F i b r i n o g e n
Occlusive
H e t e r o g e n e i t y
Vascular
Surgical
in
Disease,
C a n c e r ,
a n d
in
after
P r o c e d u r e s
IZABELLA LIPINSKA, P H . D . , BOGUSLAW LIPINSKI, P H . D . , VICTOR GUREWICH,
AND KLAUS D. HOFFMANN,
M.D.,
M.D.
From the Vascular Laboratory, Lemuel Shattuck Hospital, Department of Medicine, Tiifts University
of Medicine, Boston, Massachusetts
ABSTRACT
Lipinska, Izabella, Lipinski, Boguslaw, Gurewich, Victor, and Hoffmann,
Klaus D.: Fibrinogen heterogeneity in cancer, in occlusive vascular disease,
and after surgical procedures. Am J Clin Pathol 66: 958-966, 1976. Electrophoresis in 3.5% polyacrylamide gel was used to determine the patterns of
fibrinogen heterogeneity in healthy subjects, in postoperative patients and
in patients with cancer or occlusive vascular disease. Two major and one
minor fibrinogen fractions, differing in molecular weight, were identified,
and their concentrations in blood determined. The high-molecular-weight
(HWM) fraction was found in greatest concentration after operation, during
the period of hyperfibrinogenemia, whereas no simultaneous increase of
lower-molecular-weight (LMW and L M W ) fractions occurred, suggesting
that these were derivatives of HMW ("native") fibrinogen. No correlation
between the concentrations of the LMW and L M W fractions and fibrinolytic
activity was found, suggesting that direct degradation of HMW fibrinogen
by plasmin was unlikely. T h e high fibrinogen level in cancer patients was related to increased concentrations of HMW and LMW fractions, whereas in
the vascular-disease patients it was due exclusively to increased concentrations of LMW and LMW' fibrinogen. Serial observations indicated little
fluctuation in the concentration of these fractions, indicating a persistently
accelerated rate of conversion of HMW to LMW and LMW' fibrinogen in
occlusive vascular disease. Possible pathogenic implications are discussed
(Key words: Fibrinogen; Heterogeneity; Fibrinolysis; Electrophoresis; Vascular disease.)
UTILIZING plasma electrophoresis in 3.5% strated that plasma fibrinogen in healthy
polyacrylamide gels, we recently demon- subjects consists of two major fractions
;
differing in molecular weight. 14 Direct
Received November 11, 1975; received revised
• i- ..•
r cu •
u » „
• •
manuscript January 12, 1976; accepted for public- visualization of fibrinogen heterogeneity in
tion January 30, 1976.
blood as well as quantitation of the major
Supported by the John A. Hartford Foundation f r a c t j o n s w a s possible by this technic. The
and National Institutes or Health Research Grant
.
'
j-r-r- •
HL09203-11, U.S. Public Health Service, Washington, two tractions did not differ in their clottaDC
bility, but the clotting time of the isolated
Address reprint requests to Dr. Gurewich: Director, , • ,
,
,
• , t „,,,,„, cu •
Vascular Laboratory, St. Elizabeth's Hospital, 736 hlgh-molecular-weight (HMW) fibrinogen
Cambridge St., Boston, Massachusetts 02135.
was shorter and its capacity to form sticky
958
December 1976
959
FIBRINOGEN HETEROGENEITY
polymers non-enzymatically was considerably greater than corresponding characteristics of partially purified low-molecularweight (LMW) fibrinogen. Subunit chain
analysis indicated that the structure of the
LMW fraction was consistent with that of a
derivative of HMW fibrinogen. It was
postulated that fibrinogen undergoes continuous but limited degradation by an unknown mechanism, and that some biological properties of fibrinogen may be
determined by the concentration of the
LMW fraction.14
In the present study, the concentrations
of the HMW and LMW fractions were
determined in samples of blood from
healthy subjects and from patients who had
cancer or occlusive vascular disease, or
after surgical procedures. The purpose was
to define the normal pattern of fibrinogen
heterogeneity and to compare it with that
found in patients. Significant differences
among the groups were found. Evidence
regarding the identity of the HMW fraction with newly synthesized fibrinogen is
presented and the mechanism of its
limited degradation to LMW fibrinogen
discussed. The observations are related to
current concepts of the role of plasmin in
fibrinogen catabolism.
Patients and Methods
Patients
Healthy Subjects. Blood was taken from 50
individuals at the time of a routine physical
examination that included an electrocardiogram, hemoglobin determination,
urinalysis, and an SMA-12 chemistry profile. There were 20 men and 30 women ranging in age from 22 to 68 (mean 46) years.
Vascular-disease Patients. Blood was obtained from 34 outpatients (18 men, 16
women) who had clinical evidence of
occlusive arterial disease. All the patients
were studied at least 3 months after
their last occlusive vascular episodes.
Their ages ranged from 39 to 75 (mean 54)
years. There were 16 patients who had
chronic coronary disease, manifested by at
least one old myocardial infarction. Half of
these patients also had angina pectoris.
Twelve patients had arteriosclerosis
obliterans, with absent or diminished
peripheral pulses and intermittent claudication. Six patients had chronic cerebrovascular disease manifested by a history of
stroke or transient ischemic attacks. Among
the entire group, there were six patients
who had diabetes mellitus. No patient was
taking anticoagulants.
Cancer Patients. Blood samples were obtained from 32 patients (15 men, 17 women) hospitalized for treatment of malignant tumors. Their ages ranged from 28 to
82 (mean 60) years. Twenty-four of these
patients had evidence of diffuse metastases
(bones 11, lungs 10, liver 3). Five of the
patients were being treated with steroids
at the time their blood samples were obtained. No other chemotherapy had been
given to any of the cancer patients within
two months of the study.
Surgical Patients. Blood was obtained on
the day before the operation and on days
1, 2, 3, and 5, postoperatively from 15 patients undergoing abdominal surgery.
From eight of them, blood was also obtained on days 6 and 7. There were seven
men and eight women, ranging in age from
22 to 75 (mean 56) years.
Serial Observation. From four of the
healthy subjects and five of the vasculardisease patients, three to six blood samples
were obtained serially over periods ranging
from three to 12 months. T h e range in the
concentrations of total fibrinogen and its
three fractions was determined for each
subject.
Methods
1. Venous blood samples were collected
into 0.1 M ammonium oxalate (9:1). The
plasma was harvested after centrifugation
at 3,500 X g for 10 minutes at 4 C and
then frozen.
960
LIPINSKA ET AL.
AJ.C.P.—Vol.
66
B
FIG. 1. SDS-polyacrylamide gel (3.5%) electrophoretic patterns of a paired plasma (left) and corresponding
serum (right) from three groups of subjects:/!, vascular-disease patients; B, cancer patients; C, healthy subjects.
The locations of the three clottable fractions, HMW, LMW, and LMW'fibrinogen,are shown.
2. Fibrinogen concentration in plasma
was determined by the method of Swaim
and Feders. 24 T h e fibrinogen was clotted
with 10 NIH units of thrombin/ml and then
the clot was incubated at room temperature
for 15 minutes. Most determinations were
performed using fresh plasma, but some
plasma samples from cancer and postoperative patients had been previously
frozen for a couple of days. T h e coefficient
of variation for fibrinogen determination
is 1.2%.
3. Serum was obtained after separation
of the fibrinogen clot according to abovedescribed method and then frozen.
4. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed according to the method previously described. 14 Both plasmas and corresponding sera were diluted with 2% SDS
to the same protein concentration. Electrophoresis was performed in 3.5% gels
loaded with 0.1 ml of diluted plasma or
serum containing approximately 100 /tig of
protein. Electrophoretic separation was
conducted for 3.5 hours at 8.5 mA per
tube. T h e gels were stained for four hours
in Coomassie brilliant blue and destained
manually. Densitometric scanning of each
gel was performed (Densicord Model
542A, Photovolt, N.Y. equipped with an
integrator). The clottable protein was
identified by subtracting the integrated
areasof the protein bands in the serum gels
from the corresponding plasma gels. T h e
percentage contents of HMW, LMW and
the third fibrinogen fraction (LMW) were
calculated and multiplied by the total
fibrinogen concentration in order to obtain
the concentration of each fraction in mg/dl
plasma.
5. Euglobulin clot lysis (ECLT) was performed according to the method of Kowalski and associates.12 This determination
was made in the blood samples from the
cancer patients only. The results were expressed in arbitrary units derived from the
formula 1,300/t where t is the euglobulin
clot lysis time in minutes.
6. Fibrinogen degradation products
December 1976
FIBRINOGEN HETEROGENEITY
961
HMW
(FDP) in serum were determined by the
staphylococcal clumping test method of
Hawiger and co-workers. 10
Statistical Calculations
Student's t test was used for calculation
of the significances of differences in the
values obtained in the groups of subjects
(Hewlett Packard, Stat 1-30A). The correlation between the FDP concentration (/ug/
ml) or the ECLT in units and the LMW
t
(-)
fibrinogen concentration (mg/dl) for the (+)
cancer patients was calculated using linear
Fie. 2. Densitometry scans of thefibrinogenregion
regression analysis (Hewlet Packard, of plasma and corresponding serum of the vasculardisease patient. The clottable protein (hatched)
1-22A).
represented by HMW, LMW and LMW' fibrinogen
is indicated.
Results
96 ± 34 mg/dl, respectively (Table 1). In
Healthy Subjects. T h e electrophoretic pat- samples from eight of the healthy subjects,
tern of fibrinogen in the plasmas of healthy a third fibrinogen component (LMW')
subjects invariably showed two major bands was found at a concentration range of
migrating in close proximity (Fig. 1). A 2 0 - 6 0 mg/dl. The concentration of LMW'
serum protein migrating in position of for the entire group was 6 ± 12 mg/dl. The
HMW fibrinogen corresponds to a 2 -macro- LMW' fibrinogen fraction was found to
globulin as judged by a molecular weight migrate in the region of fibrinogen 1-9 of
of its subunits. 8 The total
fibrinogen Mosesson15 (Fig. 2).
concentration (mean ± 1 S.D.) was 305
Vascular Patients. The total fibrinogen
± 91 mg/dl. The proportion of HMW to concentration was not significantly higher
LMW fibrinogen was about 2:1. The con- than that for the healthy subjects. However,
centrations of HMW and LMW fibrino- the difference in the concentration of
gen (mean ± 1 S.D.) were 203 ± 71 and LMW fibrinogen (152 ± 65 mg/dl) was staTable 1. Mean Values ± 1 S.D. of Fibrinogen, and Statistical Significance
Total
Fibrinogen
Highmolecularweight
Fibrinogen
Lowmolecularweight
Fibrinogen
Lowmolecularweight
Fibrinogen
No.
mg/dl
mg/dl
mg/dl
mg/dl
Healthy subjects
50
305 ± 91
203 ± 71
96 ± 34
6 ± 12
Vascular disease patients
34
378 ± 133
NS
199 ± 70
NS
152 ± 65
P < 0.001
27 ± 21
P < 0.001
Cancer patients
32
585 ± 225
P < 0.02
387 ± 159
P < 0.001
176 ± 75
P < 0.001
19 ± 34
P < 0.01
962
LIPINSKAETAL.
A.J.C.P.—Vol.
66
Table 2. Mean Values ± 1 S.D. of Fibrinogen in a Group of 15 Postoperative Patients
Total
Fibrinogen
Highmolecularweight
Fibrinogen
Lowmolecularweight
Fibrinogen
Lowmolecularweight'
Fibrinogen
mg/dl
mg/dl
mg/dl
mg/dl
15
425 ± 136
242 ± 47
163 ± 36
20 ± 19
15
460 ± 160
302 ± 101
134 ± 39
24 ± 18
10
627 ± 153
447 ± 79
170 ± 52
10 ± 12
10
769 ± 223
585 ± 222
166 ± 64
18 ± 9
13
643 ± 126
446 ± 112
177 ± 52
20 ±23
8
587 ± 120
446 ± 39
127 ± 61
14 ± 12
8
619 ±81
408 ± 62
186 ± 46
25 ± 15
Number
of Determinations
Before
operation
Day after
operation
tistically highly significant (P < 0.001).
T h e ratio of HMW to LMW fibrinogen was
1.3:1. Of these patients, 27 had a detectable
LMW' fraction at a concentration range of
4 - 6 7 mg/dl. T h e concentration of LMW'
for the entire group was 27 ± 21 mg/dl,
being significantly (P < 0.001) higher than
that for the healthy subjects (Table 1, Figs.
1 and 2).
Cancer Patients. The total fibrinogen concentration was significantly (P < 0.02)
higher than that for the healthy subjects.
This difference was due to significantly
(P < 0.001) higher concentrations of both
major fractions, being 387 ± 159 and 176
± 75 mg/dl for HMW and LMW fibrinogen, respectively. Fifteen of these patients
had a detectable LMW' fraction, the concentration being 19 ± 34 mg/dl for the
entire group (Table 1, Fig. 1). The proportion of HMW to LMW fibrinogen was
nearly the same as for healthy patients
(2:1).
Surgical Patients. A significant (P
< 0.001) increase in total fibrinogen occurred on postoperative days 2 - 7 . This
increase was due predominately to HMW
fibrinogen. No significant increase in LMW
or LMW' fibrinogen occurred on any of
the postoperative days (Table 2).
Correlation with ECLT and FDP
In the cancer patients, the mean and
±S.D. ECLT was 2.85 ± 4.81 units (laboratory normal: 5.42 ± 12.5 units). There was
no correlation between the ECLT and the
LMW, LMW' or FDP concentrations. Sixteen of the cancer patients had elevated
FDP [(mean 74 /u,g/ml, range 8-1,024 n%l
ml. The mean ± 1 S.D. for the entire 32 patients was 45 ± 180 /Ag/ml. There was no
correlation between FDP and LMW or
LMW' fibrinogen concentrations.
Serial Determinations of HMW, LMW
and LMW' Fibrinogen
In the five vascular-disease patients and
four healthy subjects studied repeatedly
December 1976
FIBRINOGEN HETEROGENEITY
over time, the differences between the
groups were consistently maintained.
LMW fibrinogen with a single exception,
was invariably higher in the vascular-disease patients, who also had consistently
greater concentrations of L M W (Table 3).
Statistical Analysis
Significant differences compared with
the values in healthy subjects were obtained
as follows: Vascular-disease patients, LMW
fibrinogen (t = 3.9; P < 0.001) and LMW'
fibrinogen (t = 4.9; P < 0.001). Cancer
patients, total fibrinogen (t = 2A;P < 0.02),
HMW fibrinogen (t = 5.8; P < 0.001),
LMW fibrinogen (t = 5.1; P < 0.001), and
LMW' fibrinogen (t = 2 . 1 ; P < 0.01)
(Table 1).
Discussion
T h e heterogeneity of human fibrinogen
has been characterized by differences in
the solubilities of various clottable fractions, '•9'21-23 as well as by differences in
their eiectrophoretic mobilities4'11-13 and
molecular weights. 19,20,25 It has been proposed on the basis of their structure that
the lower-molecular-weight, higher-solubility fractions are catabolites formed by
limited plasmic digestion of fibrinogen.18,20
Utilizing electrophoresis in 3.5% gels, we
previously showed that human fibrinogen
is composed of two major fractions. 14
By this method of direct visualization of
fibrinogen the possibility of induction of
fibrinogen heterogeneity in vitro as a result
of degradation occurring during its isolation and purification can be avoided. Moreover, we have shown that plasma fibrinogen
resists degradation by naturally induced
fibrinolytic activity so long as it remains
in a soluble phase. 5 ~ 7,16 Therefore, the
presence of two major fibrinogen fractions
in plasma represents its native state of
heterogeneity.
Analysis of plasma samples from a large
group of subjects showed the existence of a
963
third fibrinogen fraction (LMW) not
previously described. T h e eiectrophoretic
mobility of this fraction was indistinguishable from that of fraction 1-9 of Mosesson.15
T h e L M W fibrinogen was found significantly less often and in lower concentrations in healthy subjects than in patients
with vascular disease or cancer. T h e patients with vascular disease had the highest
incidence of this fraction.
The relationship between blood fibrinolytic activity and the concentration of the
lower-molecular-weight fibrinogen was
studied in the cancer patients. Although as
a group their fibrinolytic activity was considerably depressed, these patients had
higher concentrations of both LMW and
L M W fibrinogen than the healthy subjects.
Among these patients, no correlation was
found between the lower-molecular-weight
fibrinogens and blood fibrinolytic activity,
suggesting that their formation is independent of this activity. Neither was there
any correlation between the LMW and
L M W fractions and the FDP concentration. It appears from these findings that
the mechanism responsible for the formation of the lower-molecular-weight fibrinogen differs from that involved in the
elaboration of FDP. T h e progressive degradation of fibrinogen into clottable and unclottable derivatives readily induced by
plasmin in vitro may not have an in-vivo
parallel.
It has been well documented that a substantial increase in total fibrinogen occurs
in most postoperative patients. 3 It is believed, though unproven, that this observation is related to increased synthesis of
fibrinogen rather than its release from a
storage site. The extravascular fibrinogen
pool is too small to account for the changes
found, having been estimated to be about
15% of the total.22 The present study shows
that the increase in fibrinogen is due entirely to a more than twofold increase in
the HMW fraction, since there was no significant change in the concentrations of the
Healthy subjects
1
Patients with vascular disease
1
95
88-113
79
63-82
164
147-193
243
210-275
42, F
85
66-105
197
162-259
196
178-264
292
275-350
78
50-130
260
215-282
25, M
176
130-230
235
139-281
174
163-196
282
250-330
545
480-600
58, M
173
168-179
158
134-202
43, M
381
365-400
55, F
204
170-276
187
180-193
225
175-350
376
320-405
45, F
255
214-307
46, M
500
440-550
45, M
145
127-160
mg/dl
mg/dl
mg/dl
283
242-334
Low-molecularweight Fibrinogen
High-molecularweight Fibrinogen
Total Fibrinogen
451
425-490
Number
of
Determinations
75, F
Age
(Yr.),
Sex
Table 3. Serial Fibrinogen Concentrations, Mean and Range
0-16
3
7
0-18
2
0-10
49
30-67
34
32-38
14
0-27
47
45-50
22
0-31
mg/dl
Low-molecularweight' Fibrinogen
December 1976
FIBRINOGEN HETEROGENEITY
LMW and L M W components. It may be
postulated from these data that the HMW
fraction represents native fibrinogen and
that formations of the LMW and LMW'
components do not take place simultaneously and are independent of the
absolute concentration of HMW fibrinogen. The above observations do not help to
explain the mechanism of formation of the
lower-molecular-weight fibrinogens, but
are consistent with the concept of their
being derivatives formed by a fairly constant degradation process.
Patients who have peripheral vascular or
coronary-artery disease tend to have higher
than normal fibrinogen concentrations. 2,17
In contrast to the postoperative patients,
the elevated fibrinogen was due exclusively
to the LMW and LMW' components, since
their HMW fibrinogen was identical to that
of the healthy subjects. Serial studies of
blood samples from five of these patients
showed the concentration of LMW fibrinogen to vary little and to remain consistently
higher than in the healthy subjects. The
conversion of HMW to lower-molecularweight fibrinogen thus appears to be consistently accelerated in vascular disease.
Quantitation of the LMW fibrinogen by
this technic may be a useful screening
test for the diagnosis of occult vascular
disease and perhaps for evaluating the effect of therapy on the disease. Additional
clinical studies are needed to determine the
specificity of the changes found in these
patients.
Although plasmin has been implicated by
a number of studies, 18,23 the exact mechanism of proteolytic degradation of fibrinogen in blood remains to be established.
Recently, we studied the effects on
fibrogen of fibrinolytic activity in blood
caused by venous occlusion,16 exercise, 6
or death. 7 Rapid fibrinolysis was induced
by each of these conditions but no conversion of HMW to LMW fibrinogen
could be demonstrated even after incubation of plasma samples for 24 hours.
965
When a potent vascular plasminogen
activator isolated from human cadavers
was added to plasma, it also failed to
cause any degradation of
fibrinogen.5
By contrast, rapid degradation of fibrin
or precipitated fibrinogen occurred. The
findings were shown to be in sharp contrast
to those obtained with streptokinase or urokinase and to those obtained with the
vascular activator in a purified system. Jt
was postulated that a solid phase was
required for fibrinogen to be degraded by
naturally occurring fibrinolytic enzymes in
blood and that in solution fibrinogen was
not degraded. 5,7
High-molecular-weight
fibrinogen
forms a fibrous, sticky precipitate and is
less soluble than the LMW fraction.14
It is possible t h a t m vivo, HMW fibrinogen
precipitates non-enzymatically on the surface of the vessel wall or blood cell
membranes. This solid phase permits binding of plasminogen activator to fibrinogen
and brings about its degradation. It has
been shown that the LMW fraction increases the solubility of
fibrinogen.14
Therefore, the formation of LMW fibrinogen may permit the precipitated fibrinogen
to redissolve and thus limit its further
degradation. According to this hypothesis,
a high concentration of LMW fibrinogen,
as in the vascular-disease patients, would
reflect enhanced intravascular fibrinogen
precipitation. Additional studies of the
biologic properties of the HMW and LMW
fractions and of the factors that determine their concentrations in the blood may
help to define the role of fibrinogen in
the pathogenesis of occlusive vascular
disease.
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966
L1PINSKA ET AL.
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