Thomas, T. A. | bioRxiv Science

bioRxiv2023

Characterization of a Novel Mouse Platelet Transfusion Model

BACKGROUNDPlatelet transfusions are increasing with advances in medical care. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. To address this, we developed a novel platelet transfusion model that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detected in clots in vivo. STUDY DESIGN AND METHODSPlatelet units stored in mouse plasma were prepared using a modified platelet rich plasma collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTSPlatelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and ADP, aggregable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. DISCUSSIONWe developed mouse platelets for transfusion analogous to human platelet units using a modified platelet rich plasma collection protocol with maximum storage of 1 day for an "old" unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

pathology↗

bioRxiv2021

Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

Proteasome Activator 28{gamma} (PA28{gamma}) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28{gamma} functions in protein degradation remains unclear. Though PA28{gamma}s mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28{gamma} regulates proteasome function. Here, we rigorously and definitively show that PA28{gamma} uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28{gamma}-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28{gamma} also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28{gamma}-20S proteasome complex at 4.4[A] validating its expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28{gamma} functions by allosterically upregulating the T-L like site in the 20S proteasome. Significance StatementThis study rigorously demonstrates that PA28g allosterically activates the b-2 proteolytic site of the 20S proteasome directly without affecting 20S gating. Further, we generated the first human 11S-human 20S proteasome cryo-EM structure of the PA28g-20S complex showing that, despite its different affects on 20S activity, it has a similar quaternary structure as the other 11S family members. The significance of these findings is paramount as the b-2 site is responsible for post-basic cleavage and suggests that PA28g is specialized to degrade positively charged DNA binding proteins. Further, b-2 upregulation via PA28g could provide a protective effect against poly-glutamine expanded proteins, like Huntingtin. This work provides a framework for PA28g drug development to treat PA28g addicted cancers and Huntingtons Disease.

biochemistry↗