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Fibrinogen and Factor VII Levels Improve With Glycemic Control in Patients With Type 1 Diabetes Mellitus Who Have Microvascular Complications
John A. D'Elia, MD;
Larry A. Weinrauch, MD;
Ray E. Gleason, PhD;
Izabela Lipinska, PhD;
Joanne Keough, RN;
Shona Pendse, MD;
Bijan Roshan, MD;
Annette T. Lee, PhD;
Geoffrey H. Tofler, MD
Arch Intern Med. 2001;161:98-101.
ABSTRACT
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To determine whether the hypercoagulable state of patients with complications
of diabetes can be reversed toward normal, a group of insulin-dependent individuals
with proteinuria was treated with intensive insulin protocols. A statistically
significant (P<.001) improvement in control of
diabetes was achieved (mean ± SEM glycosylated hemoglobin, 9.51% ±
0.35% at baseline to 8.36% ± 0.39% at 12 months; and mean ±
SEM advanced glycosylated end products, 14.8 ± 2.8 U/mL at baseline
to 8.4 ± 1.5 U/mL at 12 months). There were statistically significant
decreases in 2 procoagulant factors: mean ± SEM baseline elevated plasma
factor VII, 128.69% ± 5.63% at baseline to 106.24% ± 3.43% at
12 months (P = .002); and mean ± SEM plasma
fibrinogen, 12.3 ± 0.7 µmol/L (417.3 ± 24.7 mg/dL) at
baseline to 10.2 ± 0.7 µmol/L (348.8 ± 22.6 mg/dL) at
12 months (P = .04). Throughout the study, lipid
fractions did not change significantly. Because plasma factor VII and fibrinogen
concentrations were elevated while cholesterol and triglyceride concentrations
were not, more attention should be paid to procoagulants as markers for thromboembolic
complications in diabetic patients undergoing intensive insulin therapy.
INTRODUCTION
Patients with diabetes mellitus and impaired renal function have a high
mortality rate due to an increased incidence of cardiovascular events with
a high prevalence of symptomatic and asymptomatic coronary arterial disease.1 Because thrombosis plays an important role in acute
coronary syndromes, and elevated levels of fibrinogen and other hemostatic
factors have been found in patients with microvascular complications of diabetes,
we determined the relation between intensive insulin therapy and hemostatic
factors in these high-risk patients.
In diabetic patients, the development of nephrotic syndrome with azotemia
is associated with hyperlipidemia and an increased incidence of thromboembolic
cardiovascular events.2, 3, 4
Cholesterol, triglycerides, fibrinogen, and factor VII are synthesized in
the liver and recognized as cardiovascular risk factors; their levels are
increased in patients with type 1 diabetes mellitus.5, 6
There have been few prospective observations relating the interaction between
intensive insulin treatment and hemostatic factors in patients with type 1
diabetes mellitus who also have renal dysfunction.
PATIENTS AND METHODS
The criteria for enrollment included the following: the onset of insulin
dependence before the age of 35 years; albuminuria, with a urinary albumin
level of more than 0.1 g/d, or proteinuria, with a urinary protein level of
more than 0.3 g/d, on 2 separate 24-hour urine collections; and a creatinine
clearance of greater than 0.50 mL/s (30 mL/min). Patients were followed up
for at least 12 months as part of a multicenter study that had as its purpose
the evaluation of the effect on renal function of a regimen of 4 injections
of subcutaneous insulin per day vs a similar regimen to which a weekly intravenous
infusion of insulin had been added.7 Enrollment
required a workup of several weeks to assess the adequacy of control of diabetes
and blood pressure. All patients were maintained on a diet of 0.8 g of protein
per kilogram of body weight diet to maintain ideal body weight. At each interval,
levels of whole blood glycosylated hemoglobin, serum cholesterol, high-density
lipoprotein cholesterol, triglycerides, creatinine, and 24-hour urine for
total protein and creatinine were obtained. Under an additional protocol,
each patient at the Joslin Diabetes Center, Boston, Mass, underwent standardized
phlebotomy (at 8 AM, before any infusions) at baseline and at 6 and 12 months
to determine the plasma fibrinogen level, the fibrinolytic activity, the plasminogen
activator inhibitor (PAI-1) antigen, and plasma viscosity. Fibrinogen levels
were determined by measuring clotting times according to Clauss.8
Plasma factor VII antigen was determined by enzyme-linked immunosorbent assay
using a commercially available kit (Asserchrom VII; AG Diagnostica SIAGO,
Parsippany, NJ). Antigen levels of tissue PAI-1 were determined by enzyme-linked
immunosorbent assay using kits purchased from Biopool International, Ventura,
Calif. Fibrinolytic activity was measured in euglobulins (fibrin plate method)
as described by Brakman.9 Glycosylated hemoglobin
was measured colorimetrically following separation by high-pressure liquid
chromatography (Nichols Laboratories, San Juan Capistrano, Calif). The levels
of total cholesterol, high-density lipoprotein cholesterol, and triglycerides
were measured by the enzymatic colorimetric method with an autoanalyzer (Boehringer-Mannheim/Hitachi
Ruehl Diagnostics, Chicago, Ill). Very low-density lipoprotein cholesterol
was calculated as triglyceride divided by 5. Low-density lipoprotein cholesterol
was calculated by the Friedwald formula: low-density lipoprotein cholesterol
= total cholesterol - very low-density lipoprotein cholesterol -
high-density lipoprotein cholesterol. Levels of advanced glycosylated end
products (AGEs) were measured in plasma by enzyme-linked immunosorbent assay
using polyclonal antibodies to AGE-modified proteins (Picower Institute for
Medical Research, Manhasset, NY).10
All 23 patients recruited at the Joslin Diabetes Center were enrolled
in this study. Of these, 4 did not complete 12 months of study for medical
reasons: gangrenous cholecystitis, peripheral vascular ischemia, injury from
a motor vehicle crash, and inadequate venous access; 2 did not complete 12
months of study because of a personal decision. Thus, 17 patients completed
the study. For clarity of presentation, repeated-measures testing is only
included in statistical analysis and in Table 1 if results were available at all data points for each test.
Results from statistical analysis of all data collected (including data from
patients in whom an insufficient quantity of blood was available at one session)
were virtually identical with results presented in Table 1. Patients receiving weekly infusions of insulin were not
different from those not receiving insulin infusions for glycosylated hemoglobin
level, AGEs, or results of procoagulant studies; therefore, we combined the
groups for analysis. Two patients received cholesterol synthesis blockers
throughout the study.
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Laboratory Data for the Total Study Group*
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Frequency data were tested for significance using the  2
test for independence following the implementation of the Yates correction
for continuity. In cases in which the expected frequencies were less than
5, the Fisher exact test was used. Interrelations among the variables were
tested for significance using Pearson product moment correlations. Temporal
data were analyzed using a 2-way repeated-measures analysis of variance followed
by the Newman-Keuls test to determine the significance among time-period means.
Repeated-measures testing was performed only when all data points were present.
All data are expressed as frequencies or means, with SEMs as a measure of
dispersion. An  level of .05 was considered statistically significant.
All analyses were done using SAS statistical software (SAS Institute Inc,
Cary, NC).
RESULTS
For the 17 patients (aged 42.0 ± 1.8 years; range, 33-63 years)
described in Table 1, the glycosylated
hemoglobin level, AGEs, and hemostatic factors were outside of the normal
range at baseline. Body weight, plasma viscosity, and serum lipids did not
change significantly during the study. The levels of glycosylated hemoglobin
and AGEs decreased significantly. Plasma factor VII and plasma fibrinogen
levels also decreased significantly. The mean plasma fibrinolytic activity
was 1.4 times normal at baseline, decreasing to 0.7 times normal at 12 months,
while the level of PAI-1 did not change significantly (P = .09) from baseline to 12 months. Figure 1 depicts changes in glycemic control and hemostatic factors
during the study for individual patients. Patients in whom glycemic control
improved markedly (a glycosylated hemoglobin decrease of greater than 2% [mean,
10.6%-7.8%; a 27% decrease; and a 75% decrease in AGEs]) demonstrated decreases
of 32% in plasma factor VII and of 24% in plasma fibrinogen concentration.
Patients in whom glycemic control did not improve markedly (a glycosylated
hemoglobin improvement of less than 0.5% and a 41% decrease in AGEs) demonstrated
smaller decreases in plasma fibrinogen (13%) and factor VII (19%). The plasma
factor VII analyses were affected by technical mishandling of 4 of 51 specimens
in the 17 patients. The results were virtually identical if we used the data
from the remaining 47 samples in 17 patients as opposed to 39 samples in 13
patients (for analysis of variance).
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Individual data over a 12-month period. A, Glycosylated hemoglobin.
B, Advanced glycosylated end products. C, Plasma fibrinogen. D, Plasma factor
VII. The dark lines and brackets indicate the mean and SEM; asterisk, P<.05; and dagger, P<.02.
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COMMENT
In our patients, the baseline levels of plasma factor VII, fibrinogen,
fibrinolytic activity, and whole blood viscosity were elevated; the level
of PAI-1 was decreased. During the study, the levels of glycosylated hemoglobin
and AGEs significantly decreased with intensive efforts to maintain normal
blood glucose. Mean levels of plasma fibrinogen and factor VII decreased significantly.
Because fibrinolytic potential was increased, the elevation of plasma fibrinogen
is best explained by acceleration of synthesis in the liver. Fibrinogen synthesis
is inhibited by the administration of insulin. In 3 reports,11, 12, 13
hyperfibrinogenemia associated with insulinopenia was corrected by insulin
repletion. Normal subjects challenged by hyperinsulinemia demonstrate a reversible
increase in fibrinogen synthesis.11 In patients
with type 1 diabetes mellitus, acute withdrawal of insulin with prompt replacement
caused fibrinogen synthesis to increase, then decrease.12
In insulin-dependent patients with type 2 diabetes mellitus, intensive control
of hyperglycemia over several weeks was associated with a reversible, accelerated
turnover of fibrinogen.13
Plasma factor VII levels have been shown to increase in normal subjects
following a meal14 or with a hyperglycemic-hyperinsulinemic
clamp.15 Despite improved glycemic control
with insulin therapy, lean patients with type 2 diabetes mellitus demonstrated
no significant decrease in elevated factor VII activity over 6 months.16 Insulin-dependent patients with diabetes who have
an elevated blood glucose,17 serum triglycerides,18 or urinary albumin19
level also demonstrate increased plasma concentrations of factor VII. We hypothesize
that control of glycemia over 6 to 12 months decreased pathological activation
of hepatic synthesis of plasma factor VII and fibrinogen.
At baseline in our study, fibrinolytic activity was elevated and PAI-1
was decreased. However, at 12 months, fibrinolytic activity had been significantly
reduced without a proportional increase in PAI-1. Excessive fibrinolysis has
also been demonstrated in a group of patients with type 1 diabetes mellitus
(with similar microvascular complications) to be enhanced, with no proportional
relation to tissue plasminogen activation.20
This reduction in fibrinolytic activity without participation of PAI-1 implies
the presence of an alternative pathway. Polymorphonuclear leukocyte elastase
has been suggested as an alternative pathway of fibrin degradation in patients
with a biologically activated state,21 such
as uncontrolled diabetes.22 It is possible
that catabolism23 or inflammation24
may signal activation of immediate-phase reactants, such as elastase, from
white blood cells increasing fibrinolytic activity. We hypothesize that control
of glycemia over 6 to 12 months decreased pathological activation of an alternative
pathway that was responsible for accelerated fibrinolysis.
Our results demonstrate that improvement of glycemic control in diabetic
patients with microangiopathic complications alters procoagulant factors favoring
arterial disease. Results of the multicenter study25
have demonstrated that control of blood glucose by 4 injections of rapid-acting
insulin per day was associated with a loss of creatinine clearance of 0.13
mL/s (7.7 mL/min) per year. Patients randomized to the same insulin schedule
plus pulsatile intravenous insulin therapy 1 day per week experienced a loss
of creatinine clearance of only 0.04 mL/s (2.2 mL/min) per year (P<.04). We consider that a program of intensive insulin therapy
that can normalize levels of glycosylated hemoglobin and AGEs in diabetic
patients with microvascular complications25
will eventually be shown to protect patients from cardiovascular injury by
attenuating the inflammatory cascade that stimulates excess fibrinogen production.26 While further studies will be needed to assess the
clinical impact of these findings on thromboembolic event rates, our findings
support the recommendation that tight glycemic control "will substantially
reduce the macrovascular complications of diabetes."27
AUTHOR INFORMATION
Accepted for publication July 11, 2000.
This study was funded by Advanced Metabolic Systems, New Haven, Conn.
From the John Cook Renal Unit, Joslin Diabetes Center, Boston, Mass
(Drs D'Elia, Weinrauch, and Roshan and Ms Keough); the Clinical Research Section,
Mount Auburn Hospital, Cambridge, Mass (Dr Weinrauch); the Institute for Prevention
of Cardiovascular Disease, Beth Israel Deaconess Medical Center, Boston (Drs
Lipinska and Tofler); the Biostatistical Section, Endocrine-Hypertension Unit,
Brigham and Women's Hospital, Boston (Dr Gleason); the Picower Institute for
Medical Research, Manhasset, NY (Dr Lee); and the Harvard Medical School,
Boston (Drs D'Elia, Weinrauch, Gleason, Lipinska, Pendse, Roshan, and Tofler).
Reprints: John A. D'Elia, MD, 1 Joslin Pl, Boston, MA 02215.
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