Plasma fibronectin and platelet aggregation in diabetes mellitus

Diabetes Research and Clinical Pract~e, 2 (1986) 69-73

Elsevier

69

DRC 00071

Plasma fibronectin and platelet aggregation in diabetes mellitus Toyoshi Inoguchi, F u m i o U m e d a , Jun W a t a n a b e , Taro W a s a d a and Hiroshi Ibayashi The Thtrd Department of blternal Medicine. Faculty of Medicine, Kyushu University. Fukuoka 812. Japan

(Received 9 May 1985,accepted 10 February 1986)

Key words: Plasma fibronectin; Platelet aggregation; Diabetic vascular complications

Summary Plasma fibronectin (FN) is a high-molecular weight glycoprotein produced by endothelial cells, fibroblasts, and other mesenchymal cells. Plasma FN levels were measured in non-insulin-dependent diabetics (NIDDM) (n = 42) and compared with age-matched control subjects (n = 20). Plasma FN levels were significantly higher in the N I D D M patients (44.2 + 2.2 mg/dl, mean 4- SE) than in the control subjects (31.2 4- 2.2 mg/dl). In addition, the rate of platelet aggregation was studied in 23 of the 42 N I D D M patients. Interestingly, the plasma FN levels were significantly elevated, particularly in diabetic patients with enhanced platelet aggregation. It is suggested that elevated plasma FN may be closely related to the abnormality of platelet function in diabetics, which leads to diabetic vascular lesions.

Introduction FN is present mainly in connective tissue, basement membrane of the vascular wall and in plasma [1]. In the connective tissue, FN acts as an adhesive protein which connects the cells to extracellular matrix such as collagen substrates [2]. Plasma FN has been considered to be produced from vascular endothelial cells [3,4], fibroblasts [5-7], and other mesenchymal cells. It is known that physiologically, FN plays a stimulating role in fibrin cross-linking formation during blood clotting and wound healing process [8,9] in plasma. However, the role of plasma FN in vivo has not yet been completely established. Recent study has demonstrated that FN is found to deposit markedly in the mesangium and glomerular basement membrane in diabetic patients with

nephropathy [10]. It is suggested that plasma FN could be related to the pathogenesis of diabetic vascular lesions. In the present study, plasma FN levels and platelet aggregation rate in NIDDM patients were determined, and we discuss the relationship between plasma FN levels and abnormality in platelet function, which can lead to diabetic vascular complications.

Materials and methods Forty-two patients with non-insulin-dependent diabetes being observed at Kyushu University Hospital were studied in comparison with 20 age-matched control subjects. Clinical characteristics of the diabetic subjects are summarized in Table 1. Overnight

0168-8227/86/$03.50 © 1986ElsevierSciencePublishers B.V. (Biomedical Division)

7O TABLE 1 CHARACTERISTICS OF SUBJECTS" No.

Age (years)

Duration (years)

50.4

8.2

42tF14

5:1.9

4- 1.0

201M8 tFl9

46.4 +2.4

M28b Diabetics

Controls

FBS (mg/dl)

HbA~ (%)

Retinopathy

Proteinuria

Neuropathy

Therapy

Diet 14 Drug 10 Insulin 18

162

11.4

(+)23

(+)17

(+)29

+ 10.0

+0.6

(-)19

(-)25

(--)13

SE. b M. male: F, female a M e a n 5:

fasting whole blood was collected into a disposable tube with EDTA. Plasma was obtained immediately by centrifugation. These samples, with the addition of aprotinin at a final concentration of 40 U/ml, were stored at - 20°C until F N assay. Plasma F N was measured by Saba et al.'s immunoturbidimetric method [11]. The assay kit was obtained from Boehringer Mannheim Chemicals. For the study of platelet aggregation, whole blood containing sodium citrate at a final concentration of 0.38% was collected into siliconized plastic tubes. These samples were divided into 2 parts. One was centrifugated at 150 x g for 10 min to obtain plateletrich plasma (PRP). The other was centrifugated at 1500 x g for 15 min to obtain platelet-poor plasma (PPP). Platelet count of P R P was determined in a hemocytometer and was adjusted to 20 x 104 platelets//A using autologous PPP. Platelet aggregation was achieved using Born's turbidimetric method [12] with an Auto RAM-21 aggregometer (Rikadenki K o g y o Co., Tokyo). At 5 min after addition of collagen ( H o r m o n e Chemie, F.R.G.) into adjusted P R P at a final concentration of 1 /~g/ml, the platelet aggregation rate was determined as a percentage, assuming that the PPP represented 100% aggregation and the PRP represented 0% aggregation. Results

Plasma FN levels in N I D D M patients and control

subjects are shown in Fig. 1. Plasma F N levels were significantly elevated in diabetics (44.1 -4- 2.2 mg/dl, mean + SE) as compared with control subjects (31.2 -4- 2.2 mg/dl). There was no significant correlation between plasma F N levels and duration of diabetes, type of treatment, or fasting blood sugar and HbA1 value. Fig. 2 compares the plasma F N levels in diabetic subjects with and without diabetic complications, No significant differences were observed between the plasma FN levels of diabetics with and without retinopathy, persistent proteinuria or clinical neuropathy. Furthermore, the rate of platelet aggregation was studied in 23 diabetic subjects. As a result, platelet aggregation induced by collagen was significantly increased in diabetic 50

P
40

&

30

T

Z

U. ~

20

E m

10

C

D

Fig. I. Plasma FN levelsm NIDDM patients (D) compared with control subjects (C). Results are expressed as mean + SE.

71 Retmopathy

Protelnuna

Neuropathy

50

4o

E

3O z EL 2O ol

E_ 10 I

c

(-)

(+)

c

(-)

(+)

c

(-)

(+)

Fig. 2. Plasma FN levels in diabetic subjects with and without diabetic complicauons. Results are expressed as mean + SE. * P < 0.01; • * P < 0.05.

subjects (68 -J- 6%, mean ± SE) as compared with age-matched control subjects (39 ± 6%) [13]. The relationship between plasma FN levels and platelet aggregation rate is shown in Fig. 3. Interestingly, plasma FN levels become markedly elevated in diabetic patients with a platelet aggregation rate over 67%, which is the platelet aggregation rate (mean + 2 SD) induced by 1.0 #g/ml of collagen for control subjects in our laboratory. On the other hand, plasma FN levels in diabetics with a platelet aggregation rate of less than 66% did not significantly differ from controls.

v

Discussion Recently, it has been reported that marked FN deposits are found in the mesangium and glomerular basement membrane of diabetic patients with nephropathy. Stenman et al. [14] have indicated that FN deposits are also present in vascular atherosclerotic lesions and may play an important role in the pathogenesis of atherosclerosis. These studies suggest that FN is closely related to the pathogenesis of diabetic macro- or microangiopathy. Our study demonstrates that the plasma FN levels were

80

6O

e 60

40

P
i

z LL

~ 40

0

•

•

._T_

20

I

I

I

I

I

20

40

60

80

100

Platelet a~regatJon rate(%)

1

0

D

C

I

1

<66%

>67%

Fig. 3. Relationship between plasma FN levels and platelet aggregation rate. Results are expressed as mean + SE.

72

significantly higher in N I D D M patients both with and without diabetic complications, corresponding with the results of Davis [15]. This suggests that elevated plasma FN may cause diabetic vascular complications rather than result from vascular complications. It is possible that elevated plasma FN deposits in the vascular lesion stimulate the proliferation of mesenchymal cells [16] and the production of connective tissue matrix [17], subsequently leading to diabetic vascular complications. On the other hand, Alexander [18] reported a conflicting marked decrease in plasma FN levels in untreated or ketoacidotic diabetic subjects. However, the decrease in plasma FN may result from poor nutrition rather than from a diabetic metabolic abnormality. In the present study, ketoacidotic diabetic subjects were excluded. Significant elevation of plasma FN levels were observed in diabetic patients with platelet hyperfunction. It is noteworthy that elevated plasma FN levels can be closely related to platelet hyperaggregation, which is one factor leading to thrombus formation and acceleration of vascular lesions in diabetics [19,20]. Clearly, FN is present in platelet granules and is released during platelet aggregation [21]. Recent studies also suggested that FN could have an important role in modulating platelet function [22-25]. However, these data conflict as regards the relationship between plasma FN and platelet function. It is still unclear, therefore, whether elevated plasma FN activates platelet function or plasma FN elevation results from platelet hyperaggregation in diabetics. The present data necessitates further investigation of the mechanism of the relationship between elevated plasma FN and platelet hyperfunction in diabetics. It is concluded that elevated plasma FN levels in diabetic patients may be closely related to platelet hyperaggregation and play an important role in the pathogenesis of diabetic angiopathy. References 1 Matsuda, M., Yoshida, N., Aoki, N. and Wakabayashi, K., Distribution of cold-insoluble globulin in plasma and tissues, Ann. N.Y. Acad. Sci., 312: 74-92, 1978.

2 Klebe, R.J., Isolation of a collagen-dependent cell attachment factor, Nature, 250: 248-251, 1974. 3 Jaffe, E.A. and Mosher, D.F., Synthesis of fibronectin by cultured human endothelial cells, J. Exp. Med., 147: 17791791, 1978. 4 Macarak, E.J., Kirby, E., Kirk, T. and Kefalides, N.A., Synthesis of cold-insoluble globulin by cultured calf endothelial cells, Proc. Natl. Acad. Sci. U.S.A., 72: 2621-2625, 1978. 5 Baum, B., McDonald, J.A. and Crystal, R.G., Metabolic fate of the major cell surface protein of normal fibroblasts, Biochem. Biophys. Res. Commun., 79: 8-15, 1977. 6 Rouslaht, E. and Vaheri, A., Novel human serum protein from fibroblast plasma membrane, Nature, 248: 789-791, 1974. 7 Yamada, K.M. and Weston, J.A., Isolation of a major cell surface glycoprotein from fibroblasts, Proc. Natl. Acad. Sci. U.S.A., 71: 3492-3496, 1974. 8 Grinnell, F., Feld, M. and Minster, D., Fibroblast adhesion to fibrinogen and fibrin substrate; requirement for cold-insoluble globulin (plasma fibronectin), Cell, 19: 517-525, 1980. 9 Mosher, D.F., Fibronectin, Prog. Hemostas. Thrombos., 5: lll-151, 1980. l0 Weiss, M.A., Ooi, B.S., Ooi, Y.M., Engval, E. and Ruoslahti, E., Immunofluorescence localization of fibronectin in human kidney, Lab. Invest., 41: 340-347, 1979. 11 Saba, T.M., Albert, W.H., Blunenstock, F.F., Evanega, G., Staehler, F. and Cho, E., Evaluation of a rapid immunotubidimetric assay for opsonic fibronectin in surgical and trauma patients, J. Lab. Clin. Med., 98: 482-491, 1981. 12 Born, G.V.R., Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature, 194: 927-929, 1962. 13 Watanabe, J., Umeda, F., Wasada, T. and lbayashi, H., Effect of prostaglandin synthetase inhibitors on platelet aggregation and thromboxane production in diabetes mellitus, Tohoku J. Exp. Med., 144: 143-150. 1984. 14 Stenman, S., Smitten, K.U. and Vahei, A., Fibronectin and atherosclerosis, Acta Med. Scand., 642 (Suppl.): 165-170, 1980. 15 Davis, T.M.E., Plasma fibronectin concentrations in diabetes and the effect of short-term, improved glucose control, Diabetologia, 21: 262, 1981. 16 Peter, B.B., Stephen, I.R., Steven, A. and Ronald, G.C., Role of fibronectin as a growth factor for fibroblasts, J. Cell Biol., 97: 1925-1932, 1983. 17 Foidart, J.M., Berman, J.J., Paglia, L., Rennard, M.S.I., Abe, S., Perantoni, A. and Martin, G.R., Synthesis of fibronectin, lamin, and several collagens by a liver-derived epithelial cell line, Lab. Invest., 42: 525-532, 1980. 18 Allexander, C., Weiss, R., Kuman, D. and Nimni, M., Decreased plasma fibronectin in untreated diabetes mellitus, Diabetes, 30 (Suppl. 2): 3A, 1981. 19 Kwaan, H.C., Colwell, J.A., Cruz, S., Suwanwella, D.G. and Dobbie, J.G., Increased platelet aggregation in diabetes mellitus, J. Lab. Clin. Med., 80: 236-246, 1972.

73 20 Colwell, J.A., Raghavan, M.G.N., Halushka, P.V., Rogers, C., Wetsell, A. and Sagel, J., Platelet adhesion and aggregation in diabetes mellitus, Metabolism, 28: 394-400, 1979. 21 Zucker, M.B., Mossen, M.W., Brockman, M.,I. and Kaplan, K.L., Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation, Blood, 54: 8-12, 1979. 22 Arnesm, M.A., Hammerschmidt, D.E., Furcht, L.T. and King, R.A., A new form of Ehlers-Danlos syndrome. Fibro-

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23 Bensusan, H.B., Kah, T.L., Henry, K.G., Murray, B.A. and Culp, L.A., Evidence that fibronectin is the collagen receptor on platelet membrane, Proc. Natl. Acad, Sci. U.S.A., 75: 5864-5868, 1978. 24 Bannel, A.S., Inhibition of platelet aggregation by fibronectin, Biochem. Biophys. Res. Cornmun., 116: 135-140, 1983. 25 Houdijk, W.P.M. and Sixma, J.J., Fibronectin for platelet adhesion, Blood, 65: 598-604, 1985.