Effect of circulating tissue factor on hypercoagulability in type 2 diabetes mellitus studied by rheometry and dielectric blood coagulometry

Abstract

Background: Hypercoagulability in type 2 diabetes mellitus (T2DM) patients increases their risk of cardiovascular diseases.

Objective: The aim of this work was to investigate the hypercoagulation mechanism in T2DM patients in terms of circulating tissue factor (TF).

Methods: Whole blood coagulation tests by damped oscillation rheometry and dielectric blood coagulometry (DBCM) were performed.

Results: The average coagulation time was significantly shorter for T2DM patients than for healthy controls. In vitro addition of either anti-TF or anti-activated factor VII (FVIIa) antibody to hypercoagulable blood samples prolonged coagulation times for one group of patients, while coagulation times remained short for another group. The levels of circulating TF were estimated in the former group by measuring the coagulation times for blood samples from healthy subjects with addition of various concentrations of TF and comparing them with the coagulation times for the group. The results indicated that the levels of circulating TF were on the order of subpicomolar at most.

Conclusions: Circulating TF is at least partially responsible for a hypercoagulable group of T2DM patients, while an abnormality in the intrinsic coagulation pathway probably occurs in the other group.

Keywords: Dielectric spectroscopy; coagulation time; extrinsic/intrinsic coagulation pathway; hypercoagulable state.

Fig. 1.

Typical LDF curves, where open and filled circles correspond to the data without and with added anti-TF or anti-FVIIa antibody, respectively, and positions of ${\mathit{t}}_{\mathrm{i}}$ are indicated with arrows. Panels (a) and (b) show the curves for T2DM samples with and without significant effects of added anti-TF antibody, respectively. Panels (c) and (d): same as panels (a) and (b), respectively, but for added anti-FVIIa antibody instead of anti-TF antibody. Panel (e) corresponds to a healthy subject, and panel (f) shows a case where ${\mathit{t}}_{\mathrm{i}}$ cannot be determined because of significant artifact from fast erythrocyte sedimentation.

Fig. 2.

Typical DBCM response for a T2DM sample. Panel (a) shows the change in the dielectric dispersion curve during the progression of blood coagulation, and panel (b) is the same data normalized to the dielectric dispersion curve at the first time point. The frequency range for this measurement was 100 Hz to 110 MHz.

Fig. 3.

Normalized permittivity change at 10.7 MHz extracted from Fig. 2(b).

Fig. 4.

Comparison of ${\mathit{t}}_{\mathrm{x}}$ between T2DM and non-diabetic control samples. Open circles and open squares show data obtained by the prototype setups with the rotating and non-rotating methods, respectively (see the Section “2.3. DBCM”). Red bars show average values of ${\mathit{t}}_{\mathrm{x}}\pm \text{standard deviations}$ for T2DM from the rotating (${\mathit{t}}_{\mathrm{x}}=28.9\pm 9.6\text{ min}$, $\mathit{n}=82$) and the non-rotating (${\mathit{t}}_{\mathrm{x}}=28.2\pm 8.7\text{ min}$, $\mathit{n}=37$) methods and controls (${\mathit{t}}_{\mathrm{x}}=37.9\pm 8.4\text{ min}$, $\mathit{n}=28$). The control data taken from the previous study [19] can also be used in the present study because the same DBCM prototype system was used and operated by the same person.

Fig. 5.

DBCM responses from a healthy subject at 10 MHz normalized by the minimum and maximum values of permittivity. The black curve (1) shows the control sample without TF and antibodies, and the red curve (2) demonstrates the acceleration of the DBCM response by addition of TF to the sample blood. Simultaneous addition of TF and anti-TF antibody (3) or TF and anti-FVIIa antibody (4) prolongs the DBCM response in comparison with (2). On the other hand, addition of anti-TF antibody (5) or anti-FVIIa antibody (6) without TF shows responses similar to the control (1).

Fig. 6.

TF concentration dependence of ${\mathit{t}}_{\mathrm{x}}$ in healthy subjects (three individuals are shown by different colors). The open circle shows the normal range of prothrombin time (PT) against reagent concentration of screening PT tests with international normalized ratio (INR) and international sensitivity index (ISI) values close to one.