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Ageing and t-PA release by calcium and sodium pentosan polysulphate after oral application in rats
(1992) 6, Supp:3,424 @ Loqman GroupUKLtd1992
ZShdyks
Ageing and t-PA Release by Calcium and Sodium Pentosan Polysulphate aRer Oral Application in Rats
H.-P. KlBcking
SUMMARY A single oral dose of calcium pentosan polysulphate (10 mg/kg) induced a mean net increase in tissue-type plasminogen activator (t-PA) release of 7.2 f 4.9 IU/ml plasma/h (n = 10) in male rats, aged nine weeks (CRWIST) over 5 hours, with peak values being reached 2 hours after the administration. The mean net increase in t-PA activity amounted to 5.8 f 1.9 IU/ml plasma/h (n = 9) (not significantly different compared with young rats) in male rats (CR:WIST), aged 52 weeks, over 5 hours, with a peak value 2 hours after administration. Sodium pentosan polysulphate (NaPPS) enhanced the mean net increase in t-PA activity in young rats (9 weeks, male) by only 3.8 + 3.2 W/ml plasma/h, (n = 15), a peak value being reached 2 hours after administration. In rats aged 52 weeks (male) the mean net increase in t-PA activity measured every hour (3.5 -C 1.9 W/ml plasma; n = 8) was similar to that measured in young rats. Compared to NaPPS, CaPPS enhanced the net t-PA activity by about 50% in young rats aged 9 weeks. KEYWORDS. Calcium pentosan polysulphate (CaPPS). Sodium pentosan polysulphate (NaPPS). Tissue-type plasminogen activator (t-PA). Rats. Ageing. Fibrinolysis
INTRODUCTION
Experimental animals
Oral administration of calcium pentosan polysulphate (CaPPS, BEG0 0391), a new derivative of the sulphated polysaccharide pentosan polysulphate, led to better resorption (lo-20%) in rats compared to the values achieved with sodium pentosan polysulphate (NaPPS) (abt. l%).’ Therefore, it had to be clarified as to whether a single oral dose of CaPPS in rats caused a higher t-PA activity than NaPPS in rat plasma and whether this depended on the age of the experimental animals.
Male rats (CR:WIST, Wiga, Germany), aged 9 and 52 weeks were purchased. Prior to the experiment, the rats were anaesthetized with ethyl urethane (1.2 g/kg i.p.) One hour later the rats received an oral dose of the substance (1 ml/100 g bodyweight) dissolved in saline. Citrated blood (0.5 ml) (3.8% Na3 citrate, 1 + 9) was taken before administration of the substance and 1,2,3, 4 and 5 h afterwards out of the cannulated arteria carotis. Determination of t-PA activity in plasma
MATERIALS AND METHODS
The t-PA activity in rat plasma was determined according to the method of Chmielewska and Wiman.‘Citrated blood (1 + 9) was immediately mixed with acetate buffer and, starting within 2 min, centrifuged at 3000 x g for 15 min. After plasma separation, 10 rl of HCl (1 mol/l) was added to each 50 rtl sample of the already acidified plasma. 50 al of plasma obtained in this way was diluted with 1.75 ml sterile water. To determine the t-PA activity, 100 ctl of this diluted plasma was mixed with 100 ~1 of a solution of plasminogen substrate (S-2251,5.0 mM: plasminogen, 6 CU/ml: Tris buffer = 1:1:3). Furthermore, 50 pl of t-PA stimu-
Chemicals Calcium pentosan polysulphate (CaPPS, BEG0 0391; bene Arzneimittel GmbH Munich, Germany). Sodium pentosan polysulphate (NaPPS, bene Arzneimittel GmbH Munich, Germany).
Instituteof Pharmacology and Toxicology, Medical Academy Erfurt, Nordhauser Str. 74, D-0-5010 Erfurt, Germany
42
Fibrinolysis 43
Table 1 t-PA activity after oral administration of 10 mg CaPPS/kg to young and old male rats (CXWIST, Germany)
Table 2 t-PA activity after oral administration of 10 mg NaPpS/kg to young and old male rats (CR:WIST, Germany)
Time after oral administration (h)
Time after oral administration (h)
1 2 3 4 S
net t-PA IU/ml (n) increases (means + SD) rats, aged 9 weeks
rats, aged 52 weeks
6.6 8.5 7.4 5.1 7.6
4.3 6.9 4.6 2.5 0
+ 2 + f ?
3.4 7.0 3.0 2.3 5.1
(4) (5) (4) (3) (3)
2 + ? 2
0.3 1.0 3.9 2.4
(3)’ (3)’ (3)’ (3)’ (3)’
1 2 3 4 S
net t-PA IU/ml (n) increases (means + SD) rats, aged 9 weeks
rats, aged 52 weeks
4.3 4.7 3.1 25 0.6
2.3 + 0.7 (3)’ 5.6 2 1.3 (3)’ 15 + 1.2 (3)*
2 + + ? +
4.2 3.0 2.4 1.8 0.6
(3) (3) (3) (3) (3)
*Not significant for young versus aged rats
*Not significant for young versus aged rats
lator (3.3 mg human fibrin(ogen) fragments/l.0 ml sterile water) was added to the mixture. After mixing all the test reagents well, the sample was incubated at 370C for 120 min. The reaction was stopped by adding 50 al of acetic acid (20%) and photometrically evaluated at 405 nm. The t-PA content (IU/ml plasma) was calculated by means of a standard curve.
DISCUSSION
Statistical significance Statistical significance was calculated by Student’s t-test; p > 0.05 was considered to be of no significance.
RESULTS The net increase in t-PA activity after single oral administration of 10 mg CaPPS or NaPPS, respectively, per kg bodyweight in young (9 weeks of age) and old (52 weeks of age) male rats is given in Tables 1 and 2. Compared to old rats, CaPPS caused a twofold mean increase in t-PA activity over 5 hours in young rats. In young and old rats, peak values were reached 2 hours after CaPPS administration (Table 1). After oral administration of NaPPS the net increase in t-PA activity in young rats amounted to only 50% of that obtained with CaPPS. In old rats the net t-PA activity and the time of the maximum increase in t-PA activity were identical with the values obtained in young rats after oral administration of NaPPS (Table 2).
Buczko et al? showed that in young male rats (aged 3 months) and old male rats (aged 18 months) (CDCOBS, Charles River, Italy) epinephrine caused different results regarding the t-PA release in the perfused rat hindlegs. In old rats, in contrast to young ones, the fibrinolytic response appeared to be activated at the end of perfusion and was much more pro-nounced. The present results do not allow a corre-sponding conclusion. In our experiments oral resorption formed the prerequisite for t-PA release from the vascular endothelium. The resorption rate after oral administration of CaPPS (lo-15%) was higher than that of NaPPS (l%);l this could explain the higher net increase in t-PA activity values obtained with CaPPS in young rats compared to NaPPS. The lower net increase in t-PA activity values achieved with CaPPS in old rats are presumably due to reduced oral resorption as a symptom of old age.
REFERENCES Kliicking H-P, Hauptmann J, Richter M 1991 Profibrinolytic and anticoagulant properties of the pentosan polysulphate derivative bego 0391. Pharmazie 62: 543-544 Chmielewska J, Wiman B 1986 Determination of tissue plasminogen activator and its “fast” inhibitor in plasma. Clin Chem 32: 482-485 Bunko W, Iacoviello L, De Curtis A, Amore C, Donati M B 1991 Ageing and tissue plasminogen activator release. Thromb Haemostas 66: 745
ZShdyks
Ageing and t-PA Release by Calcium and Sodium Pentosan Polysulphate aRer Oral Application in Rats
H.-P. KlBcking
SUMMARY A single oral dose of calcium pentosan polysulphate (10 mg/kg) induced a mean net increase in tissue-type plasminogen activator (t-PA) release of 7.2 f 4.9 IU/ml plasma/h (n = 10) in male rats, aged nine weeks (CRWIST) over 5 hours, with peak values being reached 2 hours after the administration. The mean net increase in t-PA activity amounted to 5.8 f 1.9 IU/ml plasma/h (n = 9) (not significantly different compared with young rats) in male rats (CR:WIST), aged 52 weeks, over 5 hours, with a peak value 2 hours after administration. Sodium pentosan polysulphate (NaPPS) enhanced the mean net increase in t-PA activity in young rats (9 weeks, male) by only 3.8 + 3.2 W/ml plasma/h, (n = 15), a peak value being reached 2 hours after administration. In rats aged 52 weeks (male) the mean net increase in t-PA activity measured every hour (3.5 -C 1.9 W/ml plasma; n = 8) was similar to that measured in young rats. Compared to NaPPS, CaPPS enhanced the net t-PA activity by about 50% in young rats aged 9 weeks. KEYWORDS. Calcium pentosan polysulphate (CaPPS). Sodium pentosan polysulphate (NaPPS). Tissue-type plasminogen activator (t-PA). Rats. Ageing. Fibrinolysis
INTRODUCTION
Experimental animals
Oral administration of calcium pentosan polysulphate (CaPPS, BEG0 0391), a new derivative of the sulphated polysaccharide pentosan polysulphate, led to better resorption (lo-20%) in rats compared to the values achieved with sodium pentosan polysulphate (NaPPS) (abt. l%).’ Therefore, it had to be clarified as to whether a single oral dose of CaPPS in rats caused a higher t-PA activity than NaPPS in rat plasma and whether this depended on the age of the experimental animals.
Male rats (CR:WIST, Wiga, Germany), aged 9 and 52 weeks were purchased. Prior to the experiment, the rats were anaesthetized with ethyl urethane (1.2 g/kg i.p.) One hour later the rats received an oral dose of the substance (1 ml/100 g bodyweight) dissolved in saline. Citrated blood (0.5 ml) (3.8% Na3 citrate, 1 + 9) was taken before administration of the substance and 1,2,3, 4 and 5 h afterwards out of the cannulated arteria carotis. Determination of t-PA activity in plasma
MATERIALS AND METHODS
The t-PA activity in rat plasma was determined according to the method of Chmielewska and Wiman.‘Citrated blood (1 + 9) was immediately mixed with acetate buffer and, starting within 2 min, centrifuged at 3000 x g for 15 min. After plasma separation, 10 rl of HCl (1 mol/l) was added to each 50 rtl sample of the already acidified plasma. 50 al of plasma obtained in this way was diluted with 1.75 ml sterile water. To determine the t-PA activity, 100 ctl of this diluted plasma was mixed with 100 ~1 of a solution of plasminogen substrate (S-2251,5.0 mM: plasminogen, 6 CU/ml: Tris buffer = 1:1:3). Furthermore, 50 pl of t-PA stimu-
Chemicals Calcium pentosan polysulphate (CaPPS, BEG0 0391; bene Arzneimittel GmbH Munich, Germany). Sodium pentosan polysulphate (NaPPS, bene Arzneimittel GmbH Munich, Germany).
Instituteof Pharmacology and Toxicology, Medical Academy Erfurt, Nordhauser Str. 74, D-0-5010 Erfurt, Germany
42
Fibrinolysis 43
Table 1 t-PA activity after oral administration of 10 mg CaPPS/kg to young and old male rats (CXWIST, Germany)
Table 2 t-PA activity after oral administration of 10 mg NaPpS/kg to young and old male rats (CR:WIST, Germany)
Time after oral administration (h)
Time after oral administration (h)
1 2 3 4 S
net t-PA IU/ml (n) increases (means + SD) rats, aged 9 weeks
rats, aged 52 weeks
6.6 8.5 7.4 5.1 7.6
4.3 6.9 4.6 2.5 0
+ 2 + f ?
3.4 7.0 3.0 2.3 5.1
(4) (5) (4) (3) (3)
2 + ? 2
0.3 1.0 3.9 2.4
(3)’ (3)’ (3)’ (3)’ (3)’
1 2 3 4 S
net t-PA IU/ml (n) increases (means + SD) rats, aged 9 weeks
rats, aged 52 weeks
4.3 4.7 3.1 25 0.6
2.3 + 0.7 (3)’ 5.6 2 1.3 (3)’ 15 + 1.2 (3)*
2 + + ? +
4.2 3.0 2.4 1.8 0.6
(3) (3) (3) (3) (3)
*Not significant for young versus aged rats
*Not significant for young versus aged rats
lator (3.3 mg human fibrin(ogen) fragments/l.0 ml sterile water) was added to the mixture. After mixing all the test reagents well, the sample was incubated at 370C for 120 min. The reaction was stopped by adding 50 al of acetic acid (20%) and photometrically evaluated at 405 nm. The t-PA content (IU/ml plasma) was calculated by means of a standard curve.
DISCUSSION
Statistical significance Statistical significance was calculated by Student’s t-test; p > 0.05 was considered to be of no significance.
RESULTS The net increase in t-PA activity after single oral administration of 10 mg CaPPS or NaPPS, respectively, per kg bodyweight in young (9 weeks of age) and old (52 weeks of age) male rats is given in Tables 1 and 2. Compared to old rats, CaPPS caused a twofold mean increase in t-PA activity over 5 hours in young rats. In young and old rats, peak values were reached 2 hours after CaPPS administration (Table 1). After oral administration of NaPPS the net increase in t-PA activity in young rats amounted to only 50% of that obtained with CaPPS. In old rats the net t-PA activity and the time of the maximum increase in t-PA activity were identical with the values obtained in young rats after oral administration of NaPPS (Table 2).
Buczko et al? showed that in young male rats (aged 3 months) and old male rats (aged 18 months) (CDCOBS, Charles River, Italy) epinephrine caused different results regarding the t-PA release in the perfused rat hindlegs. In old rats, in contrast to young ones, the fibrinolytic response appeared to be activated at the end of perfusion and was much more pro-nounced. The present results do not allow a corre-sponding conclusion. In our experiments oral resorption formed the prerequisite for t-PA release from the vascular endothelium. The resorption rate after oral administration of CaPPS (lo-15%) was higher than that of NaPPS (l%);l this could explain the higher net increase in t-PA activity values obtained with CaPPS in young rats compared to NaPPS. The lower net increase in t-PA activity values achieved with CaPPS in old rats are presumably due to reduced oral resorption as a symptom of old age.
REFERENCES Kliicking H-P, Hauptmann J, Richter M 1991 Profibrinolytic and anticoagulant properties of the pentosan polysulphate derivative bego 0391. Pharmazie 62: 543-544 Chmielewska J, Wiman B 1986 Determination of tissue plasminogen activator and its “fast” inhibitor in plasma. Clin Chem 32: 482-485 Bunko W, Iacoviello L, De Curtis A, Amore C, Donati M B 1991 Ageing and tissue plasminogen activator release. Thromb Haemostas 66: 745