Aprotinin is a competitive inhibitor of the factor VIIa-tissue factor complex

THROMBOSIS RESEARCH 71; 205-2151993 0049-3848/93 $6.00 + .OO Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

APROTININ

IS A COMPETITIVE INHIBITOR OF THE FACTOR VIIa-TISSUE FACTOR COMPLEX J. Chabbat,

Centre

National

P. Porte, M. Tellier and M. Steinbuch de

Transfusion

Sanguine,

(Received 26.1 .1993; accepted in revised form 27.4.1993

Paris,

France

by Editor K.L. Lechner)

Abstract A highly purified preparation of human plasma factor VIIa was submitted to chromogenic assays with S-2288 factors IXa, Xa, activated protein C and thrombin being absent. Factor VIIa alone or in the presence of calcium, kept its activity even in the presence of high concentrations of aprotinin, inhibition appeared only in the presence of a factor VIIa-tissue factor complex. A two-stage amidolytic assay using activation of factor X and hydrolysis of S-2765 chromogenic purified substrate by the generated Xa was used to show a competitive inhibition with a Ki value of 30 PM. Aprotinin had no effect on factor Xa amidolytic activity per se. The factor VIIa-tissue factor complex could be adsorbed to immobilized aprotinin and removed by a chaotropic ion like KSCN 3 M. The assays with the DFP inactivated VIIa-tissue factor complex proved that the interaction involved the active site of factor VIIa. The inhibition of the VIIa-tissue factor complex was demonstrated in a clotting assay usipg aprotinin enriched normal or factor VIII deficient plasma. Key ords-:-Aprotinin-Factor VIIa-Tissue factor-Inhibition Corresponding author: Dr Chabbat J., CNTS, 4 avenue des Tropiques BP-100, 91943 Les Ulis, France.

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TISSUE FACTOR COMPLEX INHIBITOR

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The extrinsic pathway of blood coagulation is initiated by the formation of the factor VIIa-tissue factor complex which activates factor X and also factor IX. The regulation of these reactions is complex. Antithrombin III, the most important plasma inhibitor of thrombin, Xa and IXa does not significantly inhibit factor VIIa and this inhibition occurs only slowly in the presence of exogeneous heparin (1). Indeed, the extrinsic pathway is essentially regulated by a Kunitz-type protease inhibitor of plasma (2) named tissue factor pathway inhibitor (TFPI) (3). It is currently assumed to involve, in a first step, the formation of a TFPI-Xa complex and in a second step, the formation of a quaternary TFPI-Xa-VIIa-tissue factor complex in the presence of calcium (4). Another Kunitz-type protease inhibitor interacts with several plasma serine proteases, aprotinin or bovine trypsin pancreatic inhibitor (BTPI). This 6.5 kD peptide is constituted of 58 aminoacid residues. Especially, it appears like a strong inhibitor of kallicrein (5), plasmin (6), urokinase (7), leucocyte elastase (8) and activated protein C (9). In this report we describe the behaviour of VIIa and VIIa-tissue factor complex in the presence of aprotinin. MATERIALS AND METHODS The prothrombin complex concentrate (PPSB) was prepared in our Institute. Aprotinin (Iniprol) was from Laboratoires Choay (Paris, France). Chromogenic substrates, S-2238, S-2266, S-2288 and S-2765 were purchased from Kabi (Stockholm, Sweden).Thromboplastin (Thromborel) was from Behring (Marburg, Germany). Diisopropyl fluorophosphate (DFP) was from Sigma (St Louis, MO, USA). Q-Sepharose and Benzamidine-Sepharose were purchased from Pharmacia (Uppsala, Sweden). Aprotinin-Sepharose was prepared as described by Taby et al .(9).

Purification of human factor VIIa. The purification was performed by a modification

of the industrial process (11): The prepurified factor VII was adsorbed onto Q-Sepharose, washed with Tris-HCl 50 mM, NaCl 0.15 M, pH 8.0, followed by elution with the same buffer containing CaC12 5 mM as described by Broze et al (12). Traces of factor X were removed after a second chromatography on QSepharose. The elution of factor VIIa was obtained with Tris-HC15 mM, NaCl 0.2 M, pH 8.0. Traces of activated protein C (APC) and thrombin were adsorbed onto insolubilized aprotinin(9) and benzamidine-Sepharose (13) respectively.

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207

The highly purified factor VIIa had no activity on S-2765, S-2266 and S-2238. The SDS-PAGE (gradient 4-30%) analysis showed that the MW of VIIa was 53000 D. Two bands of 30000 and 23000 D were revealed in the presence of a reducing agent. Purification of human factor X. The PPSB was applied to a Q-Sepharose column, equilibrated with a buffer containing Tris-HCl 50 mM at pH 7.5. A gradient of NaCl from 0.1 to 0.5 M allowed to purify the factor X. Most factor X was eluted between 0.25 and 0.30 M NaCl. Factor VII, protein C were removed in this step but not all prothrombin and factor IX. So, this operation was followed by a chromatographic step on heparin-Sepharose as described by Bajaj et al . (14) The purified preparation did not show any chromogenic activity and the absence of factors VII and IX was revealed by ELISA. Protein determination. Protein assay was performed by the BCA method (Pierce Chemical, Rockford, IL, USA). Factors VII, IX, X and protein C antigens were evaluated by ELISA, using Asserachrom products from Stago (Asnieres, France). Chromogenic assays. Factor VIIa amidolytic activity on S-2288 was measured following the increase of absorbance at 405 nm in a buffer containing Tris-HCl 50 mM, NaCl 0.25 M, pH 8.4 at 37°C (10). For the detection of factor Xa, APC, or thrombin S-2765, S-2266 and S-2238 were used respectively as recommanded by Kabi diagnostic. Thromboplastin activity. A standard curve was established for each serie of assays. A well-defined solution of thromboplastin was used for standardization. Dilutions were made in the working buffer from 0.10 to 1.00 unit. 50 pl of the standards or samples were incubated during 5 min at 37°C in 650 ~1 of a Tris-HC150 mM, CaC12 5 mM, NaClO.15 M buffer, pH 8.4 and 100 l.tl of reagents containing 0.5 Ag U/ml factor VIIa, 0.5 Ag U/ml factor X added. The mixture was incubated 3 min at 37°C before addition of 100 pl of S-2765 chromogenic substrate. After 1 min the reaction was stopped with acetic acid and recorded at 405 nm. Coagulation time with diluted thromboplastin 50 l..tlof human normal or factor VIII deficient (Immuno AG) plasma samples were mixed with 50 pl of a 135 PM aprotinin solution. After an incubation time of 5 min at 37”C., 100 p.1 of thromborel was added and the coagulation time was measured on ST 888 (Stago) coagulation apparatus. Dilutions of thromborel (l/10 to l/4000) were made in a solution containing CaC12 20 mM, NaC133 mM and 1% human albumin.

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TISSUE FACTOR COMPLEX INHIBITOR

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RESULTS Inhibition

of factor

The effects chromogenic

VIIa

by aprotinin.

of aprotinin substrate.

Chromogenic

on factor

VIIa

were

assays.

examined

with

S-2288

Different concentrations of aprotinin (2-55 PM) were incubated at 37°C in Tris-HC150 mM, NaC10.25 M, pH 8.4 with an aliquot of factor VIIa (50 nM) during 5 min. Residual activity was measured after addition of S-2288 (0.4 mM). The experience was also performed in the presence of CaC12 5 mM or CaC12 5 mM and thromboplastin 0.15 U.

A 2-

0-

.c

.E E I

1.5

G E II ”

s

V

.

6.

s

z

?

03

2-

4 0

I

20

40

1 60

[Aprotlnln] @4

7. 0

I

8. 20

I.

I 40

8.

i 60

[Aprotinin] PM

FIG.l. Effect of aprotinin

on the amidolytic

activity of factor VIIa .

Fig. la&Inhibition of amidolytic activity of factor VIIa (50 nM); + Inhibition of amidolytic activity of factor VIIa (50 nM) in the presence of calcium (5 mM). Fig.lb-@-Inhibition of amidolytic activity of thromboplastin-factor VIIa complex (thromboplastin 0.15 U, factor VIIa 50 nM).

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TISSUE FACTOR COMPLEX INHIBITOR

These experiments demonstrated that aprotinin interacts only with the factor VIIa-thromboplastin complex (fig.1). Factor VIIa alone has a very weak amidolytic activity on S-2288 which remained unchanged in presence of aprotinin. As described for recombinant factor VIIa by et al. (lo), the activity of plasma factor VIIa increased when Pedersen calcium was added to the solution. Again the hydrolysis of the substrate was not modified in the presence of aprotinin. In contrast the activity on S-2288 of factor VIIa-thromboplastin complex was important but aprotinin inhibited it strongly. Inhibition of factor X activation. The inhibition of the factor VIIa-thromboplastin

complex by aprotinin was followed up by a chromogenic assay based on factor X activation, following the procedure described by Kondo et al. (15) with slight modifications. In our assay, thromboplastin (0.055 U) was incubated for 5 min with factor VIIa (0.8 nM), aprotinin (13.5 to 81 PM) in a buffer containing Tris-HCl 50 mM, NaCl 0.15 M, CaC12 5 mM, pH 8.4, final volume being for 400 pl. 50 ~1 of factor X (16 to 50 nM) were added to this mixture and incubated at 37°C for 2 min exactly. The addition of 50 yl EDTA (0.2 M) stopped the reaction. A sample of the incubated solution was mixed with 700 pl of S-2765 in Tris-HCl 0.05 M, NaCl 0.25 M, pH 8.4. As a control factor Xa amidolytic activity was not modified in the presence of aprotinin.

X=16

nM)

X

nM)

32

??

nM)

i0

-

40

-

S’O--

80

100

Ki = 30 ~.LM [Aprotinin] PM

FIG. 2. Inhibition of factor VIIa-tissue factor Dixon plot for the inhibition of factor The results

showed

a competitive

inhibition

with

by aprotinin, X activation. a Ki of 30 pmol/L.

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TISSUE FACTOR COMPLEX INHIBITOR

Interaction insolubilized

between aprotinin.

factor

Vlla-thromboplastin

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complex

and

Aprotinin appeared as a competitive inhibitor of factor VIIathromboplastin complex. To explain the interaction between aprotinin and the active site of factor VIIa, adsorption onto insolubilized aprotinin was performed. Because some part of thromborel was adsorbed to

insolubilized aprotinin, only the unadsorbed fraction was kept for further use. Factor VIIa (1 nM) first incubated with thromboplastin (0.45 U), was then applied to the gel (containing 5 mg aprotinin/ ml). The column was washed with the buffer containing Tris-HCl 50 mM, CaC12 5 mM, NaCl 0.5 M pH 8.0. The elution of the bound factor VIIa was obtained with KSCN 3 M. As seen in table 1, when factor VIIa alone or the DFP pretreated factor VIIa-thromboplastin complex were used, no adsorption was observed and the proteins were recovered in the effluent or in the washing solution. TABLE 1 Behaviour

of VIIa with or without thromboplastin Sepharose at pH 8.

Starting Material (%) *

on aprotinin-

Effluent

Wash

Elution

&

VIIa

100

92

2

2

VIIa/thromboplastin

100

25

11

60

DIP-VIIa/ Thromboplastin

100

90

2

4

*.

Determinations antigen.

were expressed

in percentage

recovery

These results confirmed the interaction between aprotinin active site of factor VIIa in the presence of thromboplastin.

of VIIa

and

t

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TISSUE FACTOR COMPLEX INHIBITOR

Influence deficient

on thromboplastin

of aprotinin plasma.

time of normal

211

and factor

VIII

In order to examine the effect of aprotinin on the clotting system, clotting assays were performed using prothrombin time of normal and FVIII deficient plasma with different dilutions of thromboplastin. VIII deficient plasma + aprotinin

VIII deficient plasma Normal plasma + aprotinin

1 / dilution of thromboplastin

FIG.3. Prolongation of thromboplastin coagulation time of normal deficient plasma containing aprotinin (100 uM) .

or factor VIII

The figure 3 shows the effect of aprotinin on the prothrombin time of both normal and FVIII deficient plasma. With high dilutions of of the extrinsic pathway can thus be thromboplastin the inhibition substantiated. DISCUSSION A two step model has been proposed concerning the inhibitory mechanism of TFPI (16). The first step refers to the second Kunitz domain which interacts with the active site of factor Xa (17). It has been shown that an arginine residue of the second Kunitz domain is involved for this inhibition (17). We may notice that aprotinin, unable to inhibit factor Xa, shows a lysine residue in this position fifteen. The latter is known to be very important in the inhibition of serine proteases (18).

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In the second step, the TFPI-Xa complex binds to the factor VIIa-tissue factor complex and inhibits its activity. The first Kunitz domain of TFPI is the structure involved in this inhibition (19). The results show that aprotinin is an inhibitor of the factor VIIa-tissue factor complex. Indeed factor VIIa bound to tissue factor looses its amidolytic activity in the presence of aprotinin. In contrast the inhibitor has no such effect on factors VIIa nor Xa alone. The kinetic analysis demonstrates a competitive inhibition of the activation of factor X with a Ki of 30 umol/L. The interaction with insolubilized aprotinin allows to demonstrate that the active site of factor VIIa is involved in the reaction. Indeed, DIP-factor VIIa-tissue factor complex is not retained by this matrix in opposition to the factor VIIa-tissue factor complex. Lysin is a relevant residue in the first Kunitz domain of TFPI (17) as it is in aprotinin. It has been reported that both calcium ions and tissue factor are involved in factor VIIa conformation and catalytic activity. Indeed, Nicolaisen et al. (20) showed that cathepsin G is able to degrade the light chain of factor VIIa only in the presence of calcium ions. It has been also shown that factor VIIa amidolytic activity on S-2288 is increased in the presence of calcium ions (10). Furthermore, the maximum of factor VIIa activity is obtained in the concomitant presence of these ions and tissue factor (10, 21). Our results indicate that both thromboplastin and calcium are necessary to achieve the inhibition of factor VIIa by aprotinin. These observations confirm the molecular rearrangement of VIIa occurring in the tissue factor-VIIa complex in the presence of calcium ions. This modification is essential for the inhibition by aprotinin. The normal prothrombin time observed in the presence of high amounts of thromboplastin did not allow to show any inhibition by aprotinin. small However this inhibiting effect became’ evident when concentrations of tissue factor were used as shown in a modified thromboplastin coagulation time. Using factor VIII deficient plasma it could be demonstrated that aprotinin inhibits the extrinsic pathway, and that this inhibition concerns the initial phase of the extrinsic pathway before Xa generation. Thus, it would be interesting to study the in vivo effect of aprotinin in disseminated intravascular coagulation (DIC) induced by low doses of tissue factor as previously shown for TFPI (22), giving rise to future therapeutic possibilities. The extent of DIC might indicate whether an appropriate therapeutic choice will preferably be the use of aprotinin to _ . inhibit the extrinsic pathway or the injection of activated protein C to inactivate the generated factors Va and VIIIa.

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REFERENCES

KONDO S. and KISIEL W. Regulation of factor VIIa activity in evidence that antithrombine III is the sole plasma plasma: inhibitor of human factor VIIa. Thromb. Res. 46, 325-335, 1987. WARR T.A., WARN-CRAMER B.J., RAO L.V.M. and RAPAPORT S.I. Human plasma extrinsic pathway inhibitor activity: I.Standardization of assay and evaluation of physiologic variables. Blood 74, 201-206, 1989. PALMIER M.O., HALL L.J., REISCH C.M., BALDWIN M.K., WILSON A.G.E. and WUN T.C. Clearance of recombinant tissue factor pathway inhibitor (TFPI) in rabbits.Thromb. Haemostasis 68, 3336, 1992. 4

SANDSET P.M. and ABILDGAARD U. Extrinsic pathway inhibitorthe key to feedback control of blood coagulation initiated by Haemostasis 21, 219-239, 1991. tissue thromboplastin. PISANO J.J. Chemistry and biology of the kallicrein-kinin system. Proteases and biological control. E.Reich, D.B.Rifkin and E. Shaw (Eds). Cold Spring Harbor Laboratory, 199-222, 1975. KASSEL B. Bovine trypsin-kallicrein inhibitor (Kunitz inhibitor, basic pancreatic trypsin inhibitor, polyvalent inhibitor from bovine organs). Methods in Enzymology 19. G.E. Perlman and L. Lorand (Eds) New York-London: Academic Press, 844-852, 1970.

7

LOTIENBERG R., SJAK-SHIE N., FAZLEABAS A.T. and ROBERTS R.M. Aprotinin inhibits urokinase but not tissue-type plasminogen activator. Thromb. Res. 49, 549-556, 1988.

8

LESTIENNE P. and BIETH J.G. The inhibition of human leucocyte elastase by basic pancreatic trypsin inhibitor. Arch. Biochem. Biophys. 190, 358-360, 1978.

9

TABY O., CHABBAT J. and STEINBUCH M. Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification. Thromb. Res. 59, 27-35, 1990.

10

PEDERSEN A., LUND-HANSEN, T., KOMIYAMA Y., PETERSEN L.C., OESTERGARD P.B. and KISIEL W. Inhibition of recombinant human blood coagulation factor VIIa amidolytic and proteolytic activity by zinc ions. Thromb. Haemostasis 65, 528-534, 1991.

213

214

TISSUE FACTOR COMPLEX INHIBITOR

Vol. 71, No. 3

11

CHABBAT J., HAMPIKIAN-LENIN S., TOULLY V., GAILLANDRE A., PEJAUDIER L. and STEINBUCH M. A human factor VIIa concentrate and its effects in the hemophilic dog. Thromb. Res. 54, 603-612, 1989.

12

BROZE G.J. and MAJERUS P.W. Purification and properties of human coagulation factor VII. J. Biol. Chem. 255, 1242-1247, 1980.

13

SCHMER G. The purification of bovine thrombin by affinity chromatography on benzamidin-agarose. Hoppe-Seyler’s Z Physiol.Chem. 353, 810-814, 1972.

14

BAJAJ S.P., RAPAPORT S.I. and BROWN S.F. Isolation and characterisation of human factor VII. J. Biol. Chem. 256, 253-259, 1981.

15

KONDO S., NOGUCHI M., FUNAKOSHI T., FUJIKAWA K. and KISIEL W. Inhibition of human factor VIIa-tissue factor activity by placental anticoagulant protein. Thromb. Res.48, 449-459, 1987.

16

WARM-CRAMER B.J., RAO L.V.M., MAKI S.T. and RAPAPORT S.I. Modifications of extrinsic pathway inhibitor (EPI) and factor Xa that affect their ability to interact and to inhibit factor VII/tissue factor: evidence of a two step model of inhibition. Thromb. Haemostasis 60, 453-456 ,1988.

17

GIRARD T.J., WARREN L.A., NOVOTNY W.F., LIKERT K.M., BROWN S.G., MILETICH J.P. and BROZE G.J. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 338, 5 18-520, 1989.

18

CHAUVET J. and ACHER R. Active derivatives of polypeptide inhibitor of trypsin (Kunitz and Northrop inhibitor) Biochem. Biophys. Res. Commun. 27, 230-235, 1967.

19

GIRARD T.J., MACPHAIL L.A., LIKERT K.M., NOVOTNY W.F., MILETICH J.P. and BROZE G.J. Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein. Science 248, 1421-1424, 1990.

20

NICOLAISEN E.M., PETERSEN L.C., THIM L., JACOBSEN J.K., CHRISTENSEN M. and HEDNER U. Generation of Gla domainless FVIIa by cathepsin G-mediated cleavage. FEBS. Letter 306,157160, 1992.

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21

22

TISSUE FACTOR COMPLEX INHIBITOR

LAWSON J.H., BUTENAS S. and MANN K.G. The evaluation of complex-dependent alteration in human factor VIIa. J. Biol. 267, 4834-4843, 1992.

215

Chem.

SANDSET P.M., WARN-CRAMER B.J., MAKI S.L. and RAPAPORT S.I. Depletion of extrinsic pathway inhibitor sensitizes rabbits to endotoxin-induced intravascular coagulation and the generalized Shwartzman reaction. Blood 78, 1496-1502, 1991.