Fibrinogen and risk of ischemic stroke in patients with type 2 diabetes

Background
Ischemic stroke occurs when a blood clot obstructs a vessel in the brain. Diabetes is an independent risk factor for ischemic stroke, and patients with type 2 diabetes (T2D) have 40% higher risk of developing ischemic stroke than people without T2D. Not much is known about the triggers for ischemic stroke in a T2D population, but fibrinogen plasma levels have shown to be a potential risk factor for ischemic stroke in the general population. Fibrinogen is a multifunctional, acute phase plasma protein, which plays a central role in the coagulation cascade and in inflammation. Fibrinogen is a very heterogeneous protein, with an estimate of 106 different variants. These variants affect fibrin clot characteristics differently in purified systems, making them relevant to explore in plasma in relation to stroke development in T2D.

Objectives
The PhD study aimed to systematically review the scientific evidence for an association between plasma fibrinogen and stroke in T2D. Furthermore, aims were to determine how plasma levels of fibrinogen, three fibrinogen variants (sialylated fibrinogen, fibrinogen αE, and fibrinogen γ′), and in vitro fibrin clot characteristics associate prospectively with the risk of ischemic stroke in patients with recently diagnosed T2D, and how the fibrinogen variants correlate with fibrin clot characteristics. 

Materials and methods
First, a systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines to examine the literature on association between fibrinogen and stroke in T2D patients. Next, patients enrolled in the Danish Centre for Strategic Research in T2D (DD2) cohort between 2010 and 2017 were included in a nested case-control study. DD2 is a cohort including patients recently diagnosed with T2D and includes baseline interview data, physical examination data, biological data, and a biobank. The DD2 cohort is followed in Danish Registries for disease development. A total of 144 patients who developed stroke were matched with 144 patients who did not develop stroke and baseline characteristics were compared between groups. Blood samples obtained at DD2 enrolment were analyzed for total fibrinogen and fibrinogen variants (sialylated fibrinogen, fibrinogen αE, and fibrinogen γ′) and turbidity was used to analyze in vitro fibrin clot characteristics: (steepest part of clot lysis curve (Vmax), maximum absorbance (MA), percentage of clot breakdown after 30 minutes (clot lysis), overall hemostasis potential (OHP), fiber diameter, and fiber density). Plasma fibrinogen, fibrinogen variants, and fibrin clot characteristics were compared between stroke cases and diabetes controls, and logistic regression was used to describe the prospective relationship with risk of ischemic stroke.
A control group of blood donors (n=120) was included in the study and was compared to the diabetes controls (n=144) in an unmatched case-control design.

Results
In the systematic review, we identified five studies that investigated the association between fibrinogen and risk of stroke in T2D. The five studies did not show a consistent association between fibrinogen and stroke risk in T2D. The studies had low sample size, varied designs, and poorly defined study participants, highlighting the need for larger, more rigorously defined studies to clarify the role of fibrinogen as a marker for stroke risk in T2D.
In the DD2 cohort study, total fibrinogen, absolute levels of the three fibrinogen variants (sialylated fibrinogen, fibrinogen αE, and fibrinogen γ′), and fibrin clot characteristics (V_max, MA, OHP, and fiber diameter) were higher and fiber density and clot lysis were lower in patients with T2D who over the next years developed ischemic stroke compared with matched diabetes controls, who did not develop ischemic stroke.
Baseline levels of total fibrinogen and absolute levels of fibrinogen γ´ and sialylated fibrinogen were prospectively associated with risk of ischemic stroke, but fibrinogen αE was not. Regarding fiber characteristics, V_max, MA, OHP, and fiber diameter were positively associated with risk of developing ischemic stroke in T2D patients whereas fiber density was negatively associated. Consistent correlations were observed between absolute, but not relative, levels of fibrinogen variants and clot properties.
Absolute levels of the three fibrinogen variants and clot properties (V_max, MA, OHP, and fiber diameter) were higher and relative levels of the variants were lower in the diabetes controls compared to blood donors.

Conclusions
In patients recently diagnosed with T2D, fibrinogen, absolute levels of fibrinogen γ´ and sialylated fibrinogen, and several clot properties (V_max, MA, OHP, fiber diameter, and fiber density) were prospectively associated with risk of ischemic stroke. Absolute levels of the three fibrinogen variants (sialylated fibrinogen, fibrinogen αE, and fibrinogen γ´) correlated with all the measures of clot characteristics in T2D patients. This study adds to the literature suggesting fibrinogen, but also fibrinogen variants and properties of the formed clot, as predictor variables of ischemic stroke in T2D.