Hypofibrinolysis associated with vasculopathy in non insulin dependent diabetes mellitus

THROMBOSIS RESEARCH 59; 51-59,199O 0049-3848190 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

HYPOFIBRINOLYSIS

ASSOCIATED WITH VASCULOPATHY DEPENDENT DIABETES MELLITUS

IN NON

INSULIN

L.J. Garcia Frade, H. de la Calle, M.C. Torrado, J.I. Lara, L. Cuellar, A. Garcia Avello. Haematology and Endocrinology Departments, Ramon Y Cajal Hospital, Madrid Spain (Received 12.8.1989; accepted in revised form 1.4.1990 by Editor M. Verstraete)

ABSTRACT The pathogenesis of diabetic vasculopathy has been related to modifications in hemostasis and fibrinolysis. 50 non insulin dependent diabetes mellitus patients have been studied. Euglobulin clot lysis time, fibrin plate, tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI) activity, Protein C and S, cholesterol, triglycerides and Hb Ale were determined in Diabetic patients showed decreased blood samples. with fibrinolytic activity, as measured by ECLT, to clearly increased PA1 levels. Fibrinolytic response venous occlusion was lower than normal. Vascular complications were associated both with an even higher PA1 activity and with a decreased fibrinolytic response activity and venous occlusion. Elevated PA1 to stimulus may fibrinolytic response to decreased contribute to vascular disease in diabetes.

INTRODUCTION In diabetic patients approximately 75% of mortality is due to may be either vascular disease (1). This vascular damage lower limbs) macrovascular (central nervous system, heart, similar to atherosclerosis and related to age; or microvascular with diabetes duration. Some retina) associated (kidney, giving authors note the probable relation between both types, of the generic term of diabetic vasculopathy (2). Disorders blood viscosity and coagulation-fibrinolysis lipid metabolism, system have been implicated as causal agents of this vascular damage. Keywords: occlusion,

Fibrinolysis, vasculopathy.

Diabetes mellitus, 51

t-PA, PAI, venous

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A hypercoagulable state has been described in diabetes (3). Hyper-reactive platelets, elevated values of fibrinogen, factor VIII, Von Willebrand factor (VWF:Ag, RCoF), increased concentration of fibrin soluble complexes and fibrinopeptide A, are probably associated with the metabolic disturbance. tissue plasminogen activator In the fibrinolytic system, (tPA) produced by endothelial cells and its fast acting inhibitor seem to be the main mechanism regulating plasma (PA11 fibrinolytic fibrinolytic function. In diabetic patients activity has been described either as normal or decreased (4,5,6,7). More recently (8), it has been pointed out a clear insulin dependent increase of PA-inhibitor activity in non diabetes mellitus (NIDDM). probably through non-enzymatic control, Poor metabolic glycosylation, may disturb physiological properties of proteins fibronectin, membrane (fibrinogen, antithrombin III, fibrin, gl;gl;Eroteins! and lipoproteins (91, and contribute to vascular could . It is possible that a severe metabolic control reverse these alterations. The aim of this study was to evaluate the fibrinolytic system in NIDDM compared to normal controls and the relevance of this system to the presence of vasculopathy. MATERIAL AND METHODS Sub.iects; Fifty diabetic patients (26 women and 24 men) aged 45 to 74 (58.3426.43, mean+SD), all NIDDM, were studied. The mean 39 patients were duration of diabetes was 9.24k6.22 years. with treated with insulin (33.53k2.79 Units/day), 9 agents and 2 with diet. The extent of diabetes hypoglycemic control was assessed by measuring the percent of glycosylated haemoglobin (Hb Ale). Patients were examined for clinical signs and symptoms of vascular disease, including no peripheral pulse in lower limbs, claudicatio intermittens and rest pain (12 patients were positive). The inspection of the optic fundi was positive in 8 patients for background retinopathy. Following the WHO criteria 14 patients showed arterial hypertension. A group of 98 subjects, either normal laboratory staff or "fit" patients assessed before undergoing minor surgical procedures, matched for age and sex and with normal glucose concentration formed the control group. Materials; Blood samples were obtained from patients after fasting overnight, between 8 and 9 a.m., before receiving their insulin and after an observed supine rest for a minimun of 15 minutes. From the other arm blood was sampled after 10 minutes of venous stasis (100 mm Hg). The blood was collected into tubes containing 3.8% trisodium citrate (9 vol: 1 vol) and plateletpoor plasma (PPP) was obtained by centrifuging at 4000 x g for 15 minutes. The PPP was aliquoted and stored at -3OOC. In addition, serum and blood mixed with 3.2% sodium EDTA were

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collected for measurement of cholesterol, triglycerides and HbAlc. METHODS; Fibrinogen was measured by thrombin time assay (10); Protein C and S were determined by quantitative electroimmunoassay (11); tissue plasminogen activator (t-PA) was measured by an enzyme linked immunospecific assay (ELISA) described by Rijken et al polyvinyl plates were (12) with the exception that 96-well coated with goat IgG antibodies to t-PA and stored at -4OOC. Each subsequent assay step was confined to a 1 hour incubation at 37OC; plasminogen activator-inhibitor (PAI) activity was estimated using the technique described by Speisser (13); euglobulin clot lysis time was determined according to Nilsson and Olow (14); A fibrin plate was done according to Haverkate and Traas (15) using agarose (2.5%) and human fibrinogen (10.0 mg/ml). The fibrinogen used in these experiments contained sufficient plasminogen (0.4 Units/mg of clottable fibrinogen); Glycosylated (HbAlc) was measured by haemoglobin high performance liquid chromatography; cholesterol and triglycerides were determined using a Hitachi autoanalyzer. Data analysis; Values are presented as the mean?m.s.e. The significance of differences between the mean values was evaluated using the Student's t-test. Correlation coefficients were calculated using the Pearson test. RESULTS The concentration of HbAlc (7.98+0.28 %) was higher than the normal limit range (5.7%); cholesterol (237.3726.98 vs.200.06~5.05 mg/dl) (pt0.001) and triglycerides (140.89813.34 vs.120.53'3.03 mg/dl)(p<0.05) were increased compared to control group. While HDL-cholesterol (43.5021.72 vs. 40.07+1.01 mg/dl) was not significantly, increased there were elevated levels of LDL-cholesterol (167.9126.22 vs. 125.08k2.52 mg/dl) (ptO.OO1). TABLE 1 Fibrinolytic Parameters in Diabetic Subjects and Controls NIDDM Euglobulin clot lysis time (hr) 15.60+0.90 t-PA (IU/ml) 3.1920.35 PA1 (All/ml) 45.1323.42 Protein C (%) 117.02k5.26 Protein S (%) 136.48'7.02

Control 12.3920.54 2.6520.16 30.4022.40 112.28t3.40 115.3554.03

of overall The euglobulin clot lysis time as a measure fibrinolytic activity was longer compared to the controls The t-PA antigen (15.60+0.90 vs. 12.3920.54 hr.) (p
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decreased (p
NIDDM:PAIactivity PA1 (AU/ml)

-

T

-I-

FIG.1 PAI activity (AU/ml) in presence(+) or absence (-) of peripheral vasculopathy (44.96213.38 vs. 45.1823.39) (n.s. ), 41.16f3.81) retinopathy (p
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Meanwhile, the release of plasminogen activators was lower in patients with peripheral vasculopathy, retinopathy and hypertension than in diabetics without these complications, with a high degree of significance (Fig 2). There was no correlation between t-PA release and PAI level either in the whole group or in each subgroup taken separately.

Venous occlusion test: t-PA increase t-PA (IU/ml) incr.

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+ V.D,

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FIG 2 release after venous occlusion (ILl/ml), in presence (+) or 1.15kO.24) absence (-) of peripheral vasculopathy (0 vs. 1.2950.28) (p
t-PA

Non insulin dependent diabetic patients showed a low level of elevated t-PA antigen and with clearly fibrinolytic activity, These findings are similar to the results PAI activity. with low t-PA antigen reported by Auwerx (8). Increased fibrinolytic activity may be explained by the existence of inactive t-PA/PA1 complexes.

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In patients with retinopathy or hypertension PA1 levels were increased. There is a basal inhibition of fibrinolysis that is higher in the presence of vascular complications. A poor fibrinolytic response to stimulus has previously been there is an abnormal regulation of reported in diabetes, of t-PA in the endothelium (16) and again in synthesis/release our study it is worse in diabetic patients with complications. So that, in an hypercoagulable state such as diabetes (17, 181, resulting either from an increased basal PAI hypofibrinolysis activity or from insufficient release of t-PA, may contribute to the increased risk of thrombosis. well known Elevated blood levels of proteins C and S, inhibitors of blood coagulation and implicated in fibrinolysis activation, have been previously reported for protein C (19). The relevance of the slight increase is not clear. factors Hypofibrinolysis may be one of the major contributing together with hyperviscosity and disorders of lipid metabolism, in the development of diabetic vasculopathy. Although the role of hypofibrinolysis in atherosclerosis and thrombosis has not been completely revealed. there is recent information about the importance of PA1 activity: PA1 is increased in coronariopathy infarction (20, 21) and seems to be a risk and myocardial factor for recurrent myocardial infarction (22). its Non-enzymatic glycosylation of proteins may damage It has been suggested that there is a structure and function. probable relationship between metabolic control and changes in insulin seems to be more effective than hemostasis. Human VWF, AT-III and alpha 2 porcine in the improvement of VIIIC, antiplasmin (23). Nevertheless, the increase of insulin concentration correlates positively both with PA1 synthesis by hepatocytes (24) and with PA1 plasma levels (25). Otherwise well-controlled glucaemia has been associated with decreased t-PA concentration (26). Therefore, it seems not clear that a good metabolic control allows a return to normal fibrinolysis. Until now, probably due to the difficulty of restoring physiological control, it remains to be proved that vasculopathy and fibrinolysis abnormalities are strictly related to metabolic control. Acknowledgements. This work has been supported by grant 89/0166 of "Fond0 de Investigaciones Sanitarias de la Seguridad Social". We are indebted to Mrs I. Martinez de Tejada and Mr M. Perez Pascual for their technical cooperation.

1.

2.

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