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Total and free protein S in nephrotic syndrome
THROMBOSIS RESEARCH 49; 37-42, 1988 0049-3848188 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All rights reserved.
BRIEF
COMMUNICATION
TOTAL AND FREE PROTEIN S IN NEPHROTIC SYNDROME
M.GOUAULT-HEILMANN", T.CADELHA-PARENTE*, M.LEVENT*, L.INTRATOR*, G.ROSTOKER** and G.LAGRLJE** *Laboratoire Central d'Hematologie-Immunologie et **Service de Nephrologie HOPITAL HENRI MONDOR -94010 CRETEIL, FRANCE (Received 9.7.1987; Accepted in revised form 15.10.1987 by Editor M.C. Boffa)
INTRODUCTION Thrombosis is one of the main complications in the course of nephrotic syndrome (NS) with a mean prevalence value of 30 % (1). Several coagulation abnormalities have been described in NS, including increased levels of procoagulant factors such as factors I, v, VIII, VII, X, elevated platelet count, increased platelet aggregation and/or decreased levels of coagulation inhibitors such as Antithrombin III (1 and references herein). Protein C is a vitamin K-dependent protein which, in its active form, regulates blood clot formation by inhibiting the clotting cascade at the level of factors V and VIII (2). Protein S, another vitamin K-dependent protein acts as the cofactor of the activated protein C. Both protein C and protein S inherited deficiencies are associated with recurrent venous thrombosis (3-Q). Protein S exists in plasma in two forms : fourty per cent of the total plasma protein S (TPS) are free in plasma, whereas sixty per cent are bound, in a reversible complex, to C4b-binding protein (C4BP), a regulatory protein of the complement system (5). Only free protein S (FPS) acts as the cofactor of activated protein C (6). Increased level of total protein S antigen has been reported in patients with nephrotic syndrome (7-8) whereas some authors (9) have suggested that some acquired free protein S deficiency, related to a shift from free to bound protein S, could explain the thrombotic tendency currently observed in these patients. The aim of the present study was to evaluate systematically the two forms of protein S and C4BP levels in patients with nephrotic syndrome. In addition urinary excretion of protein S was searched for. PATIENTS 32 consecutive patients (3 children, 29 adults, 18 males, 14 females, age ranging from 4 to 77 years) with chronic nephrotic syndrome of various severity were studied. Etiology of the NS was : minimal change glomerulopathy (n=l4), membranous glomerulonephritis (n=8), Shonlein-Henoch purpura (n=l), focal glomerulosclerosis (n=7), proliferative glomerulonephritis (n=2). Two patients with severe NS exhibited renal vein thrombosis. None of these patients were receiving antithrombotic drug at the time of the study.
37
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
Control group consisted in 29 healthy adult volunteers (15 males, 14 females, age ranging from 18 to 46 years) free of any medication. METHODS
Platelet poor plasma (PPP) was obtained from blood collected in sodium citrate 0.12qM (1 volume for 9 volumes of blood) and stored at -60°C until tested. A normal pooled plasma was prepared by mixing equal parts of PPP from 12 healthy subjects (6 males, 6 females) and stored at -60-C. 24 hours urinE samples were obtained from 10 patients and concentrated using Minicon B15 (Amicon Corp., Lexington, MA). Protein S antigen assay
Total (TPS:Ag) and free protein S (FPS:Ag) were measured inRthe same plasma samples by rocket electrophoresis (Asseraplate Protein S from D.Stago, Asnieres, France). Protein S complexed to C4-BP was precipitated in melting ice for 30 minutes by PEG 8000 from D.Stago (50 1.11 of a 25 % PEG solution for 300 pl of plasma), according to Comp et al (10). FPS:Ag was then assayed in the supernatant. Standard curves for TPS:Ag and FPS:Ag were obtained from normal pooled plasma treated in the same way. FPS in the normal pooled plasma was found to be 47+2.7 % (ranging from 42 to 53 %) of the TPS, with an interassay variation of 5.8.% (14 determinations). TPS:Ag and FPS:Ag in control's and patients' plasma were expressed by reference to the normal pooled plasma included in each plate and containing lU/ml of TPS:Ag and lU/ml of FPS:Ag respectively. Protein S in concentrapd urine was assayed from D.Stago) more by a "sandwich" ELISA method (Asserachrom Protein S sensitive than the Laurell's technique. The results were expressed in U/day, according to the urine's concentration factor and the 24 hours urine volume. C4BP assay was performed by rocket electrophoresis using an anti C4-BP immunserum kindly provided by J.Amiral (D.Stago). Results were expressed in percent of the normal pooled plasma level. Protein C antigen was assayed by an ELISA method (Asserachrom Protein CR, D.Stago) ; In addition, Antithrombin III biological activity was measured by amidolytic method (Stachrom AT III, D.Stago). Albumin level, proteinuria, proteinuria selectivity index, cholesterol and triglycerides levels were measured by routine techniques. Statistical
Analysis
Since values were not normally distributed, comparison of means used Mann et Whitney U test ; correlations were assessed by Spearman correlation test. RRSULTS Protein C was found increased in patients with nephrotic syndrome (1.52+0.50 U/ml) in comparison with control group (1.15~0.20 U/ml), p
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
TOTAL PROTEN 2.0 r
s ANTEN
(U/ml)
p
c------1,
.I.
.
I
:. :
:
:
:
i
. .
i
A
FREEPmrElNs r
________
A'. AA-p
1;
'AAAAAA i
:
h
.
?? ? ?? .iAi ?
7
T-p
figure 1
*
?? ??
1.0 ,_ . . .
39
:
;
AAi
A’
! ‘0-i--
i
Total protein S antigen (U/ml) in nephrotic patients and in control group (horizontal bars, mean+sd of values).
(U/ml) _
p
“““,
r-P
A A
:
.;._I_ i. 1
Free protein S antigen (U/ml) in nephrotic patients and in control group (horizontal bars, meanzsd of values).
8 8
1‘0
p8tlmtr
oontrd8
P
cGehanoFmteh(%O r--pcoJ)ol_____,
AA 200
.
Figure 3 @I-binding protein (I) in nephrotic patients and in control group (horizontal bars, mean ?? sd of values).
NEPHROTIC SYNDROME AND PROTEIN S
40
Vol. 49, No. 1
FPS:Ag was significantly decreased in male nephrotic patients (1.03+0.41 U/ml vs 1.38~0.25 U/ml, p < 0.01) whereas no difference was observed for females (Fig.2). C4BP was increased in nedhrotic patients (Fig.3) and the difference between patients (135236 %) and control group (103~26 %) is statistically significant (p(O.001). No difference was observed between males and females in patients' or control group. Urinary excretion of FPS:Ag was searched for in 10 proteinuric patients. Traces of FPS:Ag were found in 9/10 patients, including the two patients with very low level of FPS:Ag, with an excretion rate ranged from 0.02 to 2.5 U/24 hours. A weak positive correlation was found between TPS:Ag and FPS:Ag in control group (R=O.65, p(O.001) and in patients'group (R=0.39, ~(0.05). No correlation was found between FPS:Ag and C4BP neither in control group, nor in nephrotic patients. A weak negative correlation (r=-0.45, p< 0.05) was found in nephrotic patients but not in control group between FPS:Ag and the ratio C4BP/TPS:Ag (Fig.4). Figure 4
FPS:Ag
20
U/ml 8
Correlation between FPS:Ag and C4-BP/TPS ratio in nephrotic patients.
r = -0.45
t 1.5
11)
OB
!
I
I
a6
0.8
I 111
I 12
C4-0PltPs:AQ
I
1.4
I
I
1.0
12
I
ratI
No correlation was found between TPS:Ag, FPS:Ag or C4BP levels and albumin level, proteinuria, proteinuria selectivity index, cholesterol level, triglycerides levels, Protein C antigen level, AT III biological activity, whereas Protein C level was correlated negatively to albumin level and positively to proteinuria. events at the time Two patients with severe NS exhibited thrombo-embolic of the study and blood samples could be obtained before starting antithrombotic therapy. One of them had a very low level of FPS:Ag (0.35 U/ml) associated with a low level of AT III (0.48 U/ml) and a high level of protein C (1.32 U/ml). The other one had normal level of FPS:Ag (1.0 U/ml) and AT III (0.91 U/ml) but a borderline protein C level (0.63 U/ml).
DISCUSSION The high incidence of thromboembolic complications occurring during the course of nephrotic syndrome (1) and the clear relationship between thromboembolic disease and protein C (3) or S deficiencies (4) prompted us to study this natural inhibitory system in patients with nephrotic syndrome. Two points make the interpretation of the results rather complex : firstly protein S exists in plasma in two forms and only free protein S is active as
Vol.
49.
No.
1
NEPHROTIC SYNDROME AND PROTEIN S
41
a cofactor for the anticoagulant effect of activated protein C (6) ; secondly our data concerning the control group exhibit significant differences between males and females, so data must be considered according to the patients'sex. A statistically significant increase in Protein C antigen level was found in proteinuric patients and this increase is correlated with the severity of the nephrotic syndrome as previously reported (7,8,11). A statistically significant difference was found in TPS:Ag and FPS:Ag levels between males and females in control group (males having higher values), and these results are in agreement with those recently published by Boerger and ~011. (12). TPS:Ag and FPS:Ag levels in the normal female population are similar to those reported by Huisveld and al (13) in normal women. The differences between men and women we observed as others (12) could be explained by the sex-related physiological hormonal status. TPS:Ag was found elevated only in females with NS whereas FPS:Ag was found to be lower than normal values only in male nephrotic patients. Elevated levels of TPS:Ag is probably a consequence of accelerated.synthesis as suggested for other coagulation factors, but we do not have any explanation for the sex-related difference we observed. No correlation could be found between TPS:Ag level and Albumin, proteinuria, cholesterol, triglicerides levels. These results are in disagreement with previous reports (7,8). Given the multiplicity of factors affecting plasma protein concentration in nephrotic syndrome, it is not surprising to observe discrepancies in reported results as it has still been observed for other coagulation proteins (1). The decreased level of FPS:Ag in nephrotic patients cannot be explained by an urinary loss of FPS:Ag since only traces of FPS:Ag were found in concentrated urine of q/10 patients tested, despite using a sensitive ELISA method for measuring FPS:Ag. It is obvious that the molecular weight of proteins is not the unique factor responsible for their urinary excretion in proteinuric patients. In fact, other proteins with similar molecular weight such as AT III or protein C (7) have been found in urine of nephrotic patients. C&BP was found elevated in nephrotic patients, without sex-related difference, but no correlation could be found between C4BP level and FPS:Ag level. If some shift from free to bound form of protein S is an attractive hypothesis to explain low levels of FPS:Ag despite normal or high level of TPS:Ag in nephrotic syndrome (91, this shift is apparently not related only to the C4BP level. More likely the FPS:Ag level is partly dependent of both TPS:Ag level and C4BP level since a weak negative correlation has been found in this study, between FPS:Ag and the ratio C4BP/TPS:Ag. Increased hepatic synthesis rate varies probably from one protein to another one and in individual patients throughout different phases of their disease. Concerning TPS and C4BP, this can lead, in some patients, to an acquired FPS deficiency, which surprisingly is not correlated to the severity of the disease. Such an hypothesis could explain why females (with high levels of C4BP and TPS:Ag) do not exhibit very low levels of FPS:Ag. Two patients in our series exhibited thromboembolic complications. Both had a severe nephrotic syndrome ; one of them had low levels of AT III and FPS:Ag, the other one had only a borderline Protein C level. It is obvious that one coagulation abnormality cannot explain by itself the thrombotic tendency currently observed in nephrotic patients. The increased, variable, synthetic rate of coagulation proteins, the urinary loss of some of them (which is not related only to the molecular weight), a possible shift from active to inactive forms (such as free to bound protein S) in addition to alterations of rheological conditions make it difficult to predict the occurrence of thromboembolic complications in nephrotic patients.
42
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
REFERENCES 1. LLACH F. Hypercoagulability, renal vein thrombosis and other thrombotic complications of nephrotic syndrome. Kidney International, 28, 429-439, 1985.
2. CLOUSE L.H., COMP P.C. The regulation of Hemostasis : the protein C system. 1298-1303, 1986. N.Engl.J.Med., s, 3. GRIFFIN J.H., EVATT B., ZIMMERMAN T.S., KLEISS A.J. Deficiency of protein C in congenital thrombotic disease. J.Clin.Invest., 68, 1370-1373, 1981.
4. COMP P.C., ESMON C.T. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N.Engl.J.Med, 311, 1525-1528, 1984. 5. DAHLBACK B., STENFLO J. High molecular weight complex in human plasma
between vitamin K-dependent protein S and complement component C4-binding protein. Proc.Natl.Acad.Sci.USA, 2, 2512-2516, 1981. 6. COMP P-C., NIXON R.R., COOPER M.R., ESMON C.T. Familial protein S deficiency is associated with recurrent thrombosis. J.Clin.Invest., 2, 2082-2088. 1984. 7. ROSTOKER C., COUAULT-HEILMANN M., LEVENT M., ROBEVA R., LANG P., LAGRUE G.
High level of protein C and protein S in nephrotic syndrome. Nephron, 46, 220-221, 1987 (letter). 8. COSIO F., HARKER C., BATARD M., BRANDT J., GRIFFIN J.H. Plasma concentra-
tion of the natural anticoagulants protein C and protein S in patients with proteinuria. J.Lab.Clin.Med, 106, 218-222, 1985. 9. COMP P.C., VACANO S., D'ANGELO A., THURNEAU C., KAUFMAN C., ESMON C.T.
Acquired protein S deficiency occurs in pregnancy, the nephrotic syndrome and acute systemic lupus erythematosus. Blood, 66, suppl 1, Abstract 1279, 1985.
10. COMP P.C., DORAY D., PATTON D., ESMON C.T. An abnormal distribution of protein S occurs in functional protein S deficiency. Blood, 67, 504-508, 1986. 11. SALA N., OLIVER N., ESTIVILL X., MORENO R., FELEZ J., RUTLANT M. Plasmatic and urinary protein C 1eveIs in nephrotic syndrome. Thromb. Haemostas., 54, 900 (letter), 1985. 12. BOERGER L.M., MORRIS P.C., THURNAU G.R., ESMON C.T., COMP P.C. Oral contraceptives and gender affect protein S status. Blood, 69, 692-694, 1987. 13. HUISVELD I-A., HOSPERS J.E.H.? STARKENBOURG A.E., ERICH W.B.M., BOUMA B.N. Oral contraceptives reduce total protein S but not free protein S. Thromb. &, 45, 109-114, 1987.
BRIEF
COMMUNICATION
TOTAL AND FREE PROTEIN S IN NEPHROTIC SYNDROME
M.GOUAULT-HEILMANN", T.CADELHA-PARENTE*, M.LEVENT*, L.INTRATOR*, G.ROSTOKER** and G.LAGRLJE** *Laboratoire Central d'Hematologie-Immunologie et **Service de Nephrologie HOPITAL HENRI MONDOR -94010 CRETEIL, FRANCE (Received 9.7.1987; Accepted in revised form 15.10.1987 by Editor M.C. Boffa)
INTRODUCTION Thrombosis is one of the main complications in the course of nephrotic syndrome (NS) with a mean prevalence value of 30 % (1). Several coagulation abnormalities have been described in NS, including increased levels of procoagulant factors such as factors I, v, VIII, VII, X, elevated platelet count, increased platelet aggregation and/or decreased levels of coagulation inhibitors such as Antithrombin III (1 and references herein). Protein C is a vitamin K-dependent protein which, in its active form, regulates blood clot formation by inhibiting the clotting cascade at the level of factors V and VIII (2). Protein S, another vitamin K-dependent protein acts as the cofactor of the activated protein C. Both protein C and protein S inherited deficiencies are associated with recurrent venous thrombosis (3-Q). Protein S exists in plasma in two forms : fourty per cent of the total plasma protein S (TPS) are free in plasma, whereas sixty per cent are bound, in a reversible complex, to C4b-binding protein (C4BP), a regulatory protein of the complement system (5). Only free protein S (FPS) acts as the cofactor of activated protein C (6). Increased level of total protein S antigen has been reported in patients with nephrotic syndrome (7-8) whereas some authors (9) have suggested that some acquired free protein S deficiency, related to a shift from free to bound protein S, could explain the thrombotic tendency currently observed in these patients. The aim of the present study was to evaluate systematically the two forms of protein S and C4BP levels in patients with nephrotic syndrome. In addition urinary excretion of protein S was searched for. PATIENTS 32 consecutive patients (3 children, 29 adults, 18 males, 14 females, age ranging from 4 to 77 years) with chronic nephrotic syndrome of various severity were studied. Etiology of the NS was : minimal change glomerulopathy (n=l4), membranous glomerulonephritis (n=8), Shonlein-Henoch purpura (n=l), focal glomerulosclerosis (n=7), proliferative glomerulonephritis (n=2). Two patients with severe NS exhibited renal vein thrombosis. None of these patients were receiving antithrombotic drug at the time of the study.
37
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
Control group consisted in 29 healthy adult volunteers (15 males, 14 females, age ranging from 18 to 46 years) free of any medication. METHODS
Platelet poor plasma (PPP) was obtained from blood collected in sodium citrate 0.12qM (1 volume for 9 volumes of blood) and stored at -60°C until tested. A normal pooled plasma was prepared by mixing equal parts of PPP from 12 healthy subjects (6 males, 6 females) and stored at -60-C. 24 hours urinE samples were obtained from 10 patients and concentrated using Minicon B15 (Amicon Corp., Lexington, MA). Protein S antigen assay
Total (TPS:Ag) and free protein S (FPS:Ag) were measured inRthe same plasma samples by rocket electrophoresis (Asseraplate Protein S from D.Stago, Asnieres, France). Protein S complexed to C4-BP was precipitated in melting ice for 30 minutes by PEG 8000 from D.Stago (50 1.11 of a 25 % PEG solution for 300 pl of plasma), according to Comp et al (10). FPS:Ag was then assayed in the supernatant. Standard curves for TPS:Ag and FPS:Ag were obtained from normal pooled plasma treated in the same way. FPS in the normal pooled plasma was found to be 47+2.7 % (ranging from 42 to 53 %) of the TPS, with an interassay variation of 5.8.% (14 determinations). TPS:Ag and FPS:Ag in control's and patients' plasma were expressed by reference to the normal pooled plasma included in each plate and containing lU/ml of TPS:Ag and lU/ml of FPS:Ag respectively. Protein S in concentrapd urine was assayed from D.Stago) more by a "sandwich" ELISA method (Asserachrom Protein S sensitive than the Laurell's technique. The results were expressed in U/day, according to the urine's concentration factor and the 24 hours urine volume. C4BP assay was performed by rocket electrophoresis using an anti C4-BP immunserum kindly provided by J.Amiral (D.Stago). Results were expressed in percent of the normal pooled plasma level. Protein C antigen was assayed by an ELISA method (Asserachrom Protein CR, D.Stago) ; In addition, Antithrombin III biological activity was measured by amidolytic method (Stachrom AT III, D.Stago). Albumin level, proteinuria, proteinuria selectivity index, cholesterol and triglycerides levels were measured by routine techniques. Statistical
Analysis
Since values were not normally distributed, comparison of means used Mann et Whitney U test ; correlations were assessed by Spearman correlation test. RRSULTS Protein C was found increased in patients with nephrotic syndrome (1.52+0.50 U/ml) in comparison with control group (1.15~0.20 U/ml), p
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
TOTAL PROTEN 2.0 r
s ANTEN
(U/ml)
p
c------1,
.I.
.
I
:. :
:
:
:
i
. .
i
A
FREEPmrElNs r
________
A'. AA-p
1;
'AAAAAA i
:
h
.
?? ? ?? .iAi ?
7
T-p
figure 1
*
?? ??
1.0 ,_ . . .
39
:
;
AAi
A’
! ‘0-i--
i
Total protein S antigen (U/ml) in nephrotic patients and in control group (horizontal bars, mean+sd of values).
(U/ml) _
p
“““,
r-P
A A
:
.;._I_ i. 1
Free protein S antigen (U/ml) in nephrotic patients and in control group (horizontal bars, meanzsd of values).
8 8
1‘0
p8tlmtr
oontrd8
P
cGehanoFmteh(%O r--pcoJ)ol_____,
AA 200
.
Figure 3 @I-binding protein (I) in nephrotic patients and in control group (horizontal bars, mean ?? sd of values).
NEPHROTIC SYNDROME AND PROTEIN S
40
Vol. 49, No. 1
FPS:Ag was significantly decreased in male nephrotic patients (1.03+0.41 U/ml vs 1.38~0.25 U/ml, p < 0.01) whereas no difference was observed for females (Fig.2). C4BP was increased in nedhrotic patients (Fig.3) and the difference between patients (135236 %) and control group (103~26 %) is statistically significant (p(O.001). No difference was observed between males and females in patients' or control group. Urinary excretion of FPS:Ag was searched for in 10 proteinuric patients. Traces of FPS:Ag were found in 9/10 patients, including the two patients with very low level of FPS:Ag, with an excretion rate ranged from 0.02 to 2.5 U/24 hours. A weak positive correlation was found between TPS:Ag and FPS:Ag in control group (R=O.65, p(O.001) and in patients'group (R=0.39, ~(0.05). No correlation was found between FPS:Ag and C4BP neither in control group, nor in nephrotic patients. A weak negative correlation (r=-0.45, p< 0.05) was found in nephrotic patients but not in control group between FPS:Ag and the ratio C4BP/TPS:Ag (Fig.4). Figure 4
FPS:Ag
20
U/ml 8
Correlation between FPS:Ag and C4-BP/TPS ratio in nephrotic patients.
r = -0.45
t 1.5
11)
OB
!
I
I
a6
0.8
I 111
I 12
C4-0PltPs:AQ
I
1.4
I
I
1.0
12
I
ratI
No correlation was found between TPS:Ag, FPS:Ag or C4BP levels and albumin level, proteinuria, proteinuria selectivity index, cholesterol level, triglycerides levels, Protein C antigen level, AT III biological activity, whereas Protein C level was correlated negatively to albumin level and positively to proteinuria. events at the time Two patients with severe NS exhibited thrombo-embolic of the study and blood samples could be obtained before starting antithrombotic therapy. One of them had a very low level of FPS:Ag (0.35 U/ml) associated with a low level of AT III (0.48 U/ml) and a high level of protein C (1.32 U/ml). The other one had normal level of FPS:Ag (1.0 U/ml) and AT III (0.91 U/ml) but a borderline protein C level (0.63 U/ml).
DISCUSSION The high incidence of thromboembolic complications occurring during the course of nephrotic syndrome (1) and the clear relationship between thromboembolic disease and protein C (3) or S deficiencies (4) prompted us to study this natural inhibitory system in patients with nephrotic syndrome. Two points make the interpretation of the results rather complex : firstly protein S exists in plasma in two forms and only free protein S is active as
Vol.
49.
No.
1
NEPHROTIC SYNDROME AND PROTEIN S
41
a cofactor for the anticoagulant effect of activated protein C (6) ; secondly our data concerning the control group exhibit significant differences between males and females, so data must be considered according to the patients'sex. A statistically significant increase in Protein C antigen level was found in proteinuric patients and this increase is correlated with the severity of the nephrotic syndrome as previously reported (7,8,11). A statistically significant difference was found in TPS:Ag and FPS:Ag levels between males and females in control group (males having higher values), and these results are in agreement with those recently published by Boerger and ~011. (12). TPS:Ag and FPS:Ag levels in the normal female population are similar to those reported by Huisveld and al (13) in normal women. The differences between men and women we observed as others (12) could be explained by the sex-related physiological hormonal status. TPS:Ag was found elevated only in females with NS whereas FPS:Ag was found to be lower than normal values only in male nephrotic patients. Elevated levels of TPS:Ag is probably a consequence of accelerated.synthesis as suggested for other coagulation factors, but we do not have any explanation for the sex-related difference we observed. No correlation could be found between TPS:Ag level and Albumin, proteinuria, cholesterol, triglicerides levels. These results are in disagreement with previous reports (7,8). Given the multiplicity of factors affecting plasma protein concentration in nephrotic syndrome, it is not surprising to observe discrepancies in reported results as it has still been observed for other coagulation proteins (1). The decreased level of FPS:Ag in nephrotic patients cannot be explained by an urinary loss of FPS:Ag since only traces of FPS:Ag were found in concentrated urine of q/10 patients tested, despite using a sensitive ELISA method for measuring FPS:Ag. It is obvious that the molecular weight of proteins is not the unique factor responsible for their urinary excretion in proteinuric patients. In fact, other proteins with similar molecular weight such as AT III or protein C (7) have been found in urine of nephrotic patients. C&BP was found elevated in nephrotic patients, without sex-related difference, but no correlation could be found between C4BP level and FPS:Ag level. If some shift from free to bound form of protein S is an attractive hypothesis to explain low levels of FPS:Ag despite normal or high level of TPS:Ag in nephrotic syndrome (91, this shift is apparently not related only to the C4BP level. More likely the FPS:Ag level is partly dependent of both TPS:Ag level and C4BP level since a weak negative correlation has been found in this study, between FPS:Ag and the ratio C4BP/TPS:Ag. Increased hepatic synthesis rate varies probably from one protein to another one and in individual patients throughout different phases of their disease. Concerning TPS and C4BP, this can lead, in some patients, to an acquired FPS deficiency, which surprisingly is not correlated to the severity of the disease. Such an hypothesis could explain why females (with high levels of C4BP and TPS:Ag) do not exhibit very low levels of FPS:Ag. Two patients in our series exhibited thromboembolic complications. Both had a severe nephrotic syndrome ; one of them had low levels of AT III and FPS:Ag, the other one had only a borderline Protein C level. It is obvious that one coagulation abnormality cannot explain by itself the thrombotic tendency currently observed in nephrotic patients. The increased, variable, synthetic rate of coagulation proteins, the urinary loss of some of them (which is not related only to the molecular weight), a possible shift from active to inactive forms (such as free to bound protein S) in addition to alterations of rheological conditions make it difficult to predict the occurrence of thromboembolic complications in nephrotic patients.
42
NEPHROTIC SYNDROME AND PROTEIN S
Vol. 49, No. 1
REFERENCES 1. LLACH F. Hypercoagulability, renal vein thrombosis and other thrombotic complications of nephrotic syndrome. Kidney International, 28, 429-439, 1985.
2. CLOUSE L.H., COMP P.C. The regulation of Hemostasis : the protein C system. 1298-1303, 1986. N.Engl.J.Med., s, 3. GRIFFIN J.H., EVATT B., ZIMMERMAN T.S., KLEISS A.J. Deficiency of protein C in congenital thrombotic disease. J.Clin.Invest., 68, 1370-1373, 1981.
4. COMP P.C., ESMON C.T. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N.Engl.J.Med, 311, 1525-1528, 1984. 5. DAHLBACK B., STENFLO J. High molecular weight complex in human plasma
between vitamin K-dependent protein S and complement component C4-binding protein. Proc.Natl.Acad.Sci.USA, 2, 2512-2516, 1981. 6. COMP P-C., NIXON R.R., COOPER M.R., ESMON C.T. Familial protein S deficiency is associated with recurrent thrombosis. J.Clin.Invest., 2, 2082-2088. 1984. 7. ROSTOKER C., COUAULT-HEILMANN M., LEVENT M., ROBEVA R., LANG P., LAGRUE G.
High level of protein C and protein S in nephrotic syndrome. Nephron, 46, 220-221, 1987 (letter). 8. COSIO F., HARKER C., BATARD M., BRANDT J., GRIFFIN J.H. Plasma concentra-
tion of the natural anticoagulants protein C and protein S in patients with proteinuria. J.Lab.Clin.Med, 106, 218-222, 1985. 9. COMP P.C., VACANO S., D'ANGELO A., THURNEAU C., KAUFMAN C., ESMON C.T.
Acquired protein S deficiency occurs in pregnancy, the nephrotic syndrome and acute systemic lupus erythematosus. Blood, 66, suppl 1, Abstract 1279, 1985.
10. COMP P.C., DORAY D., PATTON D., ESMON C.T. An abnormal distribution of protein S occurs in functional protein S deficiency. Blood, 67, 504-508, 1986. 11. SALA N., OLIVER N., ESTIVILL X., MORENO R., FELEZ J., RUTLANT M. Plasmatic and urinary protein C 1eveIs in nephrotic syndrome. Thromb. Haemostas., 54, 900 (letter), 1985. 12. BOERGER L.M., MORRIS P.C., THURNAU G.R., ESMON C.T., COMP P.C. Oral contraceptives and gender affect protein S status. Blood, 69, 692-694, 1987. 13. HUISVELD I-A., HOSPERS J.E.H.? STARKENBOURG A.E., ERICH W.B.M., BOUMA B.N. Oral contraceptives reduce total protein S but not free protein S. Thromb. &, 45, 109-114, 1987.