Hypofibrinolysis is a common finding in patients with diabetes and a risk factor for the occurrence of micro- and macroangiopathy (1–3). Recently, a new potent inhibitor of fibrinolysis, the thrombin-activatable fibrinolysis inhibitor (TAFI) was isolated from human plasma (4). It has been reported that the plasma levels of TAFI are increased in diabetic patients, and it may play an important role in the mechanism of hypofibrinolysis observed in these patients (5).
Activated protein C (APC) is a serine protease that inhibits thrombin formation by proteolytically inactivating factors Va and VIIIa and by stimulating fibrinolysis (6,7). Thrombin stimulates the conversion of TAFI in its active form. APC may indirectly promote fibrinolysis by inhibiting thrombin generation and by inhibiting the action of plasminogen activator inhibitor-1 (7,8). Both TAFI and APC are regulated by thrombin-thrombmodulin complex on the plasma membrane of endothelium (6). This mechanism appears to be important for controlling the balance between coagulation and fibrinolysis in diabetic patients. In the present study, we investigated the plasma levels of TAFI and its relationship with APC in normotensive type 2 diabetic patients.
Forty normotensive (<140/90 mmHg) nonobese type 2 diabetic patients (28 men and 12 women, aged 54.7 ± 1.8 years [means ± SE], BMI 22.5 ± 0.4 kg/m2, diabetes duration 9.1 ± 1.1 years, systolic blood pressure 129.1 ± 2.1 mmHg, diastolic blood pressure 77.0 ± 1.6 mmHg, fasting blood glucose levels 8.59 ± 0.32 mmol/l, and HbA1c 9.1 ± 0.3%) with normal hepatic function and without any medication that may influence blood coagulation profile were enrolled in the present study. There were 30 patients with normoalbuminuria (albumin excretion rate 8.6 ± 0.6 μg/min) and 10 with microalbuminuria (47.6 ± 6.9 μg/min). No patient had cardiovascular autonomic neuropathy. Twenty six patients were being treated with diet therapy alone, 14 with oral hypoglycemic agents, but none with thiazolidine. Twenty age-matched nonobese healthy individuals (16 men and 4 women) were used as control subjects.
The plasma levels of TAFI were measured using a commercially available EIA kit (TAFI-EIA; Kordia Laboratory Supplies, Leiden, the Netherlands) (5). APC-PCI complex, a marker of ongoing protein C (PC) activation, was measured by enzyme-linked immunoassay as described (9). PC antigen was measured by solid-phase immunoassay as described (9). Total protein S (PS), which is a cofactor for activation of PC, was measured as reported (9). The plasma levels of the thrombin-antithrombin complex (TAT) were measured by EIA method as described (9). The plasma levels of d-dimer (DD) were measured by a commercial EIA kit (d-dimer test-F; Kokusai-Shiyaku, Kobe, Japan).
The ratio between the plasma concentrations of DD and TAT complex (DD/TAT), an index of fibrinolytic activity, was significantly decreased in diabetic patients compared with healthy subjects (15.3 ± 1.3 vs. 26.5 ± 2.2, P < 0.05). The plasma levels of TAFI were significantly higher (139.1 ± 10.3 vs. 99.5 ± 4.9%, P < 0.05) in diabetic patients than in normal subjects. The plasma levels of APC-PCI were significantly higher (3.36 ± 0.28 vs. 2.17 ± 0.48 pmol/l, P < 0.05) in diabetic patients than in normal subjects. The plasma levels of TAFI were positively and significantly correlated with the plasma levels of APC-PCI (r = 0.53, P < 0.001) in diabetic patients. There was significant correlation between the plasma levels of TAFI and PS in diabetic patients (r = 0.50, P < 0.005). There was no significant correlation between TAFI and PC antigen levels (r = 0.04).
The thrombomodulin-thrombin complex formed on the plasma membrane of endothelium exerts anticoagulant activity by catalyzing the conversion of PC to activated APC, which inhibits activation of blood coagulation (6,7). On the other hand, this thrombomodulin-thrombin complex may also promote coagulation by activating TAFI (6). Activated TAFI inhibits fibrinolysis by removing COOH-terminal lysine residues from fibrin. Lysine residues are high affinity binding sites for plasminogen, which is a precursor of plasmin, the key serine protease for fibrinolysis (10). In the present study, the DD/TAT ratio was significantly decreased in diabetic patients compared with healthy control subjects, suggesting the occurrence of hypofibrinolysis in diabetes. This decrease in fibrinolytic activity may be related to the increase in the plasma levels of TAFI.
Interestingly, the circulating levels of TAFI were significantly correlated with those of APC-PCI complex, a marker of APC generation. It has been reported that APC improves decrease of fibrinolytic activity induced by TAFI in vitro (11,12). The fact that circulating levels of TAFI and APC-PCI complex are significantly correlated suggests that APC may promote fibrinolysis in diabetic patients by modulating the action of TAFI. However, the significant decrease of DD/TAT in diabetic patients compared with control subjects suggests that APC may not be sufficient for suppressing the decrease in fibrinolytic activity in diabetes.
This insufficient activity of APC may be due to an imbalance between the thrombomodulin-mediated activity of both TAFI and PC in favor of the former. In brief, PC activation may be important for the regulation of TAFI-induced hypofibrinolysis in diabetes.
Address correspondence to Dr. Yutaka Yano, Third Department of Internal Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan. E-mail:.
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