tPA inhibitor, tPA:Ag, plasminogen, and α2-antiplasmin after low molecular weight heparin or standard heparin

tPA Inhibitor, tPA:Ag, Plasminogen, and q-Antiplasmin

after Low

Molecular Weight Heparin or Standard Heparin

H. Neerstrand,

P. 0stergaard.

D. Bergqvist, T. Matzsch,

U. Hedner

SliMMAR

Y. The effect on the fibrinolytic factors was investigated in a cross-over study in young healthy volunteers receiving 2500, 5000, or 10 000 XaI units of low molecular weight heparin SC, 5000 XaI units of conventional heparin SC, and compared with a control group. A variation of tPA inhibitor activity was seen with a significant decrease (p < 0.001) at 11.15 a.m., 4 h after the heparin injection, and the tPA inhibitor remained decreased at 6 and 8 h.

Also the tPA:Ag decreased but a significant decrease was not seen until at 1.15 p.m. The same decrease in tPA inhibitor activity and tPA:Ag was seen in the control group, which did not receive any heparin. Thus the decrease of tPA inhibitor as well as the shortened euglobulin clot lysis time found could not be attributed to heparin. No influence was seen on plasminogen or c+antiplasmin. It seems that increased fibrinolysis during the day may result from the variation of tPA inhibitor during the day. Therefore, when an increased fibrinolysis is seen, it is necessary to take ihe time of the blood sampling into account. Kk Y WORDS. Fibrinolysis. Diurnal variation. tPA inhibitor. tPA:Ag. molecular weight heparin.

Plasminogen. aI-antiplasmin.

Heparin. Low

(c(~-AP) and euglobulin clot lysis time (ECL). The effect on the coagulation system in the same individuals has previously been reported.’ A dose dependent inhibition of LHN-1 on the coagulation activities, factor Xa, and factor Ila, was seen with peak values at 4 h after the heparin injection.

A diurnal variation of fibrinolytic activity with a rhythm of low activity in the morning increasing during the day has been observed.‘- ’ The recent introduction of more specific assays for the various fibrinol;/tic factors and their inhibitors has made it possible to investigate the cause of this pattern. A diurnal. variation with increasing tPA activity, decreasing) tPA antigen concentration, and tPA inhibitor activity in the afternoon has been reported in normal subjects.4 The introduction of low molecular weight heparins has increased the interest in heparin and its mode of action. In recent years reports have appeared showing increased fibrinolytic activity following heparin administration.5.h.7 The aim of the present study was to investigate various librinolytic parameters in six volunteers in whom a pharmacokinetic study of a low molecular weight heparin was performed. Three different doses of low molecular weight heparin NOVO (LHN-I), peak molecular weight 4900, were compared with conventional heparin and a control group. The fibrinolytic parameters measured were tPA inhibitor activity (tPAT), tPA concentration (tPA:Ag), plasminogen activity (PLG), r,-antiplasmin activity

SUBJECTS

AND

METHODS

Six healthy volunteers, four males and two females, aged 27-39 years, weight 52-96 kg, participated in the investigation of LHN-1 and conventional heparin. One male, aged 29, weight 96 kg, was in the control group replaced by a female, aged 30 years, weight 57 kg. None of the results was in any way influenced by this substitution. The study was, except for the control group, performed as a double-blind cross-over randomised study, in which each volunteer was his own control. Except for one person, the control group consisted of the same persons as those who participated in the pharmacokinetic study of the heparins. At least I week passed between each injection of LHN-I or conventional heparin. Apart from the injections and the blood sampling, no change was made in the daily routine of the volunteers. The study was approved by the Medical Ethics Committee, University of L,und.

H. Neerstrand,

P. Bstergaard, L!. Hedner, NOVO Industri AS. Denmark. D. Bergyvist and T. Mltzsch, Department of Surgery. General Hospital. Malmii. Sweden. 39

40

tPA Inhibitor.

[PA:Ag,

Plasminogcn and r,-antiplaamin

after Low Molecular

Sweden, and performed according to the Declaration of Helsinki. The doses given were 2500, 5000. or 10 000 Xal units of LHN-1 or 5000 Xal units of conventional heparin injected subcutaneously in the thigh. Blood samples were drawn from a peripheral vein immediately before subcutaneous injection at 7.15 a.m. and l/4, l/2. 1, 2, 4, 6, 8, and 24 h after. Citrated plasma’ (final concentration 0.013 M Na-citrate) was prepared immediately after collection and frozen at - 80 ‘C. The control group was included after the pharmacokinetic study was completed.

Heparins Low molecular weight heparin NOVO (LHN-I. batch 84001) was prepared from conventional heparin of porcine mucosal origin by controlled enzymatic degradation using heparinase from Flavobacterium Heparinum. LHN-1 had a peak molecular weight of 4900 dalton determined by gel filtration. Conventional heparin NOVO (batch 5864) had a peak molecular weight of I7 500 dalton. The in vitro activities of LHN-1 was 86 XaI units/ mg, and 44 IU/mg in an APTT system. The activities of conventional heparin were 196 XaI units,‘mg and 166 IUimg in the APTT system. The preparations contained 0.9”/; sodium chloride as solvent and 10 mg/ml benzyl alcohol as preservative. The preparations were supplied in coded vials.

Analytical Methods The tPA inhibitory activity (tPA1) was measured amidolytically according to Chmielewska et al” and was expressed in IU of inhibited m-tPA (batch 840621, NOVO) per ml of plasma. The tPA concentration (tPA:Ag) was measured with an ELISA according to Bergsdorf et al.” The monoclonal antibody (tPA 1 FC) used for catching tPA was kindly supplied by J. Selmer (NOVO). As detecting antibody a polyclonal rabbit antibody against m-tPA was used (three rabbits R429931 immunised with purified m-tPA). The tPA:Ag was expressed in ngiml. The plasminogen activity (PLG) was measured in a micro titer plate using an end point amidolytic method described by Friberger et al” using streptokinase (Sigma) as a plasminogen activator. The concentration of PLG was expressed as percent of a standard plasma pool. The x,-antiplasmin activity (az-AP) was measured amidolytically according to Teger-Nilsson et al’” using a Corona Analyser (Clinicon AB) and the concentration of a,-AP was expressed as percent of a standard plasma pool. The euglobulin clot lysis time (ECL) was measured within 2 h of blood collection.”

Weight Heparm

or Standard

Heparin

Statistical Methods Two-way analysis of variance for randomised block experimental design was used to see whether there was any difference between groups and subjects. Student’s t-test was used for comparison between two blood sample points. ’4

RESULTS The tPA1 activity (Fig. I) was found to vary from day to day in the different subjects. At 7.15 a.m. (before heparin injection) tPAl varied from 4.3553.65 to 9.10+1.001U/ml (XfISD) and at 11.15 a.m. (4h after heparin injection) from 1.75 + 1.15 to 5.20+ 2.40 IU/ml. The mean tPA1 after pooling all the values was 6.25f3.05 IU!ml at 7.15 a.m. and 3.401 2.05 IU/ml at 11.15 a.m. Thus the tPA1 had decreased significantly (p < 0.001) and remained decreased at 6 h (2.70~2.15IU/ml, p 0.05). Plasminogen (Fig. 3) measured amidolytically did not show any changes between groups or subjects (p > 0.05). At 7.15 a.m., immediately before S.C. injection, plasminogen was 118 & 28’,;,, at 4 h 12 1 k 38’:;, and at 24 h 116 i 27:‘~;. a,-antiplasmin (Fig. 4) measured amidolytically had increased from 101 + 12’!&before heparin injection to 105+ lo;;; at 11.15 a.m., 4 h after injection (p < 0.05). At 24 h r,-antiplasmin was 102 + lo’:;. For all the individuals who received heparin the euglobulin clot lysis time showed a moderate fibrinolysis in 15 out of 23 subjects at 4 h compared to 8 out of 22 measured before heparin injection and the next morning.

DISCUSSION Whether the diurnal variations of the tibrinolysis previously reported4 are influenced by heparin remains to be established. Several reports showing an increased fibrinolytic activity of conventional heparin and low molecular weight heparin have appeared.5.“.7 Consequently, the fibrinolytic system (tPA:AG, tPA1, euglobulin clot lysis time, plasminogen, x,-antiplasmin) was

_ IU/ml

.B .A *E

.D

2.5 -

A : LHN-1.2.500

----

B : LHN-1.5.000

-.-.-.-

C : LHN-1.10000 D : Conv Heporln

**.**..**

E : Control

5000

0

hours

w/ml

*A .E ?? C ?? D ?? B

0 L 0

--

A : LHN-1.2.500

----.-.-.-

B : LHN-I, 5.000 C : LHN-1.10.000 D : Conv. Heparin

.*.s+*...

E : Control

I 1

I 2

5000

I 6

42

IPA Inhibitor.

tPA:Ag.

Plasminogen

and r,-antiplasmin

after Low Molecular

Weight Heparin

or Standard

Heparin

%

150

100

50

0

-

A : LHN-1,2.500

----

B : LHN -1,5.000

-a-

C : LHN -1.10.000

-a-**.-o*****

0 : Conv. Heparin E : Control

5000

0

2.4 hours

Fig. 3 Plasminogen measured amldolytically after 2500, 5000, and 10 000 Xal units of low molecular conventional heparin and a control group (mean of six volunteers).

weight heparin,

5000 Xal units of

E B -AD ‘C

100

50

0 0

-

A : LHN-1,

----

8:

2.500

LHN-1.5.000

-.-

C : LHN-

-*---***.**.*

D : Conv. Heparin E : Control

#

,

1

2

1.10.000 5000

I

I

.4

6

Fig. 4 a,-antiplasmin activity after 2500, 5000, and 10 000 XaI units of low molecular and a control group (mean of six volunteers).

weight heparin,

,*I

a

,

2L hours

5000 Xal units of conventional

heparin

Journal of Fibrinolyhis

13

___ investigated in young healthy individuals subjected to a pharmacokinetic study of LMW-heparin (LHN-I. NOVO) as well as in a control group. in which blood sampling was performed with the same time intervals but Lvithout any heparin administration. As pre\,iously reported” the maximal effect by LHN-I on the Xa inhibitory activity is obtained at 4 h after the S.C. heparin injection and is dose dependent. In our study on the fibrinolysis a moderately increased fibrinolytic activity as measured in a euglobulin clot lysis system could bc found in the plasma samples drawn at 4 h after the heparin injection. At the same time, however, a significant decrease of the tPA-inhibitory activity was seen. The same decrease was seen also in the control subjects, who were not given heparin. These results thus confirm previous findings of a diurnal variation of t PA1 activity in normals.” However, it was clear that the changes seen here were not induced by heparin. neither the conventional heparin. nor the LMW heparin. In addition to the decrease of tPA1. a decrease of the t PA:Ag during the day was previously reported.” This was confirmed by our findings of significantly 1oLvered levels of tPA:Ag at I.15 p.m. as well as at 3.15 p.m. Neither was this lowering of the tPA:Ag dependent on heparin. The only significant difference seen at noon was in r,-antiplasmin but the marginal difference will not be of any clinical relevance. Thus the result of this study indicates that the increased fibrinolysis seen in some conventional and low molecular weight heparin studies may be the result of the variation of tPA inhibitor activity during the day. Furthermore. when increased fibrinolysis is seen, it is necessary to take the time of blood sampling into

REFERENCES I

Fearnley G R, Balmforth G. Fear&y E 1957 Evidcncc ofa diurnal librinolqtic rhythm; with a smlple method ofmeasuring natural fibrinolysia. Chnical Science 16:645-650 2 Mann R D 1967 Efl’cct of age. sex, and diurnal variation on the human librinolytic system. Journal of Clinical Pathology 20: 223-226

3 Rosing D R. Brakman

4

5

6

I

8

9 IO

II

12

account.

13

,ACKNOWLEDGEMENTS The

authors thank Omta H. Liitzen and Dorte Vestcrgaard Wlnthcr for their excellent assistance and Lene Akesson for typing the manuscript. This study was supported by grants from the Swedish Medical Research Council (00759 and 05447).

Offprint orders to: Mrs H. Neerstrand, MSc (Pharm), Industri A, S. Novo Alle, DK-2840 Bagsvaerd. Denmark.

NOVA

I-1

P. Redwood D R, Goldstein R E, Bciser G D. Astrup T. Epstein S E I970 Blood tibrinolytic activity in man. Diurnal variation and the response to varying intensltics of cxcrcIsc. Circulation Rexarch 27: 171-l 84 Kluft C. Verheijcn .I H. Rijken D C. Chang G T Cr. Jie A F H, Onkelinx C 1985 Diurnal fluctuations in the activity of the fastacting t-PA inhibitor. In: Dawdson J F (‘I crl. (eds). Progrers in Fihrinolysis, Vol 7, Churchill Livingstone. Edinburgh, London. Mclbournc and New York. 117- 119 Vaircl E G. Bouty-Boyc H, Toulemonde F. Doutremepulch D, Marrh N A. Galfney P J 1983 Heparin and a low molecular weight fraction enhances thrombolysis and by this pathway cxerclses a protective effect against thrombosis. Thrombosis Rescarch 30:219%224 VinazTer H. Stemberger A. Haas S, Bliimel G 19X2 Influence of heparin: of diKerent hcparin fractions and of a low molecular \velght heparin-like substance on the mechanism of fibrinolysis. Thrombosis Research 27:34l--352 Vinarzcr H. Woler M 1985 A new Iow molecular weight heparin fragment (PK 10169): In vitro and in viva studies. Thrombosis Research 40: 135 146 Matrsch T. Bcrgqvist D. Hedner U, 0stergaard P 1986 EfYccts of an enqmatically depolymerised lo\+ molecular weight heparin as compared with conventional heparin. Submitted for publication Nilsson I M I974 Haemorrhagic and Thrombotic Diseases. Wilev J et al (eds). London, New York. Sydney. Toronto Chm&lewska J. Rinby M. Wiman B 1983 Evidence for a rapid Inhibitor to tissue plasminogcn activator in plasma. Thromhosls Research 31:427-436 Bergsdorf N. Nilsson T. WallCn P 1983 An enzyme linked nnmunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolicdisease. Thrombosis and Haemostasis 50(3):74&744 Frlherger P, Kniis M, Gustavsson S. Aurell L. Claeson G 1978 Methods for determination of plasmin, antiplasmin, and plasminogen by means ofsubstratc S-2251. Haemostasis 7: 13X-145 Teger-Nilason A C. Friberger P. Gyzander E 1977 Determination ofa new rapid plasmin inhibitor in human blood by means ofa plasmin specific trlpeptidc substrate. Scandinavian Journal of Chnical Laboratory Investlgatlon 37: 303-409 -\rmitage P 1974 Statistxal Methods in Medical Rsscarch. 3rd edlt. Black&I Scwntific Publications, Oxford. London. Edinburgh, Melbourne