-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

247 Targeted Proteomics Reveals That the Dominant Mechanism of Gpibα Loss during Platelet Storage Depends on Temperature of Storage

Program: Oral and Poster Abstracts
Type: Oral
Session: 401 Basic Science and Clinical Practice in Blood Transfusion: COVID-19 Convalescent Plasma and Transfusion Immunology I
Hematology Disease Topics & Pathways:
blood banking, blood product storage, Technology and Procedures
Saturday, December 5, 2020: 3:15 PM

Moritz Stolla, MD1,2, Yi Wang, PhD1*, Jeffrey Miles1*, Barbara Osborne, RN1*, Yu Shen1*, Lydia Fang, M.S.1*, Shawn Lawrence Bailey, BS1*, Warren Osborn1*, Junmei Chen, PhD1, Jose A. Lopez, MD, BS2,3 and Xiaoyun Fu, PhD1

1Bloodworks Northwest Research Institute, Seattle, WA
2Department of Medicine, University of Washington, Seattle, WA
3BloodworksNW Research Institute, Seattle, WA

Background: Platelet transfusion is a potentially lifesaving procedure, used for both prophylactic and therapeutic indications. Platelets can be stored at room temperature (RT) for up to 7 days in air-permeable bags. Platelet function diminishes during storage, a phenomenon known as the storage lesion. We and others have shown that platelets can be stored for extended periods of time at 4°C and still show acceptable in vitro function while limiting bacterial growth. In the present study, we used proteomics to examine the changes in human platelets stored at RT and 4°C with a focus on the glycoprotein (GP) Ib-IX-V complex, the key receptor for platelet adhesion at sites of vessel injury.

Study Design/Method: Platelet units from healthy donors were stored in 100% plasma with or without agitation (at 22°C or 4°C, respectively) at a concentration of 3x1011/L and sampled on days 0, 3, 7, and 14. Microparticles were detected by flow cytometry as described previously. For proteomic analysis, platelets were washed and digested with trypsin. Tryptic peptides were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano LC-MS/MS). MS/MS spectra were searched against the human protein database using Proteome Discoverer 2.4 software. A student t-test test was used to determine significant differences in analytes amongst the different storage groups.

Results/Finding: Under both storage conditions, GPIbα and GPV decreased significantly over storage time. However, comparison of the decline in these proteins to GPIbβ, GPIX, and other membrane proteins indicated that the mechanisms for this decline differ in the two conditions. At RT, the decrease in GPIbα and GPV appears to be largely proteolytic, given that only a minor concomitant decrease in surface level was seen in the protease-insensitive GPIX and a slight increase in GPIbβ. In addition, a comparable decrease in GPIbα level was not observed when a cytoplasmic GPIbα peptide was assayed, suggesting the extracellular portion had been proteolytically removed. In contrast, at 4°C the decline in GPIbα and GPV was accompanied by a modest decrease in GPIX, and only a small decrease in the ratio of extracellular to cytoplasmic to GPIbα peptide. These results suggested that, at 4°C, in addition to proteolysis, which was attenuated as compared to RT storage, another mechanism was responsible for removal of full-length GPIbα and other polypeptides. One such mechanism that could explain this would be loss of membrane from the platelets during 4°C storage. Indeed, we found that extracellular vesicles accumulated in the platelet supernatant during 4°C to a much higher level than at RT storage.

Summary: One of the hallmarks of the platelet storage lesion at RT is shedding of surface membrane proteins including GPIbα. Previous studies in stored mouse and human platelets revealed a cleavage mechanism dependent on the metalloproteinase ADAM17. However, whether GPIbα is lost by the same mechanism in cold-stored human platelets was unknown. Our targeted proteomics analysis confirms that proteolysis is a major cause of GPIbα loss at RT, but is a less prominent mechanism at 4°C. However, another mechanism for membrane protein loss is more prominent at the lower temperature: microvesiculation. Thus, these studies provide new insights into the platelet storage lesion and suggest that measures to prevent them will have to be tailored to the dominant mechanism operating at a particular storage temperature.

Disclosures: No relevant conflicts of interest to declare.

<< Previous Abstract | Next Abstract
*signifies non-member of ASH