Preservation Of Platelet Function Research Articles

PAR for the liver course: Preserving platelet function mitigates outcomes in cirrhosis.

PAR for the liver course: Preserving platelet function mitigates outcomes in cirrhosis.

P82: Platelet Exogenous Lipid Uptake: A Step Toward Lipid Membrane Mechanoceuticals

Background: Mechanical circulatory support (MCS) remains limited by bleeding and clotting adverse events driven by altered platelet function related to supraphysiologic shear exposure. As the primary target of platelet shear exposure is the lipid-rich plasma membrane, in prior work we demonstrated that organic solvents altering membrane properties limit SMPA while preserving platelet function to avoid bleeding. These chemical agents limiting mechanical (shear) activation have been termed “mechanoceuticals.” Natural lipids exhibit membrane altering properties similar to solvents, yet avoid the risk of toxicity, are safe for human use and offer potential therapeutic translation. As a first step here we investigated whether lipids could be taken up and incorporated into the platelet membrane. Methods: Two distinct methods were utilized - fluorescence microscopy and flow cytometry. Gel-filtered platelets (GFP) were prepared from aspirin/NSAID-free human volunteers. Platelet uptake of three differing lipids were tested: 1. cholesterol, 2. Palmitic acid (16:0 – a saturated medium chain fatty acid) and 3. Oleic acid (18:1 – partially unsaturated fatty acid). GFP were stained with Nile Red (to allow platelet visualization, fluoresces red) and aliquots incubated with one of the three lipids (30 minutes at 37.) pre-tagged with nitrobenzoxadiazole (NBD – fluoresces green). GFP was imaged via fluorescence microscopy (1000X) using red and green filters successively to visualized platelets (red), platelets with lipid uptake (green), and to confirm co-registration (green and red overlap). GFP were also incubated with NBD-tagged lipids, followed by CD-41a antibody for platelet detection and analyzed via flow cytometry. Results: Platelets were readily stained and detected via Nile Red fluorescent imaging. All lipids tested appeared to be taken up by platelets with clear incorporation in their membrane. Overlap of imaging revealed clear registration of “platelet-lipid” images (green) with platelets alone (red), with evident co-registration confirming uptake and ruling out micelle artifact. Lipids alone did not reveal detectable particle imaging (see Figure). Flow Cytometry similarly demonstrated overlap of fluorescent lipid signals with those of anti-platelet antibody (CD41-a) confirming lipid uptake by platelets. Conclusion: Platelets appear to readily uptake lipids. Both saturated and unsaturated fatty acids, as well as cholesterol are incorporated into the platelet membrane with reasonable time exposure. As these differing lipids have a range of membrane-modulating properties, such as altering fluidity and stiffness, they offer promise as potential mechanoceutical agents. As next steps, studies are now ongoing to examine the cell mechanobiological effects of these lipids and their ability to modulate shear-mediated alteration of platelet function.Figure 1. Fluorescence microscopy imaging of 100000u/uL platelets stained with Nile Red upon incubation with NBD- Cholesterol, NBD-16:0 (Palmitic acid), and NBD-18:1 (Oleic acid) lipids each at 4uM concentrations for 30 minutes at 37ο C. Magnification ×1000. Scale bar =4 μm.

Specialized Proresolving Mediators (SPMs) Help to Alleviate the Cold Platelet Storage Lesion

Introduction: Refrigeration of platelets in plasma is a promising alternative to conventional room temperature storage, but the cold temperature leads to increased levels of activation (i.e. P-Selectin and phosphatidylserine [PS]). Resolvins (Rv) are a class of specialized proresolving mediators (SPMs) that promote the resolution of inflammation and, more specifically, have been shown to regulate ADP activation of human platelets. In this study, we tested the hypothesis that the addition of Rv would help to reduce platelet activation during cold (4°C) storage and better preserve platelet function.Methods: Apheresis platelets were collected from healthy donors (n=3-4) using a Trima Accel collection system. On the day of collection (Day 0) platelets were aliquotted into 4 mini-bags (10 mL). Three of the bags received either RvE1 (100μM), RvD2 (100 μM), or Maresin 1 (MaR1; 100 μM), while the final bag received 0.1% EtOH (Vehicle control). Treated mini-bags were incubated for 15 minutes with compound or vehicle prior to long-term storage at 4°C. The following assays were performed on Day 0 and Day 7 of storage: platelet count, Multiplate aggregation (ADP, Collagen, TRAP), and flow cytometry (GPIb expression, P-Selectin, lactadherin [PS], and PAC-1 [activated GPIIb/IIIa]). Data were analyzed using paired t-tests. Significance was determined as P <0.05.Results: P-Selectin and PAC-1 expression in Day 7 Vehicle and compound-treated samples were significantly higher than Day 0 Vehicle levels (Fig. 1A, C). Compared to Vehicle at Day 7, RvD2- and MaR1-treated samples showed a significant reduction in PS expression (RvD2 vs Vehicle, P=0.031; MaR1 vs Vehicle, P=0.018) and PAC-I binding (RvD2 vs Vehicle, P=0.012; MaR1 vs Vehicle, P=0.036) (Fig. 1B, C). No differences in P-Selectin expression were observed for any of the compounds tested (Fig. 1A). TRAP response was higher in RvD2-treated samples compared to Day 7 Vehicle (P=0.047). ADP response was higher in MaR1-treated samples compared to Day 7 Vehicle (P=0.03). No differences in collagen aggregation were observed between samples. All SPM-treated bags resulted in higher platelet counts than Day 7 Vehicle.Conclusions: Cold storage of platelets at 4°C has gained increased attention as an alternative to room temperature storage due to the lower risk of bacterial contamination, better preservation of platelet function, and the potential for a prolonged shelf life; however, increased levels of platelet activation and platelet clumping in cold-stored platelets limit shelf life. Results from our study suggest that the addition of SPMs (RvE1, RvD2, and MaR1) to cold-stored platelets helps minimize platelet activation and even preserve platelet function (RvD2, MarR1) during long-term storage. A platelet product with an extended shelf life would improve platelet inventories and expand access to under-served patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Freeze-Drying of Platelet-Rich Plasma: The Quest for Standardization.

The complex biology of platelets and their involvement in tissue repair and inflammation have inspired the development of platelet-rich plasma (PRP) therapies for a broad array of medical needs. However, clinical advances are hampered by the fact that PRP products, doses and treatment protocols are far from being standardized. Freeze-drying PRP (FD-PRP) preserves platelet function, cytokine concentration and functionality, and has been proposed as a consistent method for product standardization and fabrication of an off-the-shelf product with improved stability and readiness for future uses. Here, we present the current state of experimental and clinical FD-PRP research in the different medical areas in which PRP has potential to meet prevailing medical needs. A systematic search, according to PRISMA (Preferred Reported Items for Systematic Reviews and Meta-Analyses) guidelines, showed that research is mostly focused on wound healing, i.e., developing combination products for ulcer management. Injectable hydrogels are investigated for lumbar fusion and knee conditions. In dentistry, combination products permit slow kinetics of growth factor release and functionalized membranes for guided bone regeneration.

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

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. 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. 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. 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.

Preservation of platelet function in patients with cirrhosis and thrombocytopenia undergoing esophageal variceal ligation

BackgroundThrombocytopenia is a possible risk factor for bleeding after band ligation of esophageal varices. However, elevated von Willebrand factor (VWF) in cirrhosis improves platelet function and could decrease this risk. Our objective was to assess platelet function in patients with cirrhosis undergoing esophageal variceal ligation (EVL). MethodsThe assessment consisted of platelet count, antigen and activity of VWF and VWF-cleaving protease ADAMTS-13 activity, and a platelet adhesion and aggregation test simulating vascular flow in vivo (Impact-RⓇ) prior to EVL. ResultsTotally 111 patients were divided into three groups according to platelet count: (1) < 50 × 109/L (n = 38, 34.2%); (2) 50 × 109/L to 100 × 109/L (n = 47, 42.3%); and (3) > 100 × 109/L (n = 26, 23.4%). No statistically significant difference was found in the aggregate size of platelets [group 1: 41.0 (31.8–67.3) µm2; group 2: 47.0 (33.8–71.3) µm2; and group 3: 47.0 (34.0–66.0) µm2; P = 0.60] and no significant correlation was found between aggregate size and platelet count (Spearman r = 0.07; P = 0.47). Surface coverage was 4.1% (2.8%–6.7%), 8.5% (4.0%–10.0%), and 9.0% (7.1%–12.0%) (P < 0.001) in groups 1, 2 and 3, respectively and correlated with platelet count (Spearman r = 0.39; P < 0.0001). There was no significant difference between groups in VWF or ADAMTS-13. Post-EVL bleeding occurred in six (5.4%) patients (n = 2 in group 1, n = 1 in group 2, and n = 3 in group 3; P = 0.32). Patients with bleeding had higher MELD scores [15.0 (11.3–20.3) versus 12.0 (10.0–15.0); P = 0.025], but no difference was demonstrated for platelet function parameters. ConclusionPlatelet function is preserved even in the presence of thrombocytopenia, including in the patients with post-EVL bleeding.

Far Forward Gaps in Hemorrhagic Shock and Prolonged Field Care: An Update of ALM Fluid Therapy for Field Use.

Future expeditionary missions are expected to occur in more remote austere environments where combat medics and casualties may have to wait up to 7 days before resupply or safe evacuation. Currently, there is no effective fluid therapy for hemorrhagic shock (HS) at the point-of-injury and continuum-of-care over this extended period. We have been developing a small-volume IV or IO ALM therapy for noncompressible HS and have shown in preclinical models that it extends survival to 3 days, reduces abdominal bleeding by 60%, blunts inflammation, corrects coagulopathy, preserves platelet function, and prevents immunodeficiency. The ALM-survival phenotype is associated with an upregulation of the master genes of metabolism and mitochrondrial biogenesis in heart and brain and a downregulation in the periphery. Future translational studies will investigate the timing and nature of the "switch" and extend survival to 7 days. We will also discuss some of the controversies of ALM resuscitation in pigs, present our Systems Hypothesis of Trauma (SHOT), and discuss future clinical safety trials before field use.

Minimal invasive extracorporeal circulation preserves platelet function after cardiac surgery: a prospective observational study.

Cardiac surgery on conventional cardiopulmonary bypass induces a combination of thrombocytopenia and platelet dysfunction which is strongly related to postoperative bleeding. Minimal invasive extracorporeal circulation has been shown to preserve coagulation integrity, though effect on platelet function remains unclear. We aimed to prospectively investigate perioperative platelet function in a series of patients undergoing cardiac surgery on minimal invasive extracorporeal circulation using point-of-care testing. A total of 57 patients undergoing elective cardiac surgery on minimal invasive extracorporeal circulation were prospectively recruited. Anticoagulation strategy was based on individualized heparin management and heparin level-guided protamine titration performed in all patients with a specialized point-of-care device (Hemostasis Management System - HMS Plus; Medtronic, Minneapolis, MN, USA). Platelet function was evaluated with impedance aggregometry using the ROTEM platelet (TEM International GmbH, Munich, Germany). ADPtest and TRAPtest values were assessed before surgery and after cardiopulmonary bypass. ADPtest value was preserved during surgery on minimal invasive extracorporeal circulation (58.2 ± 20 U vs. 53.6 ± 21 U; p = 0.1), while TRAPtest was found significantly increased (90 ± 27 U vs. 103 ± 38 U; p = 0.03). Postoperative ADPtest and TRAPtest values were inversely related to postoperative bleeding (correlation coefficient: -0.29; p = 0.03 for ADPtest and correlation coefficient: -0.28; p = 0.04 for TRAPtest). The preoperative use of P2Y12 inhibitors was identified as the only independent predictor of a low postoperative ADPtest value (OR = 15.3; p = 0.02). Cardiac surgery on minimal invasive extracorporeal circulation is a platelet preservation strategy, which contributes to the beneficial effect of minimal invasive extracorporeal circulation in coagulation integrity.

Acute bilirubin ditaurate exposure attenuates ex vivo platelet reactive oxygen species production, granule exocytosis and activation

BackgroundBilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. MethodsThe inhibitory potential of BRT (10–100 μM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H2DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. ResultsOur data shows ≤35 μM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 μM BRT exposure. Furthermore, platelet exposure to ≤10 μM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. ConclusionsThese data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.

Platelet enhancement of bacterial growth during room temperature storage: mitigation through refrigeration.

Due to high risk of septic transfusion reactions arising from bacterial contamination, US Food and Drug Administration regulations currently limit platelet storage to 5 days at room temperature (RT). However, blood culturing methods can take up to 7 days to detect bacteria, allowing transfusion of potentially contaminated units. Thus, cold storage (CS) may be a viable means of extending shelf life and improving safety. Platelets and fresh plasma (FP) were collected by apheresis from healthy donors, aliquoted, and challenged with Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Staphylococcus epidermidis. Aliquots were then stored at either RT or CS. Significant (p < 0.05) bacterial growth was detected at RT for most bacteria as early as Day 1 after collection, with peak growth occurring between Days 3 and 4. Growth remained static during CS. Additionally, platelets appeared to enhance bacterial replication with growth significantly lower (p < 0.05) in FP relative to RT-stored platelets. Lactic acid promoted bacterial growth when added to FP at RT. Bacterial challenge also resulted in significantly increased platelet activation (p < 0.05) and significantly reduced platelet function (p < 0.05) in RT storage relative to uninfected controls by Day 5 after collection. Conversely, CS ablated bacteria growth, limited platelet metabolism, and preserved platelet function throughout the study. These data suggest that CS presents an attractive alternative to RT to both extend storage life and reduce the risk of transfusion-related sepsis.

Effect of 3-Day Storage and Temperature on ADP-induced Platelet Aggregation

Objective: To observe platelet functions during the 3 days of platelet storage and to evaluate the effect of storage temperature towards platelet aggregation response. Methods: Two conditions of platelet storage were used in this study: at room temperature and in a cold condition at 4 °C to observe the temperature effect on platelet aggregation. The aggregation test was performed using a 96-wells plate platelet aggregation method. Results: At room temperature on day 1, the response of platelet aggregation reduced to 34.9±10.6%, which was less than half of the aggregation of fresh platelet (100%). The aggregation on day 2 reduced modestly (26.8±4.2%), in comparison with that seen on the first day. On day 3, platelet aggregation deteriorated significantly (6±1.2%), which was comparable to the control group without the presence of platelet agonist (6.1±1.4%). When platelets are stored at 4 °C, the aggregation response to ADP stimulation was slightly different to that shown by platelets stored at room temperature (22 °C). At the 4 °C, the aggregation response was 68.11±34.2%, 57.1±21.4%, and 5.9±2.6% at day 1, day 2, and day 3, respectively. The result showed that storing the blood at 4 ˚C or room temperature was not able to preserve platelet function, as the deterioration of platelet aggregation response over time was still taking place. Although was not statistically significant, our result indicated that cold storage reduced platelet responsiveness toward ADP activation. Conclusions: This current study provides evidence of the deterioration of platelet function during first 3 days of platelet storage. Moreover, we found that storing platelets in 4 °C showed no significant benefit in preserving the ADP-induced platelet aggregation capacity compared to that stored in 22 °C. Keywords: Adenosine diphosphate, platelet, platelet aggregation, platelet storage DOI: 10.15850/ijihs.v6n2.1375

Acoustophoretic purification of platelets: feasibility and impact on platelet activation and function

Purity, limited platelet activation, and preservation of platelet function are important stakes of preparation of platelet concentrates (PC) for clinical use. In fact, contaminating red blood cells and leukocytes, as well as activated and/or poorly functional platelets in PC, represents a risk of poor efficiency and adverse side effects during platelet transfusion. Therefore, optimization of preparation and storage of PC is still an active field of research. Shear-induced platelet activation is an unwanted side effect of the hard-spin (up to 5000g) step of centrifugation-based methods currently used in blood banks to prepare PC from whole blood samples. Here, we evaluated the effectiveness of an acoustic-based fractionation device for the isolation of human platelets from whole blood bags. The purity, activation status, and functionality of platelets isolated by acoustopheresis were compared with those of platelets isolated using a reference protocol known to produce limited platelet activation and consisting of two consecutive soft-spin centrifugations (120g and 1200g). Platelet concentration and purity were determined using an automated hematology analyzer. Platelet activation status and platelet reactivity to collagen and thrombin were assessed in flow cytometry by measurement of surface expression of P-selectin and activated integrin αIIbβ3. The ability of isolated platelets to incorporate into a thrombus when transfused to NOD/SCID mice was investigated by intravital microscopy using the ferric chloride-induced thrombosis model. Blood fractionation by acoustophoresis led to the elimination of more than 80% of red blood cells and leukocytes from the platelet fraction, whose mean purity was of 92.8 ± 12.8%. The activation status and reactivity to collagen and thrombin of acoustophoresis-isolated platelets were similar to those of platelets isolated by soft-spin centrifugation. Finally, acoustophoresis-isolated platelets were tethered, adhered to the vessel wall, and incorporated into a growing thrombus following ferric chloride-induced vascular injury. Together, our results indicate that acoustophoresis is a suitable method for the isolation of human platelets with minimal platelet activation and preservation of platelet function.

Cytochrome c and resveratrol preserve platelet function during cold storage

Donated platelets are stored at 22°C and discarded within 5 days because of diminished function and risk of bacterial contamination. Decline of platelet function has been attributed to decreased mitochondrial function and increased oxidative stress. Resveratrol (Res) and cytochrome c (Cyt c), in combination with hypothermic storage, may extend platelet viability. Platelets from 20 donors were pooled into four independent sets and stored at 22°C or 4°C in the absence or presence of Res (50 μM) or Cyt c (100 μM) for up to 10 days. Sequential measurement of platelet counts, coagulation function (thromboelastography), oxygen consumption, lipid peroxidation, glucose-lactate levels, pH, TCO2, and soluble platelet activation markers (CD62P/PF-4) was performed. Platelet function diminished rapidly over time at 22°C versus 4°C (adenosine diphosphate, day 10 [0.6 ± 0.5] vs. [7.8 ± 3.5], arachidonic acid: day 10 [0.5 ± 0.5] vs. [30.1 ± 27.72]). At 4°C, storage treatment with Res or Cyt c limited deterioration in platelet function up to day 10, an effect not observed at 22°C (day 10, 4°C, Con [7.8 ± 3.5] vs. Res [37.3 ± 24.19] vs. Cyt c [45.83 ± 43.06]). Mechanistic analysis revealed oxygen consumption increased in response to Cyt c at 22°C, whereas neither Cyt c or Res affected oxygen consumption at 4°C. Lipid peroxidation was only reduced at 22°C (day 7 and day 10), but remained unchanged at 4°C, or when Res or Cyt c was added. Cytosolic ROS was significantly reduced by pretreatment with Res at 4°C. Total platelet count and soluble activation markers were unchanged during storage and not affected by Res, Cyt c, or temperature. Glucose concentration, pH and TCO2 decreased while lactate levels increased during storage at 22°C but not 4°C. Platelet function is preserved by cold storage for up to 10 days. This function is enhanced by treatment with Res or Cyt c, which supports mitochondrial activity, thus potentially extending platelet shelf life.

Novel Class of Direct Thrombin Inhibitors Prevent Thrombosis By Inhibiting Fibrinogen Cleavage While Preserving Platelet Function

We have developed a new class of direct thrombin inhibitors (VE-DTIs) that act through reversible covalent enzyme inhibition. These compounds inhibit thrombin with high potency (IC50 < 10 nM) and with better than 100× selectivity against related serine proteases. In rodents, VE-DTIs show dose-dependent efficacy in preventing arterial and venous thromboses respectively in arteriovenous shunt thrombosis and venous stasis thrombosis models. They also prevent thrombin-induced pulmonary embolism in rodents. Despite their strong anticoagulation properties, VE-DTIs are associated with significantly lower bleeding times than other approved anticoagulants dabigatran, argatroban or apixaban in rodent bleeding models (tail bleeding time and saphenous vein bleeding time).We compared the effect of VE-DTIs and the DTIs argatroban and dabigatran on the coagulation cascade. In vitro, VE-DTIs inhibited thrombin-mediated fibrinogen cleavage with similar potency. Inhibition of thrombin/thrombomodulin complex-mediated activation of TAFI and protein C by VE-DTIs were also comparable. In contrast, VE-DTIs were more than 1000-fold less potent in inhibiting thrombin-mediated platelet activation than the other DTIs. Additionally, and also in contrast to argatroban and dabigatran, VE-DTIs protect rodents from the effects of thrombin-induced pulmonary embolism without significantly inhibiting platelet activation. The ability of VE-DTIs to prevent thrombosis without significantly impacting platelet function could lead to a new anticoagulant therapy with lower bleeding risk. DisclosuresSivaraja:Verseon Corporation: Employment, Equity Ownership. Sizikov:Verseon Corporation: Employment, Equity Ownership. Sandoval:Verseon Corporation: Employment, Equity Ownership. Lin:Verseon Corporation: Employment, Equity Ownership. Igoudin:Verseon Corporation: Employment, Equity Ownership. Dash:Verseon Corporation: Employment, Equity Ownership. Xu:Verseon Corporation: Employment, Equity Ownership. Clemens:Verseon Corporation: Employment, Equity Ownership. Estiarte:Verseon Corporation: Employment, Equity Ownership. Shiau:Verseon Corporation: Employment, Equity Ownership. Short:Verseon Corporation: Employment, Equity Ownership. Williams:Verseon Corporation: Employment, Equity Ownership. Datta:Verseon Corporation: Employment, Equity Ownership. Kita:Verseon Corporation: Employment, Equity Ownership.

Reducing Fibrinogen through Platelet Additive Solutions Improves Mitochondrial Function and Reduces Reactive Oxygen Species in Stored Platelets

BackgroundThe platelet storage lesion is mitigated by refrigeration in platelet additive solutions (PAS), but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Understanding underlying mechanisms responsible for enhanced hemostatic function of 4°C-stored PAS platelets will be critical for such improvements in platelet transfusion. We hypothesized that storage of platelet concentrates at 4°C leads to GPIIb-IIIa activation and thus aggregate formation through fibrinogen binding, and that this could be prevented by storing platelets in platelet additive solution (PAS) without compromising platelet function.MethodsApheresis platelets in plasma (AP) or apheresis platelets in PAS were stored at 22°C or 4°C for up to 15 days. Measurements include platelet counts, blood gases, flow cytometry analysis, intracellular reactive oxygen species (ROS) levels, and high resolution respirometry. To explore the effect of fibrinogen on storage, platelets were treated with abciximab to inhibit GPIIb-IIIa binding and aggregation. Donor fibrinogen levels were measured before being collected in PAS; samples were separated into minibags for comparison of control 4°C PAS-stored platelets to 4°C PAS-stored platelets with fibrinogen added back to original donor levels (PAS-FBN).ResultsMitochondrial respiration was lower in platelets stored at 4°C compared to 22°C in both AP and PAS; however, PAS-stored platelets retained lower mitochondrial respiration over the time course. Additionally, maximal mitochondrial oxygen utilization (oxidative burst capacity) was better preserved in 4°C-stored platelets; PAS demonstrated less overall mitochondrial exhaustion at 4°C compared to 4°C-AP samples (Figure 1). PAS-FBN resulted in reduced oxidative burst capacity over 15 days of storage which was consistent with loss of platelet function observed through flow cytometry analysis including increased phosphatidylserine expression, microparticles, and GPIb shedding. Mitochondrial intracellular ROS levels were lower in all 4°C-stored samples compared to their respective RT-stored groups. PAS-FBN resulted in significantly lower platelet counts (Day 5: BL= 1.1x106±.04plt/µL, PAS = 1.1 x106±.06plt/µL, PAS-FBN = .61 x106±.06plt/µL; P<0.001) compared to 4°C PAS-stored platelets and displayed marked increases in visible aggregates in the storage bag. PAS-stored platelets resulted in significantly lower levels of intracellular ROS compared to AP at both RT and 4°C conditions and PAS-FBN (P<0.05). 4°C-AP-stored samples treated with abciximab (AP-Ab) rescued the loss in platelet count commonly encountered in 4°C-AP storage (Day 5: BL = 1112±18, AP-4°C = 720±77, AP-Ab = 1085±28;P<0.001) and resulted in less intracellular ROS compared to untreated 4°C-AP samples at Day 5 (Figure 2).DiscussionMitochondrial damage and ROS production may contribute to loss of platelet viability during storage, whereas cold storage is known to preserve platelet function. Our previous work has shown that use of PAS in stored platelets prevents aggregate formation, enhances platelet function, results in less oxidant stress, and preserves both mitochondrial function and potential compared to AP-stored platelets. Here we demonstrate that reduction in fibrinogen levels of 4°C PAS -stored platelets plays a key role in the underlying mechanism for PAS-stored platelet enhancement. Taken together, these data indicate that cold storage combined with PAS preserves platelet viability both by maintaining energy potential and preventing degradation due to enhanced ROS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inducing mitophagy in diabetic platelets protects against severe oxidative stress.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress‐mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM. A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in “prepackaging” the complex mitophagy machinery in a short‐lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

The state of the science of whole blood: lessons learned at Mayo Clinic.

AABB Standards specify that ABO group-specific whole blood is the only acceptable choice for whole blood transfusions. Although universal donor group O stored whole blood (SWB) was used extensively by the military during the wars of the mid-twentieth century, its use has fallen out of favor and has never been used to great extent in the civilian trauma population. Interest in the use of whole blood has been renewed, particularly in light of its potential value in far-forward military and other austere environments. Evidence of preserved platelet function in SWB has heightened enthusiasm for a "one stop shop" resuscitation product providing volume, oxygen carrying capacity, and hemostatic effects. Experience with universal donor group O SWB is required to ascertain whether its use will be an advance in trauma care. Described here is the process of establishing a universal donor group O SWB at a civilian trauma center in the United States.

COX-2 Inhibition: What We Learned—A Controversial Update on Safety Data

Cyclooxygenase type 2 (COX-2)-selective nonsteroidal anti-inflammatory drugs (NSAIDs) (c2sNSAIDs) have been scrutinized relative to the less costly nonselective NSAIDs (nsNSAIDs). The conclusions reached were not always consistent with the data, and best treatment choices for patients were not always recommended. The data that were used to criticize the c2sNSAIDs are reexamined in a controversial light, demonstrating that the presence of reverse bias was often, but not always, present. A review of both Pubmed and news media articles relating to nsNSAIDs and c2sNSAIDs was conducted. References were selected on the basis of relevance to the controversies. The initial claims for the c2sNSAIDs of reduced gastrointestinal (GI) injury and preservation of platelet function were soon dwarfed by concerns regarding increased cardiovascular (CV) risk with publication of the Vioxx Gastrointestinal Outcomes Research trial for rofecoxib. Initial prothrombotic theories had a poor basis for explaining these concerns and have since largely been replaced with more credible explanations, including blood pressure elevations known to occur with all NSAIDs. Between data suggesting increased CV risk and under political pressure and public outcry, rofecoxib was withdrawn from the market in 2004. Soon, all c2sNSAIDs were under scrutiny. The Food and Drug Administration has since grouped all NSAIDs, whether c2sNSAID or nsNSAID, into one class with similar warnings regarding skin, CV, renal, and GI side effects. The entire "COX-2 debacle" is reminiscent of past events with NSAIDs. Amid this public, media, and political hysteria, it is not clear if we will see any more NSAIDs (selective or otherwise) approved in the near future.

Red Blood Cells Preserve Platelet Function and Coagulation From The Effects Of Acidemia

Despite a large body of literature regarding Acute Coagulopathy of Trauma and Massive Transfusion, there is little consensus on the appropriate diagnostic approach to establish the cause of associated uncontrolled bleeding. Thromboelastography (TEG) is widely used to characterize trauma-associated bleeding. However, the use of whole blood in this assay may obscure important changes in blood component hemostatic function. Blood varies in hematocrit from a typical level of 40% in large vessels to 10% in capillaries. Patients with persistent bleeding may present with prolonged prothrombin time (PT)/activated partial thromboplastin time (aPTT) measured in plasma, but unremarkable TEG tracings measured in whole blood drawn from a large vein. As one of the only ubiquitously accepted causes of a bleeding coagulopathy associated with trauma, acidemia was modeled in TEG to investigate 1) the differential coagulopathy detection obtained by separation of blood components before TEG testing, 2) the range of acidemia effects at various levels of the vascular tree through variable hematocrit testing, and 3) effects of replacing RBCs with plasma versus normal saline. Male donor blood (n=5) was drawn into citrate and the contact pathway was immediately further inhibited with corn trypsin inhibitor. TEG cups were preloaded with appropriate volumes of [morpholino]ethanesulfonic acid buffered saline (MBS) for pH 7.4, pH 7.1, pH 6.8 and were recalcified (15mM). R-time parameters were not significantly different when whole blood (WB) or red blood cells combined with platelet poor plasma (RBC/PPP) were activated to clot with tissue factor (1:5000 dilution induced a normal 4 minute clot time) in the simulated acidemia, whereas platelet poor plasma (PPP) showed a significant delay at pH 7.1 (p&lt;0.0001) and pH 6.8 (p&lt;0.0001) when compared to pH 7.4. Shear Elastic Modulus Strength showed only significant losses in platelet rich plasma (PRP: pH 7.4 vs pH 7.1; p=0.013, pH 7.1 vs pH 6.8; p=0.015) but not WB, RBC/PPP, or PPP. Hematocrit levels were chosen to model typical changes from large to small vessels, in the presence of 200,000 platelets/microliter, where the volume of RBCs was replaced with plasma. Results indicate that zero hematocrit conditions result in a significant R-time delay (pH 7.4 vs pH 7.1; p=0.0306, pH 7.1 vs pH 6.8; p&lt;0.0001) whereas no significant delays occur at 20%, 30%, and 40% hematocrit. Utilization of normal saline for RBC replacement did not alter any of the 20%, 30%, or 40% hematocrit R-time findings yet zero % hematocrit samples (pH 7.1; 12.9+/-1.64 minutes, pH 6.8; 34.98+/-4.03 minutes) were significantly delayed (p&lt;0.0001) when compared to plasma replacement of RBC (pH 7.1; 8.22+/-0.70 minutes, pH 6.8;13.98+/-1.54 minutes). Subsequent biochemical evaluation of whole blood (n=5) activated to clot in conditions of acidemia (pH 7.4, pH 7.1, pH 6.8) showed no significant delay or reduced peak of thrombin generation, no significant difference in factor V activation or fibrinopeptide A cleavage. Data indicate that 1) separation of blood components allows a more sensitive parsing mechanism for TEG evaluation of coagulation in acidemia, 2) simulated capillary blood containing low hematocrit demonstrates the anti-coagulant effects of acidemia obscured in whole blood from large vessels with normal hematocrit, 3) resuscitation with normal saline may exacerbate microvascular bleeding in the acidemic patient, and 4) RBCs prevent delays in clot initiation and loss of platelet force generation in the presence of acidemia. This work provides a mechanism to explain the observation that increased hematocrit reduces microvascular bleeding. Disclosures: No relevant conflicts of interest to declare.