Abstract/Summary

Background: Several platelet signalling pathways result in the activation of phospholipase C gamma 2 (PLCγ2), making it an important signalling hub. Both the ITAM-linked collagen receptor GPVI, and the hemITAM-containing podoplanin receptor CLEC-2, activate PLCγ2 via a similar mechanism. PLCγ2 is a large protein consisting of multiple domains. PLCγ2’s molecular interaction with other proteins regulates its function, however the exact mechanism is still not fully understood. PLCγ2 also plays a similar role downstream of ITAM-linked receptors in other cells, such as B- and T-cells. In 80% of leukaemia patients who acquire resistance to ibrutinib therapy, PLCγ2 mutations have been detected. Although these mutations have been shown to lead to a gain function, it’s still unknown whether these mutations might arise in platelets and what their functional effect on platelet activity might be. Aims: Characterise the structure/function relationship of PLCγ2 downstream of (hem)ITAM receptors. Methods: A PLC pharmacological inhibitor, U73122, was used in platelet function assays including aggregation, calcium release, and platelet adhesion and spreading to characterise the impact of PLCγ2 inhibition on platelet function mediated by GPVI and CLEC-2. To understand the structure/function relationship of PLCγ2 downstream of the (hem)ITAM receptors, construct with PLCγ2 mutations were transiently transfected in PLCγ2 knockout cells and U73122 was used to assess the calcium signalling in the cells. A CRISPR/Cas9 approach was also used to generate PLCγ2 mutation knock-in in WT DT40 cells. Using a laser injury model, zebrafish were used as an in vivo model to demonstrate the effect of U73122 on PLCγ2 in thrombus formation and vascular/lymphatic development. Results: GPVI mediated platelet aggregation and platelet spreading is more significantly affected by PLCγ2 inhibition than CLEC-2 mediated activation. U73122 inhibits calcium release in a similar manner downstream of both receptors. Using an overexpression system in PLCγ2 KO cells, PLCγ2 mutations lead to a gain of function as shown by increased signalling. However, this increased signalling was still susceptible to inhibition by U73122. Additionally, using the CRISPR/Cas9 approach, a PLCγ2 point mutation was successfully introduced in WT DT40 cells. Finally, U73122 leads to an extended time to occlusion and lack of thrombus formation in zebrafish. Conclusion: These results confirm the essential role for PLCγ2 downstream of platelet (hem)ITAM receptor signalling. U73122 significantly decreases GPVI and CLEC-2 mediated platelet activation. Downstream of CLEC-2, U73122 decreases signalling more significantly in cells bearing mutations in the catalytic domain suggesting that the CLEC-2 signalling pathway is more reliant on the activity of the catalytic domain of PLCγ2 than GPVI. Finally, PLCγ2 has shown to play an essential role in thrombus formation in zebrafish.