A sulphated polysaccharide - the effects in vivo and in vitro on the haemostatic system in healthy volunteers

THROMBOSIS RESEARCH 23; 309-315, 1981 0049-3848/81/150309-07$02,00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

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A SULPHATEO POLYSACCHARIDE - THE EFFECTS IN VIVO AND IN VITRO ON THE HAEMOSTATIC SYSTEM IN HEALTHY VOLUNTEERS David Bergqvist and Inga Marie Nilsson Department of Surgery and Coagulation Laboratory, Allmlnna Sjukhuset, University of Lund, MalmiS,Sweden (Received 16.2.1981; in revised form 14.5.1981. Accepted by Editor M. Samama. Received in final form by Executive Editorial Office 14.7.1981) INTRODUCTION Different glucosaminoglucans are evaluated as alternatives to low-dose heparin as prophylactic agents against postoperative deep vein thrombosis. PZ68 (prepared by Benechemie GMBH, Munich, Germany, under the name of SP54 and provided by AB Pharmacia, Uppsala, Sweden) is a sulphated polysaccharide with pentoses as base units which is prepared semisynthetically from vegetable raw materials. It is a linear macromolecule with a molecular weight of ca. 4000. It is as effective as the combination of low-dose heparin and dihydroergotamine in prophylaxis against postoperative thrombosis (1,2). The mechanism of action is not fully known and this study was undertaken on healthy volunteers to investigate the effect on the different components of the coagulation and fibrinolytic systems. MATERIALS AND METHODS Thirteen healthy volunteers of both sexes were studied, 20-35 years of age. In vivo studies. Plasma samples for the different analyses described below were taken before injection and l/2, 2, 6, and 10 hours after. The 10 hour sample was only taken after injection of PZ68. The following single subcutaneous injections were given: 1) PZ68, 1.5 mg/kg bodyweight 2) Heparin Kabi 0.75 mg/kg bodyweight (1 mg = 100 IU) 3) Heparin Kabi 1.5 mg/kg bodyweight The following tests were made (3-10): platelet count, bleeding time according to Ivy, platelet adhesiveness according to Hellem, platelet aggregation with Key words: Sulphated polysaccharide, haemostatic effect

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Born's aggregometer, APT time (General Diagnostics), thrombin time, f VIII coagulant activity (VIII:C), f VIII related antigen (VIIIR:Ag, electroimmunoP&P, f XIII (electroimmunoassay), plasminogen, assay), f IX, f V, f XII, fibrinogen, fibrin plate lysis and euglobulin clot lysis time, fibrinfibrinogen degradation products, inhibitors of plasminogen activation, CY macroglobulin. Antithrombin III was determined (a) biologically (ll), (bf immunochemically (12) and (c) with chromogenic substrate (Coatest Antithrombin /S-2238/, Kabi Diagnostica, Stockholm, Sweden) and C( -antiplasmin immunochemically with a specific antiserum kindly supplied by 6 r. Collen, Leuven, Belgium. XaI test, comprehensive and specific, was determined according to Yin et al (13), using PhadecodeTM XaI Assay (Pharmacia AB, Uppsala, Sweden). In vitro studies. PZ68 was mixed with titrated plasma to final concentrations of 6 10 and 60 ug/ml plasma and heparin to concentrations of 1 and 6 pg/ml. Tie sime normal human plasma without addition was used as control. The same parameters were analysed as for the in vivo studies except the platelet function tests. In an APT test system different concentrations of PZ68 and heparin were added, and the APT time was measured. A corresponding thrombin time test system was also used. In those systems normal plasma as well as plasma from a patient with congenital antithrombin III deficiency were used as test bases. Thrombin (Roche) in a final concentration of 1 unit/ml was used to the clotting. Finally a corresponding test was made in a pure fibrinogen-thrombin (human fibrinogen, Kabi, Stockholm, Sweden) system to exclude a direct antithrombin effect of the substance.

RESULTS f XIII, plasminogen, In vivo. VIII:C and VIIIR:Ag, f IX, P&P, f V, f XII, fibrinogen, fibrinolysis, a2-antiplasmin, inhibitor of plasminogen activation, antithrombin III, platelet function and bleeding time were not altered by PZ68 or heparin. Table I shows that the APT time is increased after PZ68 and the higher heparin dose, the effect coming faster after PZ68. Table II shows that the thrombin time is only increased by the higher heparin dose.

TABLE I The Effect of PZ68 and Heparin on APT time In Vivo, APT time in Seconds. Normal Value Less than 45 Seconds Before injection Pz68

42 (38-45)

Heparin 0.75 mg/kg

41 (39-43)

Heparin 1.5 mg/kg

40 (39-40)

I

After injection

I- I l/2 h

2h

6h

50 (45-60)

56 (50-61)

56 (50-60)

41 (38-44)

40 (35-45)

39 (36-44)

37 (35-48)

41 (40-42)

i) 50 (47-52)

10

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TABLE II The Effect of PZ68 and Heparin on Thrombin Time In Vivo. Thrombin Time in Seconds. Normal Value Less than 25 Seconds Before injec-

After injection

1

+I

1/2h

:

18-21 1

23-25

6h

,

21 (18-22)

1 -

66 (60-73)

,

10 h

21 (19-22)' 22 (20-23)

1 19 (18-20)

19-23 24

2h

21 (18-24)

-

84 (81-87)j

-

TABLE III The Effect of PZ68 and Heparin on XaI In Vivo. of the Initial Value 3efore injection

‘I

Specific

XaI Test in %

After injection 1/2h

6h

2h

1

10 h

test

PZ68

97

(85-114)

111

Heparin 0.75 mg/kg

97

(92-113)

107 (100-110)

Heparin 1.5 mg/kg

95

(96-121)

98

106

(91-117)

90

113 (110-118) 96

Comprehensive test PZ68

31 (128-134)

139 (127-147)

121 (102-140)

Heparin 0.75 mg/kg

34 (123-147)

137 (120-150)

126 (120-129)

Heparin 1.5 mg/kg

47

>155

147

100

Table III shows the results from the XaI tests. The specific XaI test is not influenced by any of the regimens but all given an increase in the comprehensive test. In vitro. No changes were seen in VIII:C and VIIIR;Ag, f IX, P&P, f V, f XIII, plasminogen, fibrinogen, fibrinolysis and a2-antiplasmin. APT f XII time is well as thrombin time were prolonged in a dose dependent way by both PZ68 and heparin (Table IV). The specific XaI was not altered whereas the comprehensive was increased, more so by heparin. The highest PZ68 and both heparin concentrations increased the biological antithrombin III. In the APT time system heparin and PZ68 are equally effective on a weight basis (Fig.1). In the thrombin time system heparin is at least ten times more effective

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TABLE IV Effect of PZ68 and Heparin

In Vitro

PZ68

b I ; Control APT time

50;

>I80

: II4

123

134 ’ 146

>155

112

II2

113

II6 ;

II8

120

94

86

94

204 ;

I37

176

182

(set)

I

22

33

XaI comprehensive

(%)

j

108

specific

(%)

:

Antithrombin

III

biol(%)

6

>I80 ) >180

94

time

1

81

36

Thrombin

(w/ml

260

1

sec. 300--

??

Heparin

x

PZ68B

250--

>300 ;

I 69

(set)

I

Heparin

:

FIG. 1

-4

200 -'

cc

___H

lOOpg/mI

The effect of ~Z68 and heparin in vitro in an APT time system

150~-

50

1

3

5

7

9

than PZ68 on a weight bases (Fig. 2). When antithrombin III deficient plasma was used as test base considerable shorter times were seen both with PZ68 and heparin. In the pure fibrinogen-thrombin system heparin as well as PZ68 have no effect. Thus a direct antithrombin effect can be excluded.

DISCUSSION In testing new compounds with a potential ability to prevent postoperative deep vein thrombosis it is necessary to elucidate the mechanism of action in detail. Recently Kakkar et al (14) have investigated a glucosaminoglucan polysulphate of bovine origin with a molecular weight between 5000 and

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FIG. 2

Heparin x PZ686

??

The effect of PZ68 and heparin in a thrombin time system in vitro

9 11 PZ68B 7 5 I I 1 I 1 1 I I I I 0.2 0.4 0.6 0.8 1.0 Heparin

13

15,000 (SSHA). The prophylactic effect against thrombi was equal to that of low-dose heparin. In operated patients this substance produced an increase in the anti-Xa activity according to Yin et al (13) whereas the antithrombin III level fell. The kaolin-clotting time was not influenced. Further studies have shown a pronounced anti-Xa effect with relatively little effect on APT (15), and a theory has been put forward that a heparin-like substance is endogenously released. We studied another small molecule, sodium pentosan polysulphate, PZ68, which is effective in preventing postoperative thrombosis in general and hip surgery (1,2). To be able to specify its place of action within the haemostatic system a study was undertaken on healthy volunteers to avoid the influence of the operative trauma. Heparin was compared in doses corresponding to low-dose heparin and heparin in therapeutic dose. Platelet count and platelet function and the components of the fibrinolytic system were not influenced. A similar substance with a lower molecular weight has in a study been shown to slightly activate the fibrinolytic system (16). The coagulation activity of factor VIII, factor VIII related antigen, the vitamin K dependent factors, factor V and factor XIII were not altered. Antithrombin III was not influenced. The influence of PZ68 on APT time was rather equal to that of heparin on a weight basis, whereas it was weaker than heparin in a thrombin time system. Our opinion on the XaI test system (13) is that the specific test measures the same effect as different antithrombin III tests and the comprehensive test measures an over all coagulation activity which much resembles the APT time. The comprehensive test does not detect congenital antithrombin III deficiency. The effect of PZ68 on this test is almost identical to that of heparin. This is in keeping with the findings that PZ68 does not alter the antithrombin III level, nor does it act as an antithrombin itself. The anti-

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Xa effect of PZ68 has recentlv been pointed out also bv other authors (17-19). The data indicate that PZ68"acts mainly by preventing the conversion of prothrombin to thrombin. It is possible that PZ68 does not have the direct effect which heparin has been shown to have on thrombin.

ACKNOWLEDGEMENT This investigation was supported by grants from the Medical Faculty, University of Lund, and the Swedish Medical Research Council (881-19X-00087-17).

REFERENCES

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