Platelet dysfunction reversal with cold-stored vs. room temperature-stored platelet transfusions.

Platelets are stored at room temperature for 5-7 days (RSP). Due to frequent and severe shortages, the FDA recently approved up to 14-day cold-stored platelets in plasma (CSP). However, the post-transfusion function of CSP is unknown and it is unclear which donors are best suited to provide either RSP or CSP. In this study, we sought to evaluate the post-transfusion platelet function and its predictors for platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). We conducted a randomized cross-over study in ten healthy humans. Subjects donated one platelet unit stored at either 22 °C or 4 °C based on randomization. Before transfusion, subjects ingested ASA to inhibit endogenous platelets. Transfusion recipients were tested for platelet function and lipid mediators. Platelet units were tested for lipid mediators only. A second round of transfusion with the alternative product was followed by an identical testing sequence. RSP reversed platelet inhibition significantly better in αIIbß3 integrin activation-dependent assays. In contrast, CSP in recipients led to significantly more thrombin generation, which was independent of platelet microparticles. Lysophosphatidylcholine-O species levels predicted the procoagulant capacity of CSP. In contrast, polyunsaturated fatty acid concentrations predicted the aggregation response of RSP. In summary, we provide the first efficacy data of extended-stored CSP in plasma. Our results suggest that identifying ideal RSP and CSP donors is possible and pave the way for larger studies in the future. Registration: NCT03787927.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Results of clinical effectiveness of conventional versus Mirasol-treated Apheresis Platelets in Patients with Hypoproliferative Thrombocytopenia (MiPLATE) trial.

BACKGROUND: The Mirasol® Pathogen Reduction Technology System was developed to reduce transfusion-transmitted diseases in platelet (PLT) products. STUDY DESIGN AND METHODS: MiPLATE trial was a prospective, multicenter, controlled, randomized, non-inferiority (NI) study of the clinical effectiveness of conventional versus Mirasol-treated Apheresis PLTs in participants with hypoproliferative thrombocytopenia. The novel primary endpoint was days of ≥Grade 2 bleeding with an NI margin of 1.6. RESULTS: After 330 participants were randomized, a planned interim analysis of 297 participants (145 MIRASOL, 152 CONTROL) receiving ≥1 study transfusion found a 2.79-relative rate (RR) in the MIRASOL compared to the CONTROL in number of days with ≥Grade 2 bleeding (95% confidence interval [CI] 1.67-4.67). The proportion of subjects with ≥Grade 2 bleeding was 40.0% (n = 58) in MIRASOL and 30.3% (n = 46) in CONTROL (RR = 1.32, 95% CI 0.97-1.81, p = .08). Corrected count increments were lower (p < .01) and the number of PLT transfusion episodes per participant was higher (RR = 1.22, 95% CI 1.05-1.41) in MIRASOL. There was no difference in the days of PLT support (hazard ratio = 0.86, 95% CI 0.68-1.08) or total number of red blood cell transfusions (RR = 1.12, 95% CI 0.91-1.37) between MIRASOL versus CONTROL. Transfusion emergent adverse events were reported in 119 MIRASOL participants (84.4%) compared to 133 (82.6%) participants in CONTROL (p = NS). DISCUSSION: This study did not support that MIRASOL was non-inferior compared to conventional platelets using the novel endpoint number of days with ≥Grade 2 bleeding in MIRASOL when compared to CONTROL.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Black dots: High-yield traction force microscopy reveals structural factors contributing to platelet forces.

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed 'black dots') that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. STATEMENT OF SIGNIFICANCE: Cells produce contractile forces during division, migration, or wound healing. Measuring cellular forces provides insight into their health, behavior, and function. We developed a technique that calculates cellular forces by seeding cells onto a pattern and quantifying how much each cell displaces the pattern. This technique is capable of measuring hundreds of cells without needing to detach them. Using this technique to evaluate human platelets, we find that platelets exerting more force tend to have more spread area, are more circular in shape, and have more uniformly distributed cytoskeletal filaments. Due to our high yield of data, we were able to apply statistical techniques that revealed combinatorial effects between these factors.

Evaluating stored platelet shape change using imaging flow cytometry.

Platelets are routinely stored at room temperature for 5-7 days before transfusion. Stored platelet quality is traditionally assessed by Kunicki's morphology score. This method requires extensive training, experience, and is highly subjective. Moreover, the number of laboratories familiar with this technique is decreasing. Cold storage of platelets has recently regained interest because of potential advantages such as reduced bacterial growth and preserved function. However, platelets exposed to cold temperatures change uniformly from a discoid to a spherical shape, reducing the morphology score outcomes to spheroid versus discoid during cooling. We developed a simpler, unbiased screening tool to measure temperature-induced platelet shape change using imaging flow cytometry. When reduced to two dimensions, spheres appear circular, while discs are detected on a spectrum from fusiform to circular. We defined circular events as having a transverse axis of >0.8 of the longitudinal axis and fusiform events ≤0.8 of the longitudinal axis. Using this assay, mouse and human platelets show a temperature and time-dependent, two-dimensional shape change from fusiform to circular, consistent with their three-dimensional change from discs to spheres. The method we describe here is a valuable tool for detecting shape change differences in response to agonists or temperature and will help screening for therapeutic measures to mitigate the cold-induced storage lesion.

What is the context? Platelets for transfusion are currently stored for 5­7 days at room temperature, increasing the risk for bacterial growthCold storage reduces the risk for bacterial growth but reduces circulation timeStored platelet quality can be assessed by the light microscopy-based Morphology Score, first described in the 1970sDownsides of the Morphology Score include subjectivity, extensive training, and reduced availability in platelet laboratories.What is new? In this study, we provide data showing that the Morphology score is reduced to a binary spheres versus discs response in cold-exposed plateletsWe developed an imaging flow cytometry-based approach to quantify platelets' response to cold based on the two-dimensional projection of the three-dimensional shapes, i.e., fusiform (discoid) versus circular (discoid and spherical)We provide validation of this approach in mouse and human plateletsWhat is the impact?This study provides an easy and unbiased tool for laboratories working on circumventing the cold-induced storage lesion or documenting spherical shape change in general.

Effect of Ado-Trastuzumab Emtansine on Autologous Platelet Kinetics and Function.

PURPOSE: Ado-trastuzumab emtansine (T-DM1) treatment results in grade 3-4 thrombocytopenia in 8%-13% of patients. Prior in vitro studies reported T-DM1 inhibition of megakaryocyte maturation as the cause of decreased platelet production. The current observational study was initiated to evaluate causes of thrombocytopenia in patients with metastatic breast cancer. MATERIALS AND METHODS: Patients with human epidermal growth factor receptor 2-positive metastatic breast cancer (N = 11) were enrolled in this postmarket safety study. 111-Indium- radiolabeled autologous platelet recoveries and survivals as well as serial platelet counts, bleeding time assays, and platelet aggregation responses to a wide range of agonists were performed at baseline (BL) and during two consecutive cycles of the drug (3.6 mg/kg IV once every 3 weeks). RESULTS: Platelet nadirs occurred earlier in cycle 2 than in cycle 1. Average nadir counts (% BL) in cycles 1 and 2 were 116,000/µL (53% ± 6%) and 115,000/µL (51% ± 9%), respectively, with return to BL by D15 in both cycles. BL platelet survival averaged 8.8 (± 0.3) days but progressively shortened to 5.5 (± 0.5) days during cycle 1 and to 4.6 (± 0.3) days during cycle 2 (P < .001 compared with BL for both cycles). Aggregation responses to all agonists decreased during the study, both in cycle 1 and cycle 2. CONCLUSION: Following T-DM1 administration, we observed statistically significant progressive decreases in platelet survivals and decreased platelet function from BL values. In distinction to published in vitro studies, these unexpected results indicate a direct toxic effect of T-DM1 on patients' autologous circulating platelets.

Evaluation of amotosalen and UVA pathogen-reduced apheresis platelets after 7-day storage.

BACKGROUND: Amotosalen/UVA pathogen-reduced platelet components (PRPCs) with storage up to 7 days are standard of care in France, Switzerland, and Austria. PRPCs provide effective hemostasis with reduced risk of transfusion-transmitted infections and transfusion-associated graft versus host disease, reduced wastage and improved availability compared with 5-day-stored PCs. This study evaluated the potency of 7-day PRPCs by in vitro characterization and in vivo pharmacokinetic analysis of autologous PCs. STUDY DESIGN AND METHODS: The in vitro characteristics of 7-day-stored apheresis PRPCs suspended in 100% plasma or 65% platelet additive solution (PAS-3)/35% plasma, thrombin generation, and in vivo radiolabeled post-transfusion recovery and survival of 7-day-stored PRPCs suspended in 100% plasma were compared with either 7-day-stored or fresh autologous conventional platelets. RESULTS: PRPCs after 7 days of storage maintained pH, platelet dose, in vitro physiologic characteristics, and thrombin generation when compared to conventional 7-day PCs. In vivo, the mean post-transfusion survival was 151.4 ± 20.1 h for 7-day PRPCs in 100% plasma (Test) versus 209.6 ± 13.9 h for the fresh autologous platelets (Control), (T-ΔC: 72.3 ± 8.8%: 95% confidence interval [CI]: 68.5, 76.1) and mean 24-h post-transfusion recovery 37.6 ± 8.4% for Test versus 56.8 ± 9.2% for Control (T-ΔC: 66.2 ± 11.2%; 95% CI: 61.3, 71.1). DISCUSSION: PRPCs collected in both 100% plasma as well as 65% PAS-3/35% plasma and stored for 7 days retained in vitro physiologic characteristics. PRPCs stored in 100% plasma for 7 days retained in vivo survival. Lower in vivo post-radiolabeled autologous platelet recovery is consistent with reported reduced count increments for allogenic transfusion.

There and back again: the once and current developments in donor-derived platelet products for hemostatic therapy.

More than 100 years ago, Duke transfused whole blood to a patient with thrombocytopenia to raise the platelet count and prevent bleeding. Since then, platelet transfusions have undergone numerous modifications from whole blood-derived platelet-rich plasma to apheresis-derived platelet concentrates. The storage time and temperature have also changed. The mandate to store platelets for a maximum of 5 to 7 days at room temperature has been challenged by recent clinical trial data, ongoing difficulties with transfusion-transmitted infections, and recurring periods of shortages that were further exacerbated by the COVID-19 pandemic. Alternative platelet storage approaches are as old as the first platelet transfusions. Cold-stored platelets may offer increased storage times (days) and improved hemostatic potential at the expense of reduced circulation time. Frozen (cryopreserved) platelets extend the storage time to years but require storage at -80°C and thawing before transfusion. Lyophilized platelets can be powder-stored for years at room temperature and reconstituted within minutes in sterile water but are probably the least explored alternative platelet product to date. Finally, whole blood offers the hemostatic spectrum of all blood components but has challenges such as ABO incompatibility. We know more than ever before about the in vitro properties of these products, and clinical trial data are accumulating. The purpose of this review is to summarize the findings of recent preclinical and clinical studies on alternative, donor-derived platelet products.

Effect of bedside filtration on aggregates from cold-stored whole blood-derived platelet-rich plasma and apheresis platelet concentrates.

BACKGROUND: The current approach to manufacture cold-stored platelets (CSP) replicates that of room temperature-stored platelets (RSP). However, this production method is associated with aggregate formation in CSP, a major pitfall that leads to significant wastage. We hypothesized that isolating platelets from whole blood as platelet-rich plasma (PRP) and storing them at a lower concentration reduces aggregates and that conventional bedside transfusion filtration removes CSP aggregates. METHODS: We collected platelets from healthy humans by apheresis (AP) and by phlebotomy, from which we generated platelet-rich plasma (PRP). We split each AP and PRP platelets into two equal aliquots, storing one at 22°C (RT-PRP and RT-AP) and the other at 4°C (4C-PRP and 4C-AP). We evaluated platelets on day 0 and day 7 of storage. After storage, we measured platelet counts, aggregates, and other key characteristics before and after filtration by a bedside filter. RESULTS: After storage, the 4C-AP platelet counts decreased significantly. 4C-PRP preserved glucose better and prevented a significant increase in lactate contrary to 4C-AP. Filtration led to significantly lower platelet counts in both 4C-PRP and 4C-AP but not in their RT counterparts. Post filtration, we observed 50% fewer aggregates only in 4C-AP, whereas 4C-PRP showed an unexpected but significant increase in aggregates. Testing confirmed activation during storage but filtration did not further activate platelets. CONCLUSION: We provide evidence that 4C-PRP is an alternative to 4C-AP and that bedside filters reduce aggregates from 4C-AP. Further studies are needed to evaluate the hemostatic potential of 4C-PRP and the management of aggregates.

From A to AB: A Caucasian Mother with High Anti-B Titer Causing Hemolytic Disease of the Newborn.

We report on a term infant with clinically significant hemolysis and hyperbilirubinemia. Testing revealed ABO incompatibility between maternal type A and infant type AB. The maternal alloantibody screen was negative. The infant's direct antiglobulin test was positive, and anti-B IgG was eluted off the infant's red blood cells (RBCs). Testing of the mother's plasma revealed a high anti-B titer. The infant was successfully treated with phototherapy and intravenous immunoglobulin. The bilirubin and hematocrit stabilized, and the infant was discharged home. This case was unusual because of its severity and unusual ABO constellation. Furthermore, this report is an exemplary educational case study on how effective collaboration between the clinical team and the blood bank laboratory is critical in reaching the correct diagnosis. In summary, the differential diagnosis of more unusual and atypical ABO-incompatible constellations must be considered when an infant presents with unexplained hemolysis.

Storage temperature determines platelet GPVI levels and function in mice and humans.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature-stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

Conformation-dependent blockage of activated VWF improves outcomes of traumatic brain injury in mice.

Traumatic brain injury-induced coagulopathy (TBI-IC) causes life-threatening secondary intracranial bleeding. Its pathogenesis differs mechanistically from that of coagulopathy arising from extracranial injuries and hemorrhagic shock, but it remains poorly understood. We report results of a study designed to test the hypothesis that von Willebrand factor (VWF) released during acute TBI is intrinsically hyperadhesive because its platelet-binding A1-domain is exposed and contributes to TBI-induced vascular leakage and consumptive coagulopathy. This hyperadhesive VWF can be selectively blocked by a VWF A2-domain protein to prevent TBI-IC and to improve neurological function with a minimal risk of bleeding. We demonstrated that A2 given through intraperitoneal injection or IV infusion reduced TBI-induced death by >50% and significantly improved the neurological function of C57BL/6J male mice subjected to severe lateral fluid percussion injury. A2 protected the endothelium from extracellular vesicle-induced injury, reducing TBI-induced platelet activation and microvesiculation, and preventing a TBI-induced hypercoagulable state. A2 achieved this therapeutic efficacy by specifically blocking the A1 domain exposed on the hyperadhesive VWF released during acute TBI. These results suggest that VWF plays a causal role in the development of TBI-IC and is a therapeutic target for this life-threatening complication of TBI.

Cold-Stored Platelets: Review of Studies in Humans.

Although numerous reviews and editorials have been published about the biologic features of platelets exposed to cold temperature and their in vitro function, none has focused on the data from studies after transfusion in healthy human participants and patients. This may, in part, be due to the paucity of well-controlled in vivo investigations of cold-stored platelets. Although numerous studies are looking into the recovery and survival of cold-stored platelets (ie, the percentage of infused platelets maintained in circulation over time), very few assess in vivo platelet function. Another caveat is that most studies were performed in the 1960s and 1970s, at a time when platelet collection and storage were different compared to today. Despite these limitations, we believe the transfusion community can take valuable information from these studies. This review is limited to data on cold-stored platelets in plasma or additive solution and does not include data on whole blood or resuspended whole blood from components because the hemostatic properties of whole blood are likely very different (the interested reader is referred to the review article focused on the hemostatic properties of platelets stored in whole blood by van der Meer et al in this special edition of Transfusion Medicine Reviews). In this review, we report that room temperature storage consistently results in a longer in vivo platelet circulation time at the expense of bacterial growth and shorter storage duration, resulting in expiration, wastage, and regional and national shortages. Cold storage of platelets universally results in moderately reduced recovery and markedly reduced survival. We found inconsistent data about the efficacy of cold-stored platelets likely due to study design differences. The analysis of the available data suggests that there is a short-lasting hemostatic effect of cold-stored platelets. Storage time or choice of anticoagulant did not have a clear effect on platelet efficacy after cold storage. In summary, more data and clinical trials are needed to better understand the effect of cold-stored platelets after transfusion into humans.

Effects of storage time prolongation on in vivo and in vitro characteristics of 4°C-stored platelets.

BACKGROUND: Cold (4°C)-stored platelets are currently under investigation for transfusion in bleeding patients. It is currently unknown how long cold-stored platelets can be stored for clinical applications. STUDY DESIGN AND METHODS: Twenty three subjects were recruited. Twenty-one subjects were available for in vivo assessment and received indium-111 radiolabeled, cold-stored platelets. We investigated 5- (n = 5), 10- (n = 6), 15- (n = 5), and 20-day-stored (n = 5) platelets and obtained samples for in vitro testing at baseline and after the designated storage time. Twenty three units were available for in vitro testing. Five- and 7-day (n = 5 each), room temperature (RT)-stored platelets served as the current clinical standard control. RESULTS: In vivo, we found a continuous decline in platelet recovery from 5 to 20 days. Platelet survival reached a low nadir after 10 days of storage. Ex vivo, we observed the maximum platelet αIIbß3 integrin response to collagen at 5 days of cold storage, and we saw a continuous decline thereafter. However, platelet integrin activation and mitochondrial membrane integrity were better preserved after 20 days at 4°C, compared to 5 days at RT. Platelet metabolic parameters suggest comparable results between 20-day cold-stored platelets and 5- or 7-day RT-stored platelets. CONCLUSION: In summary, we performed the first studies with extended, cold-stored, apheresis platelets in plasma for up to 20 days with a fresh comparator. Storing cold-stored platelets up to 20 days yields better results in vitro, but further studies in actively bleeding patients are needed to determine the best compromise between hemostatic efficacy and storage prolongation.