ALBERT

Description: Background: Platelets are required for effective treatment of severe hemorrhage. Standard-of-care storage is at room temperature (RT), but leads to a storage lesion characterized by loss of hemostatic function and increased risk of bacterial contamination. Refrigeration (4C) mitigates adverse effects; however, it results in pre-activation or priming, which may be suggestive of prothrombotic tendencies. We previously showed that 4C-stored platelets (4C-PLT) retain responses to physiological inhibitors comparable to those of fresh platelets (FR-PLT) in two static aggregation assays. To more closely mimic in-vivo conditions, we tested adhesive response in a microfluidic environment under physiologic high-shear flow. We hypothesized that 4C-PLT display superior adhesion compared to standard-of-care (RT-PLT) and that platelet hemostatic inhibition due to prostacyclin and nitric oxide (NO) would be similar to fresh. Methods: Apheresis platelets (AP) collected from 4 healthy donors were stored for 5 days at RT (22-24°C) or 4C (1-6°C). Additional whole blood was collected to obtain red blood cells (RBCs). Platelet samples were assayed on Day 1 (fresh) and Day 5 (RT-PLT and 4C-PLT) in the presence or absence of prostacyclin (10 nM Prostaglandin I2, PGI2) or an NO donor (50 uM S-Nitrosoglutathione, GSNO). Bioflux plates (Fluxion) were coated with 100 µg/ml type-1 collagen. Prior to perfusion, platelets were stained with calcein-AM (300x103PLT/ul) and RBCs were added to a hematocrit of 40%. Samples were perfused through the collagen-coated wells at an arterial shear rate of 720s-1, and compared to bovine serum album (BSA)-coated channels as a control to assess nonspecific binding. A fluorescence microscope acquired images every 30 sec for 6 min. Data were reported as fluorescence intensity units (FIU) and surface coverage (SC%) measured with Bioflux Montage (MetaMorph) software. Data were analyzed using two-way ANOVA and a post hoc Tukey test for multiple comparisons. Significance was p

Description: Background: Platelet (PLT) hemostatic responses are heavily dependent on PLT bioenergetics which drives the generation of PLT contractile forces and contribute to mechanical hemostasis. Although effective clot retraction (CR) requires PLT adhesion, aggregation and formation of strong clots, CR remains an understudied PLT function. In this work, we hypothesize that well-conserved PLT bioenergetics during storage is crucial for highly energy-dependent PLT functions including CR. To further investigate this correlation, we characterized the effect of extended PLT storage for up to 15 days on PLT bioenergetics and CR at room temperature (22°C, RT) and refrigeration (4°C, 4C). Methods: Apheresis PLT concentrates in plasma were collected from 5 healthy donors and stored for 21 days at RT with agitation, or stored stationary in the cold (4C). Fresh PLTs were treated with metabolic inhibitors for 1 hour at 37°C, recalcified with 20mM calcium chloride, and incubated at 37°C to initiate CR. The clot weights were measured post retraction, and the clots were prepared for immunohistochemistry to assess clot structure. Similarly, extent of retraction and mitochondrial respiration levels were estimated in PLTs stored for 5, 10 and 15 days. Additionally, Platelet additive solution (PAS) stored PLTs were tested as a viable alternative to plasma storage to improve metabolic exhaustion during storage. Results: 4C-stored PLTs in plasma retracted similar to fresh PLTs for up to 15 days. In comparison, RT-stored PLTs in plasma did not retract after 10 days, whereas PLTs stored in PAS at RT or 4C seemed to retain their contractile function for up to 15 days (Fig 1A). 4C-stored and fresh PLTs yielded heavier clots with a highly organized structure compared to the disorganized structure of clots from RT-stored PLTs. Mitochondrial respiration was significantly reduced over 10 days in RT- plasma stored PLTs, which was mitigated by PAS storage. Similarly, 4C storage in PAS mitigated the reduction in respiration seen in 15 days 4C- stored PLTs in plasma (Fig 1B). Conclusion: Retraction properties of 4C-stored PLTs were similar to those of fresh PLTs, together with superior clot structure and function compared to RT- stored PLTs. This suggests that 4C storage could be a suitable alternative for extended storage. In RT-stored PLTs, reduction in mitochondrial respiration did not show any correlation with the reduced ability of PLTs to retract, suggesting that preserved glycolysis in PAS- stored RT-stored PLTs enabled CR. Thus, storing PLTs in PAS at RT or 4C could potentially mitigate mitochondrial dysfunction during extended storage. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2272 Introduction: Fresh whole blood (WB) collected from a “walking blood bank” is used by the U.S. military to supplement component therapy when blood component supplies are exhausted. Currently, WB is used for emergency transfusion within 24 hours of collection, before results of pathogen testing are available. A pathogen reduction technology (PRT), which uses riboflavin and ultraviolet light to damage nucleic acids in pathogens, is being considered as a transfusion-transmitted disease (TTD) risk mitigation measure. The effect of this technology on the hemostatic properties of whole blood, particularly on clotting capacity and clot lysis, are poorly understood, and optimal storage conditions are not defined. We previously reported that activated partial thrombin time (aPTT) and prothrombin time (PT) are prolonged by treatment; however, these tests do not always correlate with clinical findings. Thromboelastography is a more robust measure of clot formation and stability over time; we previously found that maximum amplitude (MA), which represents clot strength, did not decrease with PRT treatment and was preserved by storage at 4°C. Here we explore the effects of PRT on other parameters important to clotting capacity and clot lysis, and present the effects of WB storage at 4°C compared to 22°C. Hypothesis: WB treated with PRT demonstrates similar hemostatic function to non-treated WB, and storage at 4°C reduces degradation of blood components essential to clotting capacity and clot lysis compared to 22°C. Methods: Under an IRB-approved protocol, 8 units per treatment group of WB were collected in CPD anticoagulant from healthy donors of normal hemostatic status according to standard blood donor guidelines. Pathogen reduction was performed using riboflavin and ultraviolet light (265–400nm phosphor; Mirasol® System, CaridianBCT) dosed at 80 J/mLRBC. Treatment groups included: control WB stored at 4° C (CON-04); control WB stored at 22° C (CON-22); PRT-treated WB stored at 4° C (PRT-04); and PRT-treated WB stored at 22° C (PRT-22). The hemostatic function of the blood was assessed at baseline, days 1–7, 10, 14, and 21. Factor VIII and fibrinogen were measured from assayed samples (BCS® XP system, Siemens). Thromboelastography (TEG®, Haemoscope Corp.) estimated total thrombin generation by calculating the first derivative of the TEG tracing, the Total Thrombus Generation variable (TTG). TEG was also used to measured lysis (LY30). Data were analyzed as repeated measures, followed by analysis of variance to assess interactions. Significance was set at p

Description: Background: The hemostatic and platelet functions of fresh whole blood (FWB) are well preserved when stored at 4°C for 14 days (human) or 7 days (rats). Resuscitation of either FWB or whole blood stored for 7-days at 4°C (SWB) in rats with acute traumatic coagulopathy (ATC) induced by polytrauma and hemorrhage (T/H), shows identical correction of hemodynamic and metabolic function, as well as the equivalent impact on platelet aggregation in vitro. Platelet hemostatic function is influenced by platelet number, aggregation, and platelet-leukocyte interactions. Therefore, the objective of this study is to identify the in-vivo fate and activity of platelets from FWB and SWB, and determine whether transfused platelets contribute to clot formation in tissue injury after transfusion. Method: Isoflurane anesthetized Sprague-Dawley rats underwent polytrauma, followed by 40% hemorrhage. The rats were resuscitated (20% of bled volume) 1hr after trauma using either FWB or SWB (n=5 each) collected from green fluorescence (GFP) donor rats. SWB was kept in mini-blood-bag and refrigerated at 40C for 7 days, and warmed up to 370C prior to transfusion. Sham rats had no trauma, but underwent 20% blood replacement by FWB or SWB (n=4 each). Immediately after transfusion, a liver incision was made, followed by the measurement of kidney bleeding time (both standardized incisions was made by a Surgicutt adult template device). The incisional liver sample was taken 10min after injury for immunohistochemistry stained for CD61 (platelets) and GFP. Blood samples taken from donor rats (FWB and SWB) and recipient rats at baseline, immediately before resuscitation, and 0min and 10min after transfusion were analyzed by flow cytometry using CD42d, CD45, and CD62P antibodies for defining platelets, leukocytes and activated platelets respectively. Lactadherin was used to detect phosphatidylserine (PS). Platelet-leukocyte aggregates were defined as CD42d+/CD45+. The transfused and native platelet or leukocyte was differentiated by GFP signal. Results: GFP signal intensity was equally expressed in platelets derived from either FWB or SWB. Storage at 4°C led to a significant increase in number of platelets expressing CD62P (76±2% vs. 11±4%) and PS (8.6±0.7% vs. 1.1±0.4%) as compared to FWB. SWB also showed a significant rise in intensity, but not percentage, of platelet-leukocyte aggregates (45781±6235 vs. 10978±2345 MFI). After transfusion, recipient rats showed a significant elevation in the percentage of GFP+ platelets after transfusion of FWB as compared to SWB (Sham:13.0±1.0% vs. 4.9±0.4%; T/H: 17.0±0.8% vs. 6.8±0.%). Similarly, GFP+ leukocytes aggregates from FWB were three times higher than SWB (Sham: 6.15±0.5% vs. 1.9±0.5%; T/H: 4.4±0.4% vs. 1.2±0.2%). There was greater percentage of GFP+ platelet-leukocyte aggregates in both T/H and sham rats transfused with SWB than FWB (Sham: 11.7±3.3% vs. 33.8±2.3%; T/H: 9.8±1.6% vs. 47.2±5.1%). Transfusion with SWB led to a significant increase in percentage of activated GFP+ platelets in T/H rats as compared to FWB (30.0±2.7% vs. 2.5±0.5%). However, the activity of native platelets was not significantly different between SWB and FWB in T/H rats after transfusion (3.9±1.2% vs. 3.7±0.3%). The kidney bleeding time was not significantly different in T/H rats receiving FWB and SWB (131±4 vs.127±7 seconds) under equivalent mean arterial blood pressure (82±7 vs 85±9 mmHg). The clot that formed at the site of liver incision was identified by platelet aggregates stained by the CD61 antibody. Using co-localization of CD61 and GFP, we found that the platelets from both FWB and SWB equivalently incorporated into the clot at incisional site. Conclusion: The platelets from SWB are higher in platelet activation state, clearance rate and platelet-leukocyte aggregates than FWB after transfusion in both sham rats and rats with polytrauma and hemorrhage. However, platelets from both fresh and cold-stored whole blood contribute to hemostasis of tissue injury after transfusion. This study suggests that cold-stored whole blood is an alternative resource to treat trauma patients for restoration of hemostatic function. Future study is necessary to optimize the storage of whole blood to prolong the platelet survival rate and optimize hemostatic function. This project was funded by US Army Medical Research and Material Command. Disclosures No relevant conflicts of interest to declare.

Description: Background: Platelet transfusion is vital for hemostasis in patients with severe bleeding. Currently, platelets are stored at room temperature (RT) for 5 days with gentle agitation. However, this method results in a platelet storage lesion characterized by loss of platelet function and elevated risk of bacterial contamination. It has been shown that refrigeration of platelets at 4o C mitigates adverse effects and other logistical difficulties. Recently, FDA approved the use of cold-stored apheresis platelets for the resuscitation of actively bleeding patients for up to 3 days (5. 21 CFR 606.65(e) & 610.53(c)). We hypothesized that the refrigeration of platelets stored in 65% platelet additive solution (PAS), would better preserve the platelet contractile apparatus and overall hemostatic function. We measured clot stiffness, strength, crosslinking density and platelet contractile forces (PCF) using cone and plate rheometry; and quantified the microstructural architecture of the clots. To corroborate the rheological and morphological measurements with the clotting mechanism, we assayed thrombin generation, FXIII activity, clot retraction, and cytoskeletal rearrangement. Methods: Apheresis platelet (AP) concentrates collected from 4 healthy donors were isolated and stored in 65% Platelet additive solution (PAS) for 5 days at RT (22-24°C) or 4C (4°C). Clotting function was assayed after re-suspension in fresh frozen plasma (FFP). After initiating clotting with 20mM Ca2+, 100µL of the PRP sample was placed on the rheometer plate and clot stiffness was measured for 30 mins at 37o C at a small strain of 1Hz by tracking the evolving storage modulus (G'). Normal force measurements were made to quantify the force generated in the samples as a result of platelet contractile forces. Subsequently, a progressively increasing strain of 1 to 250% was applied until the clot was irreversibly deformed to estimate the yield point (clot strength). Scanning Electron Microscopy was performed on the clots from the rheological analysis to study the clot microstructure. Thrombin generation and FXIII activity were determined using Calibrated Automated Thrombogram and activity assays, respectively. Further, the cytoskeletal rearrangement was studied by activating the washed platelets with thrombin/ TRAP for 5, 10 and 15 seconds and estimating the G-actin to F-actin turnover by Western Blots. The results were analyzed using one-way ANOVA and a post hoc Tukey test for multiple comparisons. The groups were considered statistically significant for p values less than 0.05. Results: The rheological studies on clotting indicate that cold stored platelets form clots that have comparable properties to fresh platelets. RT-stored platelets formed weaker clots (Fig. 1A, B). These findings supported by the evidence of increased FXIII cross-linking activity (Fig.1C). Further, the normal force measurements indicate reduced force generation in RT-stored platelets which imply decreased platelet contractility (Fig. 1D). We also observed differences in geometry and orientation of individual fibrin fibers between fresh and stored platelets. Our preliminary results suggest that thrombin generation potential, crosslinking density, and platelet contractility may contribute to the mechanical strength and morphological differences between fresh and platelets stored at cold and room temperature. Conclusion: Cold-stored platelets form structurally and mechanically stable clots compared to 5 day stored RT platelets as demonstrated by the rheological and microstructural studies. Together, these data suggest that 4C platelets are hemostatically more active than the current standard-of-care and may offer a better solution to patients for resuscitation of bleeding trauma patients. Figure 1 Mechanical testing of evolving clot by rheometry. (A) RT-stored platelets form clots with decreased clot stiffness (G') compared to fresh (F) and cold-stored (4C) platelets (B) Clot strength was defined as the yield point where the clot deformed irreversibly. Clots formed from RT-stored platelets offer less resistance to strain; (C) Relative Cross Linking Density was estimated as the slope of stress-strain curve obtained from amplitude sweep. RT-stored platelet clots show less crosslinking; (D) less contractile force generation in RT platelets. * p

Description: Up to 50% of preventable trauma deaths are due to truncal hemorrhage resulting in cardiovascular collapse. Lower body negative pressure (LBNP) causes central hypovolemia in humans, resulting in coagulation changes, platelet activation, and fibrinolysis. The model allows investigators to study impending hemorrhagic shock in a minimally invasive human model, but correlations to changes in clotting and lysis due to hemorrhage are not fully described. Published human data to date has not discriminated between altered coagulation due to the LBNP method versus changes attributable to incipient hemorrhagic shock. We measured multiple coagulation parameters in 16 baboons undergoing hemorrhage followed by LBNP and hypothesized that this model elicits early changes associated with acute coagulopathy due to hemorrhage. Baboons underwent incremental hemorrhage (HEM) under an ACURO-approved protocol, followed by LBNP 2-4 weeks later. Blood was sampled at baseline (T0) and after approximately 18.75% (T1), and 25% (T2) blood loss (or until presyncope), and at recovery (T3). LBNP levels were determined by matching the pulse and central venous pressures elicited by hemorrhage. Measurements included: complete blood count; blood chemistries; and coagulations tests. Parameters measured by thromboelastography (TEG) included: R-time, K-time, angle, maximum amplitude (MA), and lysis at 30 and 60 minutes (LY30, LY60). Coagulation factors were quantified over time during both experiments (STA-R Evolution, STAGO).Platelet count was measured and response to agonists (ADP, collagen, TRAP) assessed with impedance aggregometry (Multiplate, Roche Diagnostics). Markers of platelet activation were evaluated under flow cytometry (P-selectin, PAC-1, and leukocyte-platelet aggregates (CD45:CD41)). LBNP and hemorrhage caused activation of the coagulation system over time as measured by TEG R-time (HEM: T0=5.8±0.3 s; T2=4.7±0.3 s; LBNP: T0=6.5±0.3 s; T2=5.1±0.3 s; p

Description: Background: Currently, whole blood is rarely used in trauma resuscitation due, in part, to the widely held belief that refrigeration will reduce the hemostatic efficacy of stored platelets. Recently, however, Pidcoke et al. (Transfusion 2013, 53:137s) showed that hemostatic function of human whole blood was well preserved when stored at 4°C for up to 21 days. The hemostatic and resuscitative efficacy of cold-stored whole blood has not been tested in a coagulopathic animal polytrauma model. Hypothesis: We hypothesized that blood stored for 7 days at 4°C is equivalent to fresh whole blood with regard to hemostatic and coagulation function in resuscitation of severe trauma. Method: Sprague-Dawley rats (300-400g) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur. The rats were then bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. Hemorrhage was usually completed between 30-60 min. Resuscitation was started at 1hr and included the following groups: Lactated Ringer’s (LR), fresh whole blood (FWB) or FWB stored at 4°C for 7 days (sFWB). The resuscitation volume was 20% of blood volume and represents the approximate volume used in prehospital care of trauma patients in both civilian and military settings. The experiment was terminated at 2hrs. Blood samples were taken before (time 0) and 2hrs after trauma (1hr after resuscitation) to assess hemostatic function. Prothombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen were measured on ST-4 (Stago). Platelet aggregation was measured with Multiplate (Diapharma) after stimulation with ADP, collagen or thrombin (PAR4) and expressed as area under the curve per 1000 platelets. Clotting function was assessed using ROTEM (Tem International). Results: Resuscitation with FWB and sFWB led to recovery of mean arterial blood pressure to levels similar to baseline (FWB 92±2.4 to 86±3, sFWB 93±3 to 91±4). Resuscitation with LR led to a significantly lower arterial pressure (96±3 to 61±3.8mmHg, p

Description: Introduction Severe trauma with tissue damage and shock can rapidly (

Description: Background: Hemostasis is regulated by a dynamic balance between thrombin and plasmin activity, and the supply of prothrombin and plasminogen in the general circulation. This dynamic balance is important so that the formation of clots occurs at the site of injury, but does not spread to distal sites where thrombus formation can block blood flow and lead to organ dysfunction. Procoagulant and fibrinolytic activity is regulated by the supply of both active enzymes, and their respective zymogens. Disruption in the plasma levels of the enzymes and zymogens can lead to coagulopathies that have serious effects on morbidity and mortality. Polytrauma and hemorrhage can lead to an acute coagulopathy in rats that mimics the clinical hypocoagulation in both civilian and military patients with severe trauma and hemorrhage. This coagulopathy could be due an imbalance between the pro-coagulant or fibrinolytic systems. Hypothesis: We hypothesized that the dynamic balance between thrombin and plasmin activities would be altered after polytrauma and hemorrhage, favoring fibrinolysis. Method: Sprague-Dawley rats (n=8) were anesthetized with Isoflurane. Polytrauma was induced by damage to the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur. Rats were then bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. No fluid resuscitation was given. Blood samples were taken before (time 0) and at 30, 60, 120 and 240min. Rat plasmin and thrombin activity were measured by enzymatic assay. Plasminogen and prothrombin were measured after conversion to their respective active enzymes with tissue plasminogen activator or factor Xa respectively. Active enzyme was subtracted from the total to give the levels of zymogens. D-dimers were measured by ELISA. Results: There was no significant change in plasma thrombin activity after polytrauma and hemorrhage. Prothrombin levels fell slightly, but significantly over time. In contrast, plasmin activity rose significantly by 30min and continued to be elevated through the next 4hrs (235%, p