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Altered platelet and vascular prostaglandin-generation in patients with renal failure and prolonged bleeding times
THROMBOSIS @Pergamon
RESESRCH 13; 1007-1015 Press Ltd.1978. Printed
in Great Brirain OOZE-3e~8/78/1201-1007
So2.00/0
ALTERED PLATELET AND VASCULAR PROSTAGLANDIN-GENERATION IN PATIENTS WITH RENAL FAILURE AND PROLONGED BLEEDING TIMES G. Remutzi, D. Marchesi, M. Livio, A.E. Cavenaghi, G. Mecca, M.B. Donati and G. de Gaetano Divisione di Nefrologia e Dialisi, Ospedali Riuniti Bergamo, Italy and Istituto di Ricerche Farmacoloaiche "Mario Neari"-Via Eritrea. 62 20157 Mfian, Italy (Received 26.7.1978; in revised form 11.9.1978. Accepted by Editor I.M. Nilsson) ABSTRACT
Venous tissues from 15 patients with renal failure (five acute and 10 chronic] generated significantly higher PGI2-like (platelet aggregation inhibitory] activity than venous tissues from 10 normal subjects. The longer the bleeding times, the higher the values for PG12-like activity found in these patients. Both bleeding times and PGI2-like activity values returned to normal in three acute uraemic patients on restoration of their renal function. Two additional patients with acute renal failure and greatly prolonged bleeding times were under aspirin treatment at the moment of this study: venous specimens from neither of them generated measurable amounts of PGI2-like material. Malondialdehyde formation in platelets from 12 patients with chronic renal failure and prolonged bleeding times was significantly lower than in platelets from 11 controls. The defect was evident with each of the three stimuli used, i.e., collagen, arachidonic acid and thrombin. It is concluded that prostaglandin metabolism in platelets and in the vessel wall from uraemic patients is impaired in different ways, both contributing to the impaired primary haemostasis in these patients.
INTRODUCTION The pathogenesis of the bleeding tendency in uraemic patients is still un. clear. Defective platelet function resulting from retention of dialysable toxic factor(s) has long been proposed (l-4). More recently, several reports have focused attention on a contributory role of non dialysable factors in the abnormal haemostasis of patients with renal faflure (5-8). In this conte)ct,we could not obtain any evidence of defective Factor VIIK (von Hillebrand factor) in plasma and vessels from
1007
1008
~.EMIC
uraemic patients (6,9).
BLEEDIXG
AND PGs
Moreover, normal vascular fibrinolytic activity was
also found in these patients (10).
On the other hand, preliminary observa-
tions indicated increased generation of an inhibitor of platelet aggregation in the vascular tissue from these patients with renal failure (11). Some characteristics of this inhibitory activity were suggestive of it being prostacyclin (PGI2), a vascular prostaglandin endowed with potent antiaggregating and vasodilating activities (12,13). The purpose of this study was to evaluate whether this antiaggregating activity of vascular origin could be a significant factor in the abnormal bleeding associated with the uraemic condition. We present here suggestive evidence for this assumption. In addition we show that platelets from uraemic patients generated reduced amounts of prostaglandins, thus suggesting a complex role of prostaglandin metabolism in the pathogenesis of uraemic bleeding. PATIENTS AND METHODS Patients -
PG12-like activity was studied in 17 patients (13 men and four
women, aged 28-64 years) before they started haemodialysis for acute (seven patients) or chronic (10 patients) renal failure of various origin.
Bleeding
times (6) were normal ( shorter than 5 min) in two patients, moderately prolonged (5 to 8 min) in 6 and greatly prolonged (over 15 min) in 8. time could not be measured in one patient.
Bleeding
Platelet counts (6) were normal
(range 180-320 x log/l) in all but one patient who had leptospirosis and acu$ renal failure (plateletsbetween 30 and 50 x log/l). Ten male subjects (aged 20-50 years) with no apparent systemic diseases were also studied while undergoing minor plastic surgery of the forearm and acted as controls.
None of the patients or controls had received any blood
transfusions for at least two months before investigation.
Two patients (both
with acute renal failure) had been taking aspirin ( 1 g daily) during the preceeding week and were studied about four hours after the last aspirin intake. Malondialdehyde (MDA) formation was measured on platelets from 12 patients (eight men and four women, aged 18-50 years) who had been undergoing mainter: nance haemodialysis for from four months to six years. bleeding times longer than 15 min. before a haemodialytic session (6).
All of them had
Venous blood was collected immediately Eleven healthy subjects (five men and
six women, aged 20-35 years) acted as controls.
None of the patients or con-
trols had received any blood transfusion or drugs known to affect platelet
v01.13,s0.6
LTAEXIC
BLEEDIXG
d?;D PGs
1009
function (14) during the previous two months. Informed consent was obtained from all patients and controls. Methods - Venous tissue specimens were obtained and PGI2-like activity was measured as platelet aggregation inhibitory potency as previously described (11). PGI2-like activity was characterized according to current criteria (13, 15,16); namely the activity was unstable at room temperature and neutral pH, destroyed either by heating for 15 seconds at 100°C or by exposure to acidic PH.
It was stable for at least 24 hours at 4°C or at basic pil;its generation
was inhibited by incubation with acetylsalycilic acid or indomethacin, or tranylcypromine.
None of these drugs inhibited the activity once it had been
generated in the system.
Moreover, it was potentiated by inhibitors of
phosphodiesterase such as theophylline; it was distinct from PGD2 since differently from the latter prostaglandin (17), it strongly inhibited rat platelet aggregation.
Contamination with significant ADP-ase activity could
also be excluded (18). The platelet aggregation inhibitory activity was expressed as ng/ng wet tissue by extrapolation from a dose-response curve obtained on the same platelet-rich plasma with highly purified (> 99%) PGE,. It was assumed that PG12 was 30 times more potent an aggregation inhibitor than PGEl (12).
Platelet aggregation was initiated with 1 pmol/l adenosine-5'-di-
phosphate (ADP). Platelets were counted by phase contrast microscopy using tte Unopette R (Becton Dickinson, Italy) (6). Bleeding tines were measured by a semiautomatic template device (6, 11). Generation of MDA was induced by stirring platelet-rich plasma at 37"C(6) with acid soluble collagen (30 pg/ml , Harmon Chenie, Munich, West Germany)for 10 nin or with arachidonic acid sodium salt 5mM (Sigma, Mascia Brunelli,Milan, Italy) for 15 nin or with thrombin (25 NIH u/ml , Topostasine, Roche, Milan, Italy) for 5 min.
MDA was measured spectrophotometrically (Beckman MOD 25)
according to Smith et al. (19).
Results were expressed as nmolj 3~10~
platelets. RESULTS PG12-like values obtained in 10 controls and 15 patients with severe renal failure are shown in Table I. significantly (~(0.005)
Venous tissues from uraenic patients generated
higher PG12-like activity than specimens from con-
trols after both 70 and 20 min incubation. Table II
shows that longer bleeding tines were associated with higher PG12-
like activity generated within 20 min. Tissues from two patients with acute renal failure and history of daily aspirin treatment during the preceding week generated no measurable amounts of
UlUEMIC
1010
BLEEDING
AXD
PGs
TABLE I Mean values and range of PGI -like activity generated by venous tissue from 10 controls and 15 uraemic pE tients after 10 and 20 min incubation at room temperature in 0.05 M Tris-buffer, pH 7.4
PG12-LIKE ACTIVITY (ng/mg tissue) 10 min
20 min
Controls
0.57 (0.08 - 1.49)
0.59 (0.07 - 1.52)
Patients
5.08 (0.21 -33.79)
6.66 (0.63 -35.46)
4
P+
0.05
<
0.005
i ??Mann-Whitney
rank sum test (28).
TABLE II Bleeding times and venous PGI2-like activity (20 min) in 14 patients with severe renal failure
Bleeding time (min)
Number of Patients
PG12-LIKE ACTIVITY (ng/mg tissue) Mean
Range
<5
2
1.00
0.63 - 1.38
5-8
6
2.17
0.68 - 4.71
715
6
12.96
1.73 -35.46
______ PG12-like activity. Three patients with acute renal failure were studied again at the moment of by-pass removal following resolution of the renal insufficiency. In comparison with the values obtained at the beginning of haemodialytictreatment, normal bleeding times and reduction of PG12-like activity towards the upper limits of the control values were observed in all three patients when renal
So1.13,80.6
L-RAEHIC BLEEDIKG
XKD PCs
TABLE III Bleeding times, venous PGI2-like activity (20 min) and platelet counts of three patients with acute renale failure.
Patient
1
Bleeding time(min)
j A
PGI -like activity (n&mg tissue)
Bj
A
Platelet count (X109/1) a
B
Interval between A andB
B
’ ldays)
i
A:
2 1
/>15
3
6
2 2.30
26.0 7.17
1.38 1.84
250 50
290 300
27 28
4.71
1.98
220
200
15
2
Before the first hemodialysis session.
B : After
the last
hemodialysis session.
TABLE IV
Mean values and range of MDA generated by platelets from 11 controls and 12 patients with chronic renal failure in response to arachidonic acid, thrombin and collagen.
MDA (nmo1/3x108 platelets) Arachidonic acid
Thrombin
Controls
3.15 (1.30 - 4.40)
0.76 (0.24 - 1.50)
Patients
0.93 (0 - 2.25 )
(0 - 0.36 )
P+
<
0.01
+ Student's t test (28).
0.13
Collagen 0.88 (0.36 - 1.15) 0.29 (0 - 0.65)
<
0.001
I I
CWMIC
1012
function was restored.
BLEEDING
Vo1.13,No.6
AXi PGs
The low platelet count of patient No. 2 also returned
to normal (Table III). MDA generation in response to collagen, arachidonic acid or thrombin was significantly lower in platele&from
patients with chronic renal failure un-
dergoing maintenance haemodialysis than in platelets from controls (Table IV).
DISCUSSION These results indicate that venous tissues from patients with renal failure generated greater PG12-like (platelet aggregation inhibitory) activity than comparable specimens from control
subjects.
The suggestion that PGI2 might
be one of the factors contributing to the haemorrhagic diathesis of uraemic patients (11) is supported by the observation that normal or moderately prolonged bleeding times were associated with normal or moderately increased venous PG12-like activity; in contrast, greatly prolonged bleeding times (over 15 min) were associated with many-fold increase generation from venous tissues.
of PG12-like activity
This contention is further strengthened by
the fact that both bleeding times and PG12-like activity values in three acute uraemic patients returned to normal on restoration of their renal function. The pathogenesis of abnormal bleeding times in uraemic patients is however more complex than it would appear from these findings.
Indeed, venous tissues
from two patients under aspirin treatment generated no measurable amounts of PGI2-like material, yet the bleeding times of both patients were greatly prolonged (over 15 min).
It is tempting to speculate that in these two patients
the reduction of vascular PG12-like activity could not overcome the defective platelet function induced by aspirin (14), and/or the uraemic condition itself (20).
Indeed, platelets from uraemic patients with greatly prolonged
bleeding times (undergoing maintenance haemodialysis) showed a significant reduction of MDA generation, a reliable parameter of platelet activation (19), in response to different aggregating stimuli, a finding in agreement with a recent report (21).
A defect of platelet phospholipase A2 activity (22) is
unlikely since MDA formation was significantly reduced in response not only to collagen and thrombin but also to exogenous arachidonic acid. The relative contribution of platelets and the vessel
wall to the
haemorrhagic diathesis of patients with renal failure has not yet been established.
MDA is a product of platelet arachidonic acid metabolism (19)
and may reflect the generation of cyclic endoperoxides and thromboxane
A2
which are potent aggregating
agents (23,241.
PGI2 is a product of vascular
arachidonic acid metabolism, with potent antiaggregating activity (12,16,25). 1: is noteworthy that in the uraemic condition pathways appeared to be affected in
these two arachidonic acid
opposite ways which could both result in
an increased haemorrhagic risk. The pathogenesis of the altered prostaglandin metabolism in patients with renal failure is still unknown (26,27). Acknowledgements We thank Prof. S. Garattini and Prof. M.J. Silver for fruitful discussion and criticism.
We also thank drs. R. Misiani, G. Mingardi and S. Villa and
Miss E. Marchesi for valuable cooperation.
Drs. E. Poletti, P. Losapio, A.
Frontini, V. Giorgianni, R. Licini, A. Mangili, V. Brugali, C. Malgrati, L. V'illa,V. Oberti, and P. Tondini helped in collecting vascular specimens. J. Baggott, V. Pistotti, G. Brambilla, F. and V. de Ceglie helped in preparing the manuscript.
Prof. D.A. Van Dorp (Unilever Research Laboratories,
Vlaardingen, The Netherlands) kindly provided purified PGE,. This work was supported in part by the Associazione Bergamasca per lo Studio delle Malattie Renali.
REFERENCES
1.
SALZMAN, E.W., and NERI, L.L. Adhesiveness of blood platelets in uremia. Thromb. Diath. Haemorrh., -15, 84, 1966.
2.
HOROWITZ, H.I. Uremic toxins and platelet function. Archs. intern.Med., 126, 823, 1970.
3.
RABINER, S.F. Bleeding in uremia. Med. Clin. North Am., 56, 221, 1972.
4.
LINDSAY, R.M., MOORTHY, A.V., KOENS, F., and LINTON, A.L. Platelet function in dialyzed and non-dialyzed patients with chronic renal failure Clin. Nephrol., Q, 52, 1975.
5.
KAZATCHKINE, M., SULTAN, Y., CAEN, J.P, and BARIETY, J. Bleeding in renal failure: A possible cause. Br. Med. J., 2, 612, 1976.
6.
REMUZZI, G., LIVIO, M., RONCAGLIONI, M.C., MECCA, G., DONATI, M.B., and DE GAETANO, G. Bleeding in renal failure: Is von Willebrand factor implicated? Br. Med. J., 2, 359, 1977.
1014
URAEXIC
BLEEDIKG AND PGs
Vo1.13,No.6
7.
RUGGERI, Z.M., PONTICELLI, C., and MANNUCCI,P.M. Factor VIII chronic renal failure. Br. Med. J., 1, 1085, 1977.
8.
HERRMANN, R.P., MARSHALL, L.R., and HURST, P.E. Bleeding in renal failure: A possible cause. Br. Med. J., l_, 1601, 1977.
9.
REMUZZI., G., BERTANI, T., MECCA, G., DONATI, M.B., and DE GAETANO, G. Factor VIII-related protein on vascular intima of patients with chronic renal failure and prolonged bleeding times, Br. Med. J., 1, 70, 1978.
10.
REMUZZI, G., BERTANI, T., LIVIO, M., CAVENAGHI,A.E., MYSLIWIEC, M., MARCHESI, D., DONATI, M.B., DE GAETANO, G., and MECCA, G. Vascular factors in the pathogenesis of uraemic bleeding. In: Proc. XV Congr. European Dialysis and Transplant Association, Istanbul, June 4-7, 1978, in press.
11.
REMUZZI, G., CAVENAGHI, A.E., MECCA, G., DONATI, M.B., and DE GAETANO,G. Prostacyclin-like activity and bleeding in renal failure. Lancet, -II, 1195, 1977.
12.
MONCADA, S., GRYGLEWSKI, R., BUNTING, S., and VANE, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature(Lond.), -263, 663, 1976,
13.
MONCADA, S., HIGGS, E.A., and VANE, J.R. Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation. Lancet, l_, 18, 1977.
14.
DE GAETANO, G., DONATI, M.B., and GARATTINI, S. Drugs affecting platelet function tests. Their effects on haemostasis and surgical bleeding. Thromb. Diath. Haemorrh., 34_, 285, 1975.
15.
VILLA, S., and DE GAETANO, G. Prostacyclin-like activity in rat vascular tissues. Fast, long-lasting inhibitiolby treatment with lysine acetylsalicylate. Prostaglandins, 14, 1117, 1977.
16.
MacINTYRE, D.E., PEARSON, J.D., and GORDON, J.L. Localisation and stimulation of prostacyclin production in vascular cells. Nature(Lond.), -271, 549, 1978.
17.
SMITH, J.B., SILVER, M.J., INGERMAN, C.M., and KOCSIS, J.J. Prostaglandin D2 inhibits the aggregation of human platelets. Thromb. Res., 2, 291, 1974.
18.
BUNTING, S., MONCADA, S., and VANE, J.R. Antithrombotic properties of vascular endothelium. Lancet, z, 1075, 1977.
19.
SMITH, J.B., INGERMAN, C.M., and SILVER, M.J. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J. Lab. clin. Med., sg, 167, 1976.
20.
REMUZZI, G., MINGARDI, G., MECCA, G., DONATI, M.B., and DE GAETANO, G. Does aspirin precipitate major gastrointestinal bleeding in patients on haemodialysis? Lancet, 2, 359, 1977. --
and
i-01.
l?,SO.6
lTM.EXIC
BLEEDIXG
1"--i 1;
_&SD ?Gs
21.
MATTHIAS, F.R., and PALINSKI, W. Prostaglandin-endoperoxides and cyclic 3'-5'-AMP in platelets of patients with uremia. Thromb. Haemost., -38, 34, 1977.
22.
BILLS, T.K., SMITH, J.B., and SILVER, M.J. Metabolism of/l4/arachidonic acid by human platelets. Biochim. Biophys. Acta, -424, 303, T976.
23.
HAMBERG, M., SVENSSON, J., and SAMUELSSON, B. Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides Proc. Natl. Acad. Sci. USA, -72, 2994, 1975.
24.
CHARO, I.F., FEINMAN, R.D., DETWILER, T.C., SMITH, J.B., INGERMAN, C.M., and SILVER, M.J. Prostaglandin endoperoxides and thromboxane A2 can induce platelet aggregation in the absence of secretion. Nature (Lond.), -269, 66, 1977.
25.
VILLA, S., CALLIONI, A., and DE GAETANO, G. Normal prostacyclin-like activity in vascular tissues from thrombocytopenic rats. Thromb. Res., -11, 701, 1977.
26.
MORRISON, A.R., NISHIKAWA, K., and NEEDLEMAN, P. Unmasking of thromboxane A2 synthesis by ureteral obstruction in the rabbit kidney. Nature (Lond.), 267, 259, 1977.
27.
PEART, W.S. The kidney as an endocrine organ. Lancet, g,
28.
BROWNLEE, K.A. Statistical Theory and Methodology in Science and Engineering. New York: John Wiley and Sons. 1965.
Addendum - After completion of this
543, 1977.
manuscript, we became aware of a work
by Ch. Leithner et al. (Proc. XV Congress European Dialysis and Transplant Association, Istanbul, 1978, in press) confirming enhanced availability of prostacyclin-like activity in blood vessels of uraemic patients.
RESESRCH 13; 1007-1015 Press Ltd.1978. Printed
in Great Brirain OOZE-3e~8/78/1201-1007
So2.00/0
ALTERED PLATELET AND VASCULAR PROSTAGLANDIN-GENERATION IN PATIENTS WITH RENAL FAILURE AND PROLONGED BLEEDING TIMES G. Remutzi, D. Marchesi, M. Livio, A.E. Cavenaghi, G. Mecca, M.B. Donati and G. de Gaetano Divisione di Nefrologia e Dialisi, Ospedali Riuniti Bergamo, Italy and Istituto di Ricerche Farmacoloaiche "Mario Neari"-Via Eritrea. 62 20157 Mfian, Italy (Received 26.7.1978; in revised form 11.9.1978. Accepted by Editor I.M. Nilsson) ABSTRACT
Venous tissues from 15 patients with renal failure (five acute and 10 chronic] generated significantly higher PGI2-like (platelet aggregation inhibitory] activity than venous tissues from 10 normal subjects. The longer the bleeding times, the higher the values for PG12-like activity found in these patients. Both bleeding times and PGI2-like activity values returned to normal in three acute uraemic patients on restoration of their renal function. Two additional patients with acute renal failure and greatly prolonged bleeding times were under aspirin treatment at the moment of this study: venous specimens from neither of them generated measurable amounts of PGI2-like material. Malondialdehyde formation in platelets from 12 patients with chronic renal failure and prolonged bleeding times was significantly lower than in platelets from 11 controls. The defect was evident with each of the three stimuli used, i.e., collagen, arachidonic acid and thrombin. It is concluded that prostaglandin metabolism in platelets and in the vessel wall from uraemic patients is impaired in different ways, both contributing to the impaired primary haemostasis in these patients.
INTRODUCTION The pathogenesis of the bleeding tendency in uraemic patients is still un. clear. Defective platelet function resulting from retention of dialysable toxic factor(s) has long been proposed (l-4). More recently, several reports have focused attention on a contributory role of non dialysable factors in the abnormal haemostasis of patients with renal faflure (5-8). In this conte)ct,we could not obtain any evidence of defective Factor VIIK (von Hillebrand factor) in plasma and vessels from
1007
1008
~.EMIC
uraemic patients (6,9).
BLEEDIXG
AND PGs
Moreover, normal vascular fibrinolytic activity was
also found in these patients (10).
On the other hand, preliminary observa-
tions indicated increased generation of an inhibitor of platelet aggregation in the vascular tissue from these patients with renal failure (11). Some characteristics of this inhibitory activity were suggestive of it being prostacyclin (PGI2), a vascular prostaglandin endowed with potent antiaggregating and vasodilating activities (12,13). The purpose of this study was to evaluate whether this antiaggregating activity of vascular origin could be a significant factor in the abnormal bleeding associated with the uraemic condition. We present here suggestive evidence for this assumption. In addition we show that platelets from uraemic patients generated reduced amounts of prostaglandins, thus suggesting a complex role of prostaglandin metabolism in the pathogenesis of uraemic bleeding. PATIENTS AND METHODS Patients -
PG12-like activity was studied in 17 patients (13 men and four
women, aged 28-64 years) before they started haemodialysis for acute (seven patients) or chronic (10 patients) renal failure of various origin.
Bleeding
times (6) were normal ( shorter than 5 min) in two patients, moderately prolonged (5 to 8 min) in 6 and greatly prolonged (over 15 min) in 8. time could not be measured in one patient.
Bleeding
Platelet counts (6) were normal
(range 180-320 x log/l) in all but one patient who had leptospirosis and acu$ renal failure (plateletsbetween 30 and 50 x log/l). Ten male subjects (aged 20-50 years) with no apparent systemic diseases were also studied while undergoing minor plastic surgery of the forearm and acted as controls.
None of the patients or controls had received any blood
transfusions for at least two months before investigation.
Two patients (both
with acute renal failure) had been taking aspirin ( 1 g daily) during the preceeding week and were studied about four hours after the last aspirin intake. Malondialdehyde (MDA) formation was measured on platelets from 12 patients (eight men and four women, aged 18-50 years) who had been undergoing mainter: nance haemodialysis for from four months to six years. bleeding times longer than 15 min. before a haemodialytic session (6).
All of them had
Venous blood was collected immediately Eleven healthy subjects (five men and
six women, aged 20-35 years) acted as controls.
None of the patients or con-
trols had received any blood transfusion or drugs known to affect platelet
v01.13,s0.6
LTAEXIC
BLEEDIXG
d?;D PGs
1009
function (14) during the previous two months. Informed consent was obtained from all patients and controls. Methods - Venous tissue specimens were obtained and PGI2-like activity was measured as platelet aggregation inhibitory potency as previously described (11). PGI2-like activity was characterized according to current criteria (13, 15,16); namely the activity was unstable at room temperature and neutral pH, destroyed either by heating for 15 seconds at 100°C or by exposure to acidic PH.
It was stable for at least 24 hours at 4°C or at basic pil;its generation
was inhibited by incubation with acetylsalycilic acid or indomethacin, or tranylcypromine.
None of these drugs inhibited the activity once it had been
generated in the system.
Moreover, it was potentiated by inhibitors of
phosphodiesterase such as theophylline; it was distinct from PGD2 since differently from the latter prostaglandin (17), it strongly inhibited rat platelet aggregation.
Contamination with significant ADP-ase activity could
also be excluded (18). The platelet aggregation inhibitory activity was expressed as ng/ng wet tissue by extrapolation from a dose-response curve obtained on the same platelet-rich plasma with highly purified (> 99%) PGE,. It was assumed that PG12 was 30 times more potent an aggregation inhibitor than PGEl (12).
Platelet aggregation was initiated with 1 pmol/l adenosine-5'-di-
phosphate (ADP). Platelets were counted by phase contrast microscopy using tte Unopette R (Becton Dickinson, Italy) (6). Bleeding tines were measured by a semiautomatic template device (6, 11). Generation of MDA was induced by stirring platelet-rich plasma at 37"C(6) with acid soluble collagen (30 pg/ml , Harmon Chenie, Munich, West Germany)for 10 nin or with arachidonic acid sodium salt 5mM (Sigma, Mascia Brunelli,Milan, Italy) for 15 nin or with thrombin (25 NIH u/ml , Topostasine, Roche, Milan, Italy) for 5 min.
MDA was measured spectrophotometrically (Beckman MOD 25)
according to Smith et al. (19).
Results were expressed as nmolj 3~10~
platelets. RESULTS PG12-like values obtained in 10 controls and 15 patients with severe renal failure are shown in Table I. significantly (~(0.005)
Venous tissues from uraenic patients generated
higher PG12-like activity than specimens from con-
trols after both 70 and 20 min incubation. Table II
shows that longer bleeding tines were associated with higher PG12-
like activity generated within 20 min. Tissues from two patients with acute renal failure and history of daily aspirin treatment during the preceding week generated no measurable amounts of
UlUEMIC
1010
BLEEDING
AXD
PGs
TABLE I Mean values and range of PGI -like activity generated by venous tissue from 10 controls and 15 uraemic pE tients after 10 and 20 min incubation at room temperature in 0.05 M Tris-buffer, pH 7.4
PG12-LIKE ACTIVITY (ng/mg tissue) 10 min
20 min
Controls
0.57 (0.08 - 1.49)
0.59 (0.07 - 1.52)
Patients
5.08 (0.21 -33.79)
6.66 (0.63 -35.46)
4
P+
0.05
<
0.005
i ??Mann-Whitney
rank sum test (28).
TABLE II Bleeding times and venous PGI2-like activity (20 min) in 14 patients with severe renal failure
Bleeding time (min)
Number of Patients
PG12-LIKE ACTIVITY (ng/mg tissue) Mean
Range
<5
2
1.00
0.63 - 1.38
5-8
6
2.17
0.68 - 4.71
715
6
12.96
1.73 -35.46
______ PG12-like activity. Three patients with acute renal failure were studied again at the moment of by-pass removal following resolution of the renal insufficiency. In comparison with the values obtained at the beginning of haemodialytictreatment, normal bleeding times and reduction of PG12-like activity towards the upper limits of the control values were observed in all three patients when renal
So1.13,80.6
L-RAEHIC BLEEDIKG
XKD PCs
TABLE III Bleeding times, venous PGI2-like activity (20 min) and platelet counts of three patients with acute renale failure.
Patient
1
Bleeding time(min)
j A
PGI -like activity (n&mg tissue)
Bj
A
Platelet count (X109/1) a
B
Interval between A andB
B
’ ldays)
i
A:
2 1
/>15
3
6
2 2.30
26.0 7.17
1.38 1.84
250 50
290 300
27 28
4.71
1.98
220
200
15
2
Before the first hemodialysis session.
B : After
the last
hemodialysis session.
TABLE IV
Mean values and range of MDA generated by platelets from 11 controls and 12 patients with chronic renal failure in response to arachidonic acid, thrombin and collagen.
MDA (nmo1/3x108 platelets) Arachidonic acid
Thrombin
Controls
3.15 (1.30 - 4.40)
0.76 (0.24 - 1.50)
Patients
0.93 (0 - 2.25 )
(0 - 0.36 )
P+
<
0.01
+ Student's t test (28).
0.13
Collagen 0.88 (0.36 - 1.15) 0.29 (0 - 0.65)
<
0.001
I I
CWMIC
1012
function was restored.
BLEEDING
Vo1.13,No.6
AXi PGs
The low platelet count of patient No. 2 also returned
to normal (Table III). MDA generation in response to collagen, arachidonic acid or thrombin was significantly lower in platele&from
patients with chronic renal failure un-
dergoing maintenance haemodialysis than in platelets from controls (Table IV).
DISCUSSION These results indicate that venous tissues from patients with renal failure generated greater PG12-like (platelet aggregation inhibitory) activity than comparable specimens from control
subjects.
The suggestion that PGI2 might
be one of the factors contributing to the haemorrhagic diathesis of uraemic patients (11) is supported by the observation that normal or moderately prolonged bleeding times were associated with normal or moderately increased venous PG12-like activity; in contrast, greatly prolonged bleeding times (over 15 min) were associated with many-fold increase generation from venous tissues.
of PG12-like activity
This contention is further strengthened by
the fact that both bleeding times and PG12-like activity values in three acute uraemic patients returned to normal on restoration of their renal function. The pathogenesis of abnormal bleeding times in uraemic patients is however more complex than it would appear from these findings.
Indeed, venous tissues
from two patients under aspirin treatment generated no measurable amounts of PGI2-like material, yet the bleeding times of both patients were greatly prolonged (over 15 min).
It is tempting to speculate that in these two patients
the reduction of vascular PG12-like activity could not overcome the defective platelet function induced by aspirin (14), and/or the uraemic condition itself (20).
Indeed, platelets from uraemic patients with greatly prolonged
bleeding times (undergoing maintenance haemodialysis) showed a significant reduction of MDA generation, a reliable parameter of platelet activation (19), in response to different aggregating stimuli, a finding in agreement with a recent report (21).
A defect of platelet phospholipase A2 activity (22) is
unlikely since MDA formation was significantly reduced in response not only to collagen and thrombin but also to exogenous arachidonic acid. The relative contribution of platelets and the vessel
wall to the
haemorrhagic diathesis of patients with renal failure has not yet been established.
MDA is a product of platelet arachidonic acid metabolism (19)
and may reflect the generation of cyclic endoperoxides and thromboxane
A2
which are potent aggregating
agents (23,241.
PGI2 is a product of vascular
arachidonic acid metabolism, with potent antiaggregating activity (12,16,25). 1: is noteworthy that in the uraemic condition pathways appeared to be affected in
these two arachidonic acid
opposite ways which could both result in
an increased haemorrhagic risk. The pathogenesis of the altered prostaglandin metabolism in patients with renal failure is still unknown (26,27). Acknowledgements We thank Prof. S. Garattini and Prof. M.J. Silver for fruitful discussion and criticism.
We also thank drs. R. Misiani, G. Mingardi and S. Villa and
Miss E. Marchesi for valuable cooperation.
Drs. E. Poletti, P. Losapio, A.
Frontini, V. Giorgianni, R. Licini, A. Mangili, V. Brugali, C. Malgrati, L. V'illa,V. Oberti, and P. Tondini helped in collecting vascular specimens. J. Baggott, V. Pistotti, G. Brambilla, F. and V. de Ceglie helped in preparing the manuscript.
Prof. D.A. Van Dorp (Unilever Research Laboratories,
Vlaardingen, The Netherlands) kindly provided purified PGE,. This work was supported in part by the Associazione Bergamasca per lo Studio delle Malattie Renali.
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Addendum - After completion of this
543, 1977.
manuscript, we became aware of a work
by Ch. Leithner et al. (Proc. XV Congress European Dialysis and Transplant Association, Istanbul, 1978, in press) confirming enhanced availability of prostacyclin-like activity in blood vessels of uraemic patients.