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Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment
Prostaglandins Lxkoticmes and Essential Q Longman Group UK Lid 1988
Fatty Acids (1988) 31, 131438
Platelet Aggregation and Thromboxane A2 Release in Primary Biliary Cirrhosis and Effect of DPenicillamine Treatment D. P. MIKHAILIDIS, V. FONSECA, M. A. BARRADAS, EPSTEIN+, N. MCINTYRE+ and P. DANDONA
R. A. HUTTON*
J. Y. JEREMY,
0.
Department of Chemical Pathology and Human Metabolism; *Haemophilia Cenrre, Department of Haemarology; and tDeparrment of Medicine, Royal Free Hospital and School of Medicine, London N W3 2QG U.K.
Abstract - Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of Dpenicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease. Keywords: primary biliary cirrhosis; platelet count.
platelet aggregation;
Introduction
Platelet aggregation has been shown to be impaired in patients with liver disease, including cirrhosis (1). However, it is important to classify patients according to the aetiology of cirrhosis, since specific factors, such as ethanol intake (2, 3), may exert a major influence on platelet funcc
thromboxane
A,;
tion. In this regard, patients with primary biliary cirrhosis (PBC) should be assessed separately, since they may develop secondary hyperlipidaemia (4, 5) or, more rarely, Raynaud’s phenomenon (4), both of which may be associated with platelet hyperaggregability (6, 7). Furthermore, patients with PBC (or other
131 PROST.-
D-penicillamine;
132
PROSTAGLANDINS
conditions) may be treated with D-penicillamine, a drug known to cause thrombocytopenia which may not always be attributable to drug-induced depression of bone marrow activity (8-12). The present study investigates platelet function in patients with PBC and assesses whether D-penicillamine treatment influences platelet behaviour in these patients. The effect of Dpenicillamine added, in vitro. to platelets obtained from healthy volunteers. is also evaluated.
Materials and Methods A. Evaluation of platelet function in patients with PBC Selection of patients and control subjects Patients (n = 28; median age 58 years; age range: 52-70 years. 26 females, 2 males) with histologically proven PBC (13) were included in the study. All the patients had raised plasma alkaline phosphatase activity and an elevated titre of anti-mitochondrial antibodies. Plasma bilirubin was raised (>17 Fmol/l) in 15 patients. Two patients had a history of Raynaud’s phenomenon. Ten patients were being treated with D-penicillamine at the time of sampling (treated group). whereas 18 were not (untreated group). Patients were classified as having “early” (n = 10) or “late” (n = 18) disease, according to liver histological patterns (13). Samples were also obtained from 12 healthy volunteers with an age and sex distribution similar to that in the PBC group. Patients and volunteers denied taking any non-steroidal antiinflammatory drugs within the two weeks preceding sampling. Collection of samples and platelet aggregation studies Patients were sampled when attending regular out-patient appointments. Venous blood was collected in 3.8/ trisodium citrate and processed as previously described (14). Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared by centrifugation at room temperature. Following centrifugation, PRP was kept at 37 “C during all subsequent experimental proceduress, since we have shown that cooling influences platelet function (15). PRP was allowed to “equilibrate”, at 37 “C, for 15 min before aggregation was carried out, at 37 “C, using a dual channel Chronolog aggregometer (14). Platelet aggregation was expressed as the
LEUKOTRIENES
AND ESSENTIAL FA’lTY ACIDS
percentage fall in optical density 3 min after the addition of various agonists (adrenaline; adenosine diphosphate; collagen) (14, 15). The concentrations of these agonists and the number of control subjects/patients studied are shown in the appropriate tables. Platelet aggregation studies were completed within 2 h of venepuncture. Platelet counts shown in table 1 were measured in PRP using a Coulter Counter, model D (3). Assay of platelet thromboxane A2 release Platelet release of the prostanoid thromboxane A: (TXA1) was assessed by assaying thromboxane BZ (TXB2), the stable, spontaneous breakdown product of TXA? (3, 7). Briefly, platelet aggregation was induced as described above; the reaction was then stopped (3 min after adding the appropriate agonist) by mixing PRP with ethanol. The extracted sample was then stored at -70 “C until measurement of TXB? concentrations, using a specific radioimmunoassay (3, 7). Liver function tests und serum cholesterol Plasma total bilirubin, aspartate transaminase, alkaline phosphatase and albumin and serum cholesterol were measured using routine methods (SMAC Autoanalyzer, Technicon Instruments, Tarrytown, N.Y., U.S.A.) in current use at The Royal Free Hospital. B. Experiments involving PRP obtained from healthy control subjects and the addition. in vitro, of D-penicillamine. D-penicillamine base was obtained from Sigma (Poole, Dorset, U.K.). PRP was prepared from healthy volunteers as described above. The PRP was then incubated for 2 min at 37 “C with varying concentrations of D-penicillamine dissolved in 0.9% saline. Aggregation was induced by several agonists (see table 7) and the percentage fall in optical density was evaluated 3 min later. Experiments were always run in parallel. one aggregometer channel containing PRP + D-penicillamine, the other PRP + saline control. The concentrations of agonists used (see table 7) were sub-maximal, so as to allow any enhancement of aggregation to be detected (16, 17). The D-penicillamine concentrations selected for the in vitro (5-20 mg/l) experiments were similar to the plasma levels (6-8 mg/l) in those taking therapeutic doses of the drug (18).
PLATELET AGGREGATION
Statistical analysis
IN PRIMARY BILIARY CIRRHOSIS
and
expression
of
results
Results are expressed as median and range. Values in the PBC group taking D-penicillamine were compared with those in the PBC patients not taking this drug, using the unpaired nonparametric Mann-Whitney test (two-tailed) (19). Results in the patients with “early” disease were compared with those obtained in patients with “late” disease, using the same test. In the in vitro experiments, the results obtained with Dpenicillamine present were compared with the appropriate matched control samples using a paired non-parametric Wilcoxon rank sum test (two-tailed) (19). Frequency analysis was carried out using the standard chi-square test with Yates’ correction. Results A. Platelet studies in patients with PBC Platelet counts (Table 1) The platelet count in the PRP of patients with “late” PBC was significantly below that of “early” PBC (p <0.002) or controls (p <0.002). There was no significant difference between D-penicillamine treated and untreated patients, although in the treated group the count was significantly lower than that in controls (p cO.05). Table 1 PRP Platelet Counts (X1()y/l) in PBC Patients and Control Subjects. Platelet counts Median Range
Control subjects PBC + D-penicillamine PBC - no treatment “Early” PBC “Late” PBC All PBC patients For statistical
analysis,
(n (n (n (n (n (n
= = = = = =
12) IO) 18) 10) 18) 28)
326
240-396
217 241 318 193 235
91-31x 91-528 21 l-528 91-332 91-.52X
see text.
Frequency analysis of PRP platelet counts showed that for “early” PBC one out of 9 patients (11%) had platelet counts below 240 x lo’/1 (the lowest count for the control subjects); this was significantly different (p
133 (7 out of 15 patients had platelet counts below 240 x lO’/l) groups were compared. To separate the effect of disease stage from that of treatment with D-penicillamine and vice versa, we examined patient groups which were large enough in size (n = 8 approximately) and which also shared the same characteristics. These were: (a) Patients with “late” PBC - treated (n = 8) and untreated (n = 7): the median (range) PRP platelet count in the former group was 178 (90 - 284) x lo’/1 and in the latter group 200 (90 - 332) X 19’/1 respectively. (b) Patients not treated with D-penicillamine and with “early” PBC (n = 8) and “late” PBC (n = 8): the median (range) PRP platelet count in the former group was 308 (200 - 528) x lo’/1 and in the latter group 200 (90 - 332) x lo’/1 respectively. None of these differences was statistically significant, but they paralleled the changes reported for the larger patient groups described above. Platelet aggregation There was wide scatter in the results. Patients not receiving D-penicillamine (II = 15: 7 “early” PBC) showed aggregation which was not statistically different to that of controls (n = lo), whereas in patients receiving D-penicillamine (n = 10: 2 “early” PBC) platelet aggregation was significantly increased compared to the control group (see Table 2) at low concentrations of collagen and adrenaline, but not ADP. There were no significant differences between treated and untreated PBC patients. Patients with “early” PBC (n = 9; 7 untreated) (Table 3) tended to have platelets which were more aggregable than those with “late” disease (n = 16; 8 treated). This may have been partly due to thrombocytopenia (which was commoner in “late” PBC), since patients with low PRP platelet counts (cl50 x lO’/l) tended to show reduced aggregation patterns, regardless of the stage of the disease. To separate the effect of disease stage from that of treatment with D-penicillamine and vice versa, we examined patient groups which were numerically large enough and which shared the same characteristics as described for PRP counts above. These groups were: (a) Patients with “late” PBC - treated (n = 8) and untreated (n = 7): the platelet aggre-
134
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL FAll-Y AClDS
Table 2 Percentage Platelet Aggregation in Control Subjects and in PBC Patients. Results are Expressed as Median and (Range). Number of Patients = n. Patients with PBC + D-peniciilamine - D- penicillamine
Control Subjects
32* (O-76) n = 10
5 (O-76) n = 16
6 (O-23) n=9
61** (O-80) n=9
29 (O-77) n = 15
n=9
62 (44-80) n=9
55 (23-77)
n = 15
66 (SO-86) n=9
12 (O-76) n=8
0 (O-78) n = 16
3 (O-8) n=9
0.5
59** (O-85) n = 10
20 (O-88) n = 16
22 (7-60) n=9
1.0
68 (38-90) n = 10
49 (20-83) n = 15
69 (60-81) n=9
Aggregating Agent
Adrenaline (pmoI/l) 0.2 0.5
5.0 Collagen (mgfl) 0.15
ADP (pmol/l) 0.5
6 (O-59)
4 (O-64)
16 (O-31)
7 (O-36)
n=8
n=9
n = 10
70 (54-82)
66 (21-91)
72 (60-86)
n = 10
n = 16
n=9
Proportion
of patients with “Early” PBC
30%
43%
Proportion of patients with “Late” PBC
70%
57%
10.0
Statisticat analysis: PBC patients on D-penicillamine
vs controls: *p = 0.02; **p = 0.002. Mann-Whitney test (see text). PBC patients not on Dpenicillamine vs controls and PBC patients on D-peniciilamine vs PBC patients not on D-penicillamine: all comparisons = not significant. Mann Whitney test (see text).
gation responses in these patients paralleled the patterns described for the larger patient groups (which included both “late” and “early” PBC patients), but the only significant (p G.01) difference was with aggregation induced by collagen, 0.5 m$l, where median (range) percentage aggregation was 49 (O-85)% in “late” PBC + D-penicillamine and 6 (O-68)% in “late” PBC with no treatment. (b) Patients with “early” (n = 8) or “late” (n = 8) PBC not receiving treatment: platelet aggregation patterns paralleled those observed with the larger groups of patients (which included both treated and untreated patients). Median (range) aggregation differed significantly with: (i) aggregation induced by collagen 0.5 ms/l. where percentage aggre-
gation in “early” PBC-untreated was 64 (3-88)s and in “late” PBC untreated was 6 (OH%)% (p <0.04). Control subjects showed aggregation of 22 (7-60)0/c in response to the same agonist; this did not differ significantly from the values obtained from either PBC subgroup. Platelet aggregation induced by collagen 1 mg/l also differed significantly (p <0.05), values being 72 (2-83)s in “early” untreated PBC and 43 (20-58)s in “late” untreated PBC. The percentage aggregation in the “late” untreated PBC group was significantly (p
PLATELET
AGGREGATION
Percentage Platelet Aggregation in PBC Patients Classified according to Disease Stage (see text). Result are Expressed as Median and (Range). n = Number of Patients.
Table 3
Aggrqating
agent
Adrenaline 0.2
(pmol/l)
“Early”
PBC
“Late”
9 (O-65) n = 16
0.5
5X’ (10-77) n=X
43 (O-80) n = 15
5.0
69 (27-90) n=Y
57 (13-80) n = 15
9 (O-78) n=9
0 (O-.55) n = I.5
0.5
66++ (5-88) n=X
38 (O-85) n = 14
1.o
74’ (35-90) n=Y
4X (20-84) n = IS
0 (O-60) n=X
0 (O-5Y) n = I4
l(l.0
71 (57-YI) n=Y
63 (21-82) n = 17
Proportion of pts. taking D-penicillamine
33%
47%
ADP 0.5
Table 4 Platelet TXBz Release in PBC Patients Taking and not Taking D-Penicillamine and Control Subjects. Results are Expressed as Median and (Range) of TXBz (t&Ox Platelets) Released. n = Number of Subjects.
PBC
38’ (O-76) fl=Y
Collagen 0. IS
135
IN PRIMARY BILIARY CIRRHOSIS
(ms/l)
Aggregating Agent
Patients Taking D-penicillamine
with PBC Not taking D-penicillamine
Control Subjects
Adrenaline 5 pmol/l
6.6 (1.X-17.5) n=8
5.6 (
6.5 (4-11.6) n=X
Collagen 1 ms/l
9.3 (
5.1 (
8.9 (5.6-15.4) n=H
ADP IO pmol/l
2.0 (
I.0 (
2.2 (
Statistical
Analysis: Controls vs PBC + D-penicillamine, controls vs PBC only and PBC + D-penicillamine vs PBC only: all comparisons = not significant (Mann-Whitney test - see text).
Platelet Thromboxane B3 Release in “Early” and “Late” PBC (see text for definitions). Results are Expressed as Median and (Range) of TXB, (r&O’ platelets) Released. n = Number of Subjects. For Control Values see Table 4. Table 5
(pmol/l)
Stutistical
Analysis: *p <(I.02 “Early” vs “Late” PBC patients. +p <0.05; ++p
Aggregating
agent
“Early”
PBC
“Late”
PBC
Adrenaline 5 fimol/l
5.Y (1.8-17.5) n=h
6.3 (
Collagen I mg/l
6.1 (<1-13.X) n=6
6.0 (
ADP 10 pmol/l
2.3 (
2.3 (
Serum cholesterol concentrations Serum cholesterol levels were similar in the treated patients, 6.4 mmol/l (4.2-8.6) and untreated patients, 6.0 mmol/l (3.4-10.2), and between patients with “early” PBC, 6.4 mmol/i (4.9-7.5) and “late” PBC, 5.8 mmol/l (3.4-10.2). These values were not significantly different from those in the 5.9 mmol/l (3.4-7.0). Patients with controls, cholesterol above 6.5 mmol/t (the upper limit of the reference range) tended to have more enhanced aggregation patterns and TXAl release than patients with serum cholesterol in the normal range, but this difference was not statistically significant.
Liver function tests There was no obvious correlation between the liver function tests (Table 6(a), (b)) and platelet aggregation results; platelet hyperactivity was noted in patients with near normal, as well as with markedly abnormal, liver function tests. Liver function tests in all control subjects were well within the reference range.
TXBZ release (Tables 4 and 5) Aggregationinduced TXB-’ release by platelets tended to parallel aggregability, particularly at low agonist concentrations; but after taking platelet counts into consideration, no statistically significant differences were noted between “early” and “late” or treated and untreated groups or when these groups were compared to controls.
B. Experiments involving the in vitro addition of D-penicillamine base to PRP obtained from healthy control subjects (Table 7) Platelet aggregation (in samples obtained from healthy volunteers) in response to low concentrations of adrenaline and collagen (see table 7) was significantly enhanced by the addition, in
Statistical
Analysis: “Early” vs “Late” PBC; controls (see Table 4) vs “Early” or “Late” PBC. All comparisons = not significant. (Mann-Whitney test - see text).
136
PROSTAGLANDINS
Table 6(a) Liver Function “Late” PBC Patients
Tests in “Early”
and
Patients with “Early” PBC (n = 9)
Patients with “Late” PBC (n = 15)
Alk phosphatase (3.5-130 u/I)
371 (179-856)
556 (275- 1420)
Bilirubin (5- 17 ~mol/l)
IO (3-2X)
29 (g-200)*
Aspartate transaminase (S-40 U/I)
56 (38-96)
&h)**
Albumin (35-50 gil)
31 (38-44)
37 (32-44)
Liver function test (reference range)
Statistical analysis: “Early” PBC vs “Late” Whitney test. * p
PBC-
Mann-
PBC patients taking D-penicillamine (n = Y)
PBC patients not taking D-penicillarnine (n = 15)
Alk phosphatase (35-130 U/l,
514 (‘75-948)
490 (179-1420)
Bilirubin (5-17 ~mol/l)
(8-66)
1x (3-200)
Aspartate transaminase (S-40 U/I)
83 (56
(25-206)
27
Albumin
(35-50 g/l)
(Z-53,
7s 170)
38 (33-44)
Statistical analysis: Taking D-penicillamine vs not taking D-penicillamine (Mann-Whitney test): all comparisons = not significant; n = number of subjects.
vitro, of D-penicillamine (final concentration: 10 mg/l). ADP-induced aggregation, however, was not significantly enhanced. Lower concentrations of D-penicillamine (5 mg/f) did not produce significant enhancement although a trend was present (results not shown). Higher Dpenicillamine concentrations (20 mg/l) produced significant enhancement but the results are not shown since these drug concentrations are well above the therapeutic levels (6-8 m$l) achieved in vivo (18). Patients with Raynaud’s Phenomenon Patients reported to have Raynaud’s phenomenon (n = 2) did not appear to have aggregation or TXA? release patterns which were distinctly different from those in the other patients.
AND ESSENTIAL
FAITY
ACIDS
Table 7 In Vitro Effect of Adding D-Penicillaminc Base to PRP Obtained from Healthy Volunteers. Results are Expressed as Median and (Range) ‘i Aggregation. Aggregating agent
Adrenaline (0.1-1.5 pmol/l) Collagen (0.15-0.4 mg/l) ADP (0.X-2.0 ~mol/l)
Table 6(b) Liver Function Tests in PBC Patients TakingflVot Taking D-Penicillamine Liver function test (reference range)
LEUKOTRIENES
C‘ontrol experimrnr CPRP + .\crlitw) 9 (S-20)
Test experimrrlr (PRP + II~~~~t~iciii~rnliflc,)
61 (.!(I-77) (n = X) 47 (31-S) p <0.1ll (11 = 7) 35 (Z-57) p = not significant p
IO (S-20) 26 (5-Jtj)
*Final concentration of D-penicillamine = IO mg/l PRP Statistical Analysfs: control experiment vs test experiment using Wilcoxon test (see text). p values are shown with the results above. There was no significant enhancement of TXA: release in these experiments (results not shown). However. this may merely reflect the relatively low agonist concentrations whtch arc required to elicit enhancement of aggregation and which also release very \mall amounts of TXA;. n = numhcr of subjects.
Discussion Our findings show that platelet aggregation and TXBz release following the in vitro addition of adrenaline, collagen and ADP in a group of PBC patients not treated with D-penicillamine was similar to that in controls. In contrast, platelets from PBC patients receiving D-penicillamine showed significantly enhanced platelet aggrcgation in response to low concentrations of adrenaline and collagen (but not ADP) when compared with controls. Platelet TXA- release paralleled the aggregation findings but did not achieve statistical significance. Further evidence supporting the view that platelets are activated by D-penicillamine is provided by the findings of the in vitro experiments which showed that D-penicillamine. added to normal platelets, enhanced aggregation in response to low concentrations of adrenaline and collagen. but not following ADP. This pattern was similar to that observed in the cx vivo study involving the PBC patients. There was no correlation between the liver function tests and platelet function. The relatively enhanced aggregation observed in the PBC patients with “early” disease (table 3) is unlikely to be due to D-penicillamine administration. since the proportion of patients receiving this drug was greater in the “late” PBC group. Further-mote, analysis of untreated “early” and “late” PBC groups showed significantly greater
PLATELET AGGREGATION
IN PRIMARY BILIARY CIRRHOSIS
aggregation in the former group. The difference in platelet aggregability between “early” and “late” disease may relate to the significantly lower platelet counts in the latter situation, since thrombocytopenia is associated with diminished aggregation (20). TXA2 release is also influenced in a similar manner when expressed per volume of blood, but not when related to the platelet count (21). Two earlier studies (5, 22) have reported an inhibition of platelet aggregation in PBC. These observations (5, 22) would be broadly comparable to our findings if patients with “late” PBC (and therefore possibly with thrombocytopenia), and not receiving D-penicillamine had been studied since we also showed that such patients had diminished platelet aggregation when compared with control subjects, Unfortunately, our results are not directly comparable with these earlier studies, since disease stage and thrombocytopenia (a major variable in our study) were not considered in one study (5). In the other study (22). platelet counts were standardised but the disease stage was not considered. Furthermore, in this latter study (22), all the aggregation studies were conducted with luminescence reagent present. This reagent may interfere with aggregation responses, hence the recommendation by the manufacturers of the luminescence aggregometer (Coulter Electronics, Luton, Beds, U.K.) used by these authors to also assess aggregation in the absence of these reagents. Both these earlier studies (5, 22) do not appear to have assessed the effect of D-penicillamine treatment. Platelet function in PBC patients may be influenced by several factors which are not present, at least to such an extent. in other types of cirrhosis. For example, secondary hyperlipidaemia and thrombocytopenia have been reported in PBC and in patients receiving D-penicillamine (S-12, 22); PBC may also be associated with Raynaud’s phenomenon (4, 23). These variables are of interest, since secondary hyperlipidaemia and Raynaud’s phenomenon are associated with enhanced platelet aggregation (6, 7). Enhanced platelet aggregation, however, does not appear to contribute significantly to the pathogenesis of Raynaud’s phenomenon in PBC, since platelet function in these patients did not differ markedly from that in the other patients. D-penicillamine treatment is associated with thrombocytopenia but the pathogenesis remains unclear (8-12, 24). One recognised mechanism is bone marrow
137 depression (8-12, 24); however, there have also been reports of thrombotic thrombocytopenic purpura (TTP) associated with D-penicillamine therapy (8-12). Since platelet hyperactivity is thought to play a role in the pathogenesis of TTP (25), it is possible that enhanced aggregation contributes towards the development of thrombocytopenia in patients treated with Dpenicillamine by enhancing platelet consumption, especially in situations where bone marrow production of platelets is impaired. Whether inhibiting platelet function (e.g. with aspirin or dipyridamole) can be of therapeutic benefit in PBC- or D-penicillamine-associated thrombocytopenia remains to be established. To our knowledge, there is no report of increased mortality from vascular disease in PBC. This may result from the fact that mortality in these patients is probably a consequence of progression of liver disease which precedes the more long-term consequences that may result from platelet hyperactivity and mild hypercholesterolaemia. Furthermore, it has been suggested (26) that the lipid profile (elevated HDL) in PBC may in fact be protective against ischaemic heart disease. The variability of results obtained in the present study suggest that several factors influenced platelet function. Some of these factors clearly are the platelet count (low counts diminished aggregation) and treatment with Dpenicillamine (D-penicillamine treatment enhanced aggregation). A larger study would be required to ascertain whether hypercholesterolaemia and the disease stage per se make major contributions and whether other factors not considered here are also relevant. The possible role of platelet-associated immunoglobulins which have recently been reported in patients with PBC (27) must also be considered in future studies assessing platelet function. In view of the present findings and those we reported earlier in patients with alcoholic liver disease (2, 3. 28), it would seem that the first stage in any project assessing prostanoid production in chronic liver disease must be to sample as uniform a patient population as possible. Liver disease should therefore be considered as separate entities depending on aetiology. treatment and disease stage. A brief survey of the literature should reveal to any reader that this requirement does not seem to have been considered in a very large number of studies.
138
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL
FATTY
ACIDS
References I
Owen. J. S. Hutton. R. A.. Day. D. A.. Bruckdorfer. K. R. and McIntyre. N. Platelet lipid composition and platelet aggregation in human liver disease. J Lipid Res. 22: 423-430. 1981 2 Mikhailidis. D. P.. Jenkins. W. J.. Barradas. M. A.. Jeremy. J. Y. and Dandona. P Platelet function defects in chronic alcoholism. Br Med J. 2Y3: 715-71X. 19x6. D. P.. Jeremy. J. Y.. Barradas. M. A.. 3 Mikhailidis, Green. N. and Dandona. P. Effect of ethanol on vascular prostacyclin synthesis, platelet aggregation and platelet thromboxane release. Br Med J. 2X7: 13Y5-14YX. 1983. S. and Scheucr. P. J. The oresentation and 4 Sherlock. diagnosis of 100 patients with primary’biliary cirrhosis N Enel J Med. 2XY: 674-67X. lY73. S. and Aviram. M. s Baruch. Y.. Brook. G.. Eidelman. Increased concentration of high density lipoprotein in plasma and decreased platelet aggregation in primary biliary cirrhosis. Atherosclerosis. 53: 151-62. IYX4. 6 Carvalho, A. C. A.. Colman. R. W. and Lees. R. S. Platelet function in hyperbetalipoprotcinemia. N Engl J Med. 290: 434-43X. 1974. 7 Hutton. R. A.. Mikhailidis. D. P.. Bcrnstcin, R. M.. Hughes, G. R. V. and Dandona. P. Assessment of platelet function in patients with Raynaud’s syndrome. J Clin Pathol. 37: 1X2-187, lYX4. toxicity from pcnicillaminc x Camp, A. V. Haematologic in rheumatoid arthritis. J Rheumatol (Suppl 7). 8: 164-165 19x1 Y Junker. P.. Helin. G., Jensen. B. A.. Oxlund. H. and Lorcnzen. I. D-penicillamine-induced angiopathy in rats. Atherosclerosis lYX2; 45: 17-31 IO Tricc. J. M.. Pinals, R. S. and Plitman, G. I. Thrombotic thromhocytopenic purpura during penicillaminc therapy in rheumatoid arthritis. Arch Intern Med. 143: 14X7-14%+ 198.3 Weiss, A. S.. Markcnson. J. A., Weiss. M. S. and II Kammerer. W. H. Toxicity of D-penicillamine in rheumatoid arthritis. Am J Med. X4: 114-120. lY7X 12 Speth. P. A., Boerbooms, A. M.. Holdrinet, R. S.. Van de Putte. L. B. and Meyer. J. W. Thrombotic thrombocytopcnic purpura associated with D-penicillaminc treatment in rheumatoid arthritis. J Rhcumatol. Y: x12-x13. IYX? 13. Fonseca V, Epstein. 0.. Katrak. A.. Junglee. 0.. Mikhailidis. D. P.. McIntyre. N. and Dandona. P. Serum immunorcactive trypsin and pancreatic lipase in primary biliary cirrhosis. J Clin Pathol, 39: 638%640. IYX6 14. Mikhailidis. D. P.. Mikhailidis, A. M.. Woollard, M. L. and Dandona, P. Protection of prostacyclin-like activity in human plasma a non-enzymatic mechanism Clin Sci. 62: 177-181, 19X2.
IS
I6
I7
IX
IY
20 21
22
23
24
25
26 27
2X
Mikhailidis. D. P.. Hutton. R. A.. Jeremy. J. Y. and Dandona. P. Cooling decreases the efficinecy of prostaglandin inhibitors of platelet aggregation a factor of possible relevance in cold-induced pathology. Microcirculation, 2: 413423. 19X3. Barradas. M. A.. Mikhailidis. A .M.. Mikhailidis. I). P. and Dandona. P. Tine effects of fentluraminc on in vitro platclct aggregation. Br J Clin Pharmacol, 14: 466-46X. IYX2. Mikhailidia, D. I’.. Barradas. M. A.. Mikhailidia, D. I’ Mapnani, H. and Dandona. P. Comparison of the effect of a conventional heparin and a low molecular weight heparinoid on platelet function. Br J Clin Pharmacol. 17: 43-4x. 1YX-l. Bergstrom. R. F.. Kay, D. R.. Harkcom, 7‘. M. and Wagner. J. G. Pcnicillaminc kinetics in normal subjects. Clin Pharmacol Ther. 30: 4044413. 19x1. Altman. D. G.. Gore. S. M.. Gardner. M. J. and Pocock. S. J. Statistical guidelines for contributors to medical journals. Br Med J. 2X6: 14X9-lY3. 1YX.i. Born. G. V. R. and Cross. M. J. The aggregation of blood platelets. J Physiol. IhX: 17X-IY5, lY63. Carter. A. J. and Hanlcy. S. P. The effect ot platclct number and haemocrit on whole blood thromboxane synthesis. Thromh Haemostas. 53: 225-227. lYX5. Ingeberg. S.. Jacohscn. P., Fischer. E., and Bcntscn. K. D. Platelet aggregation and rclcasc of ATP in patients with hepatic cirrhosis. Stand J Gastroenterol. 20: ‘X5~‘XX. IYX.5. Reynolds. T. B.. Dcnsion. E. K.. Franke, Ii. D.. Oihcrman. F. L. and Peters, R. L. Primarv hiliarv cirrhosis with sclcrodcrma. Raynaud’s phcnomcnon and telanriectasia. Am J Med. 50: 3022313. 1971. Thomas. D.. Gallus. A. S., Brooks. P. M Tampi, R.. Gcddes. R. and Hill. W. Thrombokinctic\ in patients with rheumatoid arthritis treated with I>-penicillaminc. Ann Rheum Dis. 43: 40224Oh. IYX4. Byrne\. J. J.. and Moake. J. L. Thrombotic thrombocytopcnic purpura and the haemolytic uraemic svndrome: evolving concepts of pathogcncsis and therapy. In: Clinics in Hacmatol. Vol. IS: Thrombosis and the vcsscl wall. C. N. Chestcrman (Ed.) London: W. B. Saunders Co.. IYX6. pp 413-412 Kaplan, M. M. Primary hiliary cirrhosis. N Engl J Med. 316: 521-52x. IYX7 Bassendinc. M. F.. Collins. J. D.. Stephenson. J.. Saunders, P. and James. 0. F. W. Platelet-associated immunoglobulins in primary biliary cirrhosis: a cause of thromhocytopenia. Gut. 26: lO74-l1)7Y. IYX5. Mikhailidis. D. P.. Jenkins. W. J., Barradaa. M. A.. Jeremy. J. Y. and Dandona, P. Platelet function defects in chronic alcoholism. Br Med J. 2Y.3: 1307. IYXh.
Fatty Acids (1988) 31, 131438
Platelet Aggregation and Thromboxane A2 Release in Primary Biliary Cirrhosis and Effect of DPenicillamine Treatment D. P. MIKHAILIDIS, V. FONSECA, M. A. BARRADAS, EPSTEIN+, N. MCINTYRE+ and P. DANDONA
R. A. HUTTON*
J. Y. JEREMY,
0.
Department of Chemical Pathology and Human Metabolism; *Haemophilia Cenrre, Department of Haemarology; and tDeparrment of Medicine, Royal Free Hospital and School of Medicine, London N W3 2QG U.K.
Abstract - Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of Dpenicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease. Keywords: primary biliary cirrhosis; platelet count.
platelet aggregation;
Introduction
Platelet aggregation has been shown to be impaired in patients with liver disease, including cirrhosis (1). However, it is important to classify patients according to the aetiology of cirrhosis, since specific factors, such as ethanol intake (2, 3), may exert a major influence on platelet funcc
thromboxane
A,;
tion. In this regard, patients with primary biliary cirrhosis (PBC) should be assessed separately, since they may develop secondary hyperlipidaemia (4, 5) or, more rarely, Raynaud’s phenomenon (4), both of which may be associated with platelet hyperaggregability (6, 7). Furthermore, patients with PBC (or other
131 PROST.-
D-penicillamine;
132
PROSTAGLANDINS
conditions) may be treated with D-penicillamine, a drug known to cause thrombocytopenia which may not always be attributable to drug-induced depression of bone marrow activity (8-12). The present study investigates platelet function in patients with PBC and assesses whether D-penicillamine treatment influences platelet behaviour in these patients. The effect of Dpenicillamine added, in vitro. to platelets obtained from healthy volunteers. is also evaluated.
Materials and Methods A. Evaluation of platelet function in patients with PBC Selection of patients and control subjects Patients (n = 28; median age 58 years; age range: 52-70 years. 26 females, 2 males) with histologically proven PBC (13) were included in the study. All the patients had raised plasma alkaline phosphatase activity and an elevated titre of anti-mitochondrial antibodies. Plasma bilirubin was raised (>17 Fmol/l) in 15 patients. Two patients had a history of Raynaud’s phenomenon. Ten patients were being treated with D-penicillamine at the time of sampling (treated group). whereas 18 were not (untreated group). Patients were classified as having “early” (n = 10) or “late” (n = 18) disease, according to liver histological patterns (13). Samples were also obtained from 12 healthy volunteers with an age and sex distribution similar to that in the PBC group. Patients and volunteers denied taking any non-steroidal antiinflammatory drugs within the two weeks preceding sampling. Collection of samples and platelet aggregation studies Patients were sampled when attending regular out-patient appointments. Venous blood was collected in 3.8/ trisodium citrate and processed as previously described (14). Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared by centrifugation at room temperature. Following centrifugation, PRP was kept at 37 “C during all subsequent experimental proceduress, since we have shown that cooling influences platelet function (15). PRP was allowed to “equilibrate”, at 37 “C, for 15 min before aggregation was carried out, at 37 “C, using a dual channel Chronolog aggregometer (14). Platelet aggregation was expressed as the
LEUKOTRIENES
AND ESSENTIAL FA’lTY ACIDS
percentage fall in optical density 3 min after the addition of various agonists (adrenaline; adenosine diphosphate; collagen) (14, 15). The concentrations of these agonists and the number of control subjects/patients studied are shown in the appropriate tables. Platelet aggregation studies were completed within 2 h of venepuncture. Platelet counts shown in table 1 were measured in PRP using a Coulter Counter, model D (3). Assay of platelet thromboxane A2 release Platelet release of the prostanoid thromboxane A: (TXA1) was assessed by assaying thromboxane BZ (TXB2), the stable, spontaneous breakdown product of TXA? (3, 7). Briefly, platelet aggregation was induced as described above; the reaction was then stopped (3 min after adding the appropriate agonist) by mixing PRP with ethanol. The extracted sample was then stored at -70 “C until measurement of TXB? concentrations, using a specific radioimmunoassay (3, 7). Liver function tests und serum cholesterol Plasma total bilirubin, aspartate transaminase, alkaline phosphatase and albumin and serum cholesterol were measured using routine methods (SMAC Autoanalyzer, Technicon Instruments, Tarrytown, N.Y., U.S.A.) in current use at The Royal Free Hospital. B. Experiments involving PRP obtained from healthy control subjects and the addition. in vitro, of D-penicillamine. D-penicillamine base was obtained from Sigma (Poole, Dorset, U.K.). PRP was prepared from healthy volunteers as described above. The PRP was then incubated for 2 min at 37 “C with varying concentrations of D-penicillamine dissolved in 0.9% saline. Aggregation was induced by several agonists (see table 7) and the percentage fall in optical density was evaluated 3 min later. Experiments were always run in parallel. one aggregometer channel containing PRP + D-penicillamine, the other PRP + saline control. The concentrations of agonists used (see table 7) were sub-maximal, so as to allow any enhancement of aggregation to be detected (16, 17). The D-penicillamine concentrations selected for the in vitro (5-20 mg/l) experiments were similar to the plasma levels (6-8 mg/l) in those taking therapeutic doses of the drug (18).
PLATELET AGGREGATION
Statistical analysis
IN PRIMARY BILIARY CIRRHOSIS
and
expression
of
results
Results are expressed as median and range. Values in the PBC group taking D-penicillamine were compared with those in the PBC patients not taking this drug, using the unpaired nonparametric Mann-Whitney test (two-tailed) (19). Results in the patients with “early” disease were compared with those obtained in patients with “late” disease, using the same test. In the in vitro experiments, the results obtained with Dpenicillamine present were compared with the appropriate matched control samples using a paired non-parametric Wilcoxon rank sum test (two-tailed) (19). Frequency analysis was carried out using the standard chi-square test with Yates’ correction. Results A. Platelet studies in patients with PBC Platelet counts (Table 1) The platelet count in the PRP of patients with “late” PBC was significantly below that of “early” PBC (p <0.002) or controls (p <0.002). There was no significant difference between D-penicillamine treated and untreated patients, although in the treated group the count was significantly lower than that in controls (p cO.05). Table 1 PRP Platelet Counts (X1()y/l) in PBC Patients and Control Subjects. Platelet counts Median Range
Control subjects PBC + D-penicillamine PBC - no treatment “Early” PBC “Late” PBC All PBC patients For statistical
analysis,
(n (n (n (n (n (n
= = = = = =
12) IO) 18) 10) 18) 28)
326
240-396
217 241 318 193 235
91-31x 91-528 21 l-528 91-332 91-.52X
see text.
Frequency analysis of PRP platelet counts showed that for “early” PBC one out of 9 patients (11%) had platelet counts below 240 x lo’/1 (the lowest count for the control subjects); this was significantly different (p
133 (7 out of 15 patients had platelet counts below 240 x lO’/l) groups were compared. To separate the effect of disease stage from that of treatment with D-penicillamine and vice versa, we examined patient groups which were large enough in size (n = 8 approximately) and which also shared the same characteristics. These were: (a) Patients with “late” PBC - treated (n = 8) and untreated (n = 7): the median (range) PRP platelet count in the former group was 178 (90 - 284) x lo’/1 and in the latter group 200 (90 - 332) X 19’/1 respectively. (b) Patients not treated with D-penicillamine and with “early” PBC (n = 8) and “late” PBC (n = 8): the median (range) PRP platelet count in the former group was 308 (200 - 528) x lo’/1 and in the latter group 200 (90 - 332) x lo’/1 respectively. None of these differences was statistically significant, but they paralleled the changes reported for the larger patient groups described above. Platelet aggregation There was wide scatter in the results. Patients not receiving D-penicillamine (II = 15: 7 “early” PBC) showed aggregation which was not statistically different to that of controls (n = lo), whereas in patients receiving D-penicillamine (n = 10: 2 “early” PBC) platelet aggregation was significantly increased compared to the control group (see Table 2) at low concentrations of collagen and adrenaline, but not ADP. There were no significant differences between treated and untreated PBC patients. Patients with “early” PBC (n = 9; 7 untreated) (Table 3) tended to have platelets which were more aggregable than those with “late” disease (n = 16; 8 treated). This may have been partly due to thrombocytopenia (which was commoner in “late” PBC), since patients with low PRP platelet counts (cl50 x lO’/l) tended to show reduced aggregation patterns, regardless of the stage of the disease. To separate the effect of disease stage from that of treatment with D-penicillamine and vice versa, we examined patient groups which were numerically large enough and which shared the same characteristics as described for PRP counts above. These groups were: (a) Patients with “late” PBC - treated (n = 8) and untreated (n = 7): the platelet aggre-
134
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL FAll-Y AClDS
Table 2 Percentage Platelet Aggregation in Control Subjects and in PBC Patients. Results are Expressed as Median and (Range). Number of Patients = n. Patients with PBC + D-peniciilamine - D- penicillamine
Control Subjects
32* (O-76) n = 10
5 (O-76) n = 16
6 (O-23) n=9
61** (O-80) n=9
29 (O-77) n = 15
n=9
62 (44-80) n=9
55 (23-77)
n = 15
66 (SO-86) n=9
12 (O-76) n=8
0 (O-78) n = 16
3 (O-8) n=9
0.5
59** (O-85) n = 10
20 (O-88) n = 16
22 (7-60) n=9
1.0
68 (38-90) n = 10
49 (20-83) n = 15
69 (60-81) n=9
Aggregating Agent
Adrenaline (pmoI/l) 0.2 0.5
5.0 Collagen (mgfl) 0.15
ADP (pmol/l) 0.5
6 (O-59)
4 (O-64)
16 (O-31)
7 (O-36)
n=8
n=9
n = 10
70 (54-82)
66 (21-91)
72 (60-86)
n = 10
n = 16
n=9
Proportion
of patients with “Early” PBC
30%
43%
Proportion of patients with “Late” PBC
70%
57%
10.0
Statisticat analysis: PBC patients on D-penicillamine
vs controls: *p = 0.02; **p = 0.002. Mann-Whitney test (see text). PBC patients not on Dpenicillamine vs controls and PBC patients on D-peniciilamine vs PBC patients not on D-penicillamine: all comparisons = not significant. Mann Whitney test (see text).
gation responses in these patients paralleled the patterns described for the larger patient groups (which included both “late” and “early” PBC patients), but the only significant (p G.01) difference was with aggregation induced by collagen, 0.5 m$l, where median (range) percentage aggregation was 49 (O-85)% in “late” PBC + D-penicillamine and 6 (O-68)% in “late” PBC with no treatment. (b) Patients with “early” (n = 8) or “late” (n = 8) PBC not receiving treatment: platelet aggregation patterns paralleled those observed with the larger groups of patients (which included both treated and untreated patients). Median (range) aggregation differed significantly with: (i) aggregation induced by collagen 0.5 ms/l. where percentage aggre-
gation in “early” PBC-untreated was 64 (3-88)s and in “late” PBC untreated was 6 (OH%)% (p <0.04). Control subjects showed aggregation of 22 (7-60)0/c in response to the same agonist; this did not differ significantly from the values obtained from either PBC subgroup. Platelet aggregation induced by collagen 1 mg/l also differed significantly (p <0.05), values being 72 (2-83)s in “early” untreated PBC and 43 (20-58)s in “late” untreated PBC. The percentage aggregation in the “late” untreated PBC group was significantly (p
PLATELET
AGGREGATION
Percentage Platelet Aggregation in PBC Patients Classified according to Disease Stage (see text). Result are Expressed as Median and (Range). n = Number of Patients.
Table 3
Aggrqating
agent
Adrenaline 0.2
(pmol/l)
“Early”
PBC
“Late”
9 (O-65) n = 16
0.5
5X’ (10-77) n=X
43 (O-80) n = 15
5.0
69 (27-90) n=Y
57 (13-80) n = 15
9 (O-78) n=9
0 (O-.55) n = I.5
0.5
66++ (5-88) n=X
38 (O-85) n = 14
1.o
74’ (35-90) n=Y
4X (20-84) n = IS
0 (O-60) n=X
0 (O-5Y) n = I4
l(l.0
71 (57-YI) n=Y
63 (21-82) n = 17
Proportion of pts. taking D-penicillamine
33%
47%
ADP 0.5
Table 4 Platelet TXBz Release in PBC Patients Taking and not Taking D-Penicillamine and Control Subjects. Results are Expressed as Median and (Range) of TXBz (t&Ox Platelets) Released. n = Number of Subjects.
PBC
38’ (O-76) fl=Y
Collagen 0. IS
135
IN PRIMARY BILIARY CIRRHOSIS
(ms/l)
Aggregating Agent
Patients Taking D-penicillamine
with PBC Not taking D-penicillamine
Control Subjects
Adrenaline 5 pmol/l
6.6 (1.X-17.5) n=8
5.6 (
6.5 (4-11.6) n=X
Collagen 1 ms/l
9.3 (
5.1 (
8.9 (5.6-15.4) n=H
ADP IO pmol/l
2.0 (
I.0 (
2.2 (
Statistical
Analysis: Controls vs PBC + D-penicillamine, controls vs PBC only and PBC + D-penicillamine vs PBC only: all comparisons = not significant (Mann-Whitney test - see text).
Platelet Thromboxane B3 Release in “Early” and “Late” PBC (see text for definitions). Results are Expressed as Median and (Range) of TXB, (r&O’ platelets) Released. n = Number of Subjects. For Control Values see Table 4. Table 5
(pmol/l)
Stutistical
Analysis: *p <(I.02 “Early” vs “Late” PBC patients. +p <0.05; ++p
Aggregating
agent
“Early”
PBC
“Late”
PBC
Adrenaline 5 fimol/l
5.Y (1.8-17.5) n=h
6.3 (
Collagen I mg/l
6.1 (<1-13.X) n=6
6.0 (
ADP 10 pmol/l
2.3 (
2.3 (
Serum cholesterol concentrations Serum cholesterol levels were similar in the treated patients, 6.4 mmol/l (4.2-8.6) and untreated patients, 6.0 mmol/l (3.4-10.2), and between patients with “early” PBC, 6.4 mmol/i (4.9-7.5) and “late” PBC, 5.8 mmol/l (3.4-10.2). These values were not significantly different from those in the 5.9 mmol/l (3.4-7.0). Patients with controls, cholesterol above 6.5 mmol/t (the upper limit of the reference range) tended to have more enhanced aggregation patterns and TXAl release than patients with serum cholesterol in the normal range, but this difference was not statistically significant.
Liver function tests There was no obvious correlation between the liver function tests (Table 6(a), (b)) and platelet aggregation results; platelet hyperactivity was noted in patients with near normal, as well as with markedly abnormal, liver function tests. Liver function tests in all control subjects were well within the reference range.
TXBZ release (Tables 4 and 5) Aggregationinduced TXB-’ release by platelets tended to parallel aggregability, particularly at low agonist concentrations; but after taking platelet counts into consideration, no statistically significant differences were noted between “early” and “late” or treated and untreated groups or when these groups were compared to controls.
B. Experiments involving the in vitro addition of D-penicillamine base to PRP obtained from healthy control subjects (Table 7) Platelet aggregation (in samples obtained from healthy volunteers) in response to low concentrations of adrenaline and collagen (see table 7) was significantly enhanced by the addition, in
Statistical
Analysis: “Early” vs “Late” PBC; controls (see Table 4) vs “Early” or “Late” PBC. All comparisons = not significant. (Mann-Whitney test - see text).
136
PROSTAGLANDINS
Table 6(a) Liver Function “Late” PBC Patients
Tests in “Early”
and
Patients with “Early” PBC (n = 9)
Patients with “Late” PBC (n = 15)
Alk phosphatase (3.5-130 u/I)
371 (179-856)
556 (275- 1420)
Bilirubin (5- 17 ~mol/l)
IO (3-2X)
29 (g-200)*
Aspartate transaminase (S-40 U/I)
56 (38-96)
&h)**
Albumin (35-50 gil)
31 (38-44)
37 (32-44)
Liver function test (reference range)
Statistical analysis: “Early” PBC vs “Late” Whitney test. * p
PBC-
Mann-
PBC patients taking D-penicillamine (n = Y)
PBC patients not taking D-penicillarnine (n = 15)
Alk phosphatase (35-130 U/l,
514 (‘75-948)
490 (179-1420)
Bilirubin (5-17 ~mol/l)
(8-66)
1x (3-200)
Aspartate transaminase (S-40 U/I)
83 (56
(25-206)
27
Albumin
(35-50 g/l)
(Z-53,
7s 170)
38 (33-44)
Statistical analysis: Taking D-penicillamine vs not taking D-penicillamine (Mann-Whitney test): all comparisons = not significant; n = number of subjects.
vitro, of D-penicillamine (final concentration: 10 mg/l). ADP-induced aggregation, however, was not significantly enhanced. Lower concentrations of D-penicillamine (5 mg/f) did not produce significant enhancement although a trend was present (results not shown). Higher Dpenicillamine concentrations (20 mg/l) produced significant enhancement but the results are not shown since these drug concentrations are well above the therapeutic levels (6-8 m$l) achieved in vivo (18). Patients with Raynaud’s Phenomenon Patients reported to have Raynaud’s phenomenon (n = 2) did not appear to have aggregation or TXA? release patterns which were distinctly different from those in the other patients.
AND ESSENTIAL
FAITY
ACIDS
Table 7 In Vitro Effect of Adding D-Penicillaminc Base to PRP Obtained from Healthy Volunteers. Results are Expressed as Median and (Range) ‘i Aggregation. Aggregating agent
Adrenaline (0.1-1.5 pmol/l) Collagen (0.15-0.4 mg/l) ADP (0.X-2.0 ~mol/l)
Table 6(b) Liver Function Tests in PBC Patients TakingflVot Taking D-Penicillamine Liver function test (reference range)
LEUKOTRIENES
C‘ontrol experimrnr CPRP + .\crlitw) 9 (S-20)
Test experimrrlr (PRP + II~~~~t~iciii~rnliflc,)
61 (.!(I-77) (n = X) 47 (31-S) p <0.1ll (11 = 7) 35 (Z-57) p = not significant p
IO (S-20) 26 (5-Jtj)
*Final concentration of D-penicillamine = IO mg/l PRP Statistical Analysfs: control experiment vs test experiment using Wilcoxon test (see text). p values are shown with the results above. There was no significant enhancement of TXA: release in these experiments (results not shown). However. this may merely reflect the relatively low agonist concentrations whtch arc required to elicit enhancement of aggregation and which also release very \mall amounts of TXA;. n = numhcr of subjects.
Discussion Our findings show that platelet aggregation and TXBz release following the in vitro addition of adrenaline, collagen and ADP in a group of PBC patients not treated with D-penicillamine was similar to that in controls. In contrast, platelets from PBC patients receiving D-penicillamine showed significantly enhanced platelet aggrcgation in response to low concentrations of adrenaline and collagen (but not ADP) when compared with controls. Platelet TXA- release paralleled the aggregation findings but did not achieve statistical significance. Further evidence supporting the view that platelets are activated by D-penicillamine is provided by the findings of the in vitro experiments which showed that D-penicillamine. added to normal platelets, enhanced aggregation in response to low concentrations of adrenaline and collagen. but not following ADP. This pattern was similar to that observed in the cx vivo study involving the PBC patients. There was no correlation between the liver function tests and platelet function. The relatively enhanced aggregation observed in the PBC patients with “early” disease (table 3) is unlikely to be due to D-penicillamine administration. since the proportion of patients receiving this drug was greater in the “late” PBC group. Further-mote, analysis of untreated “early” and “late” PBC groups showed significantly greater
PLATELET AGGREGATION
IN PRIMARY BILIARY CIRRHOSIS
aggregation in the former group. The difference in platelet aggregability between “early” and “late” disease may relate to the significantly lower platelet counts in the latter situation, since thrombocytopenia is associated with diminished aggregation (20). TXA2 release is also influenced in a similar manner when expressed per volume of blood, but not when related to the platelet count (21). Two earlier studies (5, 22) have reported an inhibition of platelet aggregation in PBC. These observations (5, 22) would be broadly comparable to our findings if patients with “late” PBC (and therefore possibly with thrombocytopenia), and not receiving D-penicillamine had been studied since we also showed that such patients had diminished platelet aggregation when compared with control subjects, Unfortunately, our results are not directly comparable with these earlier studies, since disease stage and thrombocytopenia (a major variable in our study) were not considered in one study (5). In the other study (22). platelet counts were standardised but the disease stage was not considered. Furthermore, in this latter study (22), all the aggregation studies were conducted with luminescence reagent present. This reagent may interfere with aggregation responses, hence the recommendation by the manufacturers of the luminescence aggregometer (Coulter Electronics, Luton, Beds, U.K.) used by these authors to also assess aggregation in the absence of these reagents. Both these earlier studies (5, 22) do not appear to have assessed the effect of D-penicillamine treatment. Platelet function in PBC patients may be influenced by several factors which are not present, at least to such an extent. in other types of cirrhosis. For example, secondary hyperlipidaemia and thrombocytopenia have been reported in PBC and in patients receiving D-penicillamine (S-12, 22); PBC may also be associated with Raynaud’s phenomenon (4, 23). These variables are of interest, since secondary hyperlipidaemia and Raynaud’s phenomenon are associated with enhanced platelet aggregation (6, 7). Enhanced platelet aggregation, however, does not appear to contribute significantly to the pathogenesis of Raynaud’s phenomenon in PBC, since platelet function in these patients did not differ markedly from that in the other patients. D-penicillamine treatment is associated with thrombocytopenia but the pathogenesis remains unclear (8-12, 24). One recognised mechanism is bone marrow
137 depression (8-12, 24); however, there have also been reports of thrombotic thrombocytopenic purpura (TTP) associated with D-penicillamine therapy (8-12). Since platelet hyperactivity is thought to play a role in the pathogenesis of TTP (25), it is possible that enhanced aggregation contributes towards the development of thrombocytopenia in patients treated with Dpenicillamine by enhancing platelet consumption, especially in situations where bone marrow production of platelets is impaired. Whether inhibiting platelet function (e.g. with aspirin or dipyridamole) can be of therapeutic benefit in PBC- or D-penicillamine-associated thrombocytopenia remains to be established. To our knowledge, there is no report of increased mortality from vascular disease in PBC. This may result from the fact that mortality in these patients is probably a consequence of progression of liver disease which precedes the more long-term consequences that may result from platelet hyperactivity and mild hypercholesterolaemia. Furthermore, it has been suggested (26) that the lipid profile (elevated HDL) in PBC may in fact be protective against ischaemic heart disease. The variability of results obtained in the present study suggest that several factors influenced platelet function. Some of these factors clearly are the platelet count (low counts diminished aggregation) and treatment with Dpenicillamine (D-penicillamine treatment enhanced aggregation). A larger study would be required to ascertain whether hypercholesterolaemia and the disease stage per se make major contributions and whether other factors not considered here are also relevant. The possible role of platelet-associated immunoglobulins which have recently been reported in patients with PBC (27) must also be considered in future studies assessing platelet function. In view of the present findings and those we reported earlier in patients with alcoholic liver disease (2, 3. 28), it would seem that the first stage in any project assessing prostanoid production in chronic liver disease must be to sample as uniform a patient population as possible. Liver disease should therefore be considered as separate entities depending on aetiology. treatment and disease stage. A brief survey of the literature should reveal to any reader that this requirement does not seem to have been considered in a very large number of studies.
138
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL
FATTY
ACIDS
References I
Owen. J. S. Hutton. R. A.. Day. D. A.. Bruckdorfer. K. R. and McIntyre. N. Platelet lipid composition and platelet aggregation in human liver disease. J Lipid Res. 22: 423-430. 1981 2 Mikhailidis. D. P.. Jenkins. W. J.. Barradas. M. A.. Jeremy. J. Y. and Dandona. P Platelet function defects in chronic alcoholism. Br Med J. 2Y3: 715-71X. 19x6. D. P.. Jeremy. J. Y.. Barradas. M. A.. 3 Mikhailidis, Green. N. and Dandona. P. Effect of ethanol on vascular prostacyclin synthesis, platelet aggregation and platelet thromboxane release. Br Med J. 2X7: 13Y5-14YX. 1983. S. and Scheucr. P. J. The oresentation and 4 Sherlock. diagnosis of 100 patients with primary’biliary cirrhosis N Enel J Med. 2XY: 674-67X. lY73. S. and Aviram. M. s Baruch. Y.. Brook. G.. Eidelman. Increased concentration of high density lipoprotein in plasma and decreased platelet aggregation in primary biliary cirrhosis. Atherosclerosis. 53: 151-62. IYX4. 6 Carvalho, A. C. A.. Colman. R. W. and Lees. R. S. Platelet function in hyperbetalipoprotcinemia. N Engl J Med. 290: 434-43X. 1974. 7 Hutton. R. A.. Mikhailidis. D. P.. Bcrnstcin, R. M.. Hughes, G. R. V. and Dandona. P. Assessment of platelet function in patients with Raynaud’s syndrome. J Clin Pathol. 37: 1X2-187, lYX4. toxicity from pcnicillaminc x Camp, A. V. Haematologic in rheumatoid arthritis. J Rheumatol (Suppl 7). 8: 164-165 19x1 Y Junker. P.. Helin. G., Jensen. B. A.. Oxlund. H. and Lorcnzen. I. D-penicillamine-induced angiopathy in rats. Atherosclerosis lYX2; 45: 17-31 IO Tricc. J. M.. Pinals, R. S. and Plitman, G. I. Thrombotic thromhocytopenic purpura during penicillaminc therapy in rheumatoid arthritis. Arch Intern Med. 143: 14X7-14%+ 198.3 Weiss, A. S.. Markcnson. J. A., Weiss. M. S. and II Kammerer. W. H. Toxicity of D-penicillamine in rheumatoid arthritis. Am J Med. X4: 114-120. lY7X 12 Speth. P. A., Boerbooms, A. M.. Holdrinet, R. S.. Van de Putte. L. B. and Meyer. J. W. Thrombotic thrombocytopcnic purpura associated with D-penicillaminc treatment in rheumatoid arthritis. J Rhcumatol. Y: x12-x13. IYX? 13. Fonseca V, Epstein. 0.. Katrak. A.. Junglee. 0.. Mikhailidis. D. P.. McIntyre. N. and Dandona. P. Serum immunorcactive trypsin and pancreatic lipase in primary biliary cirrhosis. J Clin Pathol, 39: 638%640. IYX6 14. Mikhailidis. D. P.. Mikhailidis, A. M.. Woollard, M. L. and Dandona, P. Protection of prostacyclin-like activity in human plasma a non-enzymatic mechanism Clin Sci. 62: 177-181, 19X2.
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Mikhailidis. D. P.. Hutton. R. A.. Jeremy. J. Y. and Dandona. P. Cooling decreases the efficinecy of prostaglandin inhibitors of platelet aggregation a factor of possible relevance in cold-induced pathology. Microcirculation, 2: 413423. 19X3. Barradas. M. A.. Mikhailidis. A .M.. Mikhailidis. I). P. and Dandona. P. Tine effects of fentluraminc on in vitro platclct aggregation. Br J Clin Pharmacol, 14: 466-46X. IYX2. Mikhailidia, D. I’.. Barradas. M. A.. Mikhailidia, D. I’ Mapnani, H. and Dandona. P. Comparison of the effect of a conventional heparin and a low molecular weight heparinoid on platelet function. Br J Clin Pharmacol. 17: 43-4x. 1YX-l. Bergstrom. R. F.. Kay, D. R.. Harkcom, 7‘. M. and Wagner. J. G. Pcnicillaminc kinetics in normal subjects. Clin Pharmacol Ther. 30: 4044413. 19x1. Altman. D. G.. Gore. S. M.. Gardner. M. J. and Pocock. S. J. Statistical guidelines for contributors to medical journals. Br Med J. 2X6: 14X9-lY3. 1YX.i. Born. G. V. R. and Cross. M. J. The aggregation of blood platelets. J Physiol. IhX: 17X-IY5, lY63. Carter. A. J. and Hanlcy. S. P. The effect ot platclct number and haemocrit on whole blood thromboxane synthesis. Thromh Haemostas. 53: 225-227. lYX5. Ingeberg. S.. Jacohscn. P., Fischer. E., and Bcntscn. K. D. Platelet aggregation and rclcasc of ATP in patients with hepatic cirrhosis. Stand J Gastroenterol. 20: ‘X5~‘XX. IYX.5. Reynolds. T. B.. Dcnsion. E. K.. Franke, Ii. D.. Oihcrman. F. L. and Peters, R. L. Primarv hiliarv cirrhosis with sclcrodcrma. Raynaud’s phcnomcnon and telanriectasia. Am J Med. 50: 3022313. 1971. Thomas. D.. Gallus. A. S., Brooks. P. M Tampi, R.. Gcddes. R. and Hill. W. Thrombokinctic\ in patients with rheumatoid arthritis treated with I>-penicillaminc. Ann Rheum Dis. 43: 40224Oh. IYX4. Byrne\. J. J.. and Moake. J. L. Thrombotic thrombocytopcnic purpura and the haemolytic uraemic svndrome: evolving concepts of pathogcncsis and therapy. In: Clinics in Hacmatol. Vol. IS: Thrombosis and the vcsscl wall. C. N. Chestcrman (Ed.) London: W. B. Saunders Co.. IYX6. pp 413-412 Kaplan, M. M. Primary hiliary cirrhosis. N Engl J Med. 316: 521-52x. IYX7 Bassendinc. M. F.. Collins. J. D.. Stephenson. J.. Saunders, P. and James. 0. F. W. Platelet-associated immunoglobulins in primary biliary cirrhosis: a cause of thromhocytopenia. Gut. 26: lO74-l1)7Y. IYX5. Mikhailidis. D. P.. Jenkins. W. J., Barradaa. M. A.. Jeremy. J. Y. and Dandona, P. Platelet function defects in chronic alcoholism. Br Med J. 2Y.3: 1307. IYXh.