Platelet Storage Temperature Determines Recovery of GPVI-Function In Vivo

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.39-39
• Main Authors: Miles, Jeffrey, Usaneerungrueng, Chomkan, Obenaus, Ava M., Mollica, Molly Y, Flynn, Jake R, Bailey, Shawn Lawrence, Osborne, Barbara, Corson, Jill, Houck, Katie, Dong, Jing-fei, Sniadecki, Nathan J., Stolla, Moritz
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-141741

Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry stu