Breaking barriers in trauma research: A narrative review of opportunities to leverage veterinary trauma for accelerated translation to clinical solutions for pets and people.

Trauma is a common cause of morbidity and mortality in humans and companion animals. Recent efforts in procedural development, training, quality systems, data collection, and research have positively impacted patient outcomes; however, significant unmet need still exists. Coordinated efforts by collaborative, translational, multidisciplinary teams to advance trauma care and improve outcomes have the potential to benefit both human and veterinary patient populations. Strategic use of veterinary clinical trials informed by expertise along the research spectrum (i.e., benchtop discovery, applied science and engineering, large laboratory animal models, clinical veterinary studies, and human randomized trials) can lead to increased therapeutic options for animals while accelerating and enhancing translation by providing early data to reduce the cost and the risk of failed human clinical trials. Active topics of collaboration across the translational continuum include advancements in resuscitation (including austere environments), acute traumatic coagulopathy, trauma-induced coagulopathy, traumatic brain injury, systems biology, and trauma immunology. Mechanisms to improve funding and support innovative team science approaches to current problems in trauma care can accelerate needed, sustainable, and impactful progress in the field. This review article summarizes our current understanding of veterinary and human trauma, thereby identifying knowledge gaps and opportunities for collaborative, translational research to improve multispecies outcomes. This translational trauma group of MDs, PhDs, and DVMs posit that a common understanding of injury patterns and resulting cellular dysregulation in humans and companion animals has the potential to accelerate translation of research findings into clinical solutions.

Hyperbaric treatment of platelets is comparable to cold storage alone over 14 days.

BACKGROUND: Platelets stored at room temperature (22-24°C) for transfusion purposes have a shelf life of 5-7 days, or 72 h when stored refrigerated (1-6°C). The limited shelf life of platelet products severely compromises platelet inventory. We hypothesized that cold storage of platelets in 100% plasma using xenon gas under high pressure would extend shelf life to 14 days. STUDY DESIGN AND METHODS: Double apheresis platelet units were collected and split equally between two bags. One unit was placed in a hyperbaric chamber, pressurized to 4 bars with a xenon/oxygen gas mixture, and placed in a refrigerator for 14 days (Xe). The remaining unit was aliquoted into mini-bags (10 ml) for storage at room temperature (RTP) or in cold (CSP). Samples were assayed on days 5 (RTP) or 14 (Xe and CSP) for count, metabolism, clot strength, platelet aggregation, and activation markers. RESULTS: The platelet count in Xe samples was lower than that of RTP but significantly higher than CSP. Despite similar levels of glucose and lactate, the pH of Xe samples was significantly lower than CSP. Glycoprotein expression was better preserved by Xe storage compared to CSP, but no differences in activation were observed. Thromboelastography and aggregometry results were comparable between all groups. DISCUSSION: Cold storage of platelets in plasma with hyperbaric xenon provides no significant improvement in platelet function over cold storage alone. The use of a hyperbaric chamber and the slow off-gassing of Xe-stored units complicate platelet storage and delivery logistics.

Perfluorocarbons cause thrombocytopenia, changes in RBC morphology and death in a baboon model of systemic inflammation.

A perfluorocarbon (PFC) investigated for treatment of traumatic brain injury (TBI) delivers oxygen to support brain function, but causes transient thrombocytopenia. TBI can cause acute inflammation with resulting thrombocytopenia; an interaction between the PFC effects and TBI inflammation might exacerbate thrombocytopenia. Therefore, PFC effects on platelet (PLT) function and hemostasis in a lipopolysaccharide (LPS) model of inflammation in the baboon were studied. Animals were randomized to receive saline ±LPS, and ± one of two doses of PFC. PLT count, transmission electron microscopy, and microparticle populations were quantified at baseline (BL) and at 2, 24, 48, 72, and 96 hours; hemostatic parameters for aggregometry and for blood clotting were measured at baseline (BL) and days 3 and 4. Injection of vehicle and LPS caused thrombocytopenia within hours; PFCs caused delayed thrombocytopenia beginning 48 hours post-infusion. LPS+PFC produced a more prolonged PLT decline and decreased clot strength. LPS+PFC increased ADP-stimulated aggregation, but PFC alone did not. Microparticle abundance was greatest in the LPS+PFC groups. LPS+PFC caused diffuse microvascular hemorrhage and death in 2 of 5 baboons in the low dose LPS-PFC group and 2 of 2 in the high dose LPS-PFC group. Necropsy and histology suggested death was caused by shock associated with hemorrhage in multiple organs. Abnormal morphology of platelets and red blood cells were notable for PFC inclusions. In summary, PFC infusion caused clinically significant thrombocytopenia and exacerbated LPS-induced platelet activation. The interaction between these effects resulted in decreased hemostatic capacity, diffuse bleeding, shock and death.

Use of Specialized Pro-Resolving Mediators to Alleviate Cold Platelet Storage Lesion.

BACKGROUND: Cold-stored platelets are an attractive option for treatment of actively bleeding patients due to a reduced risk of septic complications and preserved hemostatic function compared to conventional room temperature-stored platelets. However, refrigeration causes increased platelet activation and aggregate formation. Specialized pro-resolving mediators (SPMs), cell signaling mediators biosynthesized from essential fatty acids, have been shown to modulate platelet function and activation. In this study, we sought to determine if SPMs could be used to inhibit cold-stored platelet activation. METHODS: Platelets were collected from healthy donors (n = 4-7) and treated with SPMs (resolvin E1 [RvE1], maresin 1 [MaR1], and resolvin D2 [RvD2]) or vehicle (VEH; 0.1% EtOH). Platelets were stored without agitation in the cold and assayed on Days 0 and 7 of storage for platelet activation levels using flow cytometry, platelet count, aggregation response using impedance aggregometry, and nucleotide content using mass spectrometry. RESULTS: Compared to VEH, SPM treatment inhibited GPIb shedding (all compounds), significantly reduced both PS exposure and activation of GPIIb/IIIa receptor (RvD2, MaR1), and preserved aggregation response to TRAP (RvD2, MaR1) after 7 days of storage. Similar to untreated cold-stored platelets, SPM-treated samples did not preserve platelet counts or block the release of P-Selectin. Nucleotide content was unaffected by SPM treatment in cold-stored platelets. CONCLUSIONS: SPM treatment, particularly Mar1 and RvD2, led to reduced platelet activation and preserved platelet function after 7 days of storage in the cold. Future work is warranted to better elucidate the mechanism of action of SPMs on cold platelet function and activation.

Blood component separation of pathogen-reduced whole blood by the PRP method produces acceptable red cells but platelet yields and function are diminished.

BACKGROUND: This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. STUDY DESIGN AND METHODS: PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. RESULTS: Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. CONCLUSION: Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.

Similar hemostatic responses to hypovolemia induced by hemorrhage and lower body negative pressure reveal a hyperfibrinolytic subset of non-human primates.

BACKGROUND: To study central hypovolemia in humans, lower body negative pressure (LBNP) is a recognized alternative to blood removal (HEM). While LBNP mimics the cardiovascular responses of HEM in baboons, similarities in hemostatic responses to LBNP and HEM remain unknown in this species. METHODS: Thirteen anesthetized baboons were exposed to progressive hypovolemia by HEM and, four weeks later, by LBNP. Hemostatic activity was evaluated by plasma markers, thromboelastography (TEG), flow cytometry, and platelet aggregometry at baseline (BL), during and after hypovolemia. RESULTS: BL values were indistinguishable for most parameters although platelet count, maximal clot strength (MA), protein C, thrombin anti-thrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI) activity significantly differed between HEM and LBNP. Central hypovolemia induced by either method activated coagulation; TEG R-time decreased and MA increased during and after hypovolemia compared to BL. Platelets displayed activation by flow cytometry; platelet count and functional aggregometry were unchanged. TAFI activity and protein, Factors V and VIII, vWF, Proteins C and S all demonstrated hemodilution during HEM and hemoconcentration during LBNP, whereas tissue plasminogen activator (tPA), plasmin/anti-plasmin complex, and plasminogen activator inhibitor-1 did not. Fibrinolysis (TEG LY30) was unchanged by either method; however, at BL, fibrinolysis varied greatly. Post-hoc analysis separated baboons into low-lysis (LY30 <2%) or high-lysis (LY30 >2%) whose fibrinolytic state matched at both HEM and LBNP BL. In high-lysis, BL tPA and LY30 correlated strongly (r = 0.95; P<0.001), but this was absent in low-lysis. In low-lysis, BL TAFI activity and tPA correlated (r = 0.88; P<0.050), but this was absent in high-lysis. CONCLUSIONS: Central hypovolemia induced by either LBNP or HEM resulted in activation of coagulation; thus, LBNP is an adjunct to study hemorrhage-induced pro-coagulation in baboons. Furthermore, this study revealed a subset of baboons with baseline hyperfibrinolysis, which was strongly coupled to tPA and uncoupled from TAFI activity.

An in vitro pilot study of apheresis platelets collected on Trima Accel system and stored in T-PAS+ solution at refrigeration temperature (1-6°C).

BACKGROUND: Using platelet additive solution (PAS) to dilute fibrinogen during long-term cold storage of platelets (PLTs) decreases PLT activation and increases functional PLT shelf life. We performed a randomized, paired study to assess the in vitro quality of PLTs stored in the cold in T-PAS+ for up to 18 days evaluated against PLTs stored under currently allowable conditions (5-day room temperature-stored PLTs [RTP] and 3-day cold-stored PLTs [CSP]). STUDY DESIGN AND METHODS: PLTs were collected from healthy volunteers (n = 10) and diluted to 65% T-PAS+/35% plasma before cold storage. Double-dose apheresis PLTs (in 100% plasma) were collected from the same donors and split into two bags (one bag RTP, one bag CSP). All bags were sampled on the day of collection (Day 0). CSP and RTP bags were sampled on Days 3 and 5, respectively. T-PAS+ samples were assessed on Days 3, 5, 14, 16, and 18 of storage for metabolism, hemostatic function, and activation. RESULTS: After 18 days of storage in T-PAS+, pH was 6.71 ± 0.04, PLT count was comparable to Day 3 CSP, PLT function (aggregation and clot strength) was comparable to Day 5 RTP, and PLT activation was significantly increased. CONCLUSION: Refrigerated PLTs stored in T-PAS+ for 18 days met FDA pH standards. Functional metrics suggest activity of T-PAS+-stored PLTs and the potential to contribute to hemostasis throughout 18 days of storage. Extending the shelf life of PLTs would increase access to hemostatic resuscitation for bleeding patients in military and civilian settings.

Damage Control Resuscitation.

Damage control resuscitation (DCR) is a strategy for resuscitating patients from hemorrhagic shock to rapidly restore homeostasis. Efforts are focused on blood product transfusion with whole blood or component therapy closely approximating whole blood, limited use of crystalloid to avoid dilutional coagulopathy, hypotensive resuscitation until bleeding control is achieved, empiric use of tranexamic acid, prevention of acidosis and hypothermia, and rapid definitive surgical control of bleeding. Patients receiving uncrossmatched Type O blood in the emergency department and later receiving cumulative transfusions of 10 or more red blood cell units in the initial 24-hour post-injury (massive transfusion) are widely recognized as being at increased risk of morbidity and mortality due to exsanguination. Ideally, these patients should be rapidly identified, however anticipating transfusion needs is challenging. Useful indicators of massive transfusion reviewed in this guideline include: systolic blood pressure <110 mmHg, heart rate > 105 bpm, hematocrit <32%, pH < 7.25, injury pattern (above-the-knee traumatic amputation especially if pelvic injury is present, multi-amputation, clinically obvious penetrating injury to chest or abdomen), >2 regions positive on Focused Assessment with Sonography for Trauma (FAST) scan, lactate concentration on admission >2.5, admission international normalized ratio ≥1.2-1.4, near infrared spectroscopy-derived StO2 < 75% (in practice, rarely available), BD > 6 meq/L. Unique aspects of out-of-hospital DCR (point of injury, en-route, and remote DCR) and in-hospital (Medical Treatment Facilities: Role 2b/Forward surgical teams - role 3/ combat support hospitals) are reviewed in this guideline, along with pediatric considerations.

The evolution of pediatric transfusion practice during combat operations 2001-2013.

BACKGROUND: Hemostatic resuscitation principles have significantly changed adult trauma resuscitation over the past decade. Practice patterns in pediatric resuscitation likely have changed as well; however, this evolution has not been quantified. We evaluated pediatric resuscitation practices over time within a combat trauma system. METHODS: The Department of Defense Trauma Registry was queried from 2001 to 2013 for pediatric patients (<18 years). Patients with burns, drowning, and missing injury severity score were excluded. Volumes of crystalloid, packed red blood cells (PRBC), whole blood, plasma, and platelets (PLT) given in the first 24 hours were calculated per kilogram body weight. Tranexamic acid use was also determined. Patients were divided into Early (2001-2005) and Late (2006-2013) cohorts, and subgroups of transfused (TX+) and massively transfused (MT+) patients were created. Intensive care unit and hospital length of stay and 24-hour and in-hospital mortality rates were compared. RESULTS: A total of 4,358 patients met inclusion criteria. Comparing Early versus Late, injuries from explosions, isolated or predominant head injuries, and injury severity score all increased. The proportion of TX+ patients also increased significantly (13.6% vs 37.4%, p < 0.001) as did the number of MT+ patients (2.1% vs 15.5%, p < 0.001). Transfusion of high plasma:RBC and PLT:RBC ratios increased in both the TX+ and MT+ subgroups, although overall, PLT and whole blood use was low. After adjusting for differences between groups, the odds of death was no different Early versus Late but decreased significantly in the MT+ patients with time as a continuous variable. CONCLUSION: Transfusion practice in pediatric combat casualty care shifted toward a more hemostatic approach over time. All-cause mortality was low and remained stable overall and even decreased in MT+ patients despite more injuries due to explosions, more head injuries, and greater injury severity. However, further study is required to determine the optimal resuscitation practices in critically injured children. LEVEL OF EVIDENCE: Epidemiologic study, level IV.

Immunopathological response to severe injury: platelet activation and the Th-17 immune response.

: Alterations in coagulation, inflammation and immunity are associated with major injury. As platelets have both coagulation and immune functions, the aim of this study is to correlate platelet activation with the immunoinflammatory response in trauma and burn patients. Blood samples were drawn from trauma and burn patients and healthy volunteers. Platelet (sCD40L) and coagulation (D-dimers) activation, cytokines and inflammatory markers were assessed. sCD40L, D-dimers and cytokines were elevated in both injury groups. Overall, sCD40L levels correlated with interleukin (IL)-6 and tumour necrosis factor-alpha. Subanalysis revealed a correlation between sCD40L and IL-17a in the healthy volunteers and burn groups, but not the trauma group. A parallel activation of platelets and the inflammatory response occurs postinjury. However, in trauma patients, a potential critical interrelationship between platelet activation and the Th-17 response appears to be lacking, which may contribute to coagulopathic and immunoinflammatory complications and warrants further study.

Platelets stored at 4°C contribute to superior clot properties compared to current standard-of-care through fibrin-crosslinking.

Currently, platelets for transfusion are stored at room temperature (RT) for 5-7 days with gentle agitation, but this is less than optimal because of loss of function and risk of bacterial contamination. We have previously demonstrated that cold (4°C) storage is an attractive alternative because it preserves platelet metabolic reserves, in vitro responses to agonists of activation, aggregation and physiological inhibitors, as well as adhesion to thrombogenic surfaces better than RT storage. Recently, the US Food and Drug Administration clarified that apheresis platelets stored at 4°C for up to 72 h may be used for treating active haemorrhage. In this work, we tested the hypothesis that cold-stored platelets contribute to generating clots with superior mechanical properties compared to RT-stored platelets. Rheological studies demonstrate that the clots formed from platelets stored at 4°C for 5 days are significantly stiffer (higher elastic modulus) and stronger (higher critical stress) than those formed from RT-stored platelets. Morphological analysis shows that clot fibres from cold-stored platelets were denser, thinner, straighter and with more branch points or crosslinks than those from RT-stored platelets. Our results also show that the enhanced clot strength and packed structure is due to cold-induced plasma factor XIII binding to platelet surfaces, and the consequent increase in crosslinking.

Dried plasma: state of the science and recent developments.

The early transfusion of plasma is important to ensure optimal survival of patients with traumatic hemorrhage. In military and remote or austere civilian settings, it may be impossible to move patients to hospital facilities within the first few hours of injury. A dried plasma product with reduced logistical requirements is needed to enable plasma transfusion where medically needed, instead of only where freezers and other equipment are available. First developed in the 1930s, pooled lyophilized plasma was widely used by British and American forces in WWII and the Korean War. Historical dried plasma products solved the logistical problem but were abandoned because of disease transmission. Modern methods to improve blood safety have made it possible to produce safe and effective dried plasma. Dried plasma products are available in France, Germany, South Africa, and a limited number of other countries. However, no product is available in the US. Promising products are in development that employ different methods of drying, pathogen reduction, pooling, packaging, and other approaches. Although challenges exist, the in vitro and in vivo data suggest that these products have great potential to be safe and effective. The history, state of the science, and recent developments in dried plasma are reviewed.

Making whole blood available in austere medical environments: donor performance and safety.

BACKGROUND: To provide whole blood on the battlefield can be a challenge, but a buddy system protocol is both an elegant and the only currently available means to supply blood to a Special Forces team in far-forward locations. Our aim was to investigate donor-safety associated with such a protocol. METHODS: This study was a randomized, double-blinded, controlled trial that aimed to evaluate the immediate effects of a 450 cc blood donation on physical performance in fatigued and dehydrated Special Forces soldiers. The primary outcome variables were absolute and relative maximal oxygen uptake (VO2max ), exercise tolerance time (ETT) and heart rate (HR). RESULTS: Relative VO2max decreased by 7.1% in the donation group between pre and posttest, compared to no change in the control group. Absolute VO2max decreased by 11.2 and 3.6% between pre and posttest in the donation and control groups, respectively. Mean ETT in the donation group was on average 92 seconds shorter compared to baseline, which represents a decrease of 9.5%. CONCLUSION: Donating blood after a week of strenuous physical activity is feasible for Special Forces personnel. While the donation results in some diminishment of VO2max , a 3.6%-11.2% decrease in relative VO2max , and in elevation of submaximal HR levels highly trained personnel continue to perform well both at both sub-maximal and maximal effort levels.

Whole blood for hemostatic resuscitation of major bleeding.

Recent combat experience reignited interest in transfusing whole blood (WB) for patients with life-threatening bleeding. US Army data indicate that WB transfusion is associated with improved or comparable survival compared to resuscitation with blood components. These data complement randomized controlled trials that indicate that platelet (PLT)-containing blood products stored at 4°C have superior hemostatic function, based on reduced bleeding and improved functional measures of hemostasis, compared to PLT-containing blood products at 22°C. WB is rarely available in civilian hospitals and as a result is rarely transfused for patients with hemorrhagic shock. Recent developments suggest that impediments to WB availability can be overcome, specifically the misconceptions that WB must be ABO specific, that WB cannot be leukoreduced and maintain PLTs, and finally that cold storage causes loss of PLT function. Data indicate that the use of low anti-A and anti-B titer group O WB is safe as a universal donor, WB can be leukoreduced with PLT-sparing filters, and WB stored at 4°C retains PLT function during 15 days of storage. The understanding that these perceived barriers are not insurmountable will improve the availability of WB and facilitate its use. In addition, there are logistic and economic advantages of WB-based resuscitation compared to component therapy for hemorrhagic shock. The use of low-titer group O WB stored for up to 15 days at 4°C merits further study to compare its efficacy and safety with current resuscitation approaches for all patients with life-threatening bleeding.

Shed Pleural Blood from Traumatic Hemothorax Contains Elevated Levels of Pro-Inflammatory Cytokines.

PURPOSE: The autotransfusion of unwashed (or unprocessed) shed hemothorax blood (USHB) in trauma patients is widely assumed to be beneficial; however, the inflammatory potential of shed pleural blood has not been thoroughly studied. Since previous studies have documented marked changes in coagulation function of shed pleural blood, we hypothesized that its level of inflammatory cytokines would be elevated. METHODS: A prospective observational study of trauma patients in whom cytokine levels from USHB were compared to venous samples from healthy volunteers was conducted. Differences between the cytokine content of patient-derived samples were compared to those from healthy subjects. RESULTS: There was a statistically significant increase in pro-inflammatory cytokines (IL-6, IL-8, TNFα, GM-CSF), a pro-inflammatory Th-1 cytokine (IFNγ), and anti-inflammatory Th-2 cytokines (IL-4 and IL-10) in shed pleural blood over four hours when compared with samples from healthy controls (P <0.05). Cytokine levels in USHB are approximately 10- to 100-fold higher compared with healthy control venous samples. CONCLUSIONS: USHB, even collected within the accepted four-hour window, contains significantly elevated cytokine levels, suggesting the potential for deleterious effects from autotransfusion. Randomized trials are needed to determine the safety and efficacy of autotransfusion in trauma patients.

Treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates Ebola virus in vitro.

BACKGROUND: Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called "convalescent plasma," is an effective treatment for active disease available in endemic areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV+RB) is effective against multiple enveloped, negative-sense, single-stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV+RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of Ebola virus disease (EVD). STUDY DESIGN AND METHODS: Four in vitro experiments were conducted to evaluate effects of UV+RB on green fluorescent protein EBOV (EBOV-GFP), wild-type EBOV in serum, and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1 to 3 were 4.21 log GFP units/mL, 4.96 log infectious units/mL, and 4.23 log plaque-forming units/mL. Conditions tested in the first three experiments included the following: 1-EBOV-GFP plus UV+RB; 2-EBOV-GFP plus RB only; 3-EBOV-GFP plus UV only; 4-EBOV-GFP without RB or UV; 5-virus-free control plus UV only; and 6-virus-free control without RB or UV. RESULTS: UV+RB reduced EBOV titers to nondetectable levels in both nonhuman primate serum (≥2.8- to 3.2-log reduction) and human whole blood (≥3.0-log reduction) without decreasing protective antibody titers in human plasma. CONCLUSION: Our in vitro results demonstrate that the UV+RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV+RB can improve convalescent blood product safety is indicated.

Bioenergetic profiling of platelet mitochondria during storage: 4°C storage extends platelet mitochondrial function and viability.

BACKGROUND: Platelets (PLTs) are stored at room temperature (RT) to preserve in vivo circulation time, but PLT quality is degraded. The PLT storage lesion is mitigated by refrigeration, but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Mitochondrial reactive oxygen species (ROS) generation may adversely affect PLT function and viability during storage, and refrigeration may mitigate these effects. STUDY DESIGN AND METHODS: PLTs were stored under two temperature conditions (RT, 20-24°C; or 4°C, 1-6°C) and four storage durations (baseline [BL] and Days 3, 5, and 7). Mitochondrial respiration and maximal oxygen utilization were assessed with high-resolution respirometry. Mitochondrial ROS generation was assessed using a superoxide stain. Rotational thromboelastometry (ROTEM) was performed at BL and on Day 5 to assess PLT function. Collagen-induced PLT aggregation was measured by impedance aggregometry. RESULTS: Mitochondrial ROS in 4°C-stored samples were lower compared to RT and retained a greater capacity to generate ROS after activation. Mitochondrial respiration and maximal mitochondrial utilization was conserved in PLTs stored at 4°C. ROTEM data demonstrated that net maximum clot firmness was higher in 4°C samples compared to RT and prevented fibrinolysis. The aggregation response to collagen was preserved in the 4°C samples versus RT-stored PLTs. Aggregation impairment correlated well with attenuated mitochondrial respiration and elevated production of intracellular mitochondrial ROS in the RT PLTs. CONCLUSION: Mitochondrial damage and ROS production may contribute to loss of PLT viability during storage, whereas cold storage is known to preserve PLT function. Here we demonstrate that 4°C storage results in less oxidant stress and preserves mitochondrial function and potential compared to RT.

Storage of platelets at 4°C in platelet additive solutions prevents aggregate formation and preserves platelet functional responses.

BACKGROUND: Platelet (PLT) storage has been limited to 5 days at room temperature due to metabolic decline and risk for bacterial contamination. Refrigeration preserves PLT metabolism and function as well as limits bacterial growth; however, cold storage of PLTs also leads to aggregate formation. We hypothesized that storage of PLT concentrates at 4°C leads to glycoprotein (GP)IIb-IIIa activation and thus aggregate formation through fibrinogen binding and that this could be prevented by storing PLTs in PLT additive solution (PAS) without compromising PLT function. STUDY DESIGN AND METHODS: Apheresis PLTs in plasma (AP) or apheresis PLTs in PAS were stored at 22 or 4°C for up to 15 days. Measurements include PLT counts, blood gases, aggregation response, flow cytometry analysis of integrin levels, activation markers, and microparticle formation. RESULTS: Storage of AP 4°C led to a gradual decline in PLT count and an increase in aggregate formation that was mediated by intracellular calcium leak and fibrinogen receptor activation. Storage of PAS at 4°C prevented aggregate formation due to dilution of plasma fibrinogen. PAS stored at 4°C maintained aggregation responses to multiple agonists better than 22°C controls. CONCLUSION: Storage of AP at 4°C leads to low level GPIIb-IIIa activation and results in aggregate formation over time. Separating the PLTs from the plasma component and storing them in PAS at 4°C resolves aggregate formation and preserves the metabolic and functional responses of these stored PLTs.

ENDOTHELIUM-DERIVED INHIBITORS EFFICIENTLY ATTENUATE THE AGGREGATION AND ADHESION RESPONSES OF REFRIGERATED PLATELETS.

Refrigeration of platelets (4°C) provides the possibility of improving transfusion practice over the current standard-of-care, room temperature (RT) storage. However, the increased level of platelet activation observed at 4°C in vitro is cause for concern of uncontrolled thrombosis in vivo. In this study, we assessed the safety of 4°C-stored platelets by evaluating their response to physiologic inhibitors prostacyclin (PGI2) and nitric oxide (NO). Apheresis platelets were collected from healthy donors (n = 4) and tested on Day 1 (fresh) or Day 5 (RT- and 4°C-stored) after treatment with PGI2 and NO or not for: thrombin generation; factor V (FV) activity; intracellular free calcium, cAMP and cGMP; ATP release; TRAP-induced activation; aggregation to ADP, collagen, and TRAP, and adhesion to collagen under arterial flow. Data were analyzed using two-way ANOVA and post-hoc Tukey test for multiple comparisons, with significance set at P < 0.05. Treatment with inhibitors increased intracellular cAMP and cGMP levels in fresh and stored platelets. Thrombin generation was significantly accelerated in stored platelets consistent with increased factor V levels, PS exposure, CD62P expression, intracellular free calcium, and ATP release. While treatment with inhibitors did not attenuate thrombin generation in stored platelets, activation, aggregation, and adhesion responses were inhibited by both PGI2 and NO in 4°C-stored platelets. In contrast, though RT-stored platelets were activated, they did not adhere or aggregate in response to agonists. Thus, refrigerated platelets maintain their intracellular machinery, are responsive to agonists and platelet function inhibitors, and perform hemostatically better than RT-stored platelets.