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Fibrinolysis in insulin-dependent diabetic patients with and without nephropathy
Fibrinolysis (1996) 10(5/6), 331-335 © PearsonProfessionalLtd 1996
Fibrinolysis in insulin-dependent diabetic patients with and without nephropathy B. Myrup ~, P. Rossing ~ , T. Jensen ~, J. Gram 2, C. Kluft 3, J. Jespersen 2 1Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. 2Institute for Thrombosis Research, South Jutland University Center, Esbjerg, Denmark. ~Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
Summary Objective: To investigate possible contributions from fibrinolytic disturbances to susceptibility for cardiovascular complications in insulin-dependent diabetic patients with nephropathy. Design: Cross-sectional study. Setting: Outpatient diabetic clinic in a tertiary hospital. Subjects: Insulin-dependent diabetic patients without diabetic nephropathy (normoalbuminuria, n = 17), patients with incipient diabetic nephropathy (n = 19), and patients with diabetic nephropathy (n = 13). Non-diabetic subjects served as a control group (n = 14). Results: Tissue-type plasminogen activator antigen and plasminogen activator inhibitor type 1 were lower in diabetic patients, irrespective of the level of albuminuria. Tissue-type plasminogen activator activity measured in acidified plasma by bioimmunoassay was increased in normoalbuminuria, while the level in diabetic nephropathy was not different from a non-diabetic control level, and was significantly lower than in the normoalbuminuric group. D-Dimer and soluble fibrin were elevated in the pooled group of diabetic patients and were not influenced by presence of nephropathy. The level of plasmin-o~2-antiplasmin complexes was elevated to the same extent in each group of insulin-dependent diabetic patients. Conclusion: The data suggest increased fibrinogen turnover in insulin-dependent diabetes mellitus, irrespective of the presence of nephropathy. In normoalbuminuric patients, an increase in plasminogen activation could be explained by increased activity of tissue plasminogen activator, while other explanations, e.g. fibrin accumulation, probably connected to developing atherogenesis, could be hypothesized in patients with nephropathy.
INTRODUCTION Accelerated atherosclerosis with ischemic symptoms and thromboembolic complications is a major problem in long-term management of insulin-dependent diabetes mellitus (IDDM). Thirty to forty percent of patients with IDDM develop diabetic nephropathy, characterized by increased urinary excretion of albumin, an increase in blood pressure, and decreasing kidney function. The patients with diabetic nephropathy have an extremely Received 26 August 1996 Accepted after revision 2 November 1996 Correspondence to: Dr Bjarne Myrup, Centralsygehuset i Holbaek, DK-4300 Holbaek, Denmark, Tel. +45 3131 1272; Fax. +45 5343 7603.
elevated risk for developing cardiovascular disease, 1'2 and this has led to the hypothesis that albuminuria, besides being a sign of nephropathy, also reflects widespread vascular damage.2'3 An important function of endothelium is the synthesis and release of tissue type plasminogen activator (t-PA), an essential component of the fibrinolytic system. Recent studies have shown that the fibrinolytic activity in plasma from diabetic subjects is increased. 4-6 Further elaboration has shown increased activity of t-PA4 and decreased plasminogen activator inhibitor (PAI) activity and antigen. 5,6 On the other hand, PAI activity has been found increased in IDDM,7,8 and the activity of t-PA reduced. 8 In a previous study, we have evaluated the fibrinolytic system in IDDM patients with diabetic nephropathy and found decreased level and 331
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activity of t-PA in IDDM patients with and without diabetic nephropathy. 9 In the present study, we have extended our investigations on the fibrinolytic system in groups of well characterized IDDM patients in order to try to associate possible deviations in markers of fibfinolysis and vascular wall function with the enhanced risk of cardiovascular disease in IDDM. MATERIALS AND METHODS Subjects Forty-nine patients with IDDM were included in the study. The patients were recruited from the outpatients clinic at the Steno Diabetes Center, Denmark. Patients in the age range from 18 to 60 years were allocated to three groups based on the median albumin excretion rate of three 24h urine samples. Normoalbuminuric patients were only included if the duration of diabetes was between 10 and 30 years, in order to match the duration of diabetes in patients with incipient and diabetic nephropathy. Patients receiving any other medical treatment than insulin and patients with non-diabetic kidney disease were excluded. A group of non-diabetic agematched subjects recruited from the medical staff and surroundings served as a control group. The study protocol was approved by the local ethical committee. Patients and non-diabetic control subjects were enrolled after giving informed consent. Clinical information on IDDM patients and non-diabetic control subjects included are given in Table 1. Diabetic polyneuropathy was present in one normoalbuminuric patient, in two patients with incipient nephropathy, and in one patient with diabetic nephropathy. One patient with incipient nephropathy
had a previous attack of transient cerebral ischemia. One patient with incipient nephropathy suffered from claudication. Investigation protocol Blood samples were obtained in the fasting state after 30 min of supine rest between 0800 and 0900. Following minimal stasis, a 17-gauge needle was placed in an antecubital vein. The first 10 ml of blood was not used. In an identical sequence for each patient, blood was obtained to prepare the following plasma samples: EDTA plasma (0.1 ml 0.5 mg/ml D-phenylalanyl-l-propyl-l-arginine chloromethylketon and 0.12 ml 0.34 mol/l EDTA to 10 ml blood) for the analysis of t-PA and PAId antigen, DDimer, prothrombin fragment 1+2, and PAP-complexes; citrate plasma (0.5ml 0.129mol/1 Na-citrate to 4.5m1 blood) for the analysis of PAI activity, soluble fibrin, and thrombomodulin; acidified plasma (Stabilyte, Biopool, Umea, Sweden) for the analysis of t-PA activity to reduce ex vivo complex formation with PAI-1;I° and EDTAblood (0.054ml 0.134mol/1 EDTA to 4.5ml blood) for the analysis of HbAlc. The blood samples were placed on ice immediately after sampling and remained on ice for 30 rain. After centrifugation at 2000g for 20 min at 4°C, specimens of plasma were stored in polypropylene tubes at -40°C until analysis. EDTA-blood for HbAlc analysis was stored at 4°C until analysis. After blood sampling 74 kBq ~25I-albumin was injected. Blood (100 IU heparin [Leo Pharmaceuticals, Denmark] to 10 ml blood) for measurement of transcapillary escape rate of albumin (TER) was collected after 10, 15, 20, 30, 40, 50, 55, and 60 rain. Blood was centrifuged at 2000g for 10 rain and plasma specimens of 2 ml in duplicates
Table 1 Clinical characteristics of control subjects (control) and insulin-dependent diabetic patients Insulin-dependent diabetic patients Urinary albumin excretion (mg/24 h)
Control < 30
n (women/men) Age (y) Diabetes duration (y) Albuminuria (mg/24-h) GFR* (ml/min) TER** (%/h) Retinopathy . . . . Insulin dose (IU/kg/day)
5/9 40 (25-50) -9 (3-30) -6.4 (2.7-10.6)
HbAlc (%) Blood glucose (mmol/I) BMI (kg/m 2) Trigiycerides (mmoi/I)
. -5.1 (4.5-5.5) 4.6 (4.1-5.0) 23.9 (18.6-28.7) 1.35 (0.39-4.11)
< 30
30-300
> 300
11/6 41 (23-50) 19 (13-31 ) 13 (4-30) 108 (92-137) 7.0 (4.5-9.9) 3/1 0.62 (0.36-0.93) 8.2 (6.1-10.9) 10.4 (3.8-20.5) 23.8 (19.1-28.5) 0.79 (0,52-1.10)
4/15 39 (24-56) 24 (9-47) 140 (44-246) 108 (76-152) 8.6 (3.8-14.3) 11/5 0.62 (0.27-1.01) 8.9 (6.1-11.4) 8.4 (2.7-16.4) 24.8 (20.5-37.2) 1.06 (0.51-2.37)
7/6 38 (25-54) 24 (14-37) 1126 (315-2456) 102 (54-136) 8.8 (6.2-14.2)*** 8/5 0.67 (0.43-1.12) 9.3 (7.6-10.6) . . . . . 11.1 (4.1-21.2) 23.7 (19.5-27.0) 1.28 (0.46-3.79)
Results are given as mean (range). *Glomerular filtration rate. **Transcapillary escape rate. ***P< 0,05 vs. insulin-dependent diabetic patients with albumin excretion < 30 mg/24 h. . . . . Retinopathy as number of patients with simplex retinopathy/proliferative retinopathy. * . . . . P< 0.01 vs. insulin-dependent diabetic patients with albumin excretion < 30 mg/24 h.
Fibrinolysis (1996) 10(5/6), 331-335
© Pearson Professional Ltd 1996
Fibrinolysis in insulin-dependent diabetic patients
were counted on a Cobra auto-gamma counter (Packard Instrument Inc., Downers Grove, Illinois 60515, USA). TER was calculated on logarithmical transformed radioactivity in plasma as the slope of decrease in radioactivity expressed as percentage per h. Finally, 3.7 MBq 5~Cr-EDTA was injected. Blood (100 IU heparin [Leo Pharmaceuticals, Denmark] to 10 ml blood) was collected after 180, 200, 220, and 240 min for the measurement of glomerular filtration rate. Glomerular filtration rate was calculated as described previously, 11 and corrected for body surface. Assays Urine albumin was measured by ELISA,12 and urinary albumin excretion rate was calculated. HbAlc was measured by a chromatographic technique. 13 Fasting blood glucose was measured bed-side with a One-Touch apparatus (Life Scan Inc., Milpitis, California, USA). The following parameters were measured on the above specified plasma samples, by use of commercial ELISA kits: t-PA antigen (Immulyse t-PA, Biopool); TM t-PA activity (Coatest, BIA t-PA, Chromogenix, S-43153 M61ndal, Sweden); 1° PAI-1 (TintElize PAI-1, Biopool); 15 PAI activity (Spectrolyse (131) PAI, Biopool); 16'17 soluble fibrin (Fibrinostika, Soluble Fibrin, Organon Teknika, The Netherlands); 18 D-Dimer (Coaliza, Chromogenix); 19 prothrombin fragment 1+ 2 (Enzygnost F 1+ 2, Behringwerke AG, Marburg, Germany);2° plasmin-c~2antiplasmin (PAP) (EIAAPP micro, Behringwerke AG, Marburg, Germany);2~ and thrombomodulin (Amersham Thrombomodulin, Stage, France). 22 Statistics Differences between non-diabetic subjects and normoalbuminuric patients with IDDM, and differences
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between IDDM patients with diabetic nephropathy and normoalbuminuric IDDM patients were analysed by the Mann-Whitney rank sum test. This was done in order to describe the effect of IDDM per se and the effect of present nephropathy, respectively. Correlation was analysed by Spearman rank correlation test. Linear regression was done using a Statgraphics 5.0 package (STSC Inc, Rockville, MD, USA). RESULTS A lower concentration of t-PA antigen in IDDM patients compared with non-diabetic subjects was demonstrated (Table 2). This was not influenced by the presence of diabetic nephropathy. The activity of t-PA was increased in normoalbuminuria compared with non-diabetic controls, and patients with diabetic nephropathy had a significantly lower t-PA activity than normoalbuminuric patients (Table 2). PAI-1 antigen was significantly decreased in all IDDM groups compared with non-diabetic controls (Table 2). This was independent of presence of diabetic nephropathy. The t-PA activity was negatively correlated with the level of PAI-1 antigen (r=-0.47, P= 0.0002). Results concerning soluble fibrin and D-Dimer are shown in Table 2. Soluble fibrin was elevated in IDDM with normoalbuminuria, and there were no differences between groups with different levels of albuminuria. Although soluble fibrin and D-Dimer were not elevated when results from individual groups were compared with non-diabetic controls (save soluble fibrin in normoalbuminuric IDDM patients), when data from all three groups were pooled, both levels of soluble fibrin and D-Dimer were significantly increased above control (mean 4.1 range (1.9-14.0) mg/1 vs. 3.1 (1.8-7.3) mg/1, P< 0.05, and 88 (33-260) gg/1 vs. 59 (26-101) gg/1, P<0.05, respectively). A procoagulant activity expressed as elevated prothrombin fragment 1+ 2 was found in all groups of IDDM
Table 2 Tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAl) type 1 antigen, plasminogen activator inhibitor activity, soluble fibrin, D-Dimer, prothrombin fragment 1+2 (F1+2), and c~2-antiplasmin-plasmin complex (PAP) in insulin-dependent diabetic patients and non-diabetic controls Insulin-dependent diabetic patients Urinary albumin excretion (mg/24 h)
Control < 30
< 30
30-300
> 300
t-PA antigen (ng/ml) t-PA activity (IU/ml) PAl type 1 antigen (ng/ml) PAl activity (IU/ml) Soluble fibrin (rag/I) D-Dimer (IJg/I) F1 +2 (nmol/I) PAP (IJg/I) Thrombomodulin (ng/ml)
5.4 (2.1-9.6) 0.55 (0.03-2.20) 11.0 (3.4-29.8) 15.8 (0-62.8) 3.1 (1.8-7.3) 59 (26-101 ) 0.75 (0.39-1.47) 276 (106-533) 31 (15-55)
3.3 (1.2- 5.5)* 0.67 (0.36-3.30)** 4.2 (2.3-7.0) .... 6.0 (0-20.8) 4.2 (1.9-7.9)* 78 (35-229) 1.14 (0.73-2.05)** 448 (203-982)* 30 (13-66)
4.2 (1.4-12.9)** 0.61 (0.28-3.00) 5.9 (1.1-16.7)* 3.2 (0-12.9)* 3.7 (1.9-10.0) 91 (46-260) 1.06 (0.3-2.45)** 468 (258-920)* 35 (18-60)
3.4 (1.2-8.7)** 0.50 (0.21-1.20)*** 5.8 (2.7-10.3)** 7.5 (0-15.8) 4.4 (2.2-14.0) 78 (33-147) 1.07 (0.5-1.73)* 400 (227-595)* 44 (27-65) ......
Results are mean (range), *P< 0.01 vs. control, **P< 0.05 vs. control, ***P< 0.05 vs. IDDM patients with urinary albumin excretion < 30 mg/(24 h), ****P< 0.001 vs. control.
© Pearson Professional Ltd 1996
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patients irrespectively of the level of albuminuria (Table 2). TER was elevated in diabetic nephropathy compared with normoalbuminuria, while a tendency was present in incipient nephropathy (Table 1). Thrombomodulin was increased in patients with diabetic nephropathy compared with any other group (Table 2). The level of thrombomodulin correlated positively with HbAic (r=0.43, P = 0.002), and negatively with glomerular filtration rate (only IDDM patients, r=-0.32, P=0.04). In a multiple regression model, HbA1c turned out to be the major confounder when measuring thrombomodulin in the present subjects, while glomerular filtration rate became insignificant. Consequently, thrombomodulin values were corrected for HbAlc by linear regression, but the difference between normoalbuminuric patients and patients with diabetic nephropathy was sustained (P= 0.02). There was no relation between TER and t-PA activity, D-Dimer, or PAP. DISCUSSION
The divergence in the literature concerning fibrinolysis in IDDM m a y be due to differences in selection of patients regarding diabetes duration, metabolic control, and complication status. In this and a previous study 9 we have studied the fibrinolytic system in relation to presence of diabetic nephropathy. The present results confirm reduced t-PA antigen and PAI-1 antigen in IDDM, and we demonstrated higher t-PA activity compared with a non-diabetic control level. The discrepancy between the present and our previous results 9 is explained by the recently introduced method which diminishes ex vivo complex formation of t-PA with PAI-110 and has led to a much higher measurable level of t-PA activity. Elevation of t-PA activity was only present in normoalbuminuric patients, while patients with diabetic nephropathy had a significantly lower level of t-PA activity compared with the normoalbuminuric group. In a similar study, comparable levels of t-PA antigen were reported in IDDM patients, 23 but, in contrast to our study, no difference between IDDM patients and control subjects was found. The discrepancy could be explained by differences in basic parameters of the control group since our control group was age matched. We could not confirm increasing levels of PAI activity in groups of patients with albuminuria, as was demonstrated in this otherwise comparable study. 23 In both studies there was a tendency for higher levels of triglycerides in IDDM patients with albuminuria than patients without, and PAI activity related to triglyceride in our study (all subjects: r=0.43, P<0.O01; only IDDM patients: r=0.37, P<0.O01). Thus, triglyceride levels could be a confounder in the relation between PAI activity and albuFibrinolysis (1996) 10(5/6), 331-335
minuria. We found elevated thrombomodulin in diabetic nephropathy, and this has been suggested as a marker of endothelial dysfunction. 24 Thrombomodulin is located at the endothelial surface, where it is involved in the release of t-PAY Therefore, a possible explanation of lower t-PA activity in diabetic nephropathy compared with normoalbuminuric patients may be loss of throxnbomodulin from the endothelial location. However, no relation was found to elucidate such a mechanism. It may seem a paradox that t-PA activity in diabetic nephropathy was decreased compared with normoalbuminuric patients, while no difference in PAP and D-Dimer was seen between the two groups. Since the procoagulant activity measured as prothrombin fragment 1+ 2 was similar in IDDM patients with and without nephropathy, this should also hold for fibrin production. Thus, a lower plasma t-PA activity in diabetic nephropathy may resuk in fibrin accumulation stimulating plasminogen activation by t-PA. 26 Fibrin accumulation will therefore increase until the localized t-PA activity is high enough to obtain fibrin homeostasis. In steady state conditions this will result in D-Dimer levels which reflect the formation of fibrin, and PAP levels which reflect the rate of plasminogen activation. According to this hypothesis, homeostasis in diabetic nephropathy is reached at a higher level of fibrin accumulation. Since fibrin degradation products have been shown to exert a mitogenic effect,28 fibrin accumulation could contribute to smooth muscle cell proliferation observed in atherogenesis. 27 A higher level of fibrin accumulation related to the vascular wall could occur in the development of atherosclerosis 2s linked to this process by a mitogenic effect of fibrin degradation products. 29 In conclusion, the data suggest an increase in fibrinogen turnover in IDDM. In normoalbuminuric IDDM patients, an increase in plasminogen activation could be explained by the increased activity of t-PA, while other explanations, e.g. fibrin accumulation probably connected to developing atherogenesis, could be operative in patients with nephropathy.
REFERENCES
1. Borch-JohnsenK, KreinerS. Proteinuria - a predictor of cardiovascularmortality in insulin-dependent diabetes mellitus. Br MedJ 1987; 294: 1651-1654. 2. Jensen T, Borch-JohnsenK, Kofoed-EnevoldsenA, DeckertT, Coronary heart disease in young type 1 (insulin-dependent) diabetic patients with and without diabetic nephropathy: incidence and risk factors. Diabetologia 1987; 30: 144-148. 3. DeckertT, Feldt-RasmussenB, Borch-JohnsenK,Jensen T, Kofoed-EnevoldsenA. Albuminuriareflectswidespreadvascular damage. The Steno hypothesis. Diabetologia 1989; 32: 219-226. 4. WieszorekI, Pell A C H, McIverB, MacGregorI R, Ludlam C A, FrierB M. Coagulationand fibrinolyticsystems in type I diabetes: effectsof venous occlusion and insulin-induced hypoglycaemia.Clin Sci 1993; 84: 79-86. © Pearson Professional Ltd 1996
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5. Ostermann H, Tsch6be D, Greber W, Meyer-Rfisenberg H-W, van de Loo J. Enhancement of spontaneous fibrinolytic activity in diabetic retinopathy. Thromb Haemost 1992; 68: 400-403. 6. Walmsley D, Hampton K K, Grant P J. Contrasting fibrinolytic responses in type 1 (insulin-dependent) and type 2 (non-insulindependent) diabetes. Diabetic Medicine 1991; 8: 954-959. 7. Small M, Kluft C, MacCuish A C, Lowe G D O. Tissue plasminogen activator inhibition in diabetes mellitus. Diabetes Care 1989; 12: 655-658. 8. Carmassi F, Morale M, Puccetti R, De Negri F, Moi]zani F, Navalesi RI Mariani G. Coagulation and fibfinolytic system impairment in insulin dependent diabetes mellitus. Thromb Res 1992; 67: 543-654. 9. Jensen T, Bjerre-Knudsen J, Feldt-Rasmussen B, Deckert T. Features of endothelial dysfunction in early diabetic nephropathy. Lancet 1989; 1:461-463. 10. Meijer P, Boon R, Jie A F H, Ros~n S, Kluft C. BioImmunoassay for tissue-type plasminogen activator (t-PA) in human plasma. Evaluation of blood sampling and handling procedures and comparisons with other t-PA activity methods. Fibrinolysis 1992; 6, Suppl 3: 97-99. 11. Br6chner-Mortensen. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest 1972; 30: 271-274. 12. Feldt-Rasmussen B, Dinesen B, Deckert M. Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy. Scand J Clin Lab Invest 1985, 45: 539-544. 13. Svendsen P A, Chtistiansen J S, Soegaard U, Welinder B S, Nerup J. Rapid changes in chromatographically determined haemoglobin Ale induced by short-term changes in glucose concentrations. Diabetologia 1980; 19:130-136 14. R ~ b y M, Bergsdorf T, Nflsson G, Mellbring, Winblad B, Bucht G. Age dependence of tissue plasminogen activator concentrations in plasma as studied by an improved enzymelinked Lrnmunosorbent assay. Clin Chem 1986; 32:160-165. 15. Declerck P J, Alessi M C, Verstreken M, Kmithof E K O, JuhanVague I, Collen D. Measurement of plasminogen activator inhibitor in biological fluids with a murine monoclonal antibody based enzyme-linked immunosorbent assay. Blood 1988; 71 : 220-225. 16. Chmielewska J, R~nby M, Wiman B. Evidence for a rapid inhibitory tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436.
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17. Eriksson E, ~ n b y M, Gyzander E, Risberg B. Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single point poly-D-lysine stimulated assay. Thromb Res 1988; 50: 91-101. 18. Koopman J, Haverkate F, Koopert P, Nieuwenhuizen W, Brommer E J P, Van der Werf W G C. New enzyme immunoassay of fibrin-fibrinogen degradation products in plasma using a monoclonal antibody. J Lab Clin Med 1987, 109: 75-84. 19. Declerck P J, Mombaerts P, Holvoet P, De Mol M, Collen D. Fibrinolytic response and fibrin fragment D-Dimer levels in patients with deep vein thrombosis. Thromb Haemost 1987; 58: 1024-1029. 20. Pelzer H, Schwarz, Stfiber W. Determination of human prothrombin activation fragment 1+ 2 in plasma with an antibody against a synthetic peptide. Thromb Haemost 1991; 65: 153-159. 21. Pelzer H, Pilgrim A, Schwarz A, Merte D, Keuper H, Hock H. Determination of c~2-antiplasmin-plasmin complex in human plasma with an enzyme-linked immunosorbent assay. Fibrinolysis 1993; 7: 69-74. 22. Amiral J, Adam F, Mimilla F, Chambrette B, Boffa M C. A new assay for soluble forms of thrombomodulin in plasma. Thromb Haemost 1991; 65: 947, 896 (Abstract). 23. Mahmoud R, Raccah D, Alessi M C, Aillaud M F, Juhan-Vague I, Vague P. Fibrinolysis in insulin dependent diabetic patients with or without nephropathy. Fibrinolysis 1992; 6:105-109. 24. Takahashi H, Ito S, Hanano M, Wada K, Niwano H, Seki Y, Shibaba A. Circulating thrombomodulin as a novel endothelial cell marker: comparison of its behaviour with von Willebrand factor and tissue-type plasrrdnogen activator. Am J Haematol 1992; 41: 32-39. 25. Bourin M-C, Lindahl U. Glycosaminoglycans and the regulation of blood coagulation. Biochem J 1993; 289:313-330. 26. Munkvad S. Fibrinolysis in patients with acute ischaemic heart disease. Dan Med Bull 1993; 40: 383-408. 27. Stirk C M, Kochhar A, Smith E B, Thompson W D. Presence of growth-stimulating fibrin degradation products containing fragment E in human atherosclerotic plaques. Atherosclerosis 1993; 103: 159-169. 28. Falk E, Fernandez O A. Role of thrombosis in atherosclerosis and its complications. AmJ Cardiology 1995; 75:3B-1 lB.
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Fibrinolysis in insulin-dependent diabetic patients with and without nephropathy B. Myrup ~, P. Rossing ~ , T. Jensen ~, J. Gram 2, C. Kluft 3, J. Jespersen 2 1Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. 2Institute for Thrombosis Research, South Jutland University Center, Esbjerg, Denmark. ~Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
Summary Objective: To investigate possible contributions from fibrinolytic disturbances to susceptibility for cardiovascular complications in insulin-dependent diabetic patients with nephropathy. Design: Cross-sectional study. Setting: Outpatient diabetic clinic in a tertiary hospital. Subjects: Insulin-dependent diabetic patients without diabetic nephropathy (normoalbuminuria, n = 17), patients with incipient diabetic nephropathy (n = 19), and patients with diabetic nephropathy (n = 13). Non-diabetic subjects served as a control group (n = 14). Results: Tissue-type plasminogen activator antigen and plasminogen activator inhibitor type 1 were lower in diabetic patients, irrespective of the level of albuminuria. Tissue-type plasminogen activator activity measured in acidified plasma by bioimmunoassay was increased in normoalbuminuria, while the level in diabetic nephropathy was not different from a non-diabetic control level, and was significantly lower than in the normoalbuminuric group. D-Dimer and soluble fibrin were elevated in the pooled group of diabetic patients and were not influenced by presence of nephropathy. The level of plasmin-o~2-antiplasmin complexes was elevated to the same extent in each group of insulin-dependent diabetic patients. Conclusion: The data suggest increased fibrinogen turnover in insulin-dependent diabetes mellitus, irrespective of the presence of nephropathy. In normoalbuminuric patients, an increase in plasminogen activation could be explained by increased activity of tissue plasminogen activator, while other explanations, e.g. fibrin accumulation, probably connected to developing atherogenesis, could be hypothesized in patients with nephropathy.
INTRODUCTION Accelerated atherosclerosis with ischemic symptoms and thromboembolic complications is a major problem in long-term management of insulin-dependent diabetes mellitus (IDDM). Thirty to forty percent of patients with IDDM develop diabetic nephropathy, characterized by increased urinary excretion of albumin, an increase in blood pressure, and decreasing kidney function. The patients with diabetic nephropathy have an extremely Received 26 August 1996 Accepted after revision 2 November 1996 Correspondence to: Dr Bjarne Myrup, Centralsygehuset i Holbaek, DK-4300 Holbaek, Denmark, Tel. +45 3131 1272; Fax. +45 5343 7603.
elevated risk for developing cardiovascular disease, 1'2 and this has led to the hypothesis that albuminuria, besides being a sign of nephropathy, also reflects widespread vascular damage.2'3 An important function of endothelium is the synthesis and release of tissue type plasminogen activator (t-PA), an essential component of the fibrinolytic system. Recent studies have shown that the fibrinolytic activity in plasma from diabetic subjects is increased. 4-6 Further elaboration has shown increased activity of t-PA4 and decreased plasminogen activator inhibitor (PAI) activity and antigen. 5,6 On the other hand, PAI activity has been found increased in IDDM,7,8 and the activity of t-PA reduced. 8 In a previous study, we have evaluated the fibrinolytic system in IDDM patients with diabetic nephropathy and found decreased level and 331
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activity of t-PA in IDDM patients with and without diabetic nephropathy. 9 In the present study, we have extended our investigations on the fibrinolytic system in groups of well characterized IDDM patients in order to try to associate possible deviations in markers of fibfinolysis and vascular wall function with the enhanced risk of cardiovascular disease in IDDM. MATERIALS AND METHODS Subjects Forty-nine patients with IDDM were included in the study. The patients were recruited from the outpatients clinic at the Steno Diabetes Center, Denmark. Patients in the age range from 18 to 60 years were allocated to three groups based on the median albumin excretion rate of three 24h urine samples. Normoalbuminuric patients were only included if the duration of diabetes was between 10 and 30 years, in order to match the duration of diabetes in patients with incipient and diabetic nephropathy. Patients receiving any other medical treatment than insulin and patients with non-diabetic kidney disease were excluded. A group of non-diabetic agematched subjects recruited from the medical staff and surroundings served as a control group. The study protocol was approved by the local ethical committee. Patients and non-diabetic control subjects were enrolled after giving informed consent. Clinical information on IDDM patients and non-diabetic control subjects included are given in Table 1. Diabetic polyneuropathy was present in one normoalbuminuric patient, in two patients with incipient nephropathy, and in one patient with diabetic nephropathy. One patient with incipient nephropathy
had a previous attack of transient cerebral ischemia. One patient with incipient nephropathy suffered from claudication. Investigation protocol Blood samples were obtained in the fasting state after 30 min of supine rest between 0800 and 0900. Following minimal stasis, a 17-gauge needle was placed in an antecubital vein. The first 10 ml of blood was not used. In an identical sequence for each patient, blood was obtained to prepare the following plasma samples: EDTA plasma (0.1 ml 0.5 mg/ml D-phenylalanyl-l-propyl-l-arginine chloromethylketon and 0.12 ml 0.34 mol/l EDTA to 10 ml blood) for the analysis of t-PA and PAId antigen, DDimer, prothrombin fragment 1+2, and PAP-complexes; citrate plasma (0.5ml 0.129mol/1 Na-citrate to 4.5m1 blood) for the analysis of PAI activity, soluble fibrin, and thrombomodulin; acidified plasma (Stabilyte, Biopool, Umea, Sweden) for the analysis of t-PA activity to reduce ex vivo complex formation with PAI-1;I° and EDTAblood (0.054ml 0.134mol/1 EDTA to 4.5ml blood) for the analysis of HbAlc. The blood samples were placed on ice immediately after sampling and remained on ice for 30 rain. After centrifugation at 2000g for 20 min at 4°C, specimens of plasma were stored in polypropylene tubes at -40°C until analysis. EDTA-blood for HbAlc analysis was stored at 4°C until analysis. After blood sampling 74 kBq ~25I-albumin was injected. Blood (100 IU heparin [Leo Pharmaceuticals, Denmark] to 10 ml blood) for measurement of transcapillary escape rate of albumin (TER) was collected after 10, 15, 20, 30, 40, 50, 55, and 60 rain. Blood was centrifuged at 2000g for 10 rain and plasma specimens of 2 ml in duplicates
Table 1 Clinical characteristics of control subjects (control) and insulin-dependent diabetic patients Insulin-dependent diabetic patients Urinary albumin excretion (mg/24 h)
Control < 30
n (women/men) Age (y) Diabetes duration (y) Albuminuria (mg/24-h) GFR* (ml/min) TER** (%/h) Retinopathy . . . . Insulin dose (IU/kg/day)
5/9 40 (25-50) -9 (3-30) -6.4 (2.7-10.6)
HbAlc (%) Blood glucose (mmol/I) BMI (kg/m 2) Trigiycerides (mmoi/I)
. -5.1 (4.5-5.5) 4.6 (4.1-5.0) 23.9 (18.6-28.7) 1.35 (0.39-4.11)
< 30
30-300
> 300
11/6 41 (23-50) 19 (13-31 ) 13 (4-30) 108 (92-137) 7.0 (4.5-9.9) 3/1 0.62 (0.36-0.93) 8.2 (6.1-10.9) 10.4 (3.8-20.5) 23.8 (19.1-28.5) 0.79 (0,52-1.10)
4/15 39 (24-56) 24 (9-47) 140 (44-246) 108 (76-152) 8.6 (3.8-14.3) 11/5 0.62 (0.27-1.01) 8.9 (6.1-11.4) 8.4 (2.7-16.4) 24.8 (20.5-37.2) 1.06 (0.51-2.37)
7/6 38 (25-54) 24 (14-37) 1126 (315-2456) 102 (54-136) 8.8 (6.2-14.2)*** 8/5 0.67 (0.43-1.12) 9.3 (7.6-10.6) . . . . . 11.1 (4.1-21.2) 23.7 (19.5-27.0) 1.28 (0.46-3.79)
Results are given as mean (range). *Glomerular filtration rate. **Transcapillary escape rate. ***P< 0,05 vs. insulin-dependent diabetic patients with albumin excretion < 30 mg/24 h. . . . . Retinopathy as number of patients with simplex retinopathy/proliferative retinopathy. * . . . . P< 0.01 vs. insulin-dependent diabetic patients with albumin excretion < 30 mg/24 h.
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Fibrinolysis in insulin-dependent diabetic patients
were counted on a Cobra auto-gamma counter (Packard Instrument Inc., Downers Grove, Illinois 60515, USA). TER was calculated on logarithmical transformed radioactivity in plasma as the slope of decrease in radioactivity expressed as percentage per h. Finally, 3.7 MBq 5~Cr-EDTA was injected. Blood (100 IU heparin [Leo Pharmaceuticals, Denmark] to 10 ml blood) was collected after 180, 200, 220, and 240 min for the measurement of glomerular filtration rate. Glomerular filtration rate was calculated as described previously, 11 and corrected for body surface. Assays Urine albumin was measured by ELISA,12 and urinary albumin excretion rate was calculated. HbAlc was measured by a chromatographic technique. 13 Fasting blood glucose was measured bed-side with a One-Touch apparatus (Life Scan Inc., Milpitis, California, USA). The following parameters were measured on the above specified plasma samples, by use of commercial ELISA kits: t-PA antigen (Immulyse t-PA, Biopool); TM t-PA activity (Coatest, BIA t-PA, Chromogenix, S-43153 M61ndal, Sweden); 1° PAI-1 (TintElize PAI-1, Biopool); 15 PAI activity (Spectrolyse (131) PAI, Biopool); 16'17 soluble fibrin (Fibrinostika, Soluble Fibrin, Organon Teknika, The Netherlands); 18 D-Dimer (Coaliza, Chromogenix); 19 prothrombin fragment 1+ 2 (Enzygnost F 1+ 2, Behringwerke AG, Marburg, Germany);2° plasmin-c~2antiplasmin (PAP) (EIAAPP micro, Behringwerke AG, Marburg, Germany);2~ and thrombomodulin (Amersham Thrombomodulin, Stage, France). 22 Statistics Differences between non-diabetic subjects and normoalbuminuric patients with IDDM, and differences
333
between IDDM patients with diabetic nephropathy and normoalbuminuric IDDM patients were analysed by the Mann-Whitney rank sum test. This was done in order to describe the effect of IDDM per se and the effect of present nephropathy, respectively. Correlation was analysed by Spearman rank correlation test. Linear regression was done using a Statgraphics 5.0 package (STSC Inc, Rockville, MD, USA). RESULTS A lower concentration of t-PA antigen in IDDM patients compared with non-diabetic subjects was demonstrated (Table 2). This was not influenced by the presence of diabetic nephropathy. The activity of t-PA was increased in normoalbuminuria compared with non-diabetic controls, and patients with diabetic nephropathy had a significantly lower t-PA activity than normoalbuminuric patients (Table 2). PAI-1 antigen was significantly decreased in all IDDM groups compared with non-diabetic controls (Table 2). This was independent of presence of diabetic nephropathy. The t-PA activity was negatively correlated with the level of PAI-1 antigen (r=-0.47, P= 0.0002). Results concerning soluble fibrin and D-Dimer are shown in Table 2. Soluble fibrin was elevated in IDDM with normoalbuminuria, and there were no differences between groups with different levels of albuminuria. Although soluble fibrin and D-Dimer were not elevated when results from individual groups were compared with non-diabetic controls (save soluble fibrin in normoalbuminuric IDDM patients), when data from all three groups were pooled, both levels of soluble fibrin and D-Dimer were significantly increased above control (mean 4.1 range (1.9-14.0) mg/1 vs. 3.1 (1.8-7.3) mg/1, P< 0.05, and 88 (33-260) gg/1 vs. 59 (26-101) gg/1, P<0.05, respectively). A procoagulant activity expressed as elevated prothrombin fragment 1+ 2 was found in all groups of IDDM
Table 2 Tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAl) type 1 antigen, plasminogen activator inhibitor activity, soluble fibrin, D-Dimer, prothrombin fragment 1+2 (F1+2), and c~2-antiplasmin-plasmin complex (PAP) in insulin-dependent diabetic patients and non-diabetic controls Insulin-dependent diabetic patients Urinary albumin excretion (mg/24 h)
Control < 30
< 30
30-300
> 300
t-PA antigen (ng/ml) t-PA activity (IU/ml) PAl type 1 antigen (ng/ml) PAl activity (IU/ml) Soluble fibrin (rag/I) D-Dimer (IJg/I) F1 +2 (nmol/I) PAP (IJg/I) Thrombomodulin (ng/ml)
5.4 (2.1-9.6) 0.55 (0.03-2.20) 11.0 (3.4-29.8) 15.8 (0-62.8) 3.1 (1.8-7.3) 59 (26-101 ) 0.75 (0.39-1.47) 276 (106-533) 31 (15-55)
3.3 (1.2- 5.5)* 0.67 (0.36-3.30)** 4.2 (2.3-7.0) .... 6.0 (0-20.8) 4.2 (1.9-7.9)* 78 (35-229) 1.14 (0.73-2.05)** 448 (203-982)* 30 (13-66)
4.2 (1.4-12.9)** 0.61 (0.28-3.00) 5.9 (1.1-16.7)* 3.2 (0-12.9)* 3.7 (1.9-10.0) 91 (46-260) 1.06 (0.3-2.45)** 468 (258-920)* 35 (18-60)
3.4 (1.2-8.7)** 0.50 (0.21-1.20)*** 5.8 (2.7-10.3)** 7.5 (0-15.8) 4.4 (2.2-14.0) 78 (33-147) 1.07 (0.5-1.73)* 400 (227-595)* 44 (27-65) ......
Results are mean (range), *P< 0.01 vs. control, **P< 0.05 vs. control, ***P< 0.05 vs. IDDM patients with urinary albumin excretion < 30 mg/(24 h), ****P< 0.001 vs. control.
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Myrup et al
patients irrespectively of the level of albuminuria (Table 2). TER was elevated in diabetic nephropathy compared with normoalbuminuria, while a tendency was present in incipient nephropathy (Table 1). Thrombomodulin was increased in patients with diabetic nephropathy compared with any other group (Table 2). The level of thrombomodulin correlated positively with HbAic (r=0.43, P = 0.002), and negatively with glomerular filtration rate (only IDDM patients, r=-0.32, P=0.04). In a multiple regression model, HbA1c turned out to be the major confounder when measuring thrombomodulin in the present subjects, while glomerular filtration rate became insignificant. Consequently, thrombomodulin values were corrected for HbAlc by linear regression, but the difference between normoalbuminuric patients and patients with diabetic nephropathy was sustained (P= 0.02). There was no relation between TER and t-PA activity, D-Dimer, or PAP. DISCUSSION
The divergence in the literature concerning fibrinolysis in IDDM m a y be due to differences in selection of patients regarding diabetes duration, metabolic control, and complication status. In this and a previous study 9 we have studied the fibrinolytic system in relation to presence of diabetic nephropathy. The present results confirm reduced t-PA antigen and PAI-1 antigen in IDDM, and we demonstrated higher t-PA activity compared with a non-diabetic control level. The discrepancy between the present and our previous results 9 is explained by the recently introduced method which diminishes ex vivo complex formation of t-PA with PAI-110 and has led to a much higher measurable level of t-PA activity. Elevation of t-PA activity was only present in normoalbuminuric patients, while patients with diabetic nephropathy had a significantly lower level of t-PA activity compared with the normoalbuminuric group. In a similar study, comparable levels of t-PA antigen were reported in IDDM patients, 23 but, in contrast to our study, no difference between IDDM patients and control subjects was found. The discrepancy could be explained by differences in basic parameters of the control group since our control group was age matched. We could not confirm increasing levels of PAI activity in groups of patients with albuminuria, as was demonstrated in this otherwise comparable study. 23 In both studies there was a tendency for higher levels of triglycerides in IDDM patients with albuminuria than patients without, and PAI activity related to triglyceride in our study (all subjects: r=0.43, P<0.O01; only IDDM patients: r=0.37, P<0.O01). Thus, triglyceride levels could be a confounder in the relation between PAI activity and albuFibrinolysis (1996) 10(5/6), 331-335
minuria. We found elevated thrombomodulin in diabetic nephropathy, and this has been suggested as a marker of endothelial dysfunction. 24 Thrombomodulin is located at the endothelial surface, where it is involved in the release of t-PAY Therefore, a possible explanation of lower t-PA activity in diabetic nephropathy compared with normoalbuminuric patients may be loss of throxnbomodulin from the endothelial location. However, no relation was found to elucidate such a mechanism. It may seem a paradox that t-PA activity in diabetic nephropathy was decreased compared with normoalbuminuric patients, while no difference in PAP and D-Dimer was seen between the two groups. Since the procoagulant activity measured as prothrombin fragment 1+ 2 was similar in IDDM patients with and without nephropathy, this should also hold for fibrin production. Thus, a lower plasma t-PA activity in diabetic nephropathy may resuk in fibrin accumulation stimulating plasminogen activation by t-PA. 26 Fibrin accumulation will therefore increase until the localized t-PA activity is high enough to obtain fibrin homeostasis. In steady state conditions this will result in D-Dimer levels which reflect the formation of fibrin, and PAP levels which reflect the rate of plasminogen activation. According to this hypothesis, homeostasis in diabetic nephropathy is reached at a higher level of fibrin accumulation. Since fibrin degradation products have been shown to exert a mitogenic effect,28 fibrin accumulation could contribute to smooth muscle cell proliferation observed in atherogenesis. 27 A higher level of fibrin accumulation related to the vascular wall could occur in the development of atherosclerosis 2s linked to this process by a mitogenic effect of fibrin degradation products. 29 In conclusion, the data suggest an increase in fibrinogen turnover in IDDM. In normoalbuminuric IDDM patients, an increase in plasminogen activation could be explained by the increased activity of t-PA, while other explanations, e.g. fibrin accumulation probably connected to developing atherogenesis, could be operative in patients with nephropathy.
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