title: Investigating the structure / function relationship of PLCγ2 downstream of GPVI and CLEC-2
Creators: name: Gayes, Safa
Publisher: name: University of Reading
Year: 2024

description: 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.