Plasma TFPI activity after intravenous injection of pentasaccharide (PS) and unfractionated heparin in rabbits

Thrombosis

Pergamon

Research, Vol. 75, No. 5, pp. 577-580, 1994 Copyright 8 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0049-3848/94 $6.00 + .Oo

0049-384tI(94)EOOlO2-2

BRIEF PLASMA OF

TFPI

PENTASACCHARIDE

COMMUNICATION

ACTIVITY

AFTER

(PS)

AND

INTRAVENOUS UNFRACTIONATED

INJECTION HEPARIN

IN

RABBITS Daniele Laboratoire Paris France

??

Zitoun*,

Lucienne

Bara*,

Marie-Francoise

Meyer Michel Samama** thrombose Experimentale, Faculte and ** Laboratoire Central d’Hematologie Paris France

(Received 70 February 1994 by Editor U. Hedner; revisetiaccepted

Bloch* de

Medecine, Hotel Dieu,

14 June 1994)

Tissue Factor Pathway Inhibitor (TFPI), is known, to be released in plasma after injection of intravenous or subcutaneous injection of heparin or low molecular weight heparin (1,2). Addition of protamine or polybrene in plasma can neutralize anticoagulant effects of heparin in vitro but not completely ex vivo in patients treated with heparin (3). It was believed for many years that this post-heparin anticoagulant effect was due to another activity released by heparin. More recently it has been shown that postheparin anticoagulant effect could be inhibited by anti-TFPI antibodies (4). Since we have shown in a previous work (5), that in healthy volunteers, pharmacokinetics of TFPI were more closely related to anti Ila activity than anti Xa activity we hypothetized that fragments with anti-lla activity may be required for this release, possibly from vascular wall. In if a order to determine very low molecular weight glycosaminoglycan can release TFPI in plasma, in the present activity study, we compared plasma TFPI amidolytic after in rabbits of pentasaccharide (PS), a intravenous injection synthetic fragment of very low molecular weight and with a strong and exclusive anti Xa activity, and unfractionated heparin (UFH).

unfractionated : TFPI release, pentasaccharide, Key words heparin, rabbit. Corresponding author : Dr Lucienne BARA, Universite Pierre Marie Curie, Laboratoire de thrombose Experimentale, 15 rue I’Ecole de Medecine, 75006 PARIS FRANCE

577

et de

HEPARINS AND TFPI ACTIVITY

MATERIALS

AND

Vol. 75, No. 5

METHODS

Animals : Male white New-Zealand rabbits, weight from PONCEAU establishment (EPEGNE SUR DEME. rabbits were used for each dose and each treatment.

: 2,5 to 3 kg, FRANCE), Six

Treatment : - Unfractionated heparin : (MW. 15000 D.) (FOURh IIER, DIJON, FRANCE) - Pentasaccharide (MW. 1700 D.), kindly given by Ins titut CHOAY, FRANCE). Solutions were injected intravenously in ear marg inal vein et doses 100 and 200 pg/kg rabbit and with a volume adjusted to 2 ml with saline solution. Blood was collected from carotid artery using a polyethylene catheter (BIOTROL), in plastic tube (1 volume citrate 3,8% for 9 volumes blood). Plasma was obtained by centrigugation 30 minutes at 3000 g, at 15°C. The comparison of the mean values was realized by using the STUDENT t test. Total TFPI Activity was measured by a three stage chromogenic assay as described earlier (6). Anti-Xa and anti-lla activities were measured according to previously published amidolytic assay (7).

RESULTS

Figure 1 shows that the pharmacokinetics of anti-Xa, anti-lla and injection of 100 and 200 pg/kg of TFPI activities after unfractionated heparin are closely related. These activities were minutes after injection of 100 pg/kg of dose-dependent : Five mean values anti-lla activities were UFH, anti-Xa and 0,18 +/- 0,04 and 0,17+/0,07U/ml respectively. After injection of the dose 200 pglkg, they were 0,47 +I0,13 U/ml and 0,46 +/0,12 U/ml respectively. The peak of TFPI activity as compared to TFPI baseline was 2,5 fold and 3,9 fold higher after injection of 100 and 200 pg/kg of UFH respectively. after pentasaccharide intravenous In contrast, and 200 pg/kg of PS (Figure 2), plasma anti-Xa dependent and 2 fold higher than after UFH no 0,1 1 U/ml and 0,79 +/- 0,18 U/ml ; However TFPI activity could be observed.

injection activity injection increase

of 100 was dose (0,40 +/of plasma

HEPARINS AND TFPI ACTIVITY

579

b

c

Fig. Variation injection

pg/kg-i /kg-l

0 *

ANTI

X,....

ANTI

II. _

d

1

of TFPl,, Anti-Xa, Anti Ila activities, after I.V. of unfractionated heparin (100 pg/kg-I., 200 a, b) and pentasaccharide (100 pg/kg-l$ , 2OOpg

c,d)p:o.ool*

DISCUSSION

The comparison of anti-Xa, after anti-lla and TFPI activity intravenous injection of antithrombotic doses of pentasaccharide and UFH, have shown that pharmacokinetic of TFPI activity is more closely related to anti-lla activity than pharmacokinetic of anti-Xa activity, Pentasaccharide does not release any TFPI activity in rabbit plasma when UFH can release a dose dependent TFPI activity. This could suggest that heparin fragment of MW higher than 1700 D are required to increase plasma TFPI activity. TFPI release seems to be linked to the molecular weight of injected oligosaccharides. It would be interesting in a further men. Indeed, study to determine if results similar in are discrepant results have been reported after intravenous injection of P.S to macada mulatta (8).

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REFERENCES

I- SANDSET PM., ABILDBAARD release of extrinsic pathway 803-813. 7988.

U., LARSEN ML., Heparin induces inhibitor (EPI). Thromb. Res. 50

:

2- LINDAHL AK., JACOBSEN PB;, SANDSET PM., ABILDGAARD U. Tissue factor pathway inhibitor with high anticoagulant activity in increased in post-heparin plasma and in plasma from cancer patients. Blood Coagul. Fibrin. 2 : 713-724, 1991. 3-NORDOY A., Difference protamine and polybrene as Invest. 15 . 205-210, 1963.

in heparin neutralizing tested by thrombotest.

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4- LINDAHL AK., ABILDGAARD U., LARSEN ML., AAMODT LM., Extrinsic pathway inhibitor (EPI) and NORDFANG O., BECK TC., the post-heparin coagulant effect in tissue thromboplastin induced coagulation. Thromb. Res. (Suppl XIV) ; 39-48, 1991. ZITOUN D., SAMAMA MM., COLLIGNON 5- BARA L., BLOCH MF., F., FRYDMAN A., UZAN A., BOUTHIER J. Comparative effects of enoxaparin and unfractionated heparin in healthy volunteers on prothrombin consumption in whole blood during coagulation and release of tissue factor pathway inhibitor. Throm. Res. 69 : 443452, 1993. ZITOUN D., BARA L., SAMAMA 6human, rat, rabbit plasma tissue factor Res. 72 : 269-274, 1993. 7- BARA L., Comparative (PK 10169) subcutaneous

MM. Compared pathway inhibitor.

activity of Thromb.

BILLAUD E., GRAMMOND G., KHER A., SAMAMA MM; pharmacokinetics of a low molecular weight heparin and unfractionated heparin after intravenous and administration. Thromb. Res., 39 : 631-636, 1985.

8-LOJEWSKI B., BACHER P., JESKE W., FAREED J. Molecular weight dependance on the release of tissue factor pathway inhibition after subcutaneous and intravenous actions of heparins. Thromb. and Haem. 69 (6) 1174 abst. 2263, 1993. 9- ZITOUN D., BARA L., BLOCH MF., SAMAMA MM., Intravenous injection of pentasaccharide (P.S.) does not increase TFPI activity in rabbits, Thromb. and Haem. 69 (6) 768 abst. 815, 1993.