Blood consumption in the Role 2 setting: A Department of Defense Trauma Registry analysis.

BACKGROUND: The Role 2 setting represents the most far-forward military treatment facility with limited surgical and holding capabilities. There are limited data to guide recommendations on blood product utilization at the Role 2. We describe the consumption of blood products in this setting. STUDY DESIGN AND METHODS: We analyzed data from 2007 to 2023 from the Department of Defense Trauma Registry (DODTR) that received care at a Role 2. We used descriptive and inferential statistics to characterize the volumes of blood products consumed in this setting. We also performed a secondary analysis of US military, Coalition, and US contractor personnel. RESULTS: Within our initial cohort analysis of 15,581 encounters, 17% (2636) received at least one unit of PRBCs or whole blood, of which 11% received a submassive transfusion, 4% received a massive transfusion, and 1% received a supermassive transfusion. There were 6402 encounters that met inclusion for our secondary analysis. With this group, 5% received a submassive transfusion, 2% received a massive transfusion, and 1% received a supermassive transfusion. CONCLUSIONS: We described volumes of blood products consumed at the Role 2 during recent conflicts. The maximum number of units consumed among survivors exceeds currently recommended available blood supply. Our findings suggest that rapid resupply and cold-stored chain demands may be higher than anticipated in future conflicts.

A survey of low titer O whole blood use within the trauma quality improvement program registry.

INTRODUCTION: The use of low titer O whole blood (LTOWB) has expanded although it remains unclear how many civilian trauma centers are using LTOWB. METHODS: We analyzed data on civilian LTOWB recipients in the American College of Surgeons Trauma Quality Improvement Program (TQIP) database 2020-2021. Unique facility keys were used to determine the number of centers that used LTOWB in that period. RESULTS: A total of 16,603 patients received LTOWB in the TQIP database between 2020 and 2021; 6600 in 2020, and 10,003 in 2021. The total number of facilities that reported LTOWB use went from 287/779 (37%) in 2020 to 302/795 (38%) in 2021. Between 2020 and 2021, among all level 1-3 designated trauma facilities that report to TQIP LTOWB use increased at level-1 centers (118 to 129), and level-2 centers (81 to 86), but decreased in level-3 facilities (9 to 4). Among pediatric and dual pediatric-adult designated hospitals there was a decrease in the number of pediatric level-1 centers (29 to 28) capable of administering LTOWB. Among centers with either single or dual level-1 trauma center designation with adult centers, the number that administered LTOWB to injured pediatric patients also decreased from 17 to 10, respectively. CONCLUSIONS: There was an increase in the number of facilities transfusing LTOWB between 2020 and 2021. The use of LTOWB is underutilized in children at centers that have it available. These findings inform the expansion of LTOWB use in trauma.

Time from apheresis platelet donation to cold storage: Evaluation of platelet quality and bacterial growth.

BACKGROUND: Cold storage reduces posttransfusion survival of platelets; however, it can improve platelet activation, lower risk of bacterial contamination, and extend shelf-life compared to room temperature (RT) storage. To facilitate large-scale availability, manufacturing process optimization is needed, including understanding the impact of variables on platelet potency and safety. Short time requirements from collection to storage is challenging for large blood centers to complete resuspension and qualify platelets for production. This study evaluated the impact of time from platelet component collection to cold storage on in vitro properties and bacterial growth. STUDY DESIGN AND METHODS: Double-apheresis platelet components were collected from healthy donors, suspended in 65% PAS-III/35% plasma, and split into 2 equal units. One unit was placed into cold storage within 2 h and the other unit after 8 h. Eight matched pairs were evaluated for 12 in vitro parameters. Twenty-four matched pairs were evaluated with 8 bacterial strains tested in triplicate. Samples were tested throughout 21 days of storage. RESULTS: In vitro properties were not different between 2 and 8 h units, and trends throughout storage were similar between arms. Time to cold storage did not significantly impact bacterial growth, with <1 log10 difference at all timepoints between units. DISCUSSION: Our studies showed that extending time to cold storage from 2 to 8 h from collection did not significantly increase the bacterial growth, and the platelet component quality and function is maintained. The ability to extend the time required from collection to storage will improve blood center logistics to feasibly produce CSPs.

Preliminary characterization of the properties of cold-stored apheresis platelets suspended in PAS-III with and without an 8-hour room temperature hold.

BACKGROUND: Use of extended cold storage of platelets promises to increase PLT availability and the bacterial safety of bleeding patients. No information is currently available on the preservation of apheresis PLT in vitro quality parameters when PLTs are held at room temperature early in the storage period prior to transfer to cold storage. STUDY DESIGN AND METHODS: Double units of platelets suspended in 35% plasma/65% PAS-III were collected from normal consenting research donors and rested at room temperature for 1-2 hours. One of the units was then stored at 1-6°C while the other unit was placed on an agitator at 20-24°C. Eight hours after collection, the unit stored at room temperature was transferred to 1-6°C storage without agitation. Units were sampled for an array of PLT in vitro parameters on Days 1, 7, 14, and 21. RESULTS: As expected, PLTs held for 8 hours at 20-24°C prior to 1-6°C storage had greater lactate levels and reduced glucose levels and pH compared to PLTs subjected to a 1-2-hour room temperature hold prior to cold storage (P < .05). Unexpectedly, platelets held for 8 hours at room temperature had less aggregation response to collagen, ADP, and TRAP compared to PLTs held 1-2 hours at room temperature prior to cold storage (P < .05, n = 8). CONCLUSION: Decline of aggregation response should be considered when evaluating longer than necessary room temperature holds prior to cold storage of platelets.

Sodium citrate contributes to the platelet storage lesion.

BACKGROUND: Sodium citrate has become the preferred anticoagulant used for apheresis collection and has been included in commercial platelet additive solutions (PASs) since PAS-II. It was suggested that citrate be included in PASs to prevent spontaneous aggregation. Reports in cell lines and cord blood have demonstrated that concentrations of citrate present in PAS formulations (10 mM) cause apoptosis. We evaluated whether the removal of citrate from PAS-III could improve platelet storage. STUDY DESIGN AND METHODS: Study 1 evaluated the effects of a citrate dose response on the storage of platelets in 65% PAS containing sodium chloride, sodium acetate, and phosphate. Study 2 compared the cell quality and function of platelets stored in 65% citrate-free PAS-III or PAS-III containing 10 mM of citrate. Measurements included cell count, blood gases, flow cytometry analysis of surface activation markers, and aggregation. RESULTS: Study 1 identified that inclusion of citrate in PAS resulted in a dose-dependent increase in glucose utilization, lactate formation, P-selectin expression, phosphatidylserine (PS) exposure, and reactive oxygen species (ROS) formation. Study 2 showed similar results in which platelets stored in citrate-free PAS-III benefited through better maintenance of glucose utilization with less lactate production, P-selectin expression, PS exposure, and ROS formation compared to citrate-containing PAS-III. Platelets stored in citrate-free PAS-III had aggregation responses that were at least 10% greater than those platelets stored in PAS-III. CONCLUSION: Storage of apheresis platelets in citrate-free PAS-III improved multiple storage parameters including glucose utilization, lactate production, P-selection expression, PS exposure, and ROS formation and resulted in a modest increase in aggregation.

Evaluation of a lyophilized platelet-derived hemostatic product.

BACKGROUND: Current limitations of platelet shelf life to 5 days have led to an increasingly greater demand for hemostatic agents with greater longevity. The objective of this study was to evaluate the function of a lyophilized platelet-derived hemostatic product (thrombosome [TS]) as a potential alternative to fresh platelets. METHODS: Platelets were collected from whole blood from healthy donors. TSs were reconstituted with water and added to various configurations of reassembled whole blood (platelets, plasma, and RBCs); measures included rotational thromboelastometry (ROTEM), optical aggregometry, mitochondrial function, calibrated automated thrombogram, collagen adhesion under flow (shear flow assay), and flow cytometry. RESULTS: In ROTEM, no differences were observed between maximum clot formation values for contact pathway activation thromboelastometry tests with TSs or platelet samples. Significantly decreased aggregation was observed in the TSs versus platelets (p < 0.001 for all agonists). Flow cytometry measures demonstrated significant decreases in glycoprotein Ib expression and increases in phosphatidylserine expression in the TS group (p < 0.01). The calibrated automated thrombogram assay was suggestive (lag time and peak thrombin) that the TSs might have some thrombogenic properties. Measurements of mitochondrial function revealed that TSs had no functional mitochondria. CONCLUSION: In this study, TSs were shown to have nonfunctional mitochondria. ROTEM measures revealed that the TSs had no impact on clot strength. Likewise, compared to platelets, the TSs displayed minimal aggregation, had significantly more phosphatidylserine (measure of activation status), but had the ability to adhere to a collagen surface under flow conditions and contribute to clot formation and induced greater thrombin generation.

Physiology of cold-stored platelets.

Platelet transfusions are a life-saving medical intervention used for the treatment of thrombocytopenia or hemorrhage. Extensive research has gone into trying to understand how to store platelets prior to the transfusion event. Much has been learned about storage bag materials, synthetic solutions, and how temperature impacts platelet viability and function. While room temperature storage of platelets preserves 24-hour in vivo platelet recovery and survival there is a greater risk for bacterial growth. Therefore, cold storage of platelets has become attractive due to the reduction in potential bacterial proliferation and the maintenance of platelet function beyond 5 days of storage. Cold stored platelets, however, have their own set of challenges. Cold stored platelets become activated through several mechanisms. The morphological and molecular changes that occur due to cold exposure enhance their ability to participate in the hemostatic process at the cost of rapid clearance from circulation. This review focuses on the underlying mechanisms leading to cold platelet activation and the receptor modifications involved in platelet clearance.

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.

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.

Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2).

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.

A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets.

T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

Aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures.

Aromatic residues are relatively rare within the collagen triple helix, but they appear to play a specialized role in higher-order structure and function. The role of aromatic amino acids in the self-assembly of triple-helical peptides was investigated in terms of the kinetics of self-association, the nature of aggregated species formed, and the ability of these species to activate platelet aggregation. The presence of aromatic residues on both ends of a type IV collagen model peptide is observed to greatly accelerate the kinetics of self-association, decreasing the lag time and leading to insoluble, well-defined linear fibrils as well as small soluble aggregates. Both macroscopic visible aggregates and small multimolecular complexes in solution are capable of inducing platelet aggregation through the glycoprotein VI receptor on platelets. Proline-aromatic CH...pi interactions are often observed within globular proteins and in protein complexes, and examination of molecular packing in the crystal structure of the integrin binding collagen peptide shows Phe interacts with Pro/Hyp in a neighboring triple-helical molecule. An intermolecular interaction between aromatic amino acids and imino acids within the triple helix is also supported by the observed inhibitory effect of isolated Phe amino acids on the self-association of (Pro-Hyp-Gly)(10). Given the high fraction of Pro and Hyp residues on the surface of collagen molecules, it is likely that imino acid-aromatic CH...pi interactions are important in formation of higher-order structure. We suggest that the catalysis of type I collagen fibrillogenesis by nonhelical telopeptides is due to specific intermolecular CH...pi interactions between aromatic residues in the telopeptides and Pro/Hyp residues within the triple helix.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets.

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase ß inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

Platelet ITAM signaling is critical for vascular integrity in inflammation.

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.