Introduction

Following injury, platelets are an early and essential component of coagulation biology to control hemorrhage. While platelets were initially thought to be simple cellular fragments solely for mechanical obstruction in clot formation, research demonstrates these megakaryocyte fragments to be dynamic, active, complex cellular particles central to both clot formation and breakdown, as well as to mediation of cellular interactions critical for vascular homeostasis, inflammation, and immune function [1, 2]. We will review the role of platelets in mediating hemostasis, the evidence of mechanisms of dysregulation following injury, and multiple aspects of platelet transfusion in trauma resuscitation including platelet transfusion as part of hemostatic resuscitation, the effect of platelet transfusion on coagulation and platelet function assays, risks associated with platelet transfusion and storage considerations, and future novel platelet-based therapeutics including synthetic platelet analogs to expand the supply of these biologically central cellular mediators of vascular homeostasis.

Trauma-Induced Coagulopathy

Trauma-induced coagulopathy (TIC) develops in approximately one-quarter to one-third of severely injured and hemorrhaging trauma patients [3]. Importantly, these patients suffer from worse outcomes including a lower likelihood of survival [4,5,6,7]. Mechanistically, evidence supports that TIC is a multi-factorial failure in vascular homeostasis generally characterized by an early hypocoagulable state contributing to ongoing hemorrhage, followed by a later hypercoagulable and hyperinflammatory state predisposing survivors to thromboembolic complications and organ failure [8••, 9, 10]. Although TIC is often exacerbated by concomitant dilutional and consumptive coagulopathy, TIC develops prior to resuscitative efforts and is characterized by a spectrum of adaptive and maladaptive alterations to clot formation and breakdown and cellular behavior, including that of platelets [11].

Platelets in Normal Hemostasis

In normal hemostasis, following a vascular breach and exposure of sub-endothelial collagen surfaces, platelets are activated, change shape, interact with von Willebrand Factor (vWF) and fibrinogen, and degranulate their pro-coagulant contents in the process of formation of a platelet plug. This platelet-based nidus of clot formation is a catalyst for thrombin generation, growth of fibrin mesh, and the strengthening and expansion of clot [2, 12]. Platelets additionally have a balancing role in fibrinolysis through both release of plasminogen activator inhibitor-1 (PAI-1) and alpha-2 antiplasmin to counter natural fibrinolysis, as well as through the binding of activated platelet surfaces to plasminogen and plasminogen activators, providing a surface for local fibrinolysis [13]. Furthermore, platelets maintain endothelial integrity through direct interactions with endothelial cells and the release of growth factors that stabilize vascular endothelial junctions and promote angiogenesis [14]. Finally, platelets regulate inflammatory and immune responses through the release of hundreds of recognized proteins [15], and respond to damage-associated molecular patterns (DAMPs) as part of their innate immune response [16, 17]. The platelet transcriptome also responds to trauma with differential ribonucleic acid (RNA) expression and evidence of RNA editing after injury [18•, 19].

Platelet Behavior in Trauma

Many expected adaptive platelet-based hemostatic, endothelial, inflammatory, and immune regulatory behaviors have been described in the setting of injury. However, there is also evidence to support that in the setting of severe injury, hemorrhage, and associated malperfusion, even in the setting of normal platelet counts, platelets contribute to a maladaptive response worsening coagulopathy and exacerbating the risk of subsequent thrombotic and inflammatory complications (Fig. 1) [20•]. This includes data that demonstrate robust platelet activation — including increased expression of platelet activation surface markers, release of extracellular vesicles, platelet-leukocyte interactions, and cytokine release — yet functional alterations in adhesion, aggregation, calcium mobilization, vWF interactions, and changes to platelet composition (shed platelet surface glycoproteins, histone-driven platelet structural changes, and platelet transcriptomic changes) (Table 1) [2, 8••, 21,22,23]. Dysregulation of the endothelium after trauma may also impact platelet behavior; for example, elevated catecholamines after injury were found to be associated with endotheliopathy and higher mortality [24, 25], similar to the shock-induced endotheliopathy (SHINE) described in various types of critical illness [26]. Although it is commonly identified that platelets from injured patients are “dysfunctional” due to identified impairments in their aggregation responses to stimulation in ex vivo assays [21, 27], this may be mediated by the severity of injury and degree of shock as data suggest that minor injury results in increased activation of platelets; however, severe injury and combined shock are associated with reduced activation and aggregation [28•]. Furthermore, shock-induced soluble inhibitors may contribute to functional alterations in circulating platelets after injury [20•, 28•]. Platelets activated by trauma also have been shown to balloon and release microvesicles, which coat leukocytes and have procoagulant action promoted by DAMPs such as Histone H4. These platelet-leukocyte interactions persist for several days following injury and are associated with multisystem organ dysfunction as well [29••].

Fig. 1
figure 1

Platelet and endothelial interactions. Adapted with permission from: Moore, E.E., Moore, H.B., Kornblith, L.Z. et al. Trauma-induced coagulopathy. Nat Rev Dis Primers 7, 30 (2021). Springer Nature. https://doi.org/10.1038/s41572-021-00264-3. https://www.nature.com/articles/s41572-021-00264-3

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Table 1 Platelet responses to trauma over time following injury
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Continued efforts to understand the post-injury behavior of platelets remains important in trauma resuscitation because although it is clear from clinical data that transfusion of platelets to injured and hemorrhaging trauma patients improves survival, it remains unclear what the central mechanisms of this are. This is because the biologic data supports that platelet counts and ex vivo platelet aggregation responses are relatively refractory to platelet transfusion [22], which can persist for several days following major trauma [23].

Platelet Transfusion in Trauma

Hemostatic Resuscitation in Trauma

For patients with trauma-related hemorrhage who require large volume transfusion, decades of research support that banked platelets should be transfused early as part of a hemostatic resuscitation strategy, and in a 1:1:1 balanced manner with red blood cells (RBC) and plasma [30, 31, 32••]. These hemostatic strategies of resuscitation with balanced ratios of RBC:plasma and RBC:platelets lead to earlier correction of coagulopathies and lower hemorrhage-related mortality in injured patients requiring large volume transfusion [33,34,35,36,37,38]. Both the timing and ratios of blood products are important, as early platelet transfusion within the first 6 h after injury has been shown to be most beneficial to hemostasis and mortality, both in combat and civilian populations [39,40,41,42,43,44,45]. A recent study of the American College of Surgeons Trauma Quality Improvement Program (TQIP) database demonstrated that unbalanced platelet:RBC transfusion ratios are common with nearly twice as many patients receiving unbalanced ratios, which portended worse odds of mortality than unbalanced plasma:RBC transfusion ratios. This emphasizes the importance of delivering platelets along with initial RBC and plasma resuscitation [46•]. Most data for platelet transfusion comes from in-hospital studies, and given improvements in outcomes with early balanced resuscitation, expansion to the prehospital setting may have further positive impacts on mortality in a subset of massively hemorrhaging patients [47, 48]. Furthermore, the protocolized use of balanced resuscitation for patients requiring large-volume transfusion may even reduce overall blood product consumption, improve transfusion-related costs [49], and reduce waste [50, 51]. These protocols, often referred to as massive transfusion protocols (MTP), are becoming commonplace. Recent data from patients receiving very large volumes of transfusion after injury (≥ 20u RBC in the first 24 h), coined ultramassive transfusion (UMT), suggest these patients remain at risk of failed hemostatic resuscitation. In a modern multicenter study of 17 trauma centers, nearly half of patients receiving UMT were resuscitated with unbalanced ratios of RBC:plasma or RBC:platelets, with an associated increase in mortality, demonstrating ongoing areas for improvement in these practices [52]. On the other hand, the optimal transfusion ratio of platelet:RBC for non-massively transfused patients is elusive [50, 53, 54]. Outside of the massive transfusion practices for the hemorrhaging patient, transfusion thresholds of platelet counts < 50,000/μL for acute traumatic hemorrhage and < 100,000/μL for intracranial hemorrhage remain general recommendations [55], although the support for these practices in terms of clinical outcomes remains limited [54]. However, altered platelet function and biology in trauma is independent of platelet count.

Impact of Platelet Transfusion on Platelet Function in Trauma

While the use of hemostatic resuscitation with balanced ratios of platelets to other components has improved outcomes for injured patients including survival, the impact of platelet transfusion on platelet count and function is not as clear. There is evidence that severely injured patients receiving platelet transfusion have minimal increases in their platelet counts in the first 24 h after injury, and minimal to no improvements in the aggregation responses of circulating platelets in ex vivo assays [23]. Furthermore, injured patients receiving platelet transfusions at multiple timepoints during the first 12 units of RBC transfusion had similar platelet aggregation to those who did not receive platelet transfusions [56]. However, these investigators found that injured patients who received platelet transfusions did have a decrease in observed fibrinolysis, which they hypothesized to be related to increased PAI-1 from transfused platelets. Additionally, it has been found that dilution with autologous plasma used to deliver platelets, or allogenic plasma delivered in combination with transfused platelets also results in decreased platelet aggregation in in vitro studies [57]. Similar findings have been identified in patients with traumatic brain injury, where patients taking antiplatelet agents had no improvement in outcomes after platelet transfusion [58]. While some of the data are mixed, however, on reversal of platelet inhibition after traumatic brain injury, need for neurosurgical intervention, and mortality [59,60,61], a recent systematic review and meta-analysis found that across twelve studies, platelet transfusion after traumatic brain injury in patients taking antiplatelet agents had no significant reduction in mortality, progression of hemorrhage, or need for neurosurgical interventions [62].

While it is clearly important to include platelets as part of a hemostatic resuscitation strategy based on outcomes, overall, the combined data suggests that in the setting of severe injury and shock circulating platelets are altered in their functional behaviors [28•], and that even transfused platelets may be relatively refractory in their hemostatic behavior [23]. It may be that the circulating environment is inhibitory to platelet hemostatic behaviors for both native and transfused platelets, but that the driver of clinical benefit of transfused platelets is instead due to other activities of platelets including endothelial maintenance and immune and inflammation regulation [63]. Further investigations in this area are needed.

Role of Viscoelastic and Platelet-Function Assays in Transfusion for Trauma

Initial transfusion with balanced product ratios is associated with improved patient outcomes; however, a transition to tailored resuscitation including using real-time assays of clot formation and breakdown, to achieve earlier hemostasis, and to limit product consumption is now a critical piece of hemostatic resuscitation, often known as goal-directed resuscitation. Viscoelastic assays, such as thromboelastography (TEG, Haemonetics Corp) and Rotational Thromboelastometry (ROTEM, Tem International GmbH) (Table 2), are dynamic assays that provide information about initial clot formation, rate of growth of clot, strength of clot, and breakdown of clot in a sample of whole blood taken from an injured patient (Table 1) [64]. This information can inform targeted transfusion of deficient coagulation factors and platelets in real time [65, 66•, 67, 68]. When compared to conventional coagulation assays (international normalized ratio [INR], partial thromboplastin time [PTT], platelet and fibrinogen levels), severely injured patients resuscitated with viscoelastic-directed resuscitation have been found to receive less plasma and platelet transfusions, and have improved survival [65, 69,70,71,72]. However, data suggest use of both conventional assays and viscoelastic assays in combination may be most beneficial in goal-directed resuscitation to capture a broader range of coagulation abnormalities, and patients who are coagulopathic by conventional assays have higher injury scores and worse physiologic derangements as well as higher transfusion requirements and mortality [73]. In terms of the platelet-related parameters of these viscoelastic assays, the measure of clot strength relies on the contributions from fibrin and platelets, with platelets making up a majority of the contribution to clot strength following injury [12]. Reductions in clot strength may guide platelet transfusion in the post-injury period [66•]. While not originally developed for use in trauma, TEG platelet mapping (TEG-PM) has identified platelet impairments in aggregation behavior in trauma patients [74], and is particularly associated with the severity of traumatic brain injury [75,76,77]. However, this assay is designed for identifying effects of antiplatelet medications, and has not been shown to predict massive transfusion or platelet transfusion requirements well [78]. The utility in trauma may be to identify the degree of platelet inhibition in response to arachidonic acid and adenosine diphosphate (ADP) stimulation in patients taking aspirin or clopidogrel, respectively [79]. However, in traumatic brain injury patients, TEG-PM has also been used to show reduction in platelet inhibition after platelet transfusion both in patients taking aspirin [60] and those who were not taking antiplatelet agents [59, 61].

Table 2 Viscoelastic assays used to guide platelet transfusion
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Other assays that have been used primarily in the trauma research setting to understand the effects of resuscitation and platelet transfusion include platelet aggregometer and microfluidic technologies. Multiple electrode impedance aggregometry, or Multiplate© (Verum Diagnostica GmbH, Munich, Germany), and Chronolog’s Whole Blood Aggregometer to measure the change in electrical impedance over time in whole blood in response to platelet stimulation by ADP, thrombin, collagen, or arachidonic acid, giving a proxy measurement for the degree of platelet aggregation in response to each stimulated pathway [80, 81]. These assays have been used in trauma populations to measure platelet aggregation responses following injury [21, 74, 82]. The Platelet Function Analyzer (PFA-100, Dade International Inc., Miami, FL) and Total Thrombus Formation Analysis System (T-TAS, Fujimori Kogyo, Tokyo, Japan) incorporate flow dynamics of whole blood across membranes coated with collagen and ADP or collagen and thromboplastin, respectively, to measure in vitro activity via the duration of time for a platelet plug to form and close a small aperture in the device (closure time, CT) [27, 83, 84]. The clinical role for these assays remains under investigation.

Risks Associated with Platelet Transfusion in Trauma

Despite the benefit to hemostasis and survival, there are important potential associated complications of platelet transfusions in injured patients. Data support that injured patients receiving platelet transfusions have an increased risk of developing acute respiratory distress syndrome (ARDS) [85], both early after injury [86] and later [87]. Even without receiving large volume transfusion, blunt trauma patients who received platelet transfusions had a higher risk of developing ARDS compared with those who did not receive platelets [88]. As with other blood products, patients receiving platelet transfusions are also at risk of developing transfusion-related acute lung injury (TRALI) [89] and transfusion-associated circulatory overload (TACO) [90].

Relatively common but benign reactions associated with platelet transfusions include allergic and nonhemolytic febrile transfusion reactions [54, 91], which are more common after platelet transfusions than other blood products [92], and thought to be related to cytokine release [93]. In select populations known to have severe allergic reactions, the risk may be reduced by platelet washing [94, 95], use of an inert additive solution [96, 97], or concentrating the allogenic plasma in which platelets are delivered [98]. Finally, post-transfusion purpura after platelet transfusion is rare but can occur following multiple transfusions, more often from female donors [99, 100], and is likely due to circulating anti-HPA-1a antibodies in donated platelets [101]. In the PATCH Trial, platelet transfusion was also associated with increased thromboembolic events and complications of spontaneous intracranial hemorrhage, although this study did not include trauma patients [102, 103].

Bacterial infection after platelet transfusion, although rare, is more common than after other blood products, likely due to the standard warmer storage conditions of platelets compared to other components [54, 104]. Pathogen reduction methods such as photochemical reduction with psoralen and ultraviolet-A irradiation have demonstrated effectiveness in high-risk populations with no documented bacterial infection events [104]. Transfusion of pathogen-reduced platelets is associated with decreased post-transfusion corrected counts as opposed to non-treated platelets, but these patients have similar associated bleeding events so the clinical relevance of the reduced counts may be negligible [104,105,106]. Aside from chemical and energy methods of decontaminating banked platelets, colder storage temperatures may also reduce the risk of pathogen contamination [107,108,109].

Considerations for Platelet Storage

Historically, platelets have been stored at room temperature (22 °C for 5–7 days) [110]; however, other storage modalities such as cold storage or cryopreservation, while not yet widely used, have both been studied as possible methods to extend shelf life and even improve initial hemostasis of the transfused platelets [111, 112] (Table 3).

Table 3 Platelet storage methods
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Cold storage (2 to 6 °C), compared with standard room temperature storage (20 to 24 °C) of platelets is another potential storage modality to extend platelet shelf life for up to 14 days [113•]. Cold-stored platelets have reduced circulation time after transfusion (approximately 3 days vs. 8 days for standard temperature storage) but appear to have improved hemostatic function [114, 115], with increased in vitro activation in response to stimulants [116,117,118] and reduced bleeding time in in vivo studies [119]. One pilot study of cold-stored platelet transfusion in cardiothoracic surgery patients demonstrated that post-transfusion platelet aggregation, transfusion requirements, and outcomes were similar as compared to the group receiving room temperature platelet transfusion [120••]. The CHIlled Platelet Study (CHIPS) is an ongoing randomized controlled trial to study the hemostatic efficacy of cold-stored platelet transfusion in cardiothoracic surgery patients [121••]. The Cold Stored Platelet Early Intervention in Hemorrhagic Shock (CriSP-HS) and a sub-study in traumatic brain injury (CriSP-TBI) are studying the feasibility of an early cold-stored platelet transfusion protocol, as well as impact on mortality and transfusion requirements in hemorrhagic shock and TBI, respectively [122••].

Cryopreservation of platelets can extend the shelf life of a unit of platelets from days to up to 4 years. There is relatively robust data demonstrating cryopreserved platelets (treated with dimethyl sulfoxide [DMSO] and stored at − 65 °C) can effectively establish hemostasis in bleeding patients and have not been implicated in major adverse events. However, they do result in lower post-transfusion platelet counts than with room temperature-stored platelets, including higher decrements at 24 h from transfusion [123,124,125,126]. The Cryopreserved vs. Liquid Platelet (CLIP) study in cardiothoracic surgery patients showed lower rates of significant postoperative hemorrhage, RBC transfusion, and no difference in postoperative complications for the patients who received cryopreserved platelets, although they did receive more platelet and plasma transfusions [127••]. Despite blood product shortages making the long shelf life of cryopreservation appealing, this method has yet to become commonplace in blood banking [128]. Another long-term storage method is freeze-dried lyophilization. Freeze-dried platelets, such as Thrombosomes® (CellPhire Therapeutics), are stabilized in paraformaldehyde or trehalose and then freeze-dried and stored at room temperature, where shelf life is up to 3 years, then reconstituted with saline prior to transfusion. The study of transfusion of Thrombosomes® in humans is ongoing [129, 130••] and in mouse models has been shown to contribute to clot function similarly to room temperature-stored platelets [131••].

Adjunct Therapies

The complex interactions of a plethora of other mediators with platelets during the coagulation process are also important considerations. Early interactions of platelets with von Willibrand factor promote platelet adhesion and aggregation [132]. The large increase in vWF following trauma may promote more platelet interactions but also may overwhelm the cleavage abilities of ADAMTS-13 metalloproteinase, contributing to coagulopathy by inappropriate binding of platelets to large vWF multimers [133•], reduction of ADAMTS-13 activity [134•], and downstream organ dysfunction [135, 136]. Desmopressin, or ddAVP, also increases vWF levels, promotes platelet-based hemostasis, and enhances endothelial function. Animal studies of treatment with ddAVP plus balanced blood product resuscitation after hemorrhagic shock demonstrated that ddAVP improved coagulation parameters including platelet function and clot strength, but did not impact organ damage [137•]. In human studies, administration of ddAVP has not demonstrated impact on progression of intracranial hemorrhage [138]; however, in a separate study of patients with severe traumatic brain injury, administration of ddAVP in lieu of platelet transfusion corrected platelet inhibition similarly to platelet transfusion alone [139•], suggesting that ddAVP may have similar impact on the correction of platelet dysfunction after trauma. Finally, higher serum calcium levels are associated with improved platelet activation, aggregation, and clot strength after injury [22], highlighting its importance as an adjunct during massive transfusion.

Future Directions

A deeper understanding of platelet biology, the platelet response to injury, and platelet-based therapies in trauma continues to inform new areas of study. For example, the platelet transcriptome in injured and uninjured populations is an area of ongoing exploration to identify the mechanistic and molecular bases of the commonly described post-injury platelet “dysfunction,” and uncover treatment targets [18•, 19]. Similarly, platelet-derived extracellular vesicles may also provide insights into trauma-induced coagulopathy, and recent consensus panels have provided guidance to standardize measurements to improve relevance of this test across centers [140, 141].

Beyond a better understanding of the biology, and improved assays, there are many platelet-based therapeutics of interest. For example, the transfusion of other blood-based therapies such as platelet-derived extracellular vesicles may promote thrombin-mediated clot formation in hemorrhaging patients, as well as attenuate endothelial injury following trauma [142, 143]. Furthermore, in vitro production of platelets to enhance the supply is also an area of interest. Induced pluripotent stem cells can be used to generate megakaryocytes which could create a renewable and genetically modifiable source of platelets. This opens the door to platelets that are HLA-deleted and safe against blood-borne pathogens [144]. The ex vivo production of platelets from stem cells has been explored; however, current methods are too low yield to permit clinical utility [145, 146].

Synthetic platelet analogs or “bioinspired artificial platelets” are also investigatory microparticle templates that can be modified to replicate the adhesion and aggregation mechanisms of platelet hemostasis [147•, 148]. Infusible platelet membrane (IPM, Cypress Bioscience), albumin microparticles with fibrinogen additives (Synthocyte) and liposomal microparticles with vWF-binding peptides, collagen-binding peptides, and active GPIIb-IIIa–binding fibrinogen-mimetic peptides (SynthoPlate) are a few examples of synthetic platelet analogs undergoing testing [149, 150]. While these synthetic analogs are attractive, it remains challenging to simply mimic the hemostatic, adhesive, aggregative, and other cellular functions of platelets, when these mechanisms are not fully understood in the endogenous platelet response to trauma [151].

Summary and Conclusions

Platelets are a critical component of hemostasis after trauma, both due to their mechanical hemostatic effects in formation of a platelet plug and their dynamic involvement in clot formation and breakdown, but also their biochemical activity and interaction with the endothelium and inflammatory and immune cells. While this biology after injury remains incompletely understood, platelet function is seen to be altered after trauma, particularly in the setting of severe injury and shock. Including platelet transfusions in hemostatic resuscitation with the aim of balanced transfusion is recommended to achieve the best outcomes in terms of early hemostasis and reduced mortality. Despite overall improved outcomes with early platelet transfusion in injury and hemorrhage, transfusion of platelets is associated with risk of development of ARDS, bacterial infection, and allergic and immune reactions; however, these can be mitigated with improved decontamination procedures and additive solutions in special populations. Cold storage, cryopreservation, synthetic platelets, and platelet-based adjuncts that can expand the supply and deliver safe, effective platelet-based hemostasis are current areas of investigation. Platelets are dynamic and essential components of coagulation biology and mediate a number of cellular interactions following trauma. As research continues to advance our knowledge of these complex cellular fragments, we work toward an ever more tailored and nuanced understanding of how to best diagnose and treat coagulopathy following trauma to improve patient outcomes.