Archives of Medical Research 36 (2005) 539–543
ORIGINAL ARTICLE
Plasma Prekallikrein as a Risk Factor for Diabetic Retinopathy Karolina Ke˛dzierska,a Kazimierz Ciechanowski,a Edyta Gołembiewska,a Krzysztof Safranow,b Andrzej Ciechanowicz,c Leszek Doman´ski,a Marek Mys´laka and Jacek Ro´z´an´skia a
Department of Nephrology, Transplantology and Internal Medicine, bDepartment of Biochemistry and Chemistry, c Department of Clinical Biochemistry, Pomeranian Medical University, Szczecin, Poland Received for publication October 26, 2004; accepted March 30, 2005 (ARCMED-D-044-00128).
Background. The aim of the study was to verify the hypothesis that in diabetes there is an increased activation of coagulation system leading in consequence to diabetic retinopathy. Methods. Thirty three healthy subjects (controls, 16 males and 17 females) and 35 patients with diabetes type 1 (15 males and 20 females) were examined. We monitored plasma prekallikrein (PPK), glycemia, fructosamine, glycosylated hemoglobin, activated partial thromboplastin time (PTT), INR, fibrinolysis in euglobulins time (FET), level of antithrombin III (AT III), fibrinogen (Fb) and fibrinogen degradation products (FDP). Results. In diabetic patients without retinopathy, PKK concentration was 16% higher (p ⬍0.005), in patients with background retinopathy 33% higher (p ⬍0.001), and in patients with proliferative retinopathy PKK concentration was 50% higher (p ⬍0.001) than in controls. In the subgroup of patients with proliferative retinopathy PTT was significantly shorter (p ⬍0.001), and FET was significantly longer (p ⬍0.001) than in control. In patients with diabetes higher FDP concentrations were found than in controls (p ⬍0.05). Significant correlations were found between PPK and fructosamine levels in all diabetic patients (RS ⫽ ⫹0.57 p ⬍0.001), in diabetic patients without retinopathy (RS ⫽ ⫹0.61, p ⬍0.05), and in diabetic patients with retinopathy (RS ⫽ ⫹0.62, p ⬍0.005). We found negative correlation between PPK concentration and PTT (RS ⫽ ⫺0.43, p ⬍0.001) and positive correlation between PPK concentration and FET (RS ⫽ ⫹0.59, p ⬍0.00001) in the entire study group. Conclusions. The occurrence of diabetic retinopathy is connected with higher levels of plasma prekallikrein. 쑖 2005 IMSS. Published by Elsevier Inc. Key Words: Diabetes mellitus, Microangiopathy, Prekallikrein, Retinopathy, Kallikrein-kinin system.
Introduction Diabetic retinopathy (DR) is a complication of microangiopathy type in patients with diabetes of both type 1 and 2. According to global estimates, DR morbidity ratio for all diabetes types ranges approximately from 32 to 43% (1). In Poland the estimated ratio of DR morbidity in diabetes type 1 is 54.7% and in diabetes type 2 it is 31.4%. The following factors are considered to have influence in DR pathogenesis: 1) metabolic – the toxicity of hyperglycemia which in turn
Address reprint requests to: Prof. Kazimierz Ciechanowski, MD, PhD, Department of Nephrology, Transplantology and Internal Medicine, Pomeranian Medical University, Al. Powstancow Wlkp 72, 70-111 Szczecin, Poland; E-mail:
[email protected]
0188-4409/05 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 5 .0 3 .0 50
leads to hypoxia of the retina, 2) hemodynamic and rheologic, 3) endothelial and hemostatic factors. In patients with diabetes there is the imbalance in coagulation-fibrinolysis systems, which in consequence leads to the increase of prothrombotic activity. If this involves capillaries it results in ischemia of the retina and leads to neoangiogenesis (proliferative retinopathy). It is speculated that disorders in function of vascular endothelium take part in the pathogenesis of diabetic microangiopathy (3,4). In patients with diabetes and proliferative retinopathy, the increased concentration of plasma vascular endothelial growth factor (VEGF) and von Willebrand factor (5,6), as well as increased fibrinogen concentration, were found compared to control group (7). Other authors found that activation of local proteolysis activating factors (increased expression of urokinase, PAI-1 and tissue plasminogen activator) take part in the
쑖 2005 IMSS. Published by Elsevier Inc.
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development of diabetic proliferative retinopathy (8). Fujiwara et al. (6) showed that hypercoagulability can play a significant role in the damage of retinal endothelial cells. The plasma concentration of thrombomodulin, thrombin/ antithrombin III complex and plasmin inhibitor-α2-plasmin complex, which are independent predictors of DR, can reflect that process (6). The development of this pathology is associated with the dysfunction of kallikrein-kinin system (KKS) (9). In the traditional view the stimulation of KKS took place after activation of factor XII (Hageman factor, FXII) as a result of its contact with the negatively charged surface of vascular endothelium (10–12). Circulating in the plasma prekallikrein (PK) and HK complex is a regulator of plasma KKS system. This complex binds to the receptor in endothelial cells, which is a protein complex containing cytokeratin (CK-1), urokinase plasminogen activator receptor (uPAR) and protein gC1qR (13). PK and HK complex after binding to specific receptor on the endothelial surface undergoes rapid conversion to kallikrein, independently of factor XII (14). Factor XII also presents the ability of binding to this complex, but physiologically high concentration of HK in plasma blocks the reaction. Factor XII undergoes activation only after PK activation (15). The activated kallikrein releases bradykinin, which directly leads to vasorelaxation. In addition, it converts prorenin to renin. Thus, kallikrein plays a key role in two antagonistic systems: kallikrein-kinin (vasorelaxation) and renin-angiotensin (vasoconstriction). Prolilcarboxypeptidase (PRCP) is the enzyme that regulates that entire process. It is a specific kallikrein activator and an enzyme converting angiotensin II to angiotensin II(1–7). In contrast to angiotensin II, angiotensin II(1–7) shows vasodilatatory effects (through nitric oxide [NO] and prostacyclin [PGI2]), similarly to bradykinin (16). Angiotensin II shows prothrombotic activity by the stimulation of the release of plasminogen activator inhibitor (PAI-1) (17). Because PRCP converts angiotensin II to angiotensin II(1–7) and activates PK, it can be assumed that the physiological role of KKS system is antithrombotic and fibrinolytic activity (18). According to different authors, plasma prekallikrein concentration in patients with diabetes is decreased (9) or increased (19) as a result of enhanced synthesis or impaired conversion to kallikrein. It turned out that PK concentration is a marker for the risk of hypertension and diabetic nephropathy development (19). As DR is also a microangiopathy complication, it seems that dysfunction of the kallikreinkinin system may play an important role in the development of this pathology. The aim of the study is to verify the hypothesis that suggests that in diabetes there is an increased activation of coagulation system that starts the pathological chain of reactions leading in consequence to DR. Materials and Methods Thirty three healthy subjects (16 males and 17 females) aged 20–70 years [mean 45 years, standard deviation (SD) 18,
median (Me) 45] and 35 patients with diabetes (15 males and 20 females) aged 22–70 years (mean 45 years, SD 14, Me 46) were examined. All patients had type 1 diabetes. During the study they did not experience acute metabolic disorders. Patients’ and controls’ concomitant comorbidity were stable IDH and/or arterial hypertension. Diagnoses of liver cirrhosis, nephrotic syndrome and/or renal failure were exclusion criteria. At the time of blood sampling and 3 weeks before, patients and control group did not take drugs influencing RAA or coagulation fibrinolysis system. The majority of examinations were performed on blood samples taken for other determinations necessary in the monitoring of treatment. Studies were approved by the Bioethics Committee of Pomeranian Medical University. Informed consent was obtained from all examined patients. Patients with diabetes were divided into subgroup without retinopathy (16 patients, 8 females, 8 males) with a mean age of 49 years (SD 14, Me 50), treated for diabetes for 2–30 years (mean 13.5, SD 9.5 Me 11) and subgroup with DR (19 patients, 12 females, 7 males) with mean age of 45 years (SD 13, Me 42), treated for diabetes for 7–30 years (mean 22, SD 7, Me 22). The diagnosis of DR and its stage of progression were established using ophthalmoscopy and fluorescein angiography. The subgroup of patients with DR was additionally divided into subgroup 1 with background retinopathy (9 patients, 5 females, 4 males, mean 52.5, SD 12, Me 56), treated for diabetes for 14–30 years (mean 22.5, SD 6.5, Me 24) and subgroup 2 with proliferative retinopathy (10 patients, 7 females, 3 males, mean 38.5, SD 10.5, Me 37), treated for diabetes for 7–30 years (mean 21.5, SD 7, Me 21.5). In patients with diabetes and in the control group the following parameters were monitored: glycemia, fructosamine, glycosylated hemoglobin, and coagulation system parameters such as activated partial thromboplastin time (PTT), INR, fibrinolysis in euglobulins time (FET), level of antithrombin III (AT III), fibrinogen (Fb) and fibrinogen degradation products (FDP). Determination of Plasma Prekallikrein Concentration (PPK) Plasma prekallikrein was determined using amidolytic method described in detail by Ciechanowicz (20). Prekallikreins were separated from their plasma inhibitors (C1-INH, α2-macroglobulin, antithrombin III and α-antitrypsin) by filtration on a column filled with gel QAE A-50 Sephadex (Pharmacia Ltd., Uppsala, Sweden). Samples were stored at ⫺20⬚C. After defreezing, for prekallikrein activation, 20 µL of plasma eluate was incubated at room temperature with 20 mL of trypsin (1.3 U/mL) and 20 mL of 0.5 mol/L TrisHCl-0.003 mol/L EDTA-0.003 mol/L NaN3, pH 8. After 15 min, 20 µL of ovomucoid (Sigma, St. Louis, MO) solution (4 mg/mL), specific trypsin inhibitor, was added to the mixture. After a subsequent 10 min, 20 µL of buffer 0.1 mol/L
Prekallikrein in Diabetic Retinopathy
Tris-HCl-0.003 mol/L EDTA-0.003 mol/L NaN3 (pH 8) with 0.75 mol/L NaCl and 50 mmol/L of solution S-2302 (Kabi Vitrum AB, Sweden) were added to the incubation mixture. Reaction mixture was incubated for 10 min at 37⬚C. Reaction was stopped by adding 50 mL of 50% acetic acid. Extinction of each sample was measured (spectrophotometric reader EAR 400, SLT Instruments, Salzburg, Austria) in λ ⫽ 405 nm. Prekallikrein concentration was given as a mean of two measurements of each sample reduced to an extinction value of a blind sample. The coefficient of variation of the method at normal PK level was 11%. Lower limits of detection were 0.01 units of absorbance. FET was determined with euglobulin test according to Niewiarowski (21). Prothrombin time (PT), INR, activated partial thromboplastin time (PTT), antithrombin III (AT III) concentration, fibrinogen (Fb), fibrinogen and fibrin degradation products (FDP) concentrations were determined with standard laboratory methods. Serum fructosamine concentration was determined using Fructosamine Test-La Roche (Technicon RA 1000, TexaLab Inc., Houston, TX) (22). Glycosylated hemoglobin was determined using ion-exchange liquid chromatography (23). Statistical Methods Obtained results were statistically analyzed; arithmetic mean, Me and SD were calculated. Statistical significance of the difference was calculated using Student’s t-test for paired and unpaired variables, statistical significance of the difference between groups where number of patients was lower than 11 was calculated using Mann-Whitney test.
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Correlations were obtained using linear regression equation–Spearman’s test. All calculations were performed using computer software (Statgraphics v. 5.0, Herndon, VA).
Results Mean and standard deviation of prekallikrein concentration, INR, PTT, fibrinogen concentration, FDP, AT III and FET time in 33 healthy subjects, 16 patients with diabetes and without retinopathy (non-DR) and in 19 patients with DR (9 patients with background and 10 patients with proliferative retinopathy) are presented in Table 1. The statistical power of Student’s t-test for comparison of DR (n ⫽ 16) and non-DR (n ⫽ 19) groups was enough to detect with 90% probability real differences in means equal to 0.18 units for PPK, 3.5 sec for PTT, 1.2 g/L for fibrinogen, 37% for AT III. Statistical power could not be calculated for FDP and FET because their distributions were different from normal. The relationship of examined parameters with age, gender and type of diabetes was not found. In patients with diabetes, statistically significant differences in level of glycemia, fructosamine, HbA1c, prekallikrein concentration, FDP concentration, PTT and FET were found compared to control group (Table 1). In the group of diabetic patients without retinopathy, plasma prekallikrein concentration was approximately 16% higher (p ⬍0.005), in subgroup of patients with background retinopathy 33% higher (p ⬍0.001), and in subgroup of patients with proliferative retinopathy it was 50% higher
Table 1. Comparison of coagulation and fibrinolysis parameters in control group and in patients with diabetes: diabetics without retinopathy (R), diabetics with background retinopathy and diabetic patients with proliferative retinopathy Group Diabetes Parameter Number of patients Age Disease duration (years) Glycemia (mmol/L) Fructosamine (mmol/L) HbAlc (%) PPK (units of absorbance) PTT (sec) Fibrinogen (g/L) FDP (mg/L) AT III (%) FET (min)
Control
Without R
Background R
Proliferative R
33 45 ⫾ 18 4.5 ⫾ 0.7 2.5 ⫾ 0.3 3.6 ⫾ 0.8 0.802 ⫾ 0.126 34.1 ⫾ 3.1 2.6 ⫾ 0.6 0–5 Me ⫽ 2.5 100 ⫾ 15 186 ⫾ 27
16 50 ⫾ 15 24 ⫾ 10 11.1 ⫾ 2.9† 3.50 ⫾ 0.61† 6.73 ⫾ 1.63† 0.927 ⫾ 0.156§ 31.1 ⫾ 3.5§ 2.9 ⫾ 1.8 0–80|| Me ⫽ 12.5 90 ⫾ 30 353 ⫾ 138†
9 53 ⫾ 12 23 ⫾ 7 11.8 ⫾ 3.2† 3.35 ⫾ 0.50† 7.55 ⫾ 1.32† 1.074 ⫾ 0.160*† 31.1 ⫾ 3.4§ 2.7 ⫾ 0.6 0–160|| Me ⫽ 20 90 ⫾ 30 323 ⫾ 504 †
10 39 ⫾ 11‡ 21 ⫾ 7 12.7 ⫾ 2.6† 3.91 ⫾ 0.55† 7.19 ⫾ 1.50† 1. 225 ⫾ 0.148†‡ 30.6 ⫾ 1.5† 2.7 ⫾ 1.0 0–160|| Me ⫽ 80 96 ⫾ 34 359 ⫾ 1754†
Values are expressed as mean ⫾ SD. Statistically significant differences: *p ⬍0.05 comparing to group without retinopathy; †p ⬍0.001 comparing to control; ‡p ⬍0.05 comparing to group with background R; §p ⬍0.005 comparing to control; ||p ⬍0.05 comparing to control. HbA1c, hemoglobin A1c; PPK, plasma prekallikrein; PTT, partial thromboplastin time; FDP, fibrinogen degradation products; AT III, antithrombin III; FET, fibrinolysis in euglobulins time; R, retinopathy; Me, median.
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(p ⬍0.001) than in control group (Table 1). In the subgroup of patients with proliferative retinopathy, PTT was significantly shorter (p ⬍0.001) than in control group, and time of fibrinolysis was significantly longer (p ⬍0.001)—similar to other groups of patients with diabetes. In patients with diabetes, higher FDP concentrations were found than in control group (p ⬍0.05). Statistically significant correlations were found between plasma prekallikrein and fructosamine levels in all diabetic patients (r ⫽ 0.57, p ⬍0.001), in diabetic patients without retinopathy (r ⫽ 0.61, p ⬍0.05), and in diabetic patients with retinopathy (r ⫽ 0.62, p ⬍0.005). In the examined and control groups we found negative correlation between plasma prekallikrein concentration and PTT (RS ⫽ ⫺0.43, p ⬍0.001) (Figure 1). We also found positive correlation between plasma prekallikrein concentration and FET (RS ⫽ ⫹0.59, p ⬍0.00001) (Figure 2). Discussion In our study we observed that plasma prekallikrein concentration in patients with diabetes was significantly higher than in healthy subjects. In uncomplicated diabetes, plasma prekallikrein concentration was approximately 16% higher, in patients with background retinopathy 33% higher, and in patients with proliferative retinopathy approximately 50% higher (Table 1). Jaffa et al. (19) also found higher prekallikrein concentration in patients with diabetes and hypertension. The authors suggested that such state could be caused either by increased prekallikrein synthesis in patients with diabetes or decreased conversion to kallikrein (19). In our studies we found positive correlation between plasma prekallikrein concentration and fructosamine in patients with diabetes. Plasma prekallikrein half-life is 7–10 days (2). High and statistically significant positive correlation coefficient between plasma PPK and fructosamine concentration [half-
Figure 1. Correlation between plasma prekallikrein and partial thromboplastin time in the entire study group.
Figure 2. Correlation between plasma prekallikrein and fibrinolysis in euglobulins time in the entire study group.
life approximately 20 days (20)] seems to confirm that maintained hyperglycemia stimulates prekallikrein synthesis and secretion. The results of studies by other authors concerning the relationship between PPK and DR are partially discrepant. Similar results were obtained by Federspil et al. (24). According to interpretation of these authors, increased PPK concentration in patients with diabetes can be explained by enhanced synthesis of prekallikrein and other glycoproteins in liver during chronic hyperglycemia. These authors also suggest that the higher the hyperglycemia the more enhanced is this synthesis (25). Therefore, enhanced prekallikrein synthesis in liver causes increased concentration of this protein in plasma. Contrary to our results, Ueahara et al. (9) found an 18% increase of prekallikrein concentration in the group of patients with uncomplicated diabetes, but in patients with DR they found decrease of PPK concentration to a value equal to 80% of the value found in control group. These authors (9) explained that fact with prekallikrein consumption in the process of coagulation activation leading to the development of microangiopathy. Unfortunately, the authors did not provide data characterizing liver sufficiency or nephropathy in examined patients. In this case we can suggest that liver synthesis of prekallikrein could have been impaired. In all examined patients in this study the function of the liver was normal—with normal proteinogram, bilirubin concentration ⬍1 mg/dL (17 µmol/L), normal AspAT, AlAT, ChE, ALP activity, and proteinuria not exceeding 500 mg/24 h. Differences in the obtained results of plasma prekallikrein in the above-mentioned studies (9,25) and in our study may result from different methods of plasma PPK determination. In our study we used modification of the method of PPK determination because we first separated prekallikrein from kallikrein inhibitors by filtering the plasma on Sephadex column. This eliminated the possible influence of these inhibitors on the result of the measurement (20). Jaffa et al. (19) found a higher PPK concentration in patients with
Prekallikrein in Diabetic Retinopathy
microalbuminuria and a positive correlation between PPK concentration and urinary albumin excretion in patients with diabetes. Such a relationship was not observed in patients without microalbuminuria and albumin excretion ⬍40 mg/ 24 h. This observation allowed drawing the conclusion that plasma PPK concentration was a good marker of the development of diabetic nephropathy (19). Examinations of coagulation system presented in this study comprise 35 patients with diabetes (16 patients without retinopathy and 19 patients with retinopathy). Apart from increase of PPK concentration (Table 1) in these patients, shortening of activated partial thromboplastin time (PTT), increase of fibrin degradation products (FDP) and fibrinogen concentrations and elongation of FET (Table 1) was found. Furthermore, we found a negative correlation between plasma prekallikrein concentration and PTT (RS ⫽ ⫺0.43, p ⬍0.001) and a positive correlation between PPK concentration and FET (RS ⫽ ⫹0.59 p ⬍0.00001). PTT shortening confirms increased procoagulation activity in endogenous path. Increased fibrin degradation products and fibrinogen concentrations in the serum of examined patients with diabetes reflect coagulation system activation, and FET elongation shows fibrinolysis impairment. Similar results were obtained by other authors (26,27). Activation of coagulation with simultaneous fibrinolysis attenuation can be explained by glycosylation of coagulation inhibitors and plasminogen leading to their decreased activity (28). Activation of coagulation system is said to be an important factor in the development of diabetic angiopathy (27,29). Our conclusion is that the occurrence of diabetic retinopathy is connected with higher levels of plasma prekallikrein.
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