PB0746 - Hexokinase2 regulates glycoprotein VI mediated glycolysis and platelet activation

Background: Platelets are reliant on glucose metabolism to drive multiple aspects of platelet activation. However, our understanding of the regulation of the key regulatory steps in the conversion of glucose to pyruvate and how they control platelet activation remains unclear.

Aims: We investigated the role of hexokinases (HK), the first-rate limiting enzyme in glycolysis, on platelet function and metabolism.

Methods: We used a combination of immunobiological, flow cytometric and metabolic analyses to examine HK biology in human platelets.

Results: We have found human platelets express two differentially localised isoforms of HK, HK1 and HK2. While HK1 was widely expressed, HK2 was found exclusively in the membrane fraction. Stimulation of platelets with thrombin, convulxin or U46619 resulted in increased glucose uptake, glycolysis, glycolytic capacity and used glycoATP as the primary energy source. To explore the role of HKs in platelet function we used isoform specific inhibitors of HK, 2-deoxyglucose (2-DG: panHK inhibitor) and 3-bromopyruvate (3-BP; HK2 specific inhibitor). GPVI-stimulated integrin IIb3 activation and aggregation were blocked by both inhibitors, while platelet activation in response to thrombin or U46619 were unaffected. These data suggested that HK lay downstream of GPVI and its activity was required for platelet activation. To confirm this, we found that convulxin caused a significant increase in HK activity. The change in enzyme activity was ablated by the pharmacological inhibition of Src family kinases, Syk, phosphoinositide 3-kinase and Akt (protein kinase B). Furthermore 2-DG and 3-BP modulated GPVI-induced tyrosine phosphorylation, demonstrating that HK activity and generation of ATP is required to drive activatory signalling and aggregation.

Conclusion(s): The data show for the first time that direct crosstalk between the GPVI and glycolysis pathways is required for platelet activation. Moreover, this may occur in an isoform specific manner with HK2 playing a prominent role.
This work was funded by the British Heart Foundation (RG F22/110067)