Platelets from Fresh and Cold-Stored Whole Blood Contribute to Clot Formation after Transfusion in Rats with Acute Traumatic Coagulopathy
Authors: Wu Xiaowu , Montgomery Robbie , Liu Bin , Keesee Jeffrey D , Benov Avi , Darlington Daniel N , Cap Andrew P 
Published: October 2019

Abstract
Background: The hemostatic and platelet functions of fresh whole blood (FWB) are well preserved when stored at 4°C for 14 days (human) or 7 days (rats). Resuscitation of either FWB or whole blood stored for 7-days at 4°C (SWB) in rats with acute traumatic coagulopathy (ATC) induced by polytrauma and hemorrhage (T/H), shows identical correction of hemodynamic and metabolic function, as well as the equivalent impact on platelet aggregation in vitro. Platelet hemostatic function is influenced by platelet number, aggregation, and platelet-leukocyte interactions. Therefore, the objective of this study is to identify the in-vivo fate and activity of platelets from FWB and SWB, and determine whether transfused platelets contribute to clot formation in tissue injury after transfusion.
Method: Isoflurane anesthetized Sprague-Dawley rats underwent polytrauma, followed by 40% hemorrhage. The rats were resuscitated (20% of bled volume) 1hr after trauma using either FWB or SWB (n=5 each) collected from green fluorescence (GFP) donor rats. SWB was kept in mini-blood-bag and refrigerated at 40C for 7 days, and warmed up to 370C prior to transfusion. Sham rats had no trauma, but underwent 20% blood replacement by FWB or SWB (n=4 each). Immediately after transfusion, a liver incision was made, followed by the measurement of kidney bleeding time (both standardized incisions was made by a Surgicutt adult template device). The incisional liver sample was taken 10min after injury for immunohistochemistry stained for CD61 (platelets) and GFP. Blood samples taken from donor rats (FWB and SWB) and recipient rats at baseline, immediately before resuscitation, and 0min and 10min after transfusion were analyzed by flow cytometry using CD42d, CD45, and CD62P antibodies for defining platelets, leukocytes and activated platelets respectively. Lactadherin was used to detect phosphatidylserine (PS). Platelet-leukocyte aggregates were defined as CD42d+/CD45+. The transfused and native platelet or leukocyte was differentiated by GFP signal.
Results: GFP signal intensity was equally expressed in platelets derived from either FWB or SWB. Storage at 4°C led to a significant increase in number of platelets expressing CD62P (76±2% vs. 11±4%) and PS (8.6±0.7% vs. 1.1±0.4%) as compared to FWB. SWB also showed a significant rise in intensity, but not percentage, of platelet-leukocyte aggregates (45781±6235 vs. 10978±2345 MFI). After transfusion, recipient rats showed a significant elevation in the percentage of GFP+ platelets after transfusion of FWB as compared to SWB (Sham:13.0±1.0% vs. 4.9±0.4%; T/H: 17.0±0.8% vs. 6.8±0.%). Similarly, GFP+ leukocytes aggregates from FWB were three times higher than SWB (Sham: 6.15±0.5% vs. 1.9±0.5%; T/H: 4.4±0.4% vs. 1.2±0.2%). There was greater percentage of GFP+ platelet-leukocyte aggregates in both T/H and sham rats transfused with SWB than FWB (Sham: 11.7±3.3% vs. 33.8±2.3%; T/H: 9.8±1.6% vs. 47.2±5.1%). Transfusion with SWB led to a significant increase in percentage of activated GFP+ platelets in T/H rats as compared to FWB (30.0±2.7% vs. 2.5±0.5%). However, the activity of native platelets was not significantly different between SWB and FWB in T/H rats after transfusion (3.9±1.2% vs. 3.7±0.3%). The kidney bleeding time was not significantly different in T/H rats receiving FWB and SWB (131±4 vs.127±7 seconds) under equivalent mean arterial blood pressure (82±7 vs 85±9 mmHg). The clot that formed at the site of liver incision was identified by platelet aggregates stained by the CD61 antibody. Using co-localization of CD61 and GFP, we found that the platelets from both FWB and SWB equivalently incorporated into the clot at incisional site.
Conclusion: The platelets from SWB are higher in platelet activation state, clearance rate and platelet-leukocyte aggregates than FWB after transfusion in both sham rats and rats with polytrauma and hemorrhage. However, platelets from both fresh and cold-stored whole blood contribute to hemostasis of tissue injury after transfusion. This study suggests that cold-stored whole blood is an alternative resource to treat trauma patients for restoration of hemostatic function. Future study is necessary to optimize the storage of whole blood to prolong the platelet survival rate and optimize hemostatic function. This project was funded by US Army Medical Research and Material Command.

Disclosures
No relevant conflicts of interest to declare.