Objective— Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non–vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. Approach and Results— Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E -deficient ( ApoE −/− ) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE −/− mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient ( ApoE −/− /Bgn −/0 ) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE −/− /Bgn −/0 mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE −/− /Bgn −/0 mice with the thrombin inhibitor argatroban. Ultimately, ApoE −/− /Bgn −/0 mice developed aggravated atherosclerosis. Conclusions— The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.

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Aims/hypothesis People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium–glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. Methods Male, 8-week-old LDL-receptor-deficient (Ldlr−/−) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. Results Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet–leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr−/− mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10−9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10−9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. Conclusions/interpretation We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin–platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin’s beneficial cardiovascular risk profile. Graphical abstract

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Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

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