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Fibrinogen Turnover in Progressive Systemic Sclerosis.

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343
FIBRINOGEN TURNOVER IN
PROGRESSIVE SYSTEMIC SCLEROSIS
GEOFFREY M. GRATWICK, RONALD KLEIN, JOHN S. SERGENT, and CHARLES L. CHRISTIAN
Large amounts of fibrin are seen in the intima of
the renal arterioles in progressive systemic sclerosis
(scleroderma). The half-life or half disappearance time of
plasma fibrinogen in 15 patients with scleroderma was
studied using ‘‘‘1 fibrinogen to find whether there is an
increased turnover of plasma fibrinogen paralleling this
morphologic abnormality. Patients had a more rapid fibrinogen turnover than normal controls (60.7versus 90.6
hours); the subgroup of patients with “progressive”
scleroderma had a more rapid fibrinogen half-life than
those with “stable” scleroderma (56.5 versus 73.2 hours).
The mean fibrinogen half-life of 8 patients given intravenous heparin increased to within one standard deviation
of normal, a finding that suggested that the fibrinogen
molecule in these patients was capable of normal survival.
There was a considerable variation of fibrinogen half-lives
in individual scleroderma patients over time (not seen in
the normal controls) which may be the result of intermittently increased fibrinogen consumption.
From the Departments of Medicine, The Hospital for Special
Surgery and Cornell University Medical College, New York, New
York, and the Department of Medicine, Vanderbilt University School
of Medicine, Nashville, Tennessee.
Geoffrey M. Gratwick, M.D.: The Hospital for Special Surgery, New York, New York; Ronald Klein, B.A.: Stony Brook, Long
Island, New York; John S. Sergent, M.D.: Assistant Professor of
Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; Charles L. Christian, M.D.: The Hospital for Special Surgery,
New York, New York.
Address reprint requests to Geoffrey M . Gratwick, M.D.,
The Hospital for Special Surgery, 535 East 70th Street, New York,
New York 10021.
Submitted for publication June 27, 1977; accepted August 9,
1977.
Arthritis and Rheumatism, Vol. 21, No. 3 (April 1978)
Increased amounts of fibrin are seen in renal
afferent arterioles, pulmonary arterioles, and in the perivascular areas of tendon sheaths and joints in progressive systemic sclerosis (scleroderma). This fibrin can be
demonstrated both morphologically as small, fibrous
strands and by specific immunofluorescent anti-fibrin
staining (1). The presence of fibrin is in itself not surprising; deposition of fibrin is a frequent response to inflammatory stimuli and may be found in many inflammatory
states. What is distinctive about scleroderma is the large
amount of fibrin present. It is considerably more than
that seen in vasculitic syndromes such as lupus erythematosus, and equalled only by that seen in malignant hypertension and some of the microangiopathic
hemolytic anemias (2).
In this study the authors have looked at the
plasma fibrinogen level and the fibrinogen half-lifewhich together permit an estimation of the fibrinogen
turnover-in an attempt to find a plasma abnormality
potentially corresponding to the morphologic abnormalities seen in scleroderma. Because of the large
amount of fibrin seen in perivascular areas we postulated an increased fibrinogen turnover which could in
turn be the result of three abnormalities: 1 ) increased
excretion of fibrinogen, 2) rapid utilization of fibrinogen
by an activated coagulation system, or 3 ) altered catabolism of fibrinogen. We have previously found that increased amounts of fibrinogen are not excreted in systemic lupus erythematosus and other vasculitic
syndromes (3). Standard hematologic parameters were
used to investigate the possibility of increased intra-
GRATWICK ET AL
344
vascular coagulation; they permit a reasonable b u t n o t
flawless evaluation o f t h e coagulation system because
t h e r e are states of low-grade intravascular coagulation
f o r which these tests are n o t sufficiently sensitive (4).
Although there are no specific w a y s t o evaluate t h e
tissue catabolism of fibrinogen, w e a t t e m p t e d t o evaluate t h e question indirectly b y heparinizing 8 scleroderma patients and then recalculating their fibrinogen
half-lives. By inhibiting coagulation as a potential r o u t e
o f fibrinogen degradation with heparin, w e hoped to see
whether t h e fibrinogen molecule w a s a t least c a p a b l e of
normal survival-or whether it w a s altered and more
susceptible to catabolism.
METHODS
Patients. Informed consent was obtained from 15 patients with scleroderma. Thirteen were females (age: 22-56,
mean: 41.6 years) and 2, males (age: 24-52 years). All patients
had skin disease with acrosclerosis and tightening of facial
skin; 13 had Raynaud’s phenomenon; 9 of 12 had a decrease in
vital capacity and diffusing capacity on pulmonary function
testing; and 7 of 1 1 had a decrease of esophageal motility. Ten
patients had progressive disease of from 1 to 7 years’ duration
(mean: 3.2 years) as defined by one or more of the following: a
documented decrease in pulmonary or esophageal function,
progressive muscle weakness with elevations of the CPK, in-
creasing sclerosis ot the skin by the consensus ot three rheumatologists, or severe Raynaud’s phenomenon with nonhealing
digital ulceration. Two patients died: one with normal renal
function at the time of the study died of renal failure 6 months
later and a second, with severe pulmonary function abnormalities when first studied, of pneumonia 4 months later. Five
patients had stable disease of 2 to 21 years’ duration (mean: 7.0
years), showing no progression in any of the above abnormalities over an 18-month period. Ten patients were admitted to
the Clinical Research Unit of the New York Hospital on one
or more occasions; four patients were studied while hospitalized for other reasons, and one patient was studied as an
outpatient. N o patient in this series had rapidly progressive
renal disease or malignant hypertension at the time of the
study; patients requiring hemodialysis were excluded. Controls
consisted of 6 normal male volunteers; 2 were studied twice,
one and 18 months apart, to determine the reproducibility of
the method. Six patients were studied twice over an interval
ranging from one to 18 months, and one patient was studied 3
times over a 9-month period.
Concurrent Therapy. Wherever possible, patients were
not treated with medications during the study. N o patient
received aspirin or indomethacin during the fibrinogen halflife studies; one patient was being treated with 30 mg of
prednisone and another was receiving POTABA (potassium
paraamino benzoic acid).
Laboratory Data. All patients were evaluated for prothrombin time, partial thromboplastin time, platelet count,
and fibrinogen level. Eight patients had cryofibrinogens determined and 6 fibrinogen related antigens (Thrombo-Wellco
10
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20
40
60
80
TIME
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a06.0
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I V HEPARlNlZATlON
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*
a.96
120
(HOURS)
Figure 1. Fibrinogen halfdife before and during continuous intravenous heparinizarion.
FIBRINOGEN TURNOVER
Test, Burroughs Wellcome). The serum complement was determined by the method of Kent and Fife (S), the anti-DNA
antibodies by the method of Pincus et a/. (6), and the ANA by
a n immunofluorescent test. The creatinine clearance, pulmonary function tests, and electrocardiograms were obtained
on patients when clinically indicated.
Fibrinogen Half-Life Study. I n this study the term fibrinogen half-life refers to the fibrinogen half-disappearance
time. Fibrinogen was purified from autologous plasma in sixteen instances and isologous plasma from a known normal
donor (demonstrated to be hepatitis-free in more than seventyfive such studies over the past 5 years) in 7 others. Fibrinogen
was purified by the differential salt precipitation technique and
labeled with l z 6 I monochloride (7). The fibrinogen was sterilized using a 0.22 Mm millipore filter and sterility documented
by standard culture techniques. When not given immediately,
preparations were stored at -20°C until used. I n all studies,
the radioactivity was greater than 90% clottable by bovine
thrombin.
Both patients and controls were given 500 mg of saturated solution of potassium iodide for 24 hours before injection of fibrinogen and for 10 days subsequently to block the
thyroidal uptake of radioactive iodine. An average dose of
30.3 pCi of 1261 fibrinogen was injected into the anticubital vein
and citrated samples were drawn at Vi hour and then two to
three times daily from the opposite arm for 120-140 hours.
Samples were counted in a Packard Auto Gamma Spectrophotometer and ranged from 69,000 to 134,000 counts per 5 minutes.
Fibrinogen catabolism occurs in two phases: there is
an initial rapid decay over the first 12-24 hours caused by the
elimination of labeled fibrinopeptides and split products by the
reticuloendothelial system and equilibration with extravascular areas. After 24 hours, plasma radioactivity decays exponentially. Coagulability was determined by the addition
of bovine thrombin to plasma samples; the radioactivity of the
fibrin clot (removed on a glass rod) was divided by the total
plasma radioactivity to obtain a percentage value. Fibrinogen
half-lives were calculated by the least square method, and r*
values calculated by the square of an algebraic equivalent of
the Pearson Product-Moment formula.
Heparin. Heparin was given to 8 patients by continuous intravenous infusions (EPIC, Burron Inc., Bethlehem,
Pennsylvania) at a rate of 800-1 100 units per hour to maintain
a partial thromboplastin time of two times normal or greater.
PTTs were obtained at 6, 8, 12, 24, 36 and 48 hours; no ill
effects were encountered. These patients had two fibrinogen
half-lives calculated for each dose of lZ6I fibrinogen given-an
initial half-life during the first 60-70 hours, and a second halflife during the next 50-60 hours while they were receiving
heparin (Figure I ) . A minimum of five different plasma samples was used to determine any given half-life on these patients.
RESULTS
Normal controls had a mean lzaI fibrinogen halflife of 90.6 hours (SD 6.9). The lZ6Ifibrinogen half-life of
the scleroderma patients was 60.7 hours (SD 13.4),
stable patients had a fibrinogen half-life of 73.2 hours
345
c
CONTROLS
#$
ALL PSS
PATIENTS
STABLE
DISEASE
PROGRESSIVE
DISEASE
Figure 2. Fibrinogen half-lives of control and scleroderma patients,
(SD 8.9), a n d those with evidence of progressive diseaSe
had a half-life of 56.5 hours (SD 9.5) (Figure 2). T h e
clottability of the plasma samples was 89-98%. The r2
values (the coefficient of determination) for the regression line averaged 0.97. For a two variable correlation
consisting of an average of five experimental points,
these r2 values were found t o differ significantly from
zero, thus demonstrating the nonrandomness of t h e
points.
T h e observed variation in fibrinogen half-lives
correlated with the patient's general clinical status only,
and not with the severity of articular, cutaneous, or
visceral disease. There was no correlation with the ESR
(r = O.l), plasma fibrinogen level (r = 0.18), platelets (r
= 0.05), CPK (r = 0.38), complement (r = 0.12), or any
other laboratory parameter tested. T h e prothrombin
time and partial thromboplastin time were normal in all
cases. Fibrin degradation products were negative (n =
6), cryofibrinogenemia was not observed (n = 8), a n d
antibody to ribonuclear proteins was negative (n = 4).
T h e mean plasma fibrinogen was 441 mg% (SD 71) with
a normal range of 200-400 mg% in o u r laboratory.
Seven patients were studied with homologous
plasma and had a mean half-life of 59.7 hours, whereas 8
patients studied only with autologous fibrinogen had a
mean half-life of 61.6 hours. One control h a d a half-life
of 86 hours using homologous plasma and a half-life of
87 hours using autologous plasma.
T w o of the normal controls were studied twice to
determine the reproducibility of the method. Their values were similar one and 18 months apart. Of the 7
GRATWICK ET AL
0-
12
24
.
n
36
Figure 4. Fihritrupn ha1lJil;e.so/scleroderina patients bejore and during
I V hepurirti;ution. W = hefbre heparin: j = afier heparin.
NUMBER OF MONTHS FROM BEGINNING OF STUDY
DISCUSSION
patients restudied, 4 had a decrease in their half-life, 2
an increase, and one patient both an increase and a
decrease (Figure 3). The 4 patients with a decreased
half-life had been classified as having progressive disease
and the 2 with an increase in their half-life as having
stable disease. The one patient who had both an increase
and a decrease in fibrinogen half-life had been classified
as having progressive disease; the rise in half-life could
not be correlated with any improvement in clinical
status although the fall in half-life seemed consistent
with the progressive disease in pulmonary function and
downhill course. One patient was being treated with 30
mg of prednisone the second time she was studied,
whereas another was being treated with POTABA both
times she was studied. Prednisone has been reported to
retard fibrinogen catabolism in rabbits (8) and the effect
of POTABA on fibrinogen kinetics has not been studied.
Eight patients with scleroderma were heparinized
by continuous intravenous infusion. During the first 6070 hour control period, fibrinogen half-life was 57.5
hours (SD 10.2), while the half-life increased to 87.3 hours
(SD 11.8) during the 60 hours of heparinization (Figure
4). This mean half-life is within one standard deviation
of normal. The heparinized part of this curve was different than that of the 7 patients who were not heparinized;
the latter patients had a single exponential curve with a
high r2 value for the full 100-120 hours. Heparin has
been demonstrated to have no effect on the fibrinogen
half-life of normal subjects anticoagulated with continuous intravenous heparin (9).
Although the mechanism of tissue injury in
scleroderma is unknown, there is increasing interest in
the vascular abnormalities present in the small arterioles
and capillary beds. Classically the small arterioles (150500 p ) show 1 ) pronounced intimal thickening with
mucoid infiltration, 2) adventitial fibrosis, and 3) an
increase in mucoid ground substances with the staining
properties of mucopolysaccharide and acid mucoprotein
(10). Smaller vessels (50-150 p ) show less pronounced
changes: occasional fibrin thrombi have been noted intraluminally ( 1 1 ). Capillary changes are perhaps most
marked but hardest to delineate. Norton et al. (12)
observed a decrease in the number of capillaries in skin
and skeletal muscle using electron microscopy and believed that the capillary walls were themselves abnormal, while Maricq and LeRoy have found marked dilitation of nailfold and skin capillaries and a decrease in
capillary density in these areas (13).
Several lines of evidence indicate that these vessel
changes are primary and are not the sequellae of tissue
sclerosis and atrophy. Raynaud’s phenomenon may precede evidence of tissue sclerosis, lung biopsies have
shown involvement of small arterioles without evidence
of alveolar thickening, and the capillary changes have
been noted in areas of clinically uninvolved skin. It has
been suggested that this vessel involvement, whether
primary or secondary, may give rise to coagulation abnormalities with widespread fibrin deposition. A microangiopathic hemolytic anemia was noted in 7 of 20
patients by Salyer et al. (14), and Kincaid-Smith noted
that the deposition of fibrin i n the renal arterioles was
FIBRINOGEN TURNOVER
very similar to the pattern seen in malignant hypertension, preeclampsia, and other microangiopathic hemolytic syndromes (2).
Two questions are frequently raised about fibrinogen half-life studies: 1) How reproducible are the values? 2) Is a decrease in the half-life related to an alteration in the coagulation system? The similarity of the
half-lives of 2 normal controls, when performed on multiple occasions and the concordance of the half-life values obtained with those in the literature (9), indicate
that the technique is well standardized and reproducible.
Although 4 patients with progressive scleroderma had a
decrease in their half-life over time and 2 patients with
stable disease had a return in their half-life towards
normal, we do not have enough data to link these
changes in fibrinogen half-life to the clinical prognosis.
Of note in this respect is the one patient who had an
increase in her fibrinogen half-life without any apparent
improvement in her clinical status. A possible explanation for these observed changes is that the utilization of
fibrinogen in scleroderma may be an intermittent process.
The link between an alteration in the fibrinogen
half-life and a coagulopathy is more difficult to determine. For example, it is theoretically possible that the
decreased half-life of fibrinogen in scleroderma is caused
by a preexisting abnormality in the fibrinogen molecule,
not by increased activation of the clotting system. By
heparinizing scleroderma patients and elevating their
half-lives to within one standard deviation of normal,
we can be reasonably certain that the injected fibrinogen
had potentially normal survival. We have used heparin
only to establish this point and have not sought t o
evaluate its clinical efficacy. However, it is by no means
clear that heparin has its only effect in normalizing an
activated clotting system. Heparin, a highly charged
anionic mixture, may equally well interfere with the
extravascular catabolism of fibrinogen in a currently
unknown fashion. The negative assays for fibrin degradation products were not helpful in distinguishing between the activity of coagulation and catabolic pathways, since these degradation products may not be
present in all states of low-grade intravascular coagulation (4).
The magnitude of the fibrinogen half-life change
is similar to that seen in vasculitis syndromes such as
SLE (3). The accelerated turnover may be due to increased activity of the coagulation pathways or t o
heightened catabolism. The altered intima in sclero-
347
derma may well initiate this increased coagulation or
facilitate rapid catabolism although the connection is
currently unknown.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the contribution
of Dr. Klaus Mayer and the nurses of the Clinical Research
Unit of the New York Hospital.
REFERENCES
1. Fennel RH Jr, Reddy CRRM, Vasquez JJ: Progressive
systemic sclerosis and malignant hypertension: an immunohistochemical study of renal lesions. Arch Pathol 72:
209, 1961
2. Kincaid-Smith P: Coagulation and renal disease. Kidney
Int 7:242-253, 1975
3. Sergent JS, Sherman RL, Al-Mondhiry H: Fibrinogen
catabolism in systemic lupus erythematosus. Arthritis
Rheum 19:195-199, 1976
4. Nossel H, Ti M, Kaplan K, Spanondis K, Soland T,
Butler V: The generation of fibrinopeptide A in clinical
blood samples. J Clin Invest 58: 1136-1 144, 1976
5. Kent JF, Fife EH: Precise standardization of reagents for
complement fixation. Am J Trop Med Hyg 12:103-116,
1963
6. Pincus T, Schur PH, Rose JA, et al: Measurement of
serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med 281:701-705, 1969
7. McFarlane AS: In vivo behavior of la11 fibrinogen. J Clin
Invest 42:346-361, 1963
8. Seligsohn V, Rapaport S, Shen S, Kuefler P: Effect of
corticosteroids upon fibrinogen metabolism in rabbits.
Thrombos Diasthes Haemorrh 3053 1-540, 1973
9. Collen D, Tytgat G, Claeys H, Piessens R: Metabolism
and distribution of fibrinogen. I. Fibrinogen turnover in
physiological conditions in humans. Brit J Haematol
22:681-700, 1972
10. Campbell PM, LeRoy EC: Pathogenesis of systemic sclerosis: a vascular hypothesis. Seminars Arthritis Rheum
4:351-368, 1975
11. Heptinstall RH: Pathology of the Kidney. Boston, Little,
Brown, 1974
12. Norton WL, Narda JM: Vascular disease in progressive
systemic sclerosis. Ann Int Med 73:3 17-324, 1970
13. Maricq HR, LeRoy EC: Patterns of finger capillary abnormalities in connective tissue disease by “wide field”
microscopy. Arthritis Rheum 16:619, 1973
14. Salyer WR, Salyer DC, Heptinstall RH: Scleroderma and
microangiopathic hemolytic anemia. Ann Int Med
78:895-897, 1973
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