Metformin Is Associated with Reduced Tissue Factor Procoagulant Activity in Patients with Poorly Controlled Diabetes

Abstract

Purpose: Metformin is the first-line antidiabetic drug and shown to reduce cardiovascular risk independent from its glucose lowering action. Particularly in poorly controlled diabetes, tissue factor (TF) is expressed in the vasculature and accounts for thromboembolic complications. Here, we aimed to assess the effect of metformin on TF activity and markers of vascular inflammation in poorly controlled type 2 diabetes.

Methods: In a cohort of patients with uncontrolled type 2 diabetes (glycosylated hemoglobin 8.39 ± 0.24%, 68.1 ± 2.6 mmol/mol, n = 46) of whom half of the individuals were treated with metformin and the other half did not receive metformin as part of an anti-diabetic combination therapy, we assessed TF activity and markers of vascular inflammation. In vitro, human monocytic cells (THP-1) were exposed to metformin and TF expression measured in the presence and absence of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the AMPK inhibitor compound C.

Results: In the patients, metformin treatment was associated with lower levels of TF protein (241.5 ± 19 vs. 315.4 ± 25 pg/mL, p = 0.03) and reduced TF activity (408.9 ± 49 vs. 643.8 ± 47 U/mL, p = 0.001) compared with controls. Moreover, the patients on metformin showed lower levels of vascular cell adhesion molecule (VCAM)1 (26.6 ± 1.4 vs. 35.03 ± 3.1 ng/mL, p = 0.014) and higher expression of miR-126-3p/U6sno (11.39 ± 2.8 vs. 4.26 ± 0.9, p = 0.006), a known post-transcriptional down regulator of TF and VCAM1. In vitro, metformin dose-dependently reduced lipopolysaccharide (LPS)-induced TF expression in THP-1 cells. The AMPK activator AICAR alone lowered TF expression in THP-1, while the AMPK inhibitor compound C abrogated the metformin-dependent reduction in TF expression.

Conclusions: Our data are the first to report that metformin is associated with reduced plasma TF procoagulant activity possibly explaining-at least in part-the vasculoprotective properties of metformin.

Keywords: Metformin; cardiovascular disease; coagulation; diabetes mellitus; microRNA; thrombosis; tissue factor; vascular complications; vascular inflammation.

Fig. 1

Metformin reduces tissue factor procoagulant activity in diabetes. Patient cohort: Plasma of patients with diabetes receiving metformin or not was analyzed with respect to TF protein (A), TF activity (B), leukocyte count (C), and miR-126 expression (D). n = 46; shown are mean ± SEM; differences between groups were measured by a Student’s t test or Mann–Whitney test. P-values are indicated. In vitro experiments: THP-1 cells were left untreated or incubated with the indicated concentrations of metformin for 24 h and then induced with 10 μg/mL LPS for 2 h and TF mRNA expression assessed (E). THP-1 cells were cultured in the absence or presence of metformin, AICAR, or metformin together with compound C at the indicated concentrations for 24 h. The cells were then stimulated with 10 μg/mL LPS for 2 h and TF mRNA quantified (F). HMEC-1 were left untreated or exposed to different concentrations of metformin as indicated for 72 h. miR-126 expression was then assessed under basal conditions or following stimulation with TNFα for 2 h (G). n ≥ 5; groups were compared by ANOVA with Tukey’s post hoc test (E,F) or 2-way ANOVA with Dunnett’s multiple comparison post hoc test (G). *p < 0.05, **p < 0.01, ***p < 0.0001 vs. control