Exacerbation of glycoprotein VI-dependent platelet responses in a rhesus monkey model of Type 1 diabetes

Abstract

Thrombosis is a life-threatening complication of diabetes. Platelet reactivity is crucial to thrombus formation, particularly in arterial vessels and in thrombotic complications causing myocardial infarction or ischaemic stroke, but diabetic patients often respond poorly to current antiplatelet medication. In this study, we used a nonhuman primate model of Type 1 diabetes to measure early downstream signalling events following engagement of the major platelet collagen receptor, glycoprotein (GP)VI. Diabetic monkeys were given enough insulin to maintain their blood glucose levels either at ~8 mM (well-controlled diabetes) or ~15 mM (poorly controlled diabetes). Flow cytometric analysis was used to measure platelet reactive oxygen species (ROS) generation, calcium mobilisation, receptor surface expression, and immature platelet fraction. We observed exacerbated intracellular ROS and calcium flux associated with engagement of GPVI in monkeys with poorly controlled diabetes. GPVI surface levels did not differ between healthy monkeys or the two diabetic groups. Treatment of platelets with the specific Syk inhibitor BAY61-3606 inhibited GPVI-dependent ROS and, importantly, reduced ROS generation in the poorly controlled diabetes group to that observed in healthy monkeys. These data indicate that glycaemic control is important in reducing GPVI-dependent platelet hyperreactivity and point to a potential antithrombotic therapeutic benefit of Syk inhibition in hyperglycaemic diabetes.

Figure 1

Haematological parameters for healthy and diabetic monkeys. (a) Blood glucose, (b) platelet count, (c) mean platelet volume, and (d) platelet distribution width. Data are from six monkeys in each group. *P < 0.05, ***P < 0.001.

Figure 2

Effect of well-controlled or poor glycaemic control on intracellular ROS measurements in diabetes. Flow cytometry of H₂DCF-DA-loaded monkey platelets in PRP either untreated (no add.) or treated with 10 μg/mL CRP or 5 μM TRAP. Syk inhibition by 5 μM BAY61-3606 (BAY) reduces GPVI-induced ROS generation in all monkey groups. Data are from six monkeys in each group. ***P < 0.001.

Figure 3

Influence of well- and poorly controlled diabetes on intracellular calcium mobilisation. Flow cytometry of Fluo-3-AM-loaded monkey platelets in PRP treated with 10 μg/mL CRP (a) or 10 μM TRAP (b). Data are from three to five monkeys in each group. *P < 0.05, ***P < 0.001.

Figure 4

Effect of diabetes on platelet receptor surface levels. Platelet surface expression of (a) GPVI, (b) GPIbα, (c) CD9, and (d) α _IIb β ₃. Data are from five to six monkeys in each group.