Urinary Loss of Clotting Factors Due to
Hereditary Membranous
Glomerulopathy
DAVID GREEN, M.D.,
P H . D . , JOSE ARRUDA, M.D.,
GEORGE H O N I G , M.D.,
AND ROBERT C. MUEHRCKE,
PH.D.,
M.D.
From the Department of Medicine, Northwestern University Medical School, Departments of Medicine
and Pediatrics, Abraham Lincoln School of Medicine, Chicago, Illinois, and the Department
of Medicine, West Suburban Hospital, Oak Park, Illinois
ABSTRACT
Green, David, Arruda, Jose, Honig, George, and Muehrcke, Robert C.:
Urinary loss of clotting factors due to hereditary membranous glomerulopathy. Am J Clin Pathol 65: 3 7 6 - 3 8 3 , 1976. Severe plasma deficiencies
of clotting factors IX and XII developed in a 59-year-old woman with a
nephrotic syndrome secondary to a laminated membranous glomerulopathy. Both these clotting factors were subsequently identified in the patient's urine. Chromatographic analysis of the urine revealed that the
bulk of clotting activity attributed to factors IX and XII was in early
eluting gel filtration fractions containing predominantly alpha-2 globulin
and albumin. T h e unprecedented finding of two coagulation proteins in
the urine is attributed to the marked proteinuria present in this case.
(Key words: Factors IX, XII deficiency; Hereditary membranous glomerulopathy; Nephrotic syndrome; Proteinuria.)
SIGNIFICANT
URINARY
LOSS
of
clotting
factor IX (PTC, Christmas factor), leading
to a marked plasma deficiency of this
factor, has been described to occur in patients who have the nephrotic syndrome. 5,13
In 1971, Honig and Lindley 7 found deficiencies of factor XII (Hageman factor) in
a group of children with the nephrotic
syndrome. Recently, a patient with renal
loss of both of these clotting factors was
encountered, and studies were performed
to examine this unusual form of proteinuria.
Received March 10, 1975; received revised manuscript May 5, 1975; accepted for publication May
5, 1975.
Address reprint requests to Dr. Green: Northwestern University Medical School, Department of
Medicine, 303 E. Chicago Ave., Chicago, Illinois
60611.
376
Report of a Case
A 53-year-old white woman had a 25year history of proteinuria, first noticed in a
postpartum period. Two years prior to the
present investigation she had experienced ankle swelling and facial edema.
Two months prior to admission signs of
Raynaud's disease had developed, and she
had had intermittent muscle cramps. T h e
patient specifically denied arthritis, frequent infections, or a bleeding tendency.
The family history was of interest in that
one sister has proteinuria and systemic
lupus erythematosus, and a brother had an
episode of uremia. Physical examination
revealed edema as described but was
otherwise unremarkable. T h e blood pressure was 124/74-6 mm Hg.
Laboratory studies showed normal blood
March 1976
377
URINARY LOSS OF CLOTTING FACTORS
Table 1. Clotting Factor Assays
Platelet count (103 per cu. mm.)
Platelet aggregation (collagen)
Partial thromboplastin time (sec.)
Prothrombin lime (sec.)
Factor V (%)
Factor VII (%)
Factor VIII (%)
Factor IX (%)
Factor X (%)
Factor XI (%)
Factor XII (%)
Normal
On
Admission
200-400
Normal
35-45
12
50-150
50-150
50-150
50-150
50-150
50-150
50-150
386
Norma
77
15
58
100
143
9
110
50
11
One Month
Later*
Six Months
Later*
42
56t
11
* Taking prednisone,
t Biopsy performed.
counts, but persistent proteinuria without
cellular elements or casts. Urinary protein
was 7.4 and 7.7 Gm. per 24 hours on two
occasions. Serum cholesterol was 330 mg.
per 100 ml., total lipid 1,027 mg. per 100
ml., triglycerides 132 mg. per 100 ml.,
blood urea nitrogen 20 mg. per 100 ml.,
serum creatinine 1.5 mg. per 100 ml., and
uric acid 7.5 mg. per 100 ml. Total serum
protein was 4.6 Gm. per 100 ml., with a
serum albumin of 2.8 Gm. per 100 ml.
Serum IgG was 820 mg. per 100 ml.,
serum IgA 110 mg. per 100 ml., and serum
IgM 160 mg. per 100 ml. B-1C/1A globulin
was 78 mg. per 100 ml. and serum alpha-1antitrypsin only 0.41 mg. per ml. (normal
> 1.0) (kindly performed by Dr. A. J.
Chandrasekhar). The antinuclear antibody
test was persistently positive, the direct
Coombs test weakly positive, and cryoglobulins were detected in the serum, but repeated L.E. preparations were negative.
Radiographic studies of the chest, skeleton,
and urinary tract were unremarkable.
Representative results of coagulation
studies are shown in Table 1. The partial
thromboplastin time was prolonged, while
the plasma levels of factors IX and XII
were decreased. The patient was treated
with prednisone, 20 mg. per day. After 16
days of treatment, factor IX had risen to
56% of normal and the partial thrombo-
plastin time was 42 seconds. However,
proteinuria persisted, and after a month
of prednisone treatment, the blood factor
IX level was 32%. T h e patient was observed over the next three months. T h e
proteinuria was found to wax and wane in
severity. When the coagulation factors
were near normal levels, a renal biopsy was
performed. T h e procedure was well
tolerated by the patient. During a 2V£-year
period of follow-up observation, the
proteinuria has decreased to 4 Gm. per 24
hours, total serum proteins and albumin
have increased to 6.1 and 3.1 Gm. per
100 ml., respectively, prednisone has been
discontinued, and the partial thromboplastin time has returned to normal (35
seconds).
Materials and Methods
Coagulation Factor Assays
Plastic equipment and containers were
used for the collection of all blood and
urine samples. Factor V was assayed by
the method of Shanberge and associates,14
and factor VIII by the two-stage method of
Denson, Biggs and Macfarlane. 3 Factors
VII, IX, X, XI, and XII were measured
by a one-stage kaolin-cephalin method
using known deficient plasmas as substrate.
378
GREEN
ETAL.
A.J.C.P.—Vol.
65
FIG. 1. Photomicrograph of two glomeruli, illustrating diffuse thickening of the basement membranes
with minimal hypercellularity. Hematoxylin and eosin. xlOO.
Assay of Urine for Clotting Factors
and had a length of 62 cm. and a diameter
of 1.7 cm. The eluting buffer was phosphosaline (Na 2 HP0 4 , 1.322 Gm., KH 2 P0 4 ,
0.092 Gm., NaCl, 8.76 Gm., in a volume of
1 liter, pH adjusted to 7.5 with 1 N HC1).
The protein concentration of the eluted
fractions was estimated by spectrophotometric absorbance at 280 nm. T h e protein
peaks were concentrated by filtration in
collodion bags (Bolab, Inc., Reading, Mass.)
for clotting factor assay and electrophoretic
study. Electrophoresis was performed on
cellulose acetate strips using the Model
R-100 MicroZone Electrophoresis System
(Beckman Instruments Corporation, Palo
Alto, Calif.).
A 400-ml. amount of the patient's urine
was concentrated by perevaporation to a
volume of 50 ml. and dialyzed against
three changes (1 liter each) of citratesaline solution for 24 hours at 4 C. The
dialyzed urine was then adsorbed with 100
mg. barium sulfate as described by Natelson and co-workers. 13 Antibody neutralization studies were performed using plasma
from a patient with Christmas disease
known to have a factor IX antibody (kindly
furnished by Dr. M. C. Telfer). This antibody was preincubated with the urine
eluate, normal plasma, or saline solution,
and then normal plasma was added to each
tube, followed by assay of residual factor
Results
IX. Column chromatography was performed
at room temperature with Sephadex GlOO Morphologic Examination of the Renal Biopsy
(Pharmacia Fine Chemicals, Inc., PiscatLight Microscopy (Fig. 1). Seven glomeruli
away, N. J.). T h e columns were siliconized were seen. Three glomeruli were com-
March 1976
URINARY LOSS OF CLOTTING FACTORS
379
FIG. 2. Electron micrograph, showing lamination (arrows) and forking of the glomerular basement membrane
(BM). Electron-dense deposits are present in the mesangial area (M). X4.800.
pletely hyalinized, and two were relatively
uninvolved. Examination of the remaining
glomeruli revealed thickening of the
glomerular basement membrane with
marked narrowing of the glomerular
capillary lumen. Glomerular cellularity
was increased. Synechiae connecting the
glomerular capillary tufts and Bowman's
membrane were found. There was no
evidence to suggest focal glomerulitis or
wire-loop lesions.
T h e renal tubules were relatively un-
involved. Proteinaceous casts were present
in several tubular lumina. T h e interstitium
was slightly edematous and contained focal
collections of lymphocytes, but this was not
a striking feature. No large vessels were
seen, and there was no hyalinization of
either afferent or efferent glomerular
arterioles. The findings were thought to
be consistent with a chronic glomerulonephritis of the membranoproliferative type.
Electron Microscopy (Fig. 2). T h r e e
glomeruli were examined. Electron-dense
380
GREEN
A.J.C.P. —Vol. 65
ETAL.
PLASMA
IE
o
s
g
E
FIG. 3. Protein elution patterns of
normal plasma (above) and patient's
urine (below) after chromatography
on Sephadex G-100. Factor IX and
XII activities are observed in fractions from both plasma and urine
eluates in closely proximate locations.
A small peak of factor IX activity
is also found among the very lowmolecular-weight c o m p o u n d s of
the urine.
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"deposits" were found only in the mesangial areas. The glomerular basement
membrane had a laminated appearance,
and there were small vesicular dense
bodies within the laminated membrane.
These features closely resemble those
reported for hereditary nephritis. 6,15 Other
findings included the presence of lipid
material within the tubules, mild interstitial
edema, and a slight increase in collagen
fibers. T h e absence of subepithelial deposits weighed against the diagnoses of
idiopathic membranous glomerulopathy
and lupus membranous glomerulonephritis. 4
activity was indeed due to the presence of
factor IX, antibody neutralization studies
were performed. T h e barium sulfate
eluate neutralized the equivalent of 14%
factor IX antibody, in close agreement with
the value of 11% factor IX activity found
by direct assay.
Chromatographic and Electrophoretic
Studies of the Patient's Urine
Chromatographic studies of the patient's
urine and normal plasma are shown in
Figure 3. The first peak observed in the
urine pattern consisted of aggregated
albumin, as determined by electrophoretic
analysis. Maximal clotting factor activities
Identification of Clotting Factor IX hi the Urine
were eluted in fractions just prior to the
The protein content of the concen- second protein peak. Additional factor IX
trated, dialyzed urine was 50 mg. per ml., activity, but not factor XII activity, was
and the apparent factor IX level, 25% associated with a third protein peak. A
(normal plasma standard, 100%). Since close correspondence existed between the
nonspecific urinary coagulants may account locations of urinary plasma factor IX and
for some of this activity,14 the urine was ad- factor XII activities.
sorbed with barium sulfate. Eleven per
The fractions of normal plasma and
cent factor IX activity was recovered patient urine containing maximal coagufrom the barium sulfate eluate. T o lant factor activity were concentrated and
further demonstrate that this coagulant studied by electrophoresis (Fig. 4). T h e
March 1976
FIG. 4. Protein electrophoretic patterns of normal plasma, the plasma
fraction containing the
maximum clotting factor
activities, the patient's
u r i n e , and the u r i n e
fraction containing the
maximum clotting factor
activities. Note the presence of alpha-2 globulin in both urine and
plasma fractions. T h e most
prominent protein constituent of the patient's
whole urine is alpha-1
globulin.
381
URINARY LOSS OF CLOTTING FACTORS
WHOLE PLASMA
NORMAL
PLASMA FRACTION
NORMAL
WHOLE URINE
PATIENT
URINE FRACTION
PATIENT
X <f> $
normal plasma fraction contained gamma,
beta, and alpha-2 globulin, and albumin.
T h e urine fraction was composed mainly
of alpha-2 globulin. Concentration and
electrophoresis of the third urine chromatographic peak failed to yield stainable
protein bands. Electrophoretic patterns of
whole normal plasma and the patient's
unfractionated urine are shown for comparison.
Chromatography was also performed
on concentrated samples of the patient's
urine. Under these conditions, the second
chromatographic peak showed a prominent alpha-1 band on electrophoresis. T h e
third protein peak was very much smaller
cX.
°<.
ALBUMIN
than that seen with the unconcentrated
urine, and contained much less factor IX
activity.
Discussion
Several disorders of blood coagulation,
i n c l u d i n g thrombocytosis, increased
plasma levels of fibrinogen, factors V, VII,
and VIII, accelerated thromboplastin
generation, and increased platelet aggregation, have been reported in association with
the nephrotic syndrome. 1,8_1 ° Deficiencies
of coagulation factors have been found less
frequently; isolated factor IX 5,13 and factor
XII 7 defects have been reported, and in
one instance, urinary loss of prothrombin
382
GREEN ETAL.
and factor VIII has been found, although
the plasma levels of these factors were
normal. 16 In nearly all of these accounts,
as was the case in this report, severe proteinuria (>7 Gm. daily) has been present.
T h e marked proteinuria of our patient
resulted from a laminated and forked
membranous glomerulopathy similar to
the glomerular finding in hereditary
nephritis. 6-15 Renal disease in the patient,
her sister, and a brother may thus be on the
familial basis of hereditary nephritis. In
addition, a second diagnosis in our patient,
systemic lupus erythematosus, is suspected
on the basis of a positive test for antinuclear antibodies, low serum complement, Raynaud's phenomenon, cryoglobulinemia, and the recognition of this
disease in her sister. However, the renal
lesion was not characteristic of lupus in
that subepithelial electron-dense immune
deposits were not observed. 4
The renal loss of clotting factors IX and
XII in this patient gave us an opportunity
to examine the interrelationships between
these two clotting proteins. T h e molecular
weight of factor IX has been estimated at
50,000 daltons and that of factor XII,
79,000. 1 2 Both clotting factors were
eluted in the same fractions from normal
plasma chromatographed on Sephadex
G-100, confirming their nearly identical
molecular weights. In 1964, Lewis11 reported the gel filtration characteristics of
plasma clotting factors after passage
through both Sephadex G-100 and G-200.
Her results were virtually identical to
ours—factor IX and factor XII activities emerged together from the columns
in the region between gamma globulin
and albumin.
T h e presence of factor XII in the patient's urine was indicated by the fact that
factor XII activity was eluted from the
chromatographed urine in nearly the same
position as found in the normal plasma
chromatograph. Factor IX was positively
identified in the urine by the criteria that
A.J.C.P.—Vol.
65
it could be adsorbed and eluted from
barium sulfate, and that the eluate specifically neutralized an antibody to factor IX.
That the reduced plasma concentrations
of the clotting factors were secondary to
renal loss was strongly suggested by the
direct relationship between the severity of
proteinuria, serum protein levels, and
clotting factor concentrations. When proteinuria was severe, serum albumin and
clotting factor levels were depressed. With
amelioration of the proteinuria (7.7 to 4.0
Gm. per 24 hours), the plasma levels of
both albumin and clotting factors returned
to the normal ranges.
An interesting question is why there was
selective depletion of factors IX and XII
and not other clotting proteins. For example, the molecular weights of factors
VII and IX are similar,12 and indeed,
the presence of factor VII in the urine
was suspected, since we observed some
procoagulant activity in the barium sulfate urine eluate in addition to factor IX.
Furthermore, the plasma half-life of factor
VII is only 2 - 4 hours, compared with
15-30 hours for factor IX, 2 appearing
to increase the susceptibility of factor VII
to a reduction in plasma levels through
renal loss. Yet, the plasma factor VII concentration was 100% of normal, whereas
factor IX was only 9% of normal.
One possible explanation is that there
was quantitatively a much greater urinary
loss of factor IX than factor VII. Unfortunately, the 24-hour urinary losses of
the clotting factors cannot be accurately
estimated because these proteins undergo
degradation in urine. This was demonstrated by the chromatographic analysis of
the urine, which disclosed two protein fragments with factor IX activity. In fact,
based on urinary factor IX assays, we could
account for only 1-2% of the total plasma
pool of factor IX. An alternative possibility is that extravascular reservoirs of
certain clotting factors, perhaps factor VII,
make such factors less vulnerable to deple-
March 1976
URINARY LOSS OF CLOTTING FACTORS
tion in the face of renal damage. Further
research to elucidate normal clotting
factor metabolism is needed to resolve this
question.
7.
Acknowledgments. Dr. Green was supported during
part of this study by a grant from the Schweppe
Foundation, Chicago, 111. Drs. A. Burdick, Jr., Conrad L. Pirani, and F. I. Volini, permitted publication of the case and assisted in the study.
9.
8.
10.
References
11.
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