Anthocyanin as an Antiplatelet Therapy in Diabetes: Immunopathological Assessment

Abstract

Diabetes mellitus—in particular, Type 2 diabetes mellitus (T2DM)—is one of the most prevalent chronic illnesses in many countries. Diabetes mellitus is regarded as an independent risk factor for cardiovascular disease (CVD). Cardiovascular disease is one of the main causes of mortality in patients with diabetes, mainly due to macrovascular complications. One of these macrovascular complications in diabetes is atherosclerosis, which involves a complicated pathophysiological process. In addition to hyperglycaemia, oxidative stress (OS) plays a significant role in the pathogenesis of diabetes and its associated risk of CVD. Platelet hyperactivity, in the presence of OS, has a major effect on the progression of atherosclerosis and thrombotic events. Aspirin (AS) is the most-used antiplatelet therapy for the prevention of thrombotic complications in people with T2DM. AS attenuates platelet hyperactivity. Although aspirin is a frequently used therapy for the inhibition of platelet hyperactivity, there is much evidence for AS non-responsiveness. The anthocyanin (AC) antioxidant has been shown to have an inhibitory effect on platelets and consequently may be used as a complement to other antiplatelet therapies to attenuate the negative effect of atherosclerosis and CVD in people with T2DM. Although many dietary intervention studies have shown that intake of AC-rich food may be negatively related to some CVD risk factors, the effect of pure AC on thrombotic markers such as platelet hyperactivity and haemostasis is yet to be explored. The main aim of the studies commenced for this thesis were to examine the effect of AC on thrombotic parameters and reveal the pathways by which AC might affect platelet hyperactivity, thereby providing individuals with T2DM with better protection against CVD. The aim of the first study (see Chapter 4) was to evaluate the in vitro effect of AC on platelet activation and aggregation. Fasting blood samples were collected from 13 screened and healthy volunteers after obtaining ethics clearance and signed informed consent. A full blood examination was conducted, and a dose-response curve was created by incubating platelets with five concentrations of AC (25–200 mg/L). Flow-cytometer assessed platelet activity by recording platelet surface marker expression of activation independent (CD41a) and dependent (P-selectin and PAC-1). Platelet aggregation studies were performed using the turbidimetric method by stimulating platelets using three different agonists: adenosine diphosphate (ADP), collagen and arachidonic acid (AA). The results of this study confirmed that AC at 50 mg/L significantly lowered platelet activation as expressed by the P-selectin surface activation marker and AA-stimulated platelet aggregation. However, a similar effect of AC was not detected when ADP or collagen was applied to induce platelet aggregation. Reduced AA-stimulated platelet aggregation by in vitro–adding of AC suggests that AC may reduce platelet hyperactivity, thus reducing the risk of vascular thrombosis and promoting cardioprotective effects. Following the results of Chapter 4, a subsequent study (see Chapter 5) was conducted to assess if AC had a comparable antithrombotic effect ex vivo. Twenty-six randomly recruited healthy (25–75-year-old) participants contributed to this study and consumed 320 mg of AC a day in the form of Medox® capsules for 28 days. This study was conducted in laboratories of the School of Medical Science at Griffith University. Fasting blood samples were collected pre- and post-intervention to perform platelet activation studies, which were done by measuring platelet surface marker expression of CD41a and P-selectin, and platelet–monocyte aggregates in ADP-stimulated platelets. Platelet aggregation studies were performed by stimulating platelets with various agonists such as ADP, collagen and AA. Full blood examination, coagulation and biochemistry profile analyses were also evaluated pre- and post-intervention. A flow-cytometry analysis showed that AC had a significant effect on the expression of P-selectin as measured by the platelet surface expression of CD62p. A significant decrease in ADP-stimulated platelet aggregation was detected in the blood of healthy individuals. These results endorse the idea that AC might reduce platelet aggregation by affecting a mechanism of platelet activation, specifically the P2Y2–P2Y12 receptor. Similarly, AC significantly reduced platelet activation, as a lesser concentration of fibrinogen and decreased mean platelet volume (MPV) were detected due to AC effects in normal participants. The results from Chapter 5 suggest that AC consumption may enhance protection against platelet hyperactivity–related thrombosis. Based on the results of Chapter 5, the aim of the next study (see Chapter 6) was to identify and elucidate any possible influence of AC on thrombotic risks in people with T2DM. This study involved patients with T2DM. Twenty-four patients with T2DM were recruited for this study, and they consumed 320 mg of AC a day in the form of Medox® capsules for 28 days. Blood pressure and anthropometric measures were taken before and after the intervention period. Fasting blood samples were collected pre- and post-intervention to perform platelet activation studies, which were done by measuring platelet surface marker expression of CD41a and P-selectin in ADP-stimulated platelets. Platelet aggregation, full blood examination, coagulation and biochemistry profile analyses were also evaluated pre- and post-intervention. The data from this study showed that AC had a probable lowering effect on collagen and ADP-induced platelet aggregation in T2DM. This clinical trial also demonstrated the reducing effect of AC on the TC level in the blood. The figures shown in Chapter 6 suggest that the ingestion of AC may mitigate the development of thrombotic risks due to platelet hyperactivity. Following the outcome of Chapter 6, a fourth study (see Chapter 7) was conducted to assess if AC was comparable to AS in lowering different thrombotic biomarkers as well as platelet activation and aggregation. Antiplatelet medications, such as AS, diminish platelet hyperactivity and aggregation and decrease the risk of thrombosis. These antiplatelet drugs inhibit platelet activation through different pathways. Antiplatelet agents are indicated for mitigating thrombosis, which is partly mediated by platelet hyperactivity. However, AS non-responsiveness and side effects have been reported. Antioxidants alleviate the development of atherosclerosis and mitigate the prognosis of CVD. Two groups of healthy participants consumed AC and AS for four weeks. They were tested before and after the intervention period for different parameters including full blood count, platelet activation and aggregation, biochemical tests of lipid profile, uric acid, glucose and C-reactive protein, and coagulation assay. This study (see Chapter 7) showed a significant decrease in platelet hyperactivity—as expressed by CD62p (P-selectin) caused by AC—in the participants, yet the effect of AS was more powerful. AC had a reducing effect on ADP and collagen-stimulated platelet aggregation, but AS applied a greater inhibitory effect on this. Alleviated platelet activation, along with reduced platelet aggregation, were also detected. Lower platelet degranulation correlates with a decrease in thrombus size, as P-selectin (which is expressed by platelets upon activation) is recognised to attract nearby white blood cells (WBCs) dynamically, thus increasing thrombus size. The outcomes from this study (see Chapter 7) suggest that AC could possibly be used to decrease platelet function. However, this study also showed AC to be less useful than AS in lowering the risk of thrombosis. The results achieved from the studies completed for this thesis demonstrate a positive relationship between the consumption of AC and a decrease in platelet activity, which may be instrumental in lowering the risk of thrombosis, thus providing better prevention against CVD. The hypothesised total antioxidant effect of AC may be responsible for reduced platelet activity, which is expected to delay or even prevent macrovascular or microvascular events in patients who suffer from elevated OS as a result of different diseases such as T2DM. Reduced MPV and lowered fibrinogen levels also suggest that ingestion of AC may have an effect in suspending the early stages of atherosclerosis. Thus, AC has the potential to alleviate thrombotic risk and probably reduce the risk of cardiovascular events.