentration (1 mg/rnl), it is also known to inhibit certain proteases. Therefore, there is the possibility that the activity of the rtPA that is injected into venous thrombi is significantly inhibited by arginine. If this were the case, a lower concentration of rtPA that requires less arginine for solubilization and is, therefore, less inhibited might be just as effective as the higher dose we currently use. Reducing the dose of rtPA would result in significant cost savings. Therefore, we undertook the current work to investigate whether the arginine in the rtPA that we use (Alteplase’“? does, in fact, inhibit the enzyme’s activity.

Materials

Received; Accepted

4 September after revision:

7998 4 November

1998

Correspondence lo: Dr M. K. Horne. Rm. 2C306. Building 10. National Institutes of Health, Bethesda, MD 20692. USA. Tel.: +OOl (301) 402 2457; Fax: +00 (301) 402 2046

AND

METHODS

Human fibrinogen (grade L), human plasmin, and the chromogenic substrates SPECTROZYME tPATM and SPECTROZYME PLTM were obtained from American Diagnostica Inc. (Greenwich, CT). Bovine thrombin (100 units/ml) was from Parke-Davis (Morris Plains, NJ). Plasminogen was purified from human plasma using lysine-Sepharose from Pharmacia Biotech, Inc. (Piscataway, NJ).’ RtPA (AlteplaseTM) containing 35 mg L-arginine/mg enzyme was purchased from Genentech, Inc. (San Francisco, CA). DL-arginine, D-arginine, L-arginine, and bovine serum albumin were supplied by Sigma (St. Louis, MO). All other reagents were the highest grade available from commercial sources. The effect of arginine on rtPA activity chromogenic substrate

against

a

One hundred seventy-six microliters of tris-imidazole buffer (0.22 M NaCl, 0.029 M tris-HCl, 0.029 M imidazole, pH 8.4) was placed into microtiter wells followed by the addition of 4 d rtPA (1 mg/ml stock rtPA with 35 mg/ml L-arginine). After the mixtures were warmed to 37°C for 2 min, 20 fi of SPECTROZYME tPATM (2 mM stock) was added to produce final concentrations of 20 pg/ml rtPA and 700 pgg/rnL L-arginine. Color development (A405) was monitored in a kinetic plate reader (BioTek EL 340 Bio Kinetics Microplate Reader, Windooski, VT) at 37°C for 20 min with absorbance measurements recorded every 15 s. 31

32

Hovest, Horne

Arginine (either DL, L, or D) in tris-imidazole buffer (fmal arginine concentration from 8 to 35 mg/ml in addition to the 0.7 mg/ml contributed by the rtPA stock) was substituted for the tris-imidazole buffer alone. The pH of all arginine stocks was adjusted to 8.4. To test for the effect of the increased ionic strength contributed by the arginine, experiments were performed with tris-imidazole buffer containing 0.33 M NaCI, but no arginine. The conductivity of this high NaCl buffer was identical to that of 35 mg/mL arginine in the standard u-is-imidazole buffer (18 mmho). Each arginine concentration was tested in 12 and 16 wells on separate occasions. Data curves were analyzed to determine the maximum rate of reaction in mOD/min (Delta Soft 3.0, Princeton, NJ). The effect of arginine on plasmin chromogenic substrate

activity

against

a

Stock solutions of 5 mM SPECTROZYME PLr”’ and 10 j.&l plasmin were warmed to 37°C for 2 mm and then added to microtiter wells to achieve final concentrations of 0.5 mM substrate and 0.1 j.M plasmin in 200 d of Tyrode’s buffer (140 mM NaCI, 3 mM KCI, 1 mM MgCl,, 2 mM CaCl,, 0.5 mM Na,HPO,, 1 mM NaHCO,, pH 74). Some experiments included fmal arginine (DL, L, or D) concentrations of 14 mg/ml or 28 mg/ml @H adjusted to 74). Color progression for each experiment was followed in six wells at 37°C for 7 min with A405 measured every 10 s. Maximum reaction rates were determined. The effect of arginine by rt PA

on the activation

of plasminogen

After prewarming to 37°C for 2 min 80 $ of 5 mM SPECTROZYME PI?, 80 j.J of 3.5 @¶ plasminogen, 35 ~1 of Tyrode’s buffer with or without 350 mg1m.l arginine (DL, L, or D, pH 74), and 5 $ rtPA (1 mg/ml) were combined in microtiter wells (finals concentrations: 2 mM substrate, 1.4 @l plasminogen, 25 pg/ml rtPA). Color development (A405) was monitored at 37°C for 8 min with readings taken every 10 s. Arginine concentrations (875 pg/ml derived from the rtPA stock and - 62 mg/ml when supplemented with arginine in the buffer) were tested twice in two wells. Maximum rates of reaction were determined. The effect of arginine presence of fibrin

on plasminogen

activation

in the

Fibrin clots were prepared by covering the bottom of microtiter wells with 60 $ of a mixture of fibrinogen (fmal concentration 2.4 mg/ml) and plasminogen (final concentration 0.48 m, and bovine thrombin (fmal concentration 33 units/ml). The mixtures were allowed to clot for Fibrinolysis

& Proteolysis

(1999)

13(l),

31-34

10 min. Arginine (DL, L, or D) concentrations ranging from 8.8 mg/ml to 35 mg/ml were prepared by adding 500 ~1 of rtPA (0.5 mg/ml) and varying volumes of arginine (70 mg/ml stock) and Tyrode’s buffer containing 1% bovine serum albumin to produce a final rtPA concentration of 0.25 mg/mf in a volume of 1.3 ml. These solutions and the fibrin clots were warmed to 37°C for 2 mm. Then 100 1.11 of each rtPA-arginine solution was placed on top of 12 fibrin clots. Lysis was monitored in a kinetic plate reader for 20 mm at 37°C by measuring turbidity at h405 every 15 s. All experiments were performed twice. Maximum rates (mOD/min) of fibrinolysis were determined. In similar experiments plasminogen-free clots were overlaid with 100 ~1 of solutions containing 0.6 @‘vlplasminogen and 0.15 @J rtPA with or without 35 mg/ml DL-arginine. All constituents were pre-warmed to 37°C for 2 min. Each concentration of arginine was evaluated in 12 wells in a kinetic plate reader for 40 min at 37°C. A405 (turbidity) was measured every 30 s. AU experiments were performed twice. The maximum clot lysis rate (mOD/minute) in each well was determined. The effect

of arginine

on thrombin

activity

Thrombin activity was measured by fibrinogen clotting times using fibrometers (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD). Ninety microliters of arginine (DL, L, or D) in Tyrode’s buffer @H adjusted to 74; final arginine concentration O-21 mg/ml) was added to 200 ~1 of fibrinogen (final concentration 2.4 mg/ml) and warmed to 37°C for 1 min. Then 10 ~1 of bovine thrombin (5 units/ml stock) was added. Each arginine concentration was tested 10 times. Because the ionic strength of the reaction mixtures was affected by the arginine, in some experiments 0.5 M NaCI, 10 mM tris, pH 74 was substituted for the arginine solutions. The conductivity of this buffer was equal to that of 35 mg/ml arginine in Tyrode’s buffer (25 mmho). The effect of arginine

on whole-blood

clotting

time

Aliquots (933 j.d) of freshly drawn whole blood from five normal donors were placed in three glass tubes containing 67 1.11of either Tyrode’s buffer (8 mmho), or highNaCl u-is-buffer (0.5 M NaCI, 10 mM tris, pH 74, 25 mmho), or 350 mg/ml DL-arginine in water @H 74, 25 mmho). Each tube was immediately mixed and then rocked gently every 30 s until clotting became visible. Statistical

analysis

Reaction rates with 0 and 35 mg/ml arginine were compared using the Mann-Whitney U-test for unpaired data (StatView, Abacus Concepts, Berkeley, CA). 0 Harcourt

Brace

8 Co. Ltd 1999

Arginine in coagulation and fibrinolysis in vitro

33

One milliliter volumes of 1 mg/ml rtPA were dialyzed against solutions of L-arginine (5,10, or 20 mg/ml) in trisbuffered saline for 24 h at 4°C. After dialysis the turbidity of the solutions was quantitated by measurements of optical density at h400. If a precipitate formed, the rtPA samples were redialyzed against 35 mg/ml arginine to test rtPA resolubility.

control buffer (0.22 M NaCl), rtPA activity was not affected, thereby ruling out increased ionic strength as the cause of the apparent arginine effect. In studies of rtPA activation of plasminogen there was no apparent effect of 35 mglml arginine when either fibrin or SPECTROZYME PL was used as the plasmin substrate (Table 1.1 b,c). The lack of effect of 35 mg/ml arginine on plasmin activity was also demonstrated using performed plasmin with either SPECTROZYME PL or fibrin clots as substrate (Table 1.2 a,b). Arginine (final plasma concentration 35 mg/ml), however, did prolong the clotting time of whole blood (~5) by a mean of 3.4 min (SD = 1 min, ~=5), from 5.7ti.3 min (mea&SD) to 9.1iO.9 min (mea&SD). An arginine-free control buffer with the same conductivity as the arginine stock had no effect on the clotting times. Arginine also prolonged the thrombin-induced clotting time of purified fibrinogen (Fig. 2), whereas an arginine-free buffer at the same ionic strength as 35 mg/ml arginine in standard buffer had no effect. As the arginine concentration in stock rtPA (35 mg/mg protein) was reduced by dialysis, the rtPA solution became increasingly turbid (Fig. 3) but reclarified when arginine was readded to a concentration of 35 mg/ml.

RESULTS

DISCUSSION

In all assays both L- and D-arginine were tested and produced indistinguishable results. Figure 1 demonstrates the inhibitory effect of various arginine concentrations on the reaction between rtPA and its chromogenic substrate (SPECIROZYME tPA). As arginine concentration was increased to 35 mg/ml, the enzymatic activity of rtPA diminished to 36% of the initial rate (Table 1. la). When 0.3 M NaCl tris-imidazole buffer with the same conductivity (18 mmho) as the 35 mg/ml arginine solution was substituted for the

Our results indicate that the fibrinolytic activity of rtPA is not inhibited by the concentration of arginine (35 mg/ml) that is necessary to keep 1 mg/ml of the enzyme in solution. Since fibrinolysis is actually dependent upon two enzymatic reactions - the conversion of plasminogen to plasmin by tPA and the degradation of fibrin by plasmin - we have shown that arginine interferes with neither of these. Therefore, injecting the concentrated drug directly into venous thrombi should be highly effective, as, in fact, we have observed it to be.’

0.800

0.480

I

I

I

I

I 4

I 8

I 12

1 16

-

0

nmo

I

(minutes)

Flg. 1 Arginine inhibition of rtPA activity against a chromogenic substrate (SPECTROZYME tPA). (a) 0.7 mglml arginine; (b) 6.7 mglml arginine; (c) 22.7 mglml arginine; (d) 35.7 mg/ml arginine.

Solubility

of rtPA in the presence

Table

1

Reaction

of arginine

rates

(mOD/min)

of rtPA and plasmin

Control median (range) 1. tPA tests a. chromogenic (n= 16) b. fibrinolysis

Arglnine (35 mg/ml) median (range)

Pvalue*

61

(15)

22

(10)

<0.0001

20

(10)

22

(18)

0.21

activation

282

(81)

246

(56)

0.25

2. Plasmin tests a. chromogenic substrate

201

(31)

193

(22)

0.26

5.2

(5.3)

5.4

(6.4)

0.27

substrate

(n=9) c. plasminogen

(n = 4) (n = 6) b. fibrinolysis In) = 21) *Mann-Whitney

8 Harcouri Brace & Co. Ltd 7999

U-test

for unpaired

data

Fibnhoiysis & Proteo&sis (1999) 13(l). W-34

34

Hovest, Home

1.4MO1.2MO-

3

l-

no-

1 g I5 3 b ”

2 b a 2 I-;

loo-

80-

0.8-

0.6 -

60-

0.4 -

40-

0.2-

0

5 Arginine

10 Concentration

15

20

25

(mghnl)

0: 0

5

10 Argiine

15

20

Concentration

25

30

35

(mg/ml)

Fig. 2 Arginine effect on thrombin (5 units/ml) clotting time of fibrinogen. Each point represents the mean coagulation timefl SD (IMJ-13).

Fig. 3 Solubility of rtPA in the presence of arginine. The vertical axis represents turbidity measurements at MOO.

On the other hand, arginine does inhibit rtPA activity against a small chromogenic substrate that contains arginine (Fig. 1, Table 1). This suggests that in its therapeutic role rtPA is protected from arginine inhibition by binding to its cofactor fibrin and/or by interaction with its substate plasminogen. Of interest, arginine did not inhibit the activity of plasmin against a chromogenic substrate that does riot contain argmine (Table 1.2a). Therefore, free arginine presumably competes with the arginine moiety in the tPA substrate. To pursue potential arginine roles further we tested its effect on whole-blood clotting times, which we found were significantly prolonged by a plasma concentration of 35 mg/ml of the amino acid. Although this effect could reflect inhibition of many different procoagulant reactions, we established that it at least involves inhibition of thrombin (Fig. 2). The effect of arginine on hemostatic mechanisms has apparently not been extensively studied previously. We could find very little literature on the subject. Using thromboelastography Dambisya and Lee demonstrated the L-arginine causes hypocoagulability at concentrations (eg. 1.4 @ml) much lower than those we studied; yet fib rinolysis was not affected.4 In contrast, Udvardy et al. recently reported that micromolar concentrations of arginine enhanced m-vitro lysisof plasma clots in response to added t.PA.SNeither of these groups tested the much greater concentrations of arginine that we studied. It appears, therefore, that our concern that arginine retards the effect of rtPA in lysing venous thrombi was

unfounded. On the basis of our in vitro studies and the limited amount of information in the literature, we conclude that arginine does not inhibit fibrinolysis and, in fact, has anticoagulant activity. This is fortunate since high concentrations of the amino acid are clearly required to maintain rtPA in solution in the currently available formulations (Fig. 3).

Fibhotysis

& Proteolysis (1999) 13(l), 31-34

ACKNOWLEDGEMENTS

We would like to thank Dr Richard Chang of the Radiology Department of the National Institutes of Health for the insights that led to this study. REFERENCES 1. Chang R, Home MK, Mayo DJ, Doppman JL. Pulse-spray treatment of subclavian and jugular venous thrombi with recombinant tissue plasminogen activator. J Vast Intervent Radio1 1996; 7: 845-85 1 2. Hook VYH. Arglnine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme. Life Sci 1990; 47: 1135-l 139. 3. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096. 4. Dambisya YM, Lee TL. A thromboelastography study on the in vitro effects of L-arglnine and L-NGnltro arglnine methyl ester on human whole blood coagulation and Bbrinolysis. Blood Coag Fibtinolys 1996; 7: 678-683. 5. Udvardy M, Posan E, PaIatka K, Akorjay I, Harsfalvi J. Effect of L-arginine on in vitro plasmin-generation and Bbtinolysis. Thromb Res 1997; 87: 75-82. Q liarcourt Brace & Co. Ltd 7999