Acta Med Scand 205: 535-539, 1979
Acquired Factor XI1 Deficiency in a Patient with Nephrotic Syndrome E. A. van Royen, J . E. G. d e Boer, J. M . Wilmink, C. S. P. Jenkins and Jan W . t e n Cate From the Divisions of Haemostasis and Nephrology, University Hospital Wilhelmina Gasihuis. Amsterdam, The Netherlands
ABSTRACT. A patient with nephrotic syndrome and an acquired factor XI1 deficiency associated with a factor XII-like procoagulant activity in the urine was investigated. The urinary protein with prw coagulant activity was isolated and comparative investigations revealed similar properties to plasma factor MI. It is suggested that the acquired coagulation defect may result from an insufficient biosynthetic capacity to compensate for the loss of factor XI1 in the urine. Key words: factor XII, nephrotic syndrome.
Acta Med Scand 205: 535, 1979. Deficiencies of coagulation factors are a potential risk of bleeding after kidney biopsy. Acquired factor IX and factor XI1 deficiencies have been reported in nephrotic syndrome (3, 4, 5, 11). We describe a patient suffering from nephrotic syndrome with an acquired factor XI1 deficiency associated with a factor XII-like procoagulant activity in the urine. Results from studies on the protein with procoagulant activity isolated from t h e urine supported previous findings in which factor XI1 deficiency could b e accounted for, a t least in part, by loss in the urine. MATERIALS Glassware was siliconized with a 1 % silicone solution (Siliclad, Clay Adams, Parsippany, New Yersey, USA). No further reagents were used to inhibit contact activation of factor X11. Urine from the patient was collected in siliconized bottles and stored at -30°C.
Fine Chemicals, Uppsala, Sweden), and eluted fractions were collected at 4°C. DEAE Sephadex was equilibrated in 0.01 M sodium phosphate, 0.001 M EDTA, pH 8.0, and packed in a siliconized column (diameter 5 cm, height 38 cm). A linear sodium chloride gradient was applied to the column with an elution rate of 1.5 ml/min; two buffer reservoirs were filled, one with 700 ml 0.01 M sodium phosphate, 0.001 M EDTA, pH 8.0, and the other with the same buffer containing 0.75 M sodium chloride. Sephadex G I 0 0 was equilibrated with 0.01 M sodium potassium phosphate, 0.154 M sodium chloride, pH 7.5, and packed in a siliconized column (diameter 2.5 cm, height 90 cm). The equilibration buffer was also used as eluant with an elution rate of 1.0-1.2 rnl/min. For the determination of the apparent molecular weight of the protein with factor XII-like procoagulant activity, gel filtration was performed using Sephadex G I 0 0 (column diameter 1.5 cm, height 80 cm), with 0.01 M sodium phosphate buffer, pH 7.5, as eluant. Coagulation assays Factor XI1 activity was determined by a one-stage clotting assay, using human factor XIIdeficient plasma (Dade Reagents, Merz & Dade, Bern, Switzerland). In the assay, 0.1 ml diluted urine (1 : 10, Michaelis buffer, pH 7.4) was incubated with 0.1 ml factor XII-deficient plasma and 0.1 ml of a mixture containing 4 mg kaolin and 0.2 mg phospholipid/ml. After incubation for 7 min at 37"C, 0.1 ml 0.033 M CaCI, was added and the clotting time recorded. Values are expressed in units (U),1 U representing the procoagulant activity of I ml of normal human pool plasma (assayed in a 1 : 10 dilution). Other coagulation factors were estimated by one-stage clotting assays, using human deficient substrate plasma as described previously (13). Antithrombin I11 was assayed by the method of Kahle et al. (7), using the chromogenic substrate Chromozyma TH on an AKES (Automated Kinetic Enzyme and Substrate Analyzer, Vitatron, Dieren, The Netherlands).
METHODS Gel filtration Gel filtration was performed at room temperature, using DEAE Sephadex A-50 and Sephadex (3-100 (Pharmacia
Abbreviations: BP=blood pressure, BUN=blood urea nitrogen, MW=molecular weight. Actci Med Scand 205
536
E . A . van Royen et al.
Table I. Huemostatic data before und after therapy Method
Before therapy
Bleeding time (min) Platelet count ( x 10s//n Prothrombin time (sec) Partial thromboplastin ti me (sec) Fibrinogen (g/l) Factor I1 (U) Factor V (U) Factor VII (U) Factor X (U) Factor VlII (U) Factor IX (U) Factor XI (U) Factor XI1 (U) Antithrombin 111 (U)
1.5 245 11.3 120 12.56 I .38 1.70 1.70 1.30 1.70 0.67 0.64 0.07 0.32
After therapy
180 11.2 45 5.90
-
1.58 1.40 1.80 0.64 1.10
CASE REPORT A 23-year old man was admitted to the Juliana Hospital, Amsterdam, suffering from progressive oedema and proteinuria of more than 30 glday. Serum protein content was 30 g/l, albumin 17 g/l with a marked lipaemia. Blood urea nitrogen (BUN) and creatinine were within the normal range. Investigations revealed that heavy metal intoxica-
Normal value 1-7 150-350 11.5 35-63 1.75-3.50 0.90-1.50 0.80-1.50 0.70-1.60 0.80-1.40 0.50-1 30 0.50-1 3 0 0.40-1.60 0.40-1.20 0.80-1.20
tion, amyloidosis, bilharziosis and tuberculosis could be ruled out. A kidney biopsy was scheduled and prebioptic screening of haemostatic functions revealed a marked prolongation of the kaolin-cephalin clotting time. At this time the patient was transferred to the Wilhelmina Gasthuis for further investigation. Physical examination on admission revealed a blood pressure (SP) of 150/ 100 mmHg and a pulse rate of 68/min. His body weight
+--? \
\
\ \
\ \ \
[
YCLdP;iOSPHAMIGF 5 0 3
FRllSlbllDE
160
120
80
85 -0
PRED'IISONE
JUNE 1976
JULV
Fig. 1 . Plasma factor XI1 activity, plasma albumin and proteinuria in the course of therapy.
60140
160
AUG
SEPT
OCT
'
MARCH
I977
Acquired factor XII deficiency EiS4
2.0
Els4
537
0
1.0.
0 0
: a,
1.0
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was 65.8 kg and height 1.64 m. Facial oedema, pleural fluid, ascites and extensive pitting oedema of the legs were observed. Laboratory investigations of urine samples revealed that the sediment contained many hyalin casts with some leucocytes; the protein concentration was 12 g/I (albumin 67.4%, a,-globulin 9.6%, a,-globulin 5.1 %, @globulin 11.9%, y-globulin 6.0%). Fractional clearances of IgA and a,-macroglobulin, assuming a I00 9% clearance of transfernin, were calculated to be 28.8 and 1.7%, respectively, indicating non-selective proteinuria (6). Haematological screening revealed: ESR 123 mm, Hb 8.1 mM, Hct 0.38 1/1, leucocytes 16.3x109/1 with a normal differential cell count. Serum electrolytes, BUN and creatinine were normal, while total lipids (20 g/l), cholesterol (17.1 mM) and triglycerides (9.5 mM) were increased. Serum proteins were decreased: albumin 4.6 g/l, a,-globulin 1.2 g/l, a,-globulin 17.1 g/l p- and y-globulin 7.2 g/l. Liver enzymes and complement factors Clq, C3, C4 and C5 were normal. Data from haemostatic investigations are given in Table I . After infusion of 900 ml of fresh plasma, the recovery of factor XI1 was calculated to be 38% of the administered dose. The plasma half-life of factor XI1 procoagulant activity, which was estimated by linear regression analysis of the factor XI1 procoagulant activities after infusion, was 15 hours (normal value ca. 60). A kidney biopsy was performed without correction of the factor XI1 deficiency and no haemorrhagic complications were observed. Light microscopy of the glomeruli revealed no cellular infiltration or proliferation and a normal basement membrane with no depositions, while the tubuli were found to contain protein. Immunofluorescence revealed no depositions of immunoglobulins or complement. A diagnosis of nephrotic syndrome due to minimal lesions glomerulonephritis was made. Diuretic therapy with frusemide and spironolactone was instigated. On this therapy, the oedema slowly disappeared and the body weight decreased by 25 kg. As proteinuria was not abaited, prednisolone was administered in a daily dose of 60 mg, which decreased the excretion of protein from 25 g to 6 g/day. Cyclophosphamide, administered in a daily dose of 100 mg, decreased pro-
Fig. 2. Elution profile of urinary factor XI1 following Sephadex G I 0 0 recycling chromatography. Lefr, the elution pattern after the first chromatography cycle; righr (representing the second cycle), both the protein profile and the factor XI1 clotting activity.
teinuria further to less than 1.5 glday. Plasma protein and factor XI1 procoagulant activity increased concurrently in the course of therapy (Fig. I ) . After withdrawal of all therapy (April, 1977), proteinuria remained at a level of f I .5 g/day and the following coagulation values, all within the normal range, were found: factor IX 1.4 U , factor XI I .8 U, factor XI1 0.64 U, antithrombin 111 I . 1 U. RESULTS Urine (protein concentration 12 g/l), when plasma factor XI1 procoagulant activity w a s 0.07-0.08 U and proteinuria exceeded 25 g/day, contained n o factor II/V/VII/X activity, and 0. I 1 U factor VIII, 0.01 U factor IX, 0.01 U factor XI and 0.17 U factor XII-like activity. No such clotting activity could be demonstrated in normal human urine. Incubation with kaolin significantly increased t h e procoagulant activity of the urine in the assay of factor XII. Heating of t h e urine a t 100°C for 10 min resulted in complete loss of the procoagulant activity. Factor XII-like procoagulant activity w a s concentrated by dialysis of the urine o n a collodion membrane under negative pressure. T h e procoagulant activity w a s almost completely absorbed by celite a n d following elution of t h e celite with 0.007 M NH,OH, pH 9.7, factor XII-like procoagulant activity w a s found t o b e associated with the eluate. Attempts were made t o isolate the urinary protein with factor XII-like procoagulant activity. The patient’s urine stored a t -30°C served as starting material. A cryoprecipitate, which w a s found t o have factor VIII procoagulant activity, was formed during thawing a t
4°C. After removal of the cryoprecipitate, ammonium sulphate was added t o the supernatant and the Actu Med Scand 205
538
E . A . vun Royen et ul.
ty was eluted at a volume corresponding to an apparent M w of 82000 daltons (Fig. 3). The fractional clearance of factor XI1 relative to the transferrin clearance was 57%. Assuming an M w of 82000 daltons for factor XII, the data obtained are in close agreement with the curve for the fractional clearance of IgA and a,-macroglobulin (Fig. 4). This suggests that there was no preferential loss of factor XI1 compared to other proteins.
K a v '-OI
i1
cytochrome c
DISCUSSION
1o4
mol. weight
lo5
F i g . 3. Determination of the apparent MW of urinary Factor XI1 by a plot of elution volume versus log M W .
factor XII-like procoagulant activity was found to be associated with the precipitate formed at 25'SO 9% saturation. The precipitate was sedimented and dissolved in 0.154 M sodium chloride. Following dialysis against 0.154 M sodium chloride, the dialysate was applied to a DEAE Sephadex column. Factor XII-like procoagulant activity was eluted at an ionic strength of 0. l5&0. 165. Those fractions containing factor XII-like procoagulant activity were pooled and concentrated by reprecipitation with ammonium sulphate (60% saturation), dialyzed against 0.154 M sodium chloride and applied to a Sephadex G-100 column. A recycling procedure of the eluant from this column resulted in a better separation of the proteins. Factor XII-like procoagulant activity eluted just before the second protein peak (Fig. 2 ) . Several batches of urinary factor XII-like procoagulant activity were prepared by this method. After concentration by negative pressure dialysis, I 1 preparations were pooled and submitted to rechromatography on Sephadex G-100. Fractions containing factor XII-like procoagulant activity were pooled, concentrated and used to determine the apparent molecular weight (MW) on a Sephadex G-100 column. The column was equilibrated with transferrin (MW 77000), bovine serum albumin (MW 67000), ovalbumin (MW 45000), cytochrome L' (MW 125'00). The void volume was determined using dextran blue and the total volume of the gel bed by phenol. Factor XII-like procoagulant activiActa M r d Sciind 205
Coagulation and haemostasis investigations on patients with nephrotic syndrome have revealed disparate results. Increase in platelet count, fibrinogen level and factor VIIl procoagulant activity have been observed in association with this syndrome (8, 10). However, coagulation factor deficiencies have also been observed in some patients. A deficiency of antithrombin I11 in nephrotic syndrome resulting in a thrombotic tendency has been described by Kaufmann et al. (9), while Handley and Lawrence (4) described a haemorrhagic diathesis associated with decreased levels of factor IX. Factor XI1 deficiency is not, in general, associated with a bleeding tendency but does result in a prolongation of the kaolin-cephalin clotting time and has also
100
50
20
PM
I
I
10
I
5
1
I
lo6 mol.weight
Fig. 4 . Fractional clearance of factor XII.
Acquired factor XII deficiency
been reported to occur in nephrotic syndrome (3, 5 ) . We present a patient with low levels of factor XI1 and antithrombin 111 on whom a kidney biopsy was performed without haemorrhagic complications, although the factor XI1 deficiency had not been corrected. Decreased factor XI1 procoagulant activity in association with a markedly shortened plasma half-life together with a severe proteinuria were indicative of factor XI1 loss in the urine. In the course of therapy, the low factor XI1 and antithrombin Ill levels were normalized parallel with the increase in plasma albumin. Antithrombin Ill antigen and a procoagulant activity which have been found to induce antibodies against human factor IX in rabbits have been found in the urine of patients with nephrotic syndrome (9, 11). Green et al. ( 3 ) described a patient with low levels of factors IX and XII. They demonstrated factor XI1-like procoagulant activity in the urine which showed the same elution profile as plasma factor XI1 when filtered through Sephadex G-100. However, no attempts were made to isolate the active protein. Factor XII-like procoagulant activity was also demonstrated in the urine of our patient. The factor XII-like material was labile to heat, could be absorbed to and eluted from celite and was activated by kaolin. In addition, the elution profile' after filtration through DEAE Sephadex was similar to that of plasma factor XI1 ( 2 ) . Following further purification, the active molecule was found to have an apparent MW of 82000 daltons, which is in agreement with quoted values for plasma factor XI1 (12). A proper explanation for the deficiency of antithrombin I11 and of factor XI1 in this patient is not yet apparent. The M W s of antithrombin Ill and factors 11, VII, X and XI1 are within the range of 40000-80000 daltons; therefore, MW cannot be the sole determinant of the selective loss of antithrombin Ill and factor XII. Differences in biosynthetic capacity as well as in molecular charge ( I ) of the various clotting factors may determine their plasma level in proteinuria. In our patient, the loss of factor XI1 protein, also revealed by the markedly shortened half-life, appeared to be proportional to that of other proteins such as IgA, a,-macroglobulin, since the plot of log MW against the fractional clearance of factor XI1 fitted the curve obtained for transferrin, IgA and a,-macroglobulin.
539
These findings strongly suggest that the factor XI1 deficiency in this patient resulted from an insufficient biosynthetic capacity of this protein, relative to other clotting factors, to compensate for the loss in the urine. A similar explanation may account for the deficiency of antithrombin I11 and the plasma factor VIII procoagulant activity of 1.7 U which is low for a patient with nephrotic syndrome, especially in view of the high plasma fibrinogen level. REFERENCES 1 . Brenner, B. M., Hostetter, T. H. & Humes, H. D.:
2.
3.
4. 5.
6.
7.
8.
9.
10.
II.
12.
13.
Molecular basis of proteinuria of glomerular origin. N Engl J Med 298: 826, 1978. Cochrane, C. G. & Wuepper, K. D.: The first component of the kinin forming system in human and rabbit plasma. Its relationship to clotting factor XI1 (Hageman factor). J Exp Med 134: 986, 1971. Green, D., Arruda, J., Honig, G. & Muercke, R. C.: Urinary loss of clotting factors due to hereditary membranous glomemlopathy. Am J Clin Pathol 65: 376, 1976. Handley, D. A. & Lawrence, J. R.: Factor IX deficiency in the nephrotic syndrome. Lancet I : 1079, 1967. Honig, G. R. & Indley, A.: Deficiency of Hageman factor (factor XII) in patients with the nephrotic syndrome. J Pediatr 78: 633, 1971. Joachim, G. R . , Cameron, J . S., Schwartz, M. & Becker, E. L.: Selectivity of protein excretion in patients with the nephrotic syndrome. J Clin Invest 43~2332,1964. Kahle, L. H., Schipper, H. G., Jenkins, C. S. P. & ten Cate, J. W.: Antithrombin-111. I. Evaluation of an automated antithrombin 111 method. Thromb Res 12: 1003, 1978. Kanfer, A., Kleinknecht, D., Broyer, M. & Josso, E.: Coagulation studies in 45 cases of nephrotic syndrome without uremia. Thromb Diath Haemorrh (Stuttg) 24: 562, 1970. Kaufmann, R. H., de Graeff, J., Brutel de la Rivikre, G. & van Es, L. A,: Unilateral renal vein thrombosis and nephrotic syndrome. Report of a case with protein selectivity and antithrombin-111 clearance studies. Am J Med 60: 1048, 1976. Kendall, A. G., Lohmann, R. C. & Dossetor, J. B.: Nephrotic syndrome a hypercoagulable state. Arch Intern Med 127: 1021, 1971. Natelson, E. A., Lynch, E. C., Hettig, R. A. & Alfrey, C . P.: Acquired factor IX deficiency in the nephrotic syndrome. Ann Intern Med 73: 373, 1970. Revak, S. D., Cochrane, C. G., Johnston, A. R. & Hugli, T. E.: Structural changes accompanying enzymatic activation of human Hageman factor. J Clin Invest 54: 619, 1974. van Royen, E. A,, Treffers, P. E. & ten Cate, J. W.: Hypertonic saline induced abortion as pathophysiologic model of low grade intravascular coagulation. Scand J Haematol 13: 166, 1974. Actu Med Scund 205
Acquired Factor XI1 Deficiency in a Patient with Nephrotic Syndrome E. A. van Royen, J . E. G. d e Boer, J. M . Wilmink, C. S. P. Jenkins and Jan W . t e n Cate From the Divisions of Haemostasis and Nephrology, University Hospital Wilhelmina Gasihuis. Amsterdam, The Netherlands
ABSTRACT. A patient with nephrotic syndrome and an acquired factor XI1 deficiency associated with a factor XII-like procoagulant activity in the urine was investigated. The urinary protein with prw coagulant activity was isolated and comparative investigations revealed similar properties to plasma factor MI. It is suggested that the acquired coagulation defect may result from an insufficient biosynthetic capacity to compensate for the loss of factor XI1 in the urine. Key words: factor XII, nephrotic syndrome.
Acta Med Scand 205: 535, 1979. Deficiencies of coagulation factors are a potential risk of bleeding after kidney biopsy. Acquired factor IX and factor XI1 deficiencies have been reported in nephrotic syndrome (3, 4, 5, 11). We describe a patient suffering from nephrotic syndrome with an acquired factor XI1 deficiency associated with a factor XII-like procoagulant activity in the urine. Results from studies on the protein with procoagulant activity isolated from t h e urine supported previous findings in which factor XI1 deficiency could b e accounted for, a t least in part, by loss in the urine. MATERIALS Glassware was siliconized with a 1 % silicone solution (Siliclad, Clay Adams, Parsippany, New Yersey, USA). No further reagents were used to inhibit contact activation of factor X11. Urine from the patient was collected in siliconized bottles and stored at -30°C.
Fine Chemicals, Uppsala, Sweden), and eluted fractions were collected at 4°C. DEAE Sephadex was equilibrated in 0.01 M sodium phosphate, 0.001 M EDTA, pH 8.0, and packed in a siliconized column (diameter 5 cm, height 38 cm). A linear sodium chloride gradient was applied to the column with an elution rate of 1.5 ml/min; two buffer reservoirs were filled, one with 700 ml 0.01 M sodium phosphate, 0.001 M EDTA, pH 8.0, and the other with the same buffer containing 0.75 M sodium chloride. Sephadex G I 0 0 was equilibrated with 0.01 M sodium potassium phosphate, 0.154 M sodium chloride, pH 7.5, and packed in a siliconized column (diameter 2.5 cm, height 90 cm). The equilibration buffer was also used as eluant with an elution rate of 1.0-1.2 rnl/min. For the determination of the apparent molecular weight of the protein with factor XII-like procoagulant activity, gel filtration was performed using Sephadex G I 0 0 (column diameter 1.5 cm, height 80 cm), with 0.01 M sodium phosphate buffer, pH 7.5, as eluant. Coagulation assays Factor XI1 activity was determined by a one-stage clotting assay, using human factor XIIdeficient plasma (Dade Reagents, Merz & Dade, Bern, Switzerland). In the assay, 0.1 ml diluted urine (1 : 10, Michaelis buffer, pH 7.4) was incubated with 0.1 ml factor XII-deficient plasma and 0.1 ml of a mixture containing 4 mg kaolin and 0.2 mg phospholipid/ml. After incubation for 7 min at 37"C, 0.1 ml 0.033 M CaCI, was added and the clotting time recorded. Values are expressed in units (U),1 U representing the procoagulant activity of I ml of normal human pool plasma (assayed in a 1 : 10 dilution). Other coagulation factors were estimated by one-stage clotting assays, using human deficient substrate plasma as described previously (13). Antithrombin I11 was assayed by the method of Kahle et al. (7), using the chromogenic substrate Chromozyma TH on an AKES (Automated Kinetic Enzyme and Substrate Analyzer, Vitatron, Dieren, The Netherlands).
METHODS Gel filtration Gel filtration was performed at room temperature, using DEAE Sephadex A-50 and Sephadex (3-100 (Pharmacia
Abbreviations: BP=blood pressure, BUN=blood urea nitrogen, MW=molecular weight. Actci Med Scand 205
536
E . A . van Royen et al.
Table I. Huemostatic data before und after therapy Method
Before therapy
Bleeding time (min) Platelet count ( x 10s//n Prothrombin time (sec) Partial thromboplastin ti me (sec) Fibrinogen (g/l) Factor I1 (U) Factor V (U) Factor VII (U) Factor X (U) Factor VlII (U) Factor IX (U) Factor XI (U) Factor XI1 (U) Antithrombin 111 (U)
1.5 245 11.3 120 12.56 I .38 1.70 1.70 1.30 1.70 0.67 0.64 0.07 0.32
After therapy
180 11.2 45 5.90
-
1.58 1.40 1.80 0.64 1.10
CASE REPORT A 23-year old man was admitted to the Juliana Hospital, Amsterdam, suffering from progressive oedema and proteinuria of more than 30 glday. Serum protein content was 30 g/l, albumin 17 g/l with a marked lipaemia. Blood urea nitrogen (BUN) and creatinine were within the normal range. Investigations revealed that heavy metal intoxica-
Normal value 1-7 150-350 11.5 35-63 1.75-3.50 0.90-1.50 0.80-1.50 0.70-1.60 0.80-1.40 0.50-1 30 0.50-1 3 0 0.40-1.60 0.40-1.20 0.80-1.20
tion, amyloidosis, bilharziosis and tuberculosis could be ruled out. A kidney biopsy was scheduled and prebioptic screening of haemostatic functions revealed a marked prolongation of the kaolin-cephalin clotting time. At this time the patient was transferred to the Wilhelmina Gasthuis for further investigation. Physical examination on admission revealed a blood pressure (SP) of 150/ 100 mmHg and a pulse rate of 68/min. His body weight
+--? \
\
\ \
\ \ \
[
YCLdP;iOSPHAMIGF 5 0 3
FRllSlbllDE
160
120
80
85 -0
PRED'IISONE
JUNE 1976
JULV
Fig. 1 . Plasma factor XI1 activity, plasma albumin and proteinuria in the course of therapy.
60140
160
AUG
SEPT
OCT
'
MARCH
I977
Acquired factor XII deficiency EiS4
2.0
Els4
537
0
1.0.
0 0
: a,
1.0
a
-
z
_O
was 65.8 kg and height 1.64 m. Facial oedema, pleural fluid, ascites and extensive pitting oedema of the legs were observed. Laboratory investigations of urine samples revealed that the sediment contained many hyalin casts with some leucocytes; the protein concentration was 12 g/I (albumin 67.4%, a,-globulin 9.6%, a,-globulin 5.1 %, @globulin 11.9%, y-globulin 6.0%). Fractional clearances of IgA and a,-macroglobulin, assuming a I00 9% clearance of transfernin, were calculated to be 28.8 and 1.7%, respectively, indicating non-selective proteinuria (6). Haematological screening revealed: ESR 123 mm, Hb 8.1 mM, Hct 0.38 1/1, leucocytes 16.3x109/1 with a normal differential cell count. Serum electrolytes, BUN and creatinine were normal, while total lipids (20 g/l), cholesterol (17.1 mM) and triglycerides (9.5 mM) were increased. Serum proteins were decreased: albumin 4.6 g/l, a,-globulin 1.2 g/l, a,-globulin 17.1 g/l p- and y-globulin 7.2 g/l. Liver enzymes and complement factors Clq, C3, C4 and C5 were normal. Data from haemostatic investigations are given in Table I . After infusion of 900 ml of fresh plasma, the recovery of factor XI1 was calculated to be 38% of the administered dose. The plasma half-life of factor XI1 procoagulant activity, which was estimated by linear regression analysis of the factor XI1 procoagulant activities after infusion, was 15 hours (normal value ca. 60). A kidney biopsy was performed without correction of the factor XI1 deficiency and no haemorrhagic complications were observed. Light microscopy of the glomeruli revealed no cellular infiltration or proliferation and a normal basement membrane with no depositions, while the tubuli were found to contain protein. Immunofluorescence revealed no depositions of immunoglobulins or complement. A diagnosis of nephrotic syndrome due to minimal lesions glomerulonephritis was made. Diuretic therapy with frusemide and spironolactone was instigated. On this therapy, the oedema slowly disappeared and the body weight decreased by 25 kg. As proteinuria was not abaited, prednisolone was administered in a daily dose of 60 mg, which decreased the excretion of protein from 25 g to 6 g/day. Cyclophosphamide, administered in a daily dose of 100 mg, decreased pro-
Fig. 2. Elution profile of urinary factor XI1 following Sephadex G I 0 0 recycling chromatography. Lefr, the elution pattern after the first chromatography cycle; righr (representing the second cycle), both the protein profile and the factor XI1 clotting activity.
teinuria further to less than 1.5 glday. Plasma protein and factor XI1 procoagulant activity increased concurrently in the course of therapy (Fig. I ) . After withdrawal of all therapy (April, 1977), proteinuria remained at a level of f I .5 g/day and the following coagulation values, all within the normal range, were found: factor IX 1.4 U , factor XI I .8 U, factor XI1 0.64 U, antithrombin 111 I . 1 U. RESULTS Urine (protein concentration 12 g/l), when plasma factor XI1 procoagulant activity w a s 0.07-0.08 U and proteinuria exceeded 25 g/day, contained n o factor II/V/VII/X activity, and 0. I 1 U factor VIII, 0.01 U factor IX, 0.01 U factor XI and 0.17 U factor XII-like activity. No such clotting activity could be demonstrated in normal human urine. Incubation with kaolin significantly increased t h e procoagulant activity of the urine in the assay of factor XII. Heating of t h e urine a t 100°C for 10 min resulted in complete loss of the procoagulant activity. Factor XII-like procoagulant activity w a s concentrated by dialysis of the urine o n a collodion membrane under negative pressure. T h e procoagulant activity w a s almost completely absorbed by celite a n d following elution of t h e celite with 0.007 M NH,OH, pH 9.7, factor XII-like procoagulant activity w a s found t o b e associated with the eluate. Attempts were made t o isolate the urinary protein with factor XII-like procoagulant activity. The patient’s urine stored a t -30°C served as starting material. A cryoprecipitate, which w a s found t o have factor VIII procoagulant activity, was formed during thawing a t
4°C. After removal of the cryoprecipitate, ammonium sulphate was added t o the supernatant and the Actu Med Scand 205
538
E . A . vun Royen et ul.
ty was eluted at a volume corresponding to an apparent M w of 82000 daltons (Fig. 3). The fractional clearance of factor XI1 relative to the transferrin clearance was 57%. Assuming an M w of 82000 daltons for factor XII, the data obtained are in close agreement with the curve for the fractional clearance of IgA and a,-macroglobulin (Fig. 4). This suggests that there was no preferential loss of factor XI1 compared to other proteins.
K a v '-OI
i1
cytochrome c
DISCUSSION
1o4
mol. weight
lo5
F i g . 3. Determination of the apparent MW of urinary Factor XI1 by a plot of elution volume versus log M W .
factor XII-like procoagulant activity was found to be associated with the precipitate formed at 25'SO 9% saturation. The precipitate was sedimented and dissolved in 0.154 M sodium chloride. Following dialysis against 0.154 M sodium chloride, the dialysate was applied to a DEAE Sephadex column. Factor XII-like procoagulant activity was eluted at an ionic strength of 0. l5&0. 165. Those fractions containing factor XII-like procoagulant activity were pooled and concentrated by reprecipitation with ammonium sulphate (60% saturation), dialyzed against 0.154 M sodium chloride and applied to a Sephadex G-100 column. A recycling procedure of the eluant from this column resulted in a better separation of the proteins. Factor XII-like procoagulant activity eluted just before the second protein peak (Fig. 2 ) . Several batches of urinary factor XII-like procoagulant activity were prepared by this method. After concentration by negative pressure dialysis, I 1 preparations were pooled and submitted to rechromatography on Sephadex G-100. Fractions containing factor XII-like procoagulant activity were pooled, concentrated and used to determine the apparent molecular weight (MW) on a Sephadex G-100 column. The column was equilibrated with transferrin (MW 77000), bovine serum albumin (MW 67000), ovalbumin (MW 45000), cytochrome L' (MW 125'00). The void volume was determined using dextran blue and the total volume of the gel bed by phenol. Factor XII-like procoagulant activiActa M r d Sciind 205
Coagulation and haemostasis investigations on patients with nephrotic syndrome have revealed disparate results. Increase in platelet count, fibrinogen level and factor VIIl procoagulant activity have been observed in association with this syndrome (8, 10). However, coagulation factor deficiencies have also been observed in some patients. A deficiency of antithrombin I11 in nephrotic syndrome resulting in a thrombotic tendency has been described by Kaufmann et al. (9), while Handley and Lawrence (4) described a haemorrhagic diathesis associated with decreased levels of factor IX. Factor XI1 deficiency is not, in general, associated with a bleeding tendency but does result in a prolongation of the kaolin-cephalin clotting time and has also
100
50
20
PM
I
I
10
I
5
1
I
lo6 mol.weight
Fig. 4 . Fractional clearance of factor XII.
Acquired factor XII deficiency
been reported to occur in nephrotic syndrome (3, 5 ) . We present a patient with low levels of factor XI1 and antithrombin 111 on whom a kidney biopsy was performed without haemorrhagic complications, although the factor XI1 deficiency had not been corrected. Decreased factor XI1 procoagulant activity in association with a markedly shortened plasma half-life together with a severe proteinuria were indicative of factor XI1 loss in the urine. In the course of therapy, the low factor XI1 and antithrombin Ill levels were normalized parallel with the increase in plasma albumin. Antithrombin Ill antigen and a procoagulant activity which have been found to induce antibodies against human factor IX in rabbits have been found in the urine of patients with nephrotic syndrome (9, 11). Green et al. ( 3 ) described a patient with low levels of factors IX and XII. They demonstrated factor XI1-like procoagulant activity in the urine which showed the same elution profile as plasma factor XI1 when filtered through Sephadex G-100. However, no attempts were made to isolate the active protein. Factor XII-like procoagulant activity was also demonstrated in the urine of our patient. The factor XII-like material was labile to heat, could be absorbed to and eluted from celite and was activated by kaolin. In addition, the elution profile' after filtration through DEAE Sephadex was similar to that of plasma factor XI1 ( 2 ) . Following further purification, the active molecule was found to have an apparent MW of 82000 daltons, which is in agreement with quoted values for plasma factor XI1 (12). A proper explanation for the deficiency of antithrombin I11 and of factor XI1 in this patient is not yet apparent. The M W s of antithrombin Ill and factors 11, VII, X and XI1 are within the range of 40000-80000 daltons; therefore, MW cannot be the sole determinant of the selective loss of antithrombin Ill and factor XII. Differences in biosynthetic capacity as well as in molecular charge ( I ) of the various clotting factors may determine their plasma level in proteinuria. In our patient, the loss of factor XI1 protein, also revealed by the markedly shortened half-life, appeared to be proportional to that of other proteins such as IgA, a,-macroglobulin, since the plot of log MW against the fractional clearance of factor XI1 fitted the curve obtained for transferrin, IgA and a,-macroglobulin.
539
These findings strongly suggest that the factor XI1 deficiency in this patient resulted from an insufficient biosynthetic capacity of this protein, relative to other clotting factors, to compensate for the loss in the urine. A similar explanation may account for the deficiency of antithrombin I11 and the plasma factor VIII procoagulant activity of 1.7 U which is low for a patient with nephrotic syndrome, especially in view of the high plasma fibrinogen level. REFERENCES 1 . Brenner, B. M., Hostetter, T. H. & Humes, H. D.:
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Molecular basis of proteinuria of glomerular origin. N Engl J Med 298: 826, 1978. Cochrane, C. G. & Wuepper, K. D.: The first component of the kinin forming system in human and rabbit plasma. Its relationship to clotting factor XI1 (Hageman factor). J Exp Med 134: 986, 1971. Green, D., Arruda, J., Honig, G. & Muercke, R. C.: Urinary loss of clotting factors due to hereditary membranous glomemlopathy. Am J Clin Pathol 65: 376, 1976. Handley, D. A. & Lawrence, J. R.: Factor IX deficiency in the nephrotic syndrome. Lancet I : 1079, 1967. Honig, G. R. & Indley, A.: Deficiency of Hageman factor (factor XII) in patients with the nephrotic syndrome. J Pediatr 78: 633, 1971. Joachim, G. R . , Cameron, J . S., Schwartz, M. & Becker, E. L.: Selectivity of protein excretion in patients with the nephrotic syndrome. J Clin Invest 43~2332,1964. Kahle, L. H., Schipper, H. G., Jenkins, C. S. P. & ten Cate, J. W.: Antithrombin-111. I. Evaluation of an automated antithrombin 111 method. Thromb Res 12: 1003, 1978. Kanfer, A., Kleinknecht, D., Broyer, M. & Josso, E.: Coagulation studies in 45 cases of nephrotic syndrome without uremia. Thromb Diath Haemorrh (Stuttg) 24: 562, 1970. Kaufmann, R. H., de Graeff, J., Brutel de la Rivikre, G. & van Es, L. A,: Unilateral renal vein thrombosis and nephrotic syndrome. Report of a case with protein selectivity and antithrombin-111 clearance studies. Am J Med 60: 1048, 1976. Kendall, A. G., Lohmann, R. C. & Dossetor, J. B.: Nephrotic syndrome a hypercoagulable state. Arch Intern Med 127: 1021, 1971. Natelson, E. A., Lynch, E. C., Hettig, R. A. & Alfrey, C . P.: Acquired factor IX deficiency in the nephrotic syndrome. Ann Intern Med 73: 373, 1970. Revak, S. D., Cochrane, C. G., Johnston, A. R. & Hugli, T. E.: Structural changes accompanying enzymatic activation of human Hageman factor. J Clin Invest 54: 619, 1974. van Royen, E. A,, Treffers, P. E. & ten Cate, J. W.: Hypertonic saline induced abortion as pathophysiologic model of low grade intravascular coagulation. Scand J Haematol 13: 166, 1974. Actu Med Scund 205
