ACTA

Acta Diabetol 29: 78-81, 1992

DIABETOLOGICA 9 Springer-Verlag 1992

Fibrinogen and yon Willebrand factor in type II diabetes mellitus Isabella Catalano 1, Giovanni Davi z, Francesco Gennaro 1, Giuseppe Montalto 1, Giuseppina Marino 1, Carlo Giammarresi 1, Antonina Ganci ~, Anna Cal~ 1, and Alberto Notarbartolo 1 Patologia Medica II, Universitfi di Palermo, Palermo, Italy z Ematologia, Universitfi di Chieti, Chieti, Italy

Abstract. A hypercoagulable state m a y contribute to the formation of early vascular lesions in diabetes. The yon Willebrand factor is required for the attachment of platelets to the subendothelium; fibrinogen is required for platelet aggregation. This study was designed to assess in type II diabetic patients plasma levels of fibrinogen and von Willebrand factor to see if these variables are associated with platelet aggregation responses to adenosine diphosphate (ADP). Fibrinogen and the von Willebrand factor were significantly increased in diabetics but only fibrinogen was significantly related to platelet aggregation for ADP. Strict metabolic control does not reduce the increased concentrations o f these two proteins. Hyperfibrinogenaemia was related to the presence of macrovascular disease. Therefore measurements of plasma fibrinogen could be added to the cardiovascular risk factor p r o f l e of diabetic patients. Intervention studies are also needed to reduce the increased incidence of thrombotic diseases in patients with diabetes mellitus.

Key words: Diabetes mellitus - Fibrinogen - von Willebrand factor - Platelet aggregation

Introduction Diabetes mellitus is associated with early vascular complications. It has been suggested that a hypercoagulable state associated with metabolic disturbances [1] may contribute to vascular lesions. Blood platelets might initiate the formation of atheroma by attaching to denuded subendothelial structures. The von Willebrand factor (vWF) is required for the attachment of platelets to the subendothelium [2] and also has a key role in platelet aggregation at high shear stress. Because vWF-deficient pigs show a remarkable resistance to atherosclerosis, the possibe role of v W F as a risk factor in cardiovascular disease in man has been investigated [3]. Correspondence to." J. Catalano, Via S. Botticelli, 21, 1-90100 Paler-

mo, Italy

Fibrinogen is a major blood glycoprotein that plays an essential role in haemostasis and maintenance of blood viscosity. Fibrinogen is important for effective in vitro and in vivo platelet function as supported by studies on patients with G l a n z m a n n ' s thromboasthenia, which might help in explaining the thrombogenic potential of fibrinogen. Moreover, several epidemiological studies have indicated an independent association of cardiovascular mortality with fibrinogen levels [4-6]. This cross-sectional study was designed to assess plasm a levels of fibrinogen and v W F related antigen (vWF Ag) in diabetic patients in order to investigate whether these variables are associated with platelet aggregation response to adenosine diphosphate (ADP). In fact, diabetic patients show increased binding of fibrinogen to platelets and effective inhibition of binding to diabetic platelets corrects the hyperaggregable state [7, 8].

Subjects and methods One hundred and twenty-five patients with type II diabetes mellitus (69 women and 56 men; mean age 65.1 _+12.3 years). 40 young healthy volunteers (20 women and 20 men; mean age 29.5_+15.0 years) and 40 healthy subjects of equivalent age (18 women and 22 men; mean age 62.2_+ 13.4 years) were studied between June 1987 and October 1991 (Table 1). Type II diabetes was defined in accordance with the criteria of the American Diabetes Association [9]. All had non-insulin-dependent diabetes with a mean duration of 16.2 years (range: 7-26 years). Forty-nine patients had a history of and/or a clinical examination showing evidence of maerovaseular complications. Twenty-seven patients had stable angina pectoris or previous myocardial infarction, 16 had peripheral vascular disease and 6 had a history of previous episodes of stroke. Patients with coronary heart disease were in a stable phase, as judged on the basis of clinical symptoms, electrocardiographic monitoring during exercise, Holter monitoring, and echocardiographic observations. Patients with peripheral vascular disease were in Fontaine's stage II (intermittent claudication, ankle-arm pressure index < 0.85, and no resting pain), with a constant level of pain while walking. Their disorder had been diagnosed on the basis of clinical symptoms, ability to walk long distances, cycle ergometry, and Doppler echographic study of the lower limbs. Patients with cerebrovascular disease had a clinical history of and positive results on carotid Doppler testing and Doppler echography.

79

Isabella Catalano et al.: Fibrinogen and von Willebrand factor in type II diabetes mellitus Table 1. Summary of demographic data on subjects studied

Number Sex (M/F) Age (years) Duration of diabetes (years)

Young controls

Controls of equivalent age

Diabetes (all)

Diabetes without vascular disease

Diabetes with vascular disease

40 19/21 29.5 • 15.0 -

40 18/22 62.2 • 13.4 -

125 56/69 65.1 • 12.3 16.2_+ 9.5

76 32/44 60.3 • 12.5 12.2• 8.7

49 25/24 72.0 • 7.8 21.2•

Values are given as mean _+1 SD

They had relatively stable disease, with one or two episodes of transient ischaemia per month before they were admitted to hospital. In none of the patients had vascular disease progressed significantly during the previous 12 months, as established at outpatient visits. Moreover, all had mild-to-moderate symptoms compatible with a virtually normal life style. All patients continued cardiovascular drug therapy (beta-blockers, calcium-channel blockers, diuretics) during the study period and were also asked to abstain from aspirin-like drugs. At the time of the study, all patients were being treated with three daily injections of insulin (intermediate-acting and regular insulin).

Plasma von Willebrand factor antigen (%)

Study design

ACso for ADP (gM)

A cross-sectional comparison of plasma levels of fibrinogen and vWF was performed between patients and controls. Patients were admitted to hospital and fasted overnight. Before they received insulin, blood was drawn for measurement of plasma levels of fibrinogen and vWF. Paired measurements of these plasma proteins and ADP-indueed platelet aggregation were carried out in 62 diabetic patients while they were on standard therapy.

Table 2. Indexes in patients with type II diabetes

Plasma fibrinogen (rag/d1)

Statistical analysis The data were analysed by non-parametric methods to avoid assumptions on the distribution of the measured variables [15]. An analysis of variance was performed by the Kruskall-Wallismethod. Subsequent pairwise comparisons were made by the Mann-Whitney U test. All values are reported as mean • I SD. Statistical significance was defined as P < 0.05.

Controls of equivalent age n = 40

All diabetics (n=)

287 ___51

301 •

430 __122

(125)

103 •

138 • 47

(62)

0.76_+0.27

(0.62)

98 •

1.56___0.28 1.45•

Values are given as mean • 1 SD * P<0.001, ** P<0.005 versus controls of equivalent age

Table 3. Distribution of the parameters according to the presence

of vascular complications Diabetes with- (n=) out vascular disease

Diabetes with (n=) vascular disease

Plasma fibrinogen (rag/d1)

376 •

(76)*

512 •

(49)

Plasma von Willebrand factor antigen (%)

113 •

(34)*

174 •

(49)

ACso for ADP (gM)

0.85•

(34)**

0.61 •

(28)

Biochemical analyses Blood was drawn between 8 and 9 a.m. and collected in 3.8% sodium citrate (1 ml per 9 ml blood). Platelet-rich and platelet-poor plasma was prepared as previously described [10]. Platelet aggregation was measured in an Elvi 840 (Logos) aggregometer by Born's method [11]. Percent aggregation was determined assuming that platelet-poor plasma blank represented 100% aggregation and platelet-rich plasma blank represented 0% aggregation. Threshold aggregating concentration with ADP was defined as the lowest concentration of the agent that caused at least a 50% increase in light transmittance within 3 rain. Plasma fibrinogen was measured with a commercially available nephelometric assay (Behringwerke, Marburg, FRG). The normal range was 200-450 mg/dl. Plasma vWF Ag was measured by ELISA (Boehringer Mannheim, Mannheim, FRG). The normal range was 60-150%. The measurements were done without prior knowledge of the clinical diagnosis [12, 13]. Blood glucose levels were measured by a glucose oxidase method. Measurement of the extent of glycation of serum proteins, as determined by the reduction of alkaline nitro blue tetrazolium salts (fructosamine test), was used as an integrated glycaemic index, reflecting glycaemic control during the 2 weeks preceding blood analysis [14].

Young controls n = 40

Values are given as mean • 1 SD * P<0.001,

** P < 0 . 0 2

Results

F i b r i n o g e n a n d v W F A g were significantly increased in diabetic patients, with or w i t h o u t clinically evident vascular disease (Table 2). M o r e o v e r , platelets f r o m the p a t i e n t s with type II diabetes mellitus required significantly less A D P to aggregate. A m o n g the patients in w h o m paired m e a s u r e m e n t s were o b t a i n e d (n = 62), a strong a n d significant direct c o r r e l a t i o n (r = 0.62; P < 0.001) was f o u n d b e t w e e n A D P i n d u c e d platelet a g g r e g a t i o n a n d f i b r i n o g e n levels. T h e highest c o n c e n t r a t i o n s of f i b r i n o g e n a n d v W F Ag were f o u n d in the diabetic patients with evidence o f m a c r o v a s cular c o m p l i c a t i o n s (Table 3).

80

Isabella Catalano et al.: Fibrinogen and yon Willebrand factor in type II diabetes mellitus

Table 4. Distribution of plasma fibrinogen levels in type II diabetics with macrovascular complications Type of macrovascular complications

Mean + SD (mg/dl)

Range (mg/dl)

None (n=76) All with VD (n=49) CHD (n = 16) PMI (n=ll) CVD (n= 6) PVD (n = 16)

376_+_+ 94

240-693

512• 486 + 86" 483__+ 92* 556-t- 52* 543 + 151 *

303-765 320-651 370-633 490-634 303-765

Values are given as mean + 1 SD. VD, Vascular disease; CHD, stable angina pectoris; PMI, previous myocardial infarction; CVD, previous stroke; PVD, peripheral vascular disease. * P < 0.001 vs diabetics without macrovascular complications

Table 5. Fibrinogen levels in type II diabetics according to metabolic control

Strict metabolic control (n = 50) Poor metabolic control (n = 75)

Serum fructosamine (mmol/1)

Fibrinogen (mg/dl)

2.61 _ 0.13

437 • 99

*

NS 444 • 114

3.48 • 0.47

Values are given as mean • 1 SD * P<0.001; NS, not significant

As reported in Table 4, diabetic patients had elevated fibrinogen levels irrespective of the type of macrovascular complications. Patients with peripheral vascular disease and those with previous stroke had higher fibrinogen values. No differences in plasma fibrinogen and vWF Ag were observed in diabetics who achieved strict metabolic control (with significant reduction in serum fructosamine) compared diabetic patients in whom intensive insulin treatment did not result in better metabolic control (Table 5).

Discussion

The results of recent prospective studies ascribe a well-defined epidemiological relevance to the concept of hypercoagulability. As a matter of fact, increased plasma levels of factor VII and fibrinogen affect the natural history of stroke and coronary heart disease. They should thus be measured when evaluating thrombotic risk [16]. In our study plasma fibrinogen and vWF Ag levels were increased in diabetic patients and the differences become highly significant for diabetics with vascular complications, with only a slight increase in those with no vascular lesions. Moreover, the sensitivity of platelets to A D P correlates only with the plasma levels of fibrinogen, as previously shown for patients with ischaemic heart disease [17].

Previous observations suggested that haemostatic abnormalities exist prior to vascular disease [18, 19] and our results indicate that fibrinogen and vWF levels increase with the progression of macroangiopathy and are unrelated to defective metabolic control. vWF Ag found to be elevated in children with diabetes of short duration and with no vascular alterations [20]. However vWF Ag is not a useful parameter in predicting arterial thrombotic disease [21], because only extremely low vWF activity is protective against atherosclerosis. Therefore, even small amounts of vWF permit the interaction between platelets and endothelium, which initiates atherosclerosis. In our study hyperfibrinogenaemia was related to the presence of macrovascular disease, particularly to peripheral artery disease (PAD), as previously observed in PAD patients without diabetes [21]. This implies that high fibrinogen levels may greatly enhance the injurious effects of established risk factors in complications from atherosclerosis. Until a prospective study is carried out, it is not clear if high fibrinogen levels are simply the consequence of acute phase reactions. In fact, hyperfibrinogenaemia, (fibrinogen being an acute-phase protein), may be caused by diabetic microvascular disease [22], lipid metabolism alterations, environmental factors (e.g. smoking) and by genetic factors [23]. Hyperfibrinogenaemia was correlated with the increase of platelet sensitivity to ADP, as observed in diabetic patients whose platelets show increased fibrinogen binding to platelets [7, 8]. However, we have to consider that maximal aggregation of platelets is achieved in response to concentration of fibrinogen far below those with a predictive value for stroke or myocardial infarction. Apart from the fact that platelet aggregation is a function of plasma fibrinogen concentration, we must remember that fibrinogen is a determinant of blood viscosity, and that fibrinogen and fibrin are important constituents of atheroma [24]. A great deal of evidence points to a role for fibrinogen in various cardiovascular disorders. Our data indicate that plasma fibrinogen should be added to the cardiovascular risk factor profile of diabetic patients. A variety of different interventions have now reported lower plasma fibrinogen levels in humans [25]. However, our present knowledge precludes any definite statements about what effect might be expected from therapies targeted selectively against fibrinogen.

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Vol. 35, No. 7, 1992 Full instructions to authors A5 Review C. N. Hales, D.J.P. Barker Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis 595 Originals A. A. F.Sima, W.-X. Zhang, P.V. Cherian, S. Chakrabarti Impaired visual evoked potential and primary axonopathy of the optic nerve in the diabetic BB/W-rat 602

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A. Kowluru, R.A. Kowluru, A. Yamazaki Functional alterations of G-proteins in diabetic rat retina: a possible explanation for the early visual abnormalities in diabetes mellitus 624 K. C. Chiu, M.A. Province, G. K. Dowse, P. Z. Zimmet, G. Wagner, S. Serjeantson, M. A. Permutt A genetic marker at the glucokinase gene locus for Type 2 (non-insulin-dependent) diabetes mellitus in Mauritian Creoles 632 F. Shimada, Y. Suzuki, M. Taira, N. Hashimoto, O. Nozaki, H. Makino, S. Yoshida Abnormal messenger ribonucleic acid (mRNA) transcribed from a mutant insulin receptor gene in a patient with type A insulin resistance 639 A. Elamin, H. Altahir, B. Ismail, T. Tuvemo Clinical pattern of childhood Type 1 (insulindependent) diabetes mellitus intheSudan 645

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S. Lemieux, J.-P. Despr6s, A. Nadeau, D. Prud'homme, A. Tremblay, C. Bouchard Heterogeneous glycaemic and insulinaemic responses to oral glucose in non-diabetic men: interactions between duration of obesity, body fat distribution and family history of diabetes mellitus 653

A. Veves, H. J. Murray, M. J. Young, A. J. M. Boulton The risk of foot ulceration in diabetic patients with high foot pressure: a prospective study 660 E. Hagstr6m-Toft, P.Arner, U. Johansson, L. S. Eriksson, U. Ungerstedt, J. Bolinder Effect of insulin on human adipose tissue metabolism in situ. Interactions with betaadrenoceptors 664 G. Dahlquist, B. K~illen Maternal-child blood group incompatibility and other perinatal events increase the risk for early-onset Type 1 (insulin-dependent) diabetes mellitus 671 K. Osei, D.A. Cottrell, M. L. Henry, R. J. Tesi, R, M. Ferguson, T. M. O'Dorisio Minimal model analysis of insulin sensitivity and glucose-mediated glucose disposal in -type 1 (insulin-dependent) diabetic pancreas allograft recipients 676 J. Sturis, K. S. Polonsky, E. T. Shapiro, J. D. Blackman, N. M. O'Meara, E.Van Cauter Abnormalities in the ultradian oscillations of insulin secretion and glucose levels in Type 2 (non-insulin-dependent) diabetic patients 681 P. H. Hendriksen, P. L. Oey, G. H. Wieneke, B. Bravenboer, J. D. Banga Subclinical diabetic neuropathy: similarities between electrophysiological results of patients with Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes mellitus 690