THROMBOSIS @)Pergemon
RESEARCH 16: 517-525 Press Ltd.1979. Printed
in Great Britain 0049-3848/79/1101-0517
#02.00/O
FAMILIAL DEFICIENCY OF A PLASMA FACTOR STIMULATING VASCULAR PROSTACYCLIN ACTIVITY Giuseppe REMUZZI", Donatella MARCHESI', Rocco MISIANI', Giuliano MECCA', Giovanni de GAETANO+ and Maria Benedetta DONATI* "Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo BERGAMO, Italy and 'Istituto di Ricerche Farmacologiche "Mario Negri" - Via Eritrea, 62 20157 MILAN, Italy (Received 24.5.1979; in revised form 10.7.1979. Accepted by Editor H. Stormorken) ABSTRACT We previously reported successful treatment with plasma in patients with thrombotic microangiopathy and defective vascular PG12 activity and suggested that a defect in a plasma factor stimulating PGI synthesis might be implicated in the pathogenesis. We report ere that in one of these patients the plasma defect was still detectable one year after clinical remission (without recurrence). Two of this patient's four children had a similar, though less severe, plasma defect. The proposita is a 54-year-old woman with a clinical and laboratory picture of haemolytic uraemic syndrome. Unlike normal plasma, the patient's plasma had a low capacity to stimulate PG12 production by rat aortic rings (previously washed until their endogenous PGI2 activity was exhausted). After plasma treatment the patient's plasma behaved normally in this respect, but again appeared deficient at out-patient follow-up. PGI2 stimulating activity was normal in two daughters but consistently low (20-50% of control) in both patient's sons. None of them had any history or clinical signs of microangiopathicdisorders. Detection of this
2
plasma defect in apparently hea1th.vsubjects and in patients who have recovered from thrombotic microangiopathyepisodes could have clinical implications.
INTRODUCTION The haemolytic uraemic syndrome (H.U.S) is a severe disorder characterized by thrombocytopenia, microangiopathic haemolytic anaemia, renal impairement; fluctuating neurological signs may also be present,. Histologically this syndrome is characterized by microthrombi occluding terminal arterioles and capillaries. This pathological event is possibly the consequence of uncontrolled intravascular platelet aggregation and fibrin deposition. The close clinical and pathological correlation between H.U.S. and _--_-----Key words: Haemolytic uraemic syndrome - Prostacyclin - Plasma factors -Congenital disorders 517
518
VASCULAR
PROSTACYCLIN
STIMULATOR
vo1.16,No.3/4
thrombotic thrombocytopenic purpura (T.T.P.) has often been noted and there is no method at present which clearly differentiates H.U.S. from T.T.P. (1). The occurrence together of some of the abnormalities of H.U.S. or T.T.P. has also been reported in patientswith malignant hypertension, post partum renal failure and systemic diseases such as disseminated lupus erythematosus and scleroderma (1). We have recently suggested (2,3) that patients with H.U.S. and allied conditions lack a factor normally present in human plasma (4,5) stimulating prostacyclin (PGI ), a potent endogenous inhibitor of platelet aggregation (6). Defective PiI could favour widespread formation of platelet thrombi in the microcircula 2. ion. In two patients vascular PG12 activity, absent during relapse, was restored after infusion of normal plasma (7). We report here that in one of these patients the plasma defect was still detectable one year after clinical remission (without recurrence). In addition two sons of the patient have a similar - though less severe - defect. Both to date enjoy apparent good health. MATERIALS AND METHODS Blood samples were collected by clean venipuncture into plastic tubes. Platelet-free plasma was obtained by differential centrifugation of titrated venous blood (8). One volume of 0.126 M trisodium citrate was used for 9 volumes of normal blood; this proportion was appropriately adjusted according to the haematocrit value, in order to ensure comparable anticoagulant concentrations in all tested plasma samples. Assessment of Capacity of Plasma to Stimulate Vascular Prostacyclin Activity ___--__-__---_-_ ____ ____________________~~~~~~~~~~~~~~~~~~~~ -----------The capacity of plasma to stimulate generation of prostacyclin activity was tested using 'exhausted' rat aortic rings (5,9,10). For this purpose rings (4 mg wet weight) of rat thoracic aorta were incubated with 200 ul isotonic saline (10 min at room temperature); the supernatant solution was then removed, replaced by fresh isotonic saline and the incubation continued for further 10 min. This procedure was repeated until no antiaggregating (prostacyclin) activity could be detected in the supernatant solution. The 'exhausted' rings were then incubated with 400 ~1 plasma (30 min at 37°C); in these conditions plasma from normal subjects stimulated rings to generate again prostacyclin activity (5,lO). The capacity of test plasma to stimulate prostacyclin activity was expressed as percent of the activity displayed by plasma from control tested simultaneously. Prostacyclin activity of rat aortic rings was evaluated as platelet aggregation inhibitory activity according to the bioassay proposed by Moncada et al. (9) in an Elvi 840 aggregometer (Elvi Logos, Milan) (11). The vascular specimens were incubated in 200 ul tris buffer (0.15 mol/l, pH 9.0) for 10 min at room temperature. 100 ~1 samples of the supernatant fluid from the incubation mixture were then incubated for 1 min at 37°C in 250 ~1 platelet-rich plasma (300 x log platelets/l) obtained by centrifugation of titrated venous blood from a normal volunteer (8,ll). Platelet aggregation was started with 1 pmol/l adenosine5'-diphosphate disodium salt (Sigma, Milan). Platelet aggregation inhibitory activity was expressed as ng/mg wet tissue by extrapolation from a doseresponse curve obtained on the same PPP with highly purified PG12 (obtained through the courtesy of Dr. J. Pike, Upjohn, Kalamazoo, Michigan, USA). PG12 activity was characterized according to current criteria (4,6); the activity was heat labile and was unstable at room temperature and at neutral pH but was stable for at least 24 hours at 4'C or at basic pH; its generation was inhibited by acetylsalicylic acid, or indomethacin, although neither drug inhibited the activity once it had been generated in the system; moreover it
Vol.~6,No.3/4
VASCULAR PROSTACYCLIN STIMULATOR
5’19
was potentiated by phosphodiesterase inhibitors such as theophylline; it was distinguished from PGD2 by the fact that it also strongly inhibited rat platelet aggregation; contamination with significant ADP-ase activity could also be excluded on the basis of incubation experiments with ADP. Complete inhibition of vascular antiaggregating activity was obtained following incubation with an antiserum which antagonizedauthentic PG12 (the antiserum was a gift from Drs. M.J. Silver and 3.8. Smith, Cardeza Foundation, Philadelphia, Pa., USA). CASE REPORTS AND LABORATORY RESULTS The proposita is a 54-year-old woman admitted to the Nephrology Division of our Hospital for acute renal failure. The diagnosis of haemolytic uraemic syndrome was made on the basis of thrombocytopenia, microangiopathic haemolytic anaemia, neurological derangement and bioptic features of renal cortical necrosis. After plasma exchange and infusion, marked improvement was seen in clinical conditions and laboratory test results. One month later, the patient was discharged (7). Table I summarizes laboratory parameters recorded at intervals since admission. Platelets, red cells and leucocyte counts were or gradually became normal. A similar trend was observed for haemoglobin, haenmtocrit, total protein, total transferrin, and bilirubin. However, signs of sustained., though modest, haemolysis were consistently present, as indicated by low haptoglobin levels, increased reticulocyte count and high normal values of sideraemia (and low normal values of unsaturated transferrin). At admission the patient's plasma had a very low capacity to stimulate the production of PGI activity by vascular tissues. After plasma treatment, the patient‘s plasma ehaved normally in this respect. However, a marked deficiency of PG12 stimulating capacity was again found in patient's plasma at subsequent out-patient follow-up (Table II) and a congenital origin of the defect was suspected. The patient's family was therefore studied. The genealogical tree is represented in Fig. 1. None of the subjects examined had any history or clinical signs of microangiopathic disorders. The main laboratory findings in the proposita's four children are assembled in Table I. Most of the parameters studied were within the normal range except for the haptoglobin level, which was low normal in all four subjects and sideraemia, which was high normal in two (E.B. and D.B.) or increased in one (G.B.). The capacity of plasma to stimulate vascular PG12 activity was normal in one daughter (A.B.),borderline in the other (E.B.) but consistently reduced in both sons (D.B. and G.B.) (Fig. 1 and Table II).
2
_DISCUSSION Recurrent microangiopathic disorders have been reported in the same subject or in members of the same family (12,13). In some cases these disorders have been suggested to be hereditary or of familial origin. We have previously described a defect in a plasma activity regulating platelet/vesselwall interaction in patients with thrombotic microangiopathy who recovered after treatment with plasma (2,3). We report here that such a defect persisted in one of these patients during a one-year follow-up, while the patient was asymptomatic with a compensated'haemolytic state. This would be in agreement with previous descriptions of recurrent episodes in the same individual (1,12). Byrnes and Khurana (14) reported remission in one patient with thrombotic thrombocytopenic purpura after the administration of plasma. More recently, Upshaw (15)
240
3.60
1.20
23
15
Total transferrin (g/l)
Unsat.transferrin (g/l)
Total bilirubin (mg/l)
Conjugated bilirubin (ms/l)
Schistocytes
6.0
13
20
1.30
3.50
220
(0.10
53
numerous numerous
Serum creatinine (mg/dl) 11.0
co.10
Serum iron (pg/dl)
54
Haptoglobin (g/l)
Albumin (X)
6.7
1.40
1.50
few
4.5
15
few
3.1
8
13
3.00
3.20 20
180
0.60
55
7.2
3
34
12.2
7.0
3.8
170
RESEARCH 16: 517-525 Press Ltd.1979. Printed
in Great Britain 0049-3848/79/1101-0517
#02.00/O
FAMILIAL DEFICIENCY OF A PLASMA FACTOR STIMULATING VASCULAR PROSTACYCLIN ACTIVITY Giuseppe REMUZZI", Donatella MARCHESI', Rocco MISIANI', Giuliano MECCA', Giovanni de GAETANO+ and Maria Benedetta DONATI* "Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo BERGAMO, Italy and 'Istituto di Ricerche Farmacologiche "Mario Negri" - Via Eritrea, 62 20157 MILAN, Italy (Received 24.5.1979; in revised form 10.7.1979. Accepted by Editor H. Stormorken) ABSTRACT We previously reported successful treatment with plasma in patients with thrombotic microangiopathy and defective vascular PG12 activity and suggested that a defect in a plasma factor stimulating PGI synthesis might be implicated in the pathogenesis. We report ere that in one of these patients the plasma defect was still detectable one year after clinical remission (without recurrence). Two of this patient's four children had a similar, though less severe, plasma defect. The proposita is a 54-year-old woman with a clinical and laboratory picture of haemolytic uraemic syndrome. Unlike normal plasma, the patient's plasma had a low capacity to stimulate PG12 production by rat aortic rings (previously washed until their endogenous PGI2 activity was exhausted). After plasma treatment the patient's plasma behaved normally in this respect, but again appeared deficient at out-patient follow-up. PGI2 stimulating activity was normal in two daughters but consistently low (20-50% of control) in both patient's sons. None of them had any history or clinical signs of microangiopathicdisorders. Detection of this
2
plasma defect in apparently hea1th.vsubjects and in patients who have recovered from thrombotic microangiopathyepisodes could have clinical implications.
INTRODUCTION The haemolytic uraemic syndrome (H.U.S) is a severe disorder characterized by thrombocytopenia, microangiopathic haemolytic anaemia, renal impairement; fluctuating neurological signs may also be present,. Histologically this syndrome is characterized by microthrombi occluding terminal arterioles and capillaries. This pathological event is possibly the consequence of uncontrolled intravascular platelet aggregation and fibrin deposition. The close clinical and pathological correlation between H.U.S. and _--_-----Key words: Haemolytic uraemic syndrome - Prostacyclin - Plasma factors -Congenital disorders 517
518
VASCULAR
PROSTACYCLIN
STIMULATOR
vo1.16,No.3/4
thrombotic thrombocytopenic purpura (T.T.P.) has often been noted and there is no method at present which clearly differentiates H.U.S. from T.T.P. (1). The occurrence together of some of the abnormalities of H.U.S. or T.T.P. has also been reported in patientswith malignant hypertension, post partum renal failure and systemic diseases such as disseminated lupus erythematosus and scleroderma (1). We have recently suggested (2,3) that patients with H.U.S. and allied conditions lack a factor normally present in human plasma (4,5) stimulating prostacyclin (PGI ), a potent endogenous inhibitor of platelet aggregation (6). Defective PiI could favour widespread formation of platelet thrombi in the microcircula 2. ion. In two patients vascular PG12 activity, absent during relapse, was restored after infusion of normal plasma (7). We report here that in one of these patients the plasma defect was still detectable one year after clinical remission (without recurrence). In addition two sons of the patient have a similar - though less severe - defect. Both to date enjoy apparent good health. MATERIALS AND METHODS Blood samples were collected by clean venipuncture into plastic tubes. Platelet-free plasma was obtained by differential centrifugation of titrated venous blood (8). One volume of 0.126 M trisodium citrate was used for 9 volumes of normal blood; this proportion was appropriately adjusted according to the haematocrit value, in order to ensure comparable anticoagulant concentrations in all tested plasma samples. Assessment of Capacity of Plasma to Stimulate Vascular Prostacyclin Activity ___--__-__---_-_ ____ ____________________~~~~~~~~~~~~~~~~~~~~ -----------The capacity of plasma to stimulate generation of prostacyclin activity was tested using 'exhausted' rat aortic rings (5,9,10). For this purpose rings (4 mg wet weight) of rat thoracic aorta were incubated with 200 ul isotonic saline (10 min at room temperature); the supernatant solution was then removed, replaced by fresh isotonic saline and the incubation continued for further 10 min. This procedure was repeated until no antiaggregating (prostacyclin) activity could be detected in the supernatant solution. The 'exhausted' rings were then incubated with 400 ~1 plasma (30 min at 37°C); in these conditions plasma from normal subjects stimulated rings to generate again prostacyclin activity (5,lO). The capacity of test plasma to stimulate prostacyclin activity was expressed as percent of the activity displayed by plasma from control tested simultaneously. Prostacyclin activity of rat aortic rings was evaluated as platelet aggregation inhibitory activity according to the bioassay proposed by Moncada et al. (9) in an Elvi 840 aggregometer (Elvi Logos, Milan) (11). The vascular specimens were incubated in 200 ul tris buffer (0.15 mol/l, pH 9.0) for 10 min at room temperature. 100 ~1 samples of the supernatant fluid from the incubation mixture were then incubated for 1 min at 37°C in 250 ~1 platelet-rich plasma (300 x log platelets/l) obtained by centrifugation of titrated venous blood from a normal volunteer (8,ll). Platelet aggregation was started with 1 pmol/l adenosine5'-diphosphate disodium salt (Sigma, Milan). Platelet aggregation inhibitory activity was expressed as ng/mg wet tissue by extrapolation from a doseresponse curve obtained on the same PPP with highly purified PG12 (obtained through the courtesy of Dr. J. Pike, Upjohn, Kalamazoo, Michigan, USA). PG12 activity was characterized according to current criteria (4,6); the activity was heat labile and was unstable at room temperature and at neutral pH but was stable for at least 24 hours at 4'C or at basic pH; its generation was inhibited by acetylsalicylic acid, or indomethacin, although neither drug inhibited the activity once it had been generated in the system; moreover it
Vol.~6,No.3/4
VASCULAR PROSTACYCLIN STIMULATOR
5’19
was potentiated by phosphodiesterase inhibitors such as theophylline; it was distinguished from PGD2 by the fact that it also strongly inhibited rat platelet aggregation; contamination with significant ADP-ase activity could also be excluded on the basis of incubation experiments with ADP. Complete inhibition of vascular antiaggregating activity was obtained following incubation with an antiserum which antagonizedauthentic PG12 (the antiserum was a gift from Drs. M.J. Silver and 3.8. Smith, Cardeza Foundation, Philadelphia, Pa., USA). CASE REPORTS AND LABORATORY RESULTS The proposita is a 54-year-old woman admitted to the Nephrology Division of our Hospital for acute renal failure. The diagnosis of haemolytic uraemic syndrome was made on the basis of thrombocytopenia, microangiopathic haemolytic anaemia, neurological derangement and bioptic features of renal cortical necrosis. After plasma exchange and infusion, marked improvement was seen in clinical conditions and laboratory test results. One month later, the patient was discharged (7). Table I summarizes laboratory parameters recorded at intervals since admission. Platelets, red cells and leucocyte counts were or gradually became normal. A similar trend was observed for haemoglobin, haenmtocrit, total protein, total transferrin, and bilirubin. However, signs of sustained., though modest, haemolysis were consistently present, as indicated by low haptoglobin levels, increased reticulocyte count and high normal values of sideraemia (and low normal values of unsaturated transferrin). At admission the patient's plasma had a very low capacity to stimulate the production of PGI activity by vascular tissues. After plasma treatment, the patient‘s plasma ehaved normally in this respect. However, a marked deficiency of PG12 stimulating capacity was again found in patient's plasma at subsequent out-patient follow-up (Table II) and a congenital origin of the defect was suspected. The patient's family was therefore studied. The genealogical tree is represented in Fig. 1. None of the subjects examined had any history or clinical signs of microangiopathic disorders. The main laboratory findings in the proposita's four children are assembled in Table I. Most of the parameters studied were within the normal range except for the haptoglobin level, which was low normal in all four subjects and sideraemia, which was high normal in two (E.B. and D.B.) or increased in one (G.B.). The capacity of plasma to stimulate vascular PG12 activity was normal in one daughter (A.B.),borderline in the other (E.B.) but consistently reduced in both sons (D.B. and G.B.) (Fig. 1 and Table II).
2
_DISCUSSION Recurrent microangiopathic disorders have been reported in the same subject or in members of the same family (12,13). In some cases these disorders have been suggested to be hereditary or of familial origin. We have previously described a defect in a plasma activity regulating platelet/vesselwall interaction in patients with thrombotic microangiopathy who recovered after treatment with plasma (2,3). We report here that such a defect persisted in one of these patients during a one-year follow-up, while the patient was asymptomatic with a compensated'haemolytic state. This would be in agreement with previous descriptions of recurrent episodes in the same individual (1,12). Byrnes and Khurana (14) reported remission in one patient with thrombotic thrombocytopenic purpura after the administration of plasma. More recently, Upshaw (15)
240
3.60
1.20
23
15
Total transferrin (g/l)
Unsat.transferrin (g/l)
Total bilirubin (mg/l)
Conjugated bilirubin (ms/l)
Schistocytes
6.0
13
20
1.30
3.50
220
(0.10
53
numerous numerous
Serum creatinine (mg/dl) 11.0
co.10
Serum iron (pg/dl)
54
Haptoglobin (g/l)
Albumin (X)
6.7
1.40
1.50
few
4.5
15
few
3.1
8
13
3.00
3.20 20
180
0.60
55
7.2
3
34
12.2
7.0
3.8
170
