A role for SHARPIN in platelet linear protein ubiquitination and function – By Moore et al. (2021).

Background

Src homology 3 and multiple ankyrin repeat domains protein (SHANK)-associated RH domain-interacting protein (SHARPIN). SHARPIN modulates immune and inflammatory responses. A spontaneous null mutation in the SHARPIN gene in mice results in chronic proliferative dermatitis, multi-organ inflammation, and increased expression of inflammatory cytokines.

The molecular mechanism behind these events could be SHARPIN’s role in regulating the activation of NfkB as part of the linear ubiquitin chain assembly complex (LUBAC), responsible for regulating many cell signaling pathways.

Ubiquitination and NfKB play a role in platelet function. Additionally, SHARPIN negatively regulates integrin and can also regulate platelet function by activating the NfkB pathway.

Until now, there’s little information about linear ubiquitination or SHARPIN in mouse platelets. So, researchers at the University of Bristol (UK), evaluated platelet function in mice with impaired SHARPIN expression.

Methods

To assess the role of SHARPIN in regulating platelet function, the researchers used C57BL/KaLawRij-SHARPINcpdm/RijSunJ (cpdm) mouse line. The cpdm mutation results in the loss of SHARPIN protein expression. The group evaluated if these mice had altered thrombus formation. For that, they tested the ability of cpdm/cpdm platelet to adhere to fibrilar collagen under arterial shear.

The experiment involved perfusing whole blood anticoagulated with 40 μM PPACK and 5 U/ml clinical-grade heparin and labeled with DiOC into a Cellix Vena 8 biochip coated with fibrilar collagen (120 dynes/cm2, 3 min). The researchers washed and fixed the biochips before capturing the images using a 40× oil immersion objective on a Leica SPE single-channel confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope.

Results

Accumulation on collagen under shear is reduced in cpdm/cpdm platelets

SHARPIN loss reduced platelet accumulation and thrombus volume in whole blood under non-coagulating conditions compared to the control (Fig. 1 C).

Platelets from cpdm/cpdm mice do not express SHARPIN, have reduced linear ubiquitination and show reduced accumulation on collagen under shear. Washed platelets were treated as indicated before lysis in 4× NuPAGE sample buffer containing 0.5 M DTT before SDS-PAGE/Western blotting. A) SHARPIN and TALIN (control) expression in wild-type and cpdm/cpdm platelets. B) Detection of linear (Met1-linked) ubiquitination by LUB9 antibody in basal and stimulated wild-type and cpdm/cpdm platelets. i representative blot, ii summary densitometry n=3 mean +/-s.e., C) Whole blood anticoagulated with 40 μM PPACK and 5 U/ml clinical-grade heparin and labelled with DiOC[was drawn over a Cellix Vena8 biochip coated with fibrillar collagen (120 dynes/cm2, 3 min). Biochips were washed and fixed before images captured using a 40× oil immersion objective on a Leica SPE single channel confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. Accumulation of cpdm/cpdm platelets was reduced in comparison to wt/wt platelets resulting in a reduction in thrombus volume (n = 4 mean ± s.e.). For blotting data 2-way ANOVA (variable 1 = genotype, variable 2 = agonist stimulation) followed by Bonferroni’s multiple comparison test was used to test statistical significance. For in vitro flow data a Mann Whitney test was used to test statistical significance. α = 0.05. Three randomly chosen fields of view were analysed per sample. Quantification was performed using Volocity® 6.1.1 Quantitation software (Perkin Elmer Inc., San Jose, CA, USA). Representative images were captured using ImageJ 1.52k (Z-projection). α = 0.05. *: p<0.05, **: p<0.01.

Other experiments in this study

The researchers conducted a series of experiments to analyse the effects of SHARPIN loss on mice’s platelet function and signaling.

Main findings of these experiments

• Platelets from cpdm/cpdm mice don’t express SHARPIN and have reduced linear ubiquitination.

• Cpdm/cpdm mice showed increased white blood cell count without altering red blood cell count. Platelet count moderately decreased, and mean volume increased in these animals. Also, their platelets expressed less integrin β1.

• Platelet function in cpdm/cpdm mice is reduced in response to the GPVI agonist (CRP-XL).

• U46619-mediated platelet function is impaired in cpmd/cpdm platelets.

• GPVI and U46619-mediated signaling is altered in cpdm/cpdm platelets.

• Activation of human platelets induces IKK-dependent phosphorylation but not degradation of IκBα, a primary downstream substrate of the IkB kinase complex, required for NfKB translocation/activation in nucleated cells.

These results indicate that SHARPIN loss and linear ubiquitination results in impaired thrombosis and platelet functional responses to CRP and U46619. Suppression of platelet function does not depend on inhibition of the NfkB pathway.

For more details, access the complete study here.

How to get started

Are you thinking about running similar experiments in your lab? This is the minimum experimental setup you’ll need:

• Vena8 Fluoro+ biochips — to mimic human blood vessels and model blood clots.

• Mirus Evo pump — to control flow rates in the biochip. You may set the shear rate to model thrombosis in microcapillaries or other vessels.

• Microenvironmental chamber — a temperature-controlled frame that keeps the biochip at 370C. The microenvironmental chamber sits on the microscope stage

• Inverted microscope — we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option

• Digital camera — to capture images and video recordings. We supply the Prime BSI Express with the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

• Image Pro Cell Analysis software — for your image and video analysis

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. We have options that suit all budgets. You can check them out on our eShop.

References

1- Moore, S. F., Zhao, X., Mallah, S., Poole, A. W., Mundell, S. J., Hutchinson, J. L., & Hers, I. (2021). A role for SHARPIN in platelet linear protein ubiquitination and function. bioRxiv.