Making the cut: Protein breakdown in platelets during storage
What is this research about?
Proteins are responsible for most of the work in cells. Based on their functions, locations, and interactions with each other, proteins keep cells, tissues, organs and bodies healthy and working well. Within cells, proteins are built up and broken down as needed. Each protein is made of individual building blocks called amino acids. To build proteins, amino acids bond with each other to make short chains, called peptides, and longer chains, called proteins. Conversely,proteolysis is the process of cutting proteins into peptides or individual amino acids. During proteolysis, bonds between amino acids are cut by proteins called proteases.
Targeted, specific cutting of proteins may contribute to the loss of platelet function during storage.
Proteolysis is an important part of normal cell processes and occurs for many reasons. For example, proteins can be completely broken down to prevent the build up of unnecessary or damaged proteins. In this way, proteolysis works like a shredder and completely destroys unwanted proteins. Proteolysis can also be a controlled and specific process during which the protein is cut at very specific sites. The resulting protein or peptide is shorter but stable and often functional. In this case, proteolysis can be compared to a scissors in skilled hands, carefully tailoring the form and function of specific proteins.
Platelets, red blood cells and plasma prepared by Canadian Blood Services for transfusion contain thousands of different types of proteins. Platelets are important for blood clotting and are often transfused as supportive therapy for cancer patients. Unlike red blood cells and plasma, which can be stored for longer, platelets have a short shelf life of just five days. In part, this is because they must be stored at room temperature, which puts them at greater risk of bacterial contamination. However, this is not the only reason to limit platelet storage time. The function of platelets declines during their short storage time. Known as the platelet storage lesion, this phenomenon is much studied but not yet fully understood. The platelet storage lesion affects everything from platelet shape to the platelet’s ability to metabolize and involves changes in the platelet’s proteins.
In this study, researchers at the University of British Columbia centre for blood research explored platelet storage lesion from a new perspective. They focused on what happens to proteins, and, specifically, which proteins get cut, where they get cut, and how these protein changes might affect protein and platelet function.
What did the researchers do?
Platelets were collected, processed and stored under the standard conditions used at Canadian Blood Services. These control platelets were compared to platelets stored in the presence of a drug (marimastat) that blocks the action of a group of protein-cutting proteases called metalloproteinases. A special technique developed at the University of British Columbia called TAILS was used to examine proteolysis of platelet proteins during storage. Using TAILS the researchers examined all proteins in platelets and found out which proteins are cut, at precisely what location within the protein and at what point during the storage period.
What did the researchers find?
Nearly 3,000 proteins and over 7,500 unique peptides were identified in platelets.
Over 3,000 N-termini were found. The N-terminus is a physical starting point of a protein or a peptide; its position defines the structural and functional boundaries of a protein and may indicate that the protein or peptide has been cut by proteases.
Proteins involved in cell structure, metabolism and cell communication were shown to be cut during platelet storage.
The majority (77 per cent) of the cut proteins produced stable fragments. This suggests that the process is not simply breakdown or degradation of unnecessary proteins, but a regulated, controlled process.
Experiments with protease blocking drugs showed that metalloproteinases were responsible for much of the proteolysis seen in platelets during storage.
How can you use this research?
Proteolysis of certain proteins has been linked to platelet storage lesion before, but this wide-ranging approach provided a broad view of which proteins are cut in platelets during storage and where they are cut. Using the TAILS approach developed at the University of British Columbia, the study showed wide-spread proteolysis in platelets during storage. Importantly, the results showed that proteolysis in stored platelets is not just protein shredding. Instead, much of the proteolysis is tailored, i.e. targeted, specific processing of proteins that results in stable proteins or peptides that may function differently from the original parent protein.
With the advent of new technologies to reduce the risk of bacterial contamination of platelets comes the possibility of longer storage times for platelets. For this to become a reality, a better understanding of the platelet storage lesion is needed. This study shows the important role that proteolysis plays in platelets during storage. The findings suggest that metalloproteinases could be targeted to stop the loss of platelet function that currently prevents extended storage.
The drug used in this study, marimastat, was developed as an anti-cancer drug and used in clinical trials in the early 2000s. Although found to be clinically safe, it failed to improve survival and caused painful side effects and its use was discontinued. Whether storage of platelets with metalloproteinase inhibitors or other proteolysis inhibitors could safely improve the quality and function of platelets for transfusion is an intriguing prospect that remains to be tested.
About the research team
Dr. Anna Prudova is a research associate in Dr. Christopher Overall’s laboratory at the University of British Columbia centre for blood research, Vancouver, BC. Dr. Ulrich Eckhard and Nikolaus Fortelny are a postdoctoral fellow and a graduate student, respectively, in Dr. Overall’s laboratory at the centre for blood research. Dr. Christopher Overall is a professor in the department of oral biological and medical sciences and the department of biochemistry and molecular biology at the University of British Columbia, Vancouver, BC. Dr. Katherine Serrano is a research associate with Canadian Blood Services centre for innovation and a clinical assistant professor at the department of pathology and laboratory medicine at the University of British Columbia. Dr. Dana Devine is the Canadian Blood Services chief medical and scientific officer and a professor in the department of pathology and laboratory medicine at the University of British Columbia.
This research unit is derived from the following publication(s)
Acknowledgements: Dr. Prudova was supported by a University of British Columbia centre for blood research strategic training program in transfusion science and the centre for blood research collaborative award. Dr. Eckhard was supported by a postdoctoral fellowship from the Michael Smith Foundation for Health Research. Dr. Overall holds a Canada research chair in protease proteomics and systems biology.
This research received financial support from Canadian Blood Services, funded by the provincial and territorial Ministries of Health and Health Canada, and from Canadian Institutes of Health Research. Infrastructure funding at the University of British Columbia centre for blood research is from the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research. The views expressed herein do not necessarily represent the view of the federal government. Canadian Blood Services is grateful to blood donors for making this research possible.