PB0820 - Decreased clot size following stasis and modulated platelet-derived ROS in GPVI deficiency

Background: Platelet receptor GPVI plays a crucial role in primary haemostasis. Altered platelet bioenergetics contributes to increased thrombosis risk and is a feature of metabolic diseases, including diabetes.

Aims: To characterise functionality of GPVI-/- vs WT clots and determine the role of GPVI on reactive oxygen species (ROS) formation in hyperglycaemia.

Methods: Clot retraction and rotational thromboelastometry (ROTEM) analyses were performed using whole blood from wild-type (WT) and GPVI-deficient (GPVI-/-) mice on standard diet. Clot weight from IVC stasis-induced thrombosis were compared at 48hr. Human platelets were isolated in buffer containing 5 or 25mM glucose corresponding to normo- and acute hyperglycaemia, respectively, and incubated with GPVI-signalling inhibitors for 1hr. Murine platelets were isolated from WT and GPVI-/- mice with access to liquid high-sugar diet (30% sucrose in drinking water) vs controls ad libitum for 8-weeks. Platelet mitochondrial density was determined using citrate synthase activity assay and platelet ROS was measured using commercially available DCFDA assay.

Results: Clot weight and RBC incorporation were decreased in GPVI-/- versus WT mice (42±11% and 38±16%, respectively, p< 0.05). A significantly reduced thrombus weight after IVC ligation was observed in GPVI-/- vs WT mice (0.87±0.03 and 1.18±0.07, respectively, p< 0.01). No changes in clot firmness were observed following ROTEM analysis. Tyrosine kinase inhibitors PRT-060318 (Syk) and dasatinib (Src) significantly reduced ROS in normo-glycaemia conditions (38±8% and 34±19%, respectively, p< 0.05). PRT-060318 also decreased ROS in acute hyperglycaemia. No effect of tyrosine kinase inhibitors on mitochondrial density was observed. Following 8-week liquid high-sucrose diet, WT mice showed significantly increased ROS and mitochondrial density (34±4% and 37±9%, respectively, p< 0.05) compared to normal drinking water. High-sucrose liquid diet did not alter ROS or mitochondrial density in GPVI-/- mice.

Conclusion(s): Our data suggest that targeting GPVI could modulate hyperglycaemia-induced increased platelet activity, contributing to reduction in thrombosis risk in patients with diabetes.