Lyophilized Platelets Research Articles

Sidestream dark field video microscopy demonstrates shelf-stable blood products preserve the endothelial glycocalyx in a canine hemorrhagic shock model.

To utilize sidestream dark field video microscopic technology to evaluate the endothelium in a canine hemorrhagic shock and resuscitation model. 6 purpose-bred adult dogs were anesthetized, instrumented, and subjected to hemorrhagic shock from September 2021 through June 2022. Each dog was resuscitated with 5 resuscitation strategies in an experimental crossover design study: (1) lactated Ringer's solution (LRS) and hydroxyethyl starch (HES) solution; (2) canine chilled whole blood (CWB); (3) canine fresh frozen plasma (FFP) and packed RBCs (pRBC); (4) canine freeze-dried plasma (FDP) and hemoglobin-based oxygen carrier (HBOC); or (5) HBOC/FDP and canine lyophilized platelets. Sidestream dark field video microscopic evaluation was performed at 5 time points: commencement, after hemorrhage, after shock, after resuscitation (T135), and conclusion (T180). There was a significant difference between the perfused boundary region (PBR) measurements when comparing the LRS/HES resuscitation arm to the CWB and FFP/pRBC resuscitation arms at T180. A significant difference in PBR was appreciated in the LRS/HES arm at T135 and T180 compared to its baseline. No other significant differences in PBR were appreciated when resuscitation arms were compared longitudinally or to each other. Shelf-stable blood products preserved the endothelial glycocalyx similarly to CWB and pRBC/FFP as evaluated by sidestream dark field video microscopy. Lactated Ringer and HES solutions did not adequately preserve the endothelial glycocalyx compared to CWB and pRBC/FFP. Shelf-stable blood products are a viable option to preserve the endothelial glycocalyx when used during hemorrhagic resuscitation in dogs.

Comparison of shelf-stable and conventional resuscitation products in a canine model of hemorrhagic shock.

Treatment of severe hemorrhagic shock typically involves hemostatic resuscitation with blood products. However, logistical constraints often hamper the wide distribution of commonly used blood products like whole blood. Shelf-stable blood products and blood substitutes are poised to be able to effectively resuscitate individuals in hemorrhagic shock when more conventional blood products are not readily available. Purpose-bred adult dogs (n = 6) were anesthetized, instrumented, and subjected to hemorrhagic shock (mean arterial pressure <50 mm Hg or 40% blood volume loss). Then each dog was resuscitated with one of five resuscitation products: (1) lactated ringers solution and hetastarch (LRS/Heta), (2) canine chilled whole blood (CWB), (3) fresh frozen plasma (FFP) and packed red blood cells (pRBC), (4) canine freeze-dried plasma (FDP) and hemoglobin-based oxygen carrier (HBOC), or (5) HBOC/FDP and canine lyophilized platelets (LyoPLT). Each dog was allowed to recover after the hemorrhage resuscitation event and was then subjected to another hemorrhage event and resuscitated with a different product until each dog was resuscitated with each product. At the time when animals were determined to be out of shock as defined by a shock index <1, mean arterial pressure (mmHg) values (mean ± standard error) were higher for FFP/pRBC (n = 5, 83.7 ± 4.5) and FDP/HBOC+LyoPLT (n = 4, 87.8 ± 2.1) as compared with WB (n = 4, 66.0 ± 13.1). A transient increase in creatinine was seen in dogs resuscitated with HBOC and FDP. Albumin and base excess increased in dogs resuscitated with HBOC and FDP products compared with LRS/heta and CWB ( p < 0.01). Combinations of shelf-stable blood products compared favorably to canine CWB for resolution of shock. Further research is needed to ascertain the reliability and efficacy of these shelf-stable combinations of products in other models of hemorrhage that include a component of tissue damage as well as naturally occurring trauma.

Clearance of Trehalose Lyophilized Platelets in Mice

Abstract The utilization of stored platelet transfusion has emerged as an effective approach in the management of thrombocytopenia. However, the limited availability of fresh platelets in veterinary medicine due to the challenging storage requirements poses a significant constraint. Lyophilized platelets offer extended shelf-life and convenient storage options, enhancing their accessibility for thrombocytopenic patients. However, only a limited number of studies have examined the clearance and survival rate of transfused lyophilized platelets, particularly regarding the lyophilization technique utilizing trehalose as a platelet stabilization agent. The objective of this study was to assess the recovery and survival rate of trehalose lyophilized platelets within the circulatory system. To investigate these parameters, CMFDA-labeled lyophilized platelets were administered to mice, and their recovery and survival rates were analyzed. Flow cytometric analysis revealed the rapid clearance of lyophilized platelets from the systemic circulation. The immediate post-infusion percent recovery of labeled platelet particles was 42.7 ± 8.15 %. The average survival rates at post-infusion time points at 15, 30, 45, and 60 minutes were 28.2 ± 4.31, 14.5 ± 3.56, 5.1 ± 2.02, and 0.82 ± 0.57, respectively. The calculated mean half-life was 8.39 ± 0.44 minutes. The most pronounced decrease in labeled lyophilized platelet count occurred during the 30-minute timeframe immediately following infusion. Subsequently, over 99% of lyophilized platelets were eliminated after 60 minutes post-infusion. These findings indicate that higher dosages and more frequent administration of trehalose lyophilized platelets might be necessary to achieve a therapeutic effect comparable to that of fresh platelets.

Dose-dependent effect of canine lyophilized platelet on an in vitro hemodilution model

The transfusion of stored platelets has emerged as an efficient method for treating dogs with thrombocytopenia. However, the availability of fresh platelets is limited in veterinary medicine due to demanding storage conditions. Lyophilized platelets have long shelf lives and can be easily stored, increasing their accessibility for thrombocytopenic dogs. Due to the lack of research and information on the dose effect, canine lyophilized platelets are used at a clinical dosage without research-based evidence. This study was to evaluate the dose effect of lyophilized canine platelets on blood coagulability. Three different concentrations of lyophilized canine platelets were added to in vitro hemodilution blood model, increasing the platelet count by 25, 50, and 100 × 106/ml and coagulation profiles were analyzed. The coagulability was evaluated via the plasma fibrinogen concentration, coagulation time, thromboelastography (TEG), and platelet function analyzer (PFA). Higher concentrations of lyophilized platelets showed dose-dependent association with decreased aPTT and R-time of TEG and increased alpha angle and MA of TEG. These results showed the potential that the higher dose of canine lyophilized platelets better improve blood coagulability than the standard dose and provided the basis for further safety and clinical studies.

1172: EVALUATION OF SHELF-STABLE BLOOD PRODUCTS FOR RESUSCITATION IN A CANINE MODEL OF HEMORRHAGIC SHOCK

Introduction: Hemorrhagic shock is a major cause of morbidity and mortality in trauma patients. Current resuscitation methods use blood products that require temperature control and have a limited lifespan, which present significant limitations for use in rural areas and combat zones. This study examined whether shelf stable blood product resuscitation strategies leveraging freeze-dried plasma (FDP), hemoglobin based oxygen carrier (HBOC), and lyophilized platelets (LP) provide an alternative to conventional resuscitation strategies in a canine model. We hypothesized that a combination of HBOC and FDP with or without LP would be non-inferior to chilled whole blood (CWB) for hemorrhagic shock resuscitation. Methods: Seven dogs were anesthetized and subjected to controlled hemorrhage of up to 40% of their blood volume (T0) over one hour to achieve a mean arterial blood pressure of 35-50 mmHg. They were maintained in shock for 45 minutes and were then randomized to receive: CWB, FDP+HBOC, or FDP+HBOC+LP over 30 minutes (T105-135). Citrated blood was collected at T0, T105, T135, and T180 for thromboelastography (TEG 5000). Samples were run in duplicates with kaolin activation to measure reaction time (r), kinetic time (k), alpha angle (alpha), and maximum amplitude (MA). Between-group comparisons were performed using a Kruskal-Wallis ANOVA and a Bonferroni correction. Results are reported as mean +/- standard deviation. Results: There was no difference in baseline parameters between groups or in TEG values between groups at the end of shock T0 and T105. There was a difference in alpha and MA between groups at T135 (p= 0.02 for both) and T180 (p=0.02 and 0.002, respectively). Specifically, alpha and MA were lower in the FDP+HBOC+LP group (alpha: 54.9 +/- 6.6 deg; MA: 43.5 +/- 11.5 mm) than in the CWB group (alpha: 64.4 +/- 5.2 deg; MA: 58.2 +/- 5.1 mm). There was no difference in r, alpha, and k between groups at any time point. Conclusions: In this canine model of controlled hemorrhage, TEG parameters between FDP+HBOC and CWB were not different. We demonstrated evidence of reduced coagulation function in FDP+HBOC+LP. The biological relevance of those findings should be determined in an uncontrolled hemorrhage model. Resuscitation with shelf-stable products appeared safe and satisfactory in this model.

Lyophilized platelets inhibit platelet aggregation with simultaneous paradoxical promotion of platelet adhesion.

Lyophilized platelets have been explored as a potential hemostatic agent due to their long-term ambient storage capabilities that make them readily available in various scenarios. Additionally, their high biocompatibility and the key role of platelet interactions in various clinical conditions make them a promising platform for drug delivery. To explore these applications and for wider clinical deployment, the interactions between lyophilized platelets and fresh platelets must be examined. This project characterized receptor expression on the lyophilized platelet surface and their ability to bind fibrinogen using flow cytometry. The effect of lyophilized platelets on aggregation of unaltered platelets was assessed using light transmission aggregometry while the effect on adhesion was evaluated using static and microfluidic assays. Lyophilized platelets maintained significant levels of GPIIb and GPVI receptors on their surface, though the expression was reduced from fresh platelets. Additionally, lyophilized platelets maintained GPIb expression similar to fresh platelets. Furthermore, 15.8% of the lyophilized platelets exhibited the active conformation of the GPIIb/IIIa receptor, indicating a significant increase over fresh platelets. Lyophilized platelets also exhibited an increase in exposed phosphatidylserine and fibrinogen binding. Despite the effect of lyophilized platelets in promoting the adhesion of fresh platelets on a collagen-coated surface, their net effect was inhibitory on platelet aggregation. This study demonstrates that lyophilized platelets can have paradoxical effects on platelet adhesion and aggregation, which could have an impact for clinical applications. Detailed characterization and engineering of these effects will be important for their continued development as a drug delivery platform.

Lyophilized platelets versus cryopreserved platelets for management of bleeding in thrombocytopenic dogs: A multicenter randomized clinical trial

BackgroundThrombocytopenia in dogs is common in critical care medicine, but availability of fresh platelet concentrates in veterinary medicine can be limiting. Lyophilized platelets have long shelf‐lives and can be easily transported, stored, and administered in various settings.ObjectiveTo evaluate the efficacy and safety of a novel trehalose‐stabilized canine lyophilized platelet product in thrombocytopenic dogs with clinically‐evident bleeding.AnimalsEighty‐eight dogs with platelet counts <50 × 103/μL and a standardized bleeding assessment tool (DOGiBAT) score ≥2.MethodsMulticenter, randomized, non‐blinded, non‐inferiority clinical trial comparing dimethyl sulfoxide (DMSO)‐stabilized cryopreserved platelet concentrates (CPP) with trehalose‐stabilized lyophilized platelets (LP) for control of bleeding in thrombocytopenic dogs. Dogs were randomized to receive 3 × 109 platelets/kg of LP or CPP. Primary outcome measures were change in DOGiBAT score, platelet count, need for additional red cell transfusion and all‐cause mortality.ResultsFifty dogs received LP and 38 received CPP. Baseline demographics and clinical characteristics of both groups were comparable. At 1‐hour post‐transfusion, LP were superior for change in DOGiBAT score, and non‐inferior at 24‐hours post‐transfusion. The LP were non‐inferior to CPP for change in platelet count, need for additional red blood cell units, and survival to discharge. The LP were superior for change in hematocrit at 1‐hour post‐transfusion, and non‐inferior at 24‐hours. No adverse effects were noted in either group.Conclusions and Clinical ImportanceA novel trehalose‐stabilized canine LP product appears to be logistically superior and is clinically non‐inferior to DMSO‐stabilized canine CPP for management of bleeding in thrombocytopenic dogs.

Lyophilized Platelet Transfusion Does Not Constitute An Immunologic “Second Hit” In a Non-Human Primate Hemorrhagic Shock Model

AbstractAbstract 3360 Background-Apheresed platelet concentrates accumulate biologic response modulators (BRMs) with prolonged storage. BRMs have been localized to the supernatant fraction of stored blood products and BRM levels correlate with length of storage. BRMs, and hence transfusion, have been demonstrated to function as a recipient immune “second hits”, in the construct of the two hit model of multiple organ dysfunction and failure. Furthermore, contemporary practices for apheresed platelet processing and storage restricts transfusibility to five days or less from time of collection. Platelet processing by lyophilization offers the potential for long term platelet storage and transfusibility. Lyophilized platelets (LP) are preserved in a metabolically inactive state until rehydrated in sterile water immediately prior to infusion and therefore, intuitively, of limited BRM activity. We hypothesized that LP transfusion will not constitute an immunologic “second hit”. Methods-Rhesus Macaques were anesthetized and subjected to grade III hemorrhagic shock to induce a systemic inflammatory profile consistent with an immunological “first hit”. To determine the “second hit” capability of LP, 2 × 1010 reconstituted LP (a weight based equivalent dose of an average size human receiving 1 six pack of platelets) were infused at 15 minutes (T=15) following the initiation of shock. In parallel experiments, normal saline (NS) or 2 × 1010 fresh (day 1 – 3) apheresed human platelets (FAP) were infused at T=15 15 minutes following initiation of shock to serve as controls. Subsequently, volume resuscitation with additional normal saline, per ATLS guidelines, was performed and all animals survived for 480 minutes (T=480) post-shock initiation. Physiologic parameters were continually monitored, serum collected immediately preceding shock (T=0), T=240 and T=480 for TNF α, IL-1β, IL-6, and IL-8 analysis. Supernatants from the LP and FAP administered during the study were assayed for pro-inflammatory cytokines. All cytokine detection was performed on a Luminex IS 100. Analysis: paired sample t-test and one-way ANOVA; results reported as mean (SEM). Results-Grade III hemorrhagic shock, determined by a reduction in MAP (43% (4.5), p<0.05) and blood loss index (24% (3.9), p<0.05), resulted in a significant pro-inflammatory response determined by significant changes in the serum levels of IL-6 and IL-8 from T=0 to T=240 and T=480 (p<0.05) in NS controls. No statistically significant differences were detected in serum concentration of TNFα, IL-1β, IL-6, and IL-8 at T=240 and T=480 amongst the three groups (NS, LP, FAP). The supernatants from transfused LP and FAP contained undetectable levels of GMCSF, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, and IL-15. However, the FAP supernatant contained increased concentrations of in TNFα (255%), MCP1 (405%), IP-10 (817%) and IL-8 (864%) as compared to LP. Conclusions-The model utilized in this study effectively constitutes an immunological first hit as determined by altered systemic levels of IL6 and IL8 in response to the hemorrhagic shock. As expected based upon the nature of LP processing and storage, the supernatant of the infused LPs was lacking in BRMs even though infused LP was > 6 months post-collection. The infusion of fresh apheresed platelets did not constitute an immunologic second hit in this study; given the age (day 1 – 3) of the FAP utilized this result is consistent with previous reports and is a sound negative control and excludes a xenogeneic pro-inflammatory response to the infusion of human platelets. The inflammatory profile of LP treated animals was not different than NS control or FAP treated animals, in conjunction with the above indicate that lyophilized platelet transfusion does not constitute an immunologic second hit in the setting of hemorrhagic shock. Disclosures:No relevant conflicts of interest to declare.

A Ristocetin-Independent ELISA for the Measurement of VWF Activity.

AbstractAbstract 1411Von Willebrand Disease (VWD) is a relatively common bleeding disorder characterized by both quantitative and qualitative deficiencies in von Willebrand Factor. The correct diagnosis and classification of VWD is made on the basis of a personal and family bleeding history and the results of a panel of laboratory tests. Evaluations of both VWF antigen level and function are required. Most commonly VWF function is evaluated based on VWF binding to platelets through the platelet surface glycoprotein GPIba in a platelet aggregation assay (VWF:RCo). The VWF:RCo assay requires the use of the antibiotic ristocetin to induce a conformational change in VWF which allows VWF to bind to GPIba on the surface of fresh or fixed platelets in the absence of VWF denaturation. When evaluating 172 normal controls, Flood et. al. (Blood, 2010) elegantly demonstrated that 63% of African-American controls and 17% of Caucasian controls had a single nucleotide polymorphism, D1472H, which altered the ability of these individuals to bind ristocetin and resulted in abnormal VWF:RCo results in the absence of any bleeding abnormalities.GTI Diagnostics, Inc. (Waukesha, Wisconsin) has developed a ristocetin-independent fluorescent ELISA for the quantitative measurement of VWF activity in plasma, specifically VWF binding to immobilized GPIba. This VWF activity ELISA, referred to as the GTI IbCo Assay, uses microwells coated with recombinant GPIba containing two “gain-of-function” mutations. The mutant GPIba is capable of spontaneous binding to VWF in the absence of ristocetin or VWF denaturation (Flood et. al, Blood, 2010). A brief description of the assay follows. In the absence of ristocetin, calibrators, controls, and samples are added to the microwells and VWF binding to GPIba is allowed to take place. Unbound material is washed from the wells and a biotinylated anti-VWF detection antibody is added to the wells. The microwells are washed to remove any unbound detection antibody. A streptavidin-labeled horseradish peroxidase conjugate is added to the microwells and incubated. After a final wash step, a fluorescent peroxidase substrate is added to the wells. The reaction is quenched after 30 minutes and the fluorescence is read using an excitation wavelength between 315 and 340 nm and an emission wavelength between 370 and 470 nm. The reportable result (U/dL) of VWF binding to GPIba is determined from the standard curve. All assay incubation steps are 30 minutes and conducted at room temperature, therefore the assay can be completed in < 3 hours.A small scale method comparison study was conducted on 11 plasma samples purchased from George King Bio-Medical, Inc. The plasma samples are Factor Assay ConTrol (FACT) samples and extensively characterized by George King Bio-Medical, Inc for 25 different coagulation parameters including VWF:RCo using a Chrono-Log platelet aggregometer and Helena Laboratories lyophilized platelets. The 11 samples were tested in the GTI IbCo Assay and the results obtained were compared to those obtained with the comparative method, in this case the VWF:RCo values provided by the supplier. For standardization of the GTI IbCo Assay, a IbCo value of 100 U/dL was assigned to the ISTH Secondary Coagulation Standard Lot #3. As illustrated in the table below, the samples tested included samples with varying levels VWF:RCo activity. When the results were compared, there was good correlation and agreement between the two methods. Larger method comparison studies will be conducted.Sample NumberVWF:RCo by Platelet Aggregation (%)GTI IbCo Assay (U/dL)GK110099GK2100104GK39885GK489109GK59599GK69891GK73844GK83635GK958GK10126GK1145Based on this limited data set the GTI IbCo Assay demonstrated good correlation and agreement with the “gold standard” Ristocetin Cofactor Assay by platelet aggregation. The GTI IbCo Assay is able to measure the functional activity of VWF, specifically in regards to VWF binding to GPIba in the absence of ristocetin in a simple ELISA format. The GTI IbCo Assay removes the need for fresh or lyophilized platelets from the traditional assay and also eliminates the ristocetin dependence from the VWF activity measurement. By removing any dependence on ristocetin to induce the VWF to GPIba interaction we have also removed any possible assay discrepancies based on a patient's inability to interact with ristocetin. Disclosures:Stapleton:GTI Diagnostics: Employment. Bub:GTI Diagnostics: Employment. Chance:GTI Diagnostics, Inc: Employment.

Canine platelet transfusions

To review potential platelet storage options, guidelines for administration of platelets, and adverse events associated with platelet transfusions. Data sources included original research publications and scientific reviews. Transfusion of platelet concentrates (PCs) plays a key role in the management of patients with severe thrombocytopenia. Currently PCs are stored at 22 degrees C under continuous gentle agitation for up to 5 days. Chilling of platelets is associated with rapid clearance of transfused platelets, and galactosylation of platelets has proven unsuccessful in prolonging platelet survival. Although approved by the American Association of Blood Banks, cryopreservation of human platelets in 6% DMSO largely remains a research technique. Pre-storage leukoreduction of PCs has reduced but not eliminated acute inflammatory transfusion reactions, with platelet inflammatory mediators contributing to such reactions. Canine plateletpheresis allows collection of a concentrate with a high platelet yield, typically 3-4.5 x 10(11) versus <1 x 10(11) for whole blood-derived platelets, improving the ability to provide sufficient platelets to meet the recipient's transfusion needs. Cryopreservation of canine platelets in 6% DMSO offers immediate availability of platelets, with an acceptable posttransfusion in vivo platelet recovery and half-life of 50% and 2 days, respectively. While data on administration of rehydrated lyophilized platelets in bleeding animal models are encouraging, due to a short lifespan (min) posttransfusion, their use will be limited to control of active bleeding, without a sustained increase in platelet count. Fresh PC remains the product of choice for control of bleeding due to severe thrombocytopenia or thrombopathia. While cryopreservation and lyophilization of canine platelets offer the benefits of immediate availability and long-term storage, the compromise is decreased in vivo recovery and survival of platelets and some degree of impaired function, though such products could still be life saving.

Survival of baboon biotin‐X‐N‐hydroxysuccinimide and 111In‐oxine‐labelled autologous fresh and lyophilized reconstituted platelets

In accordance with Food and Drug Administration (FDA) regulations, platelets can be stored in the liquid state at 22 degrees C for only 5 days. Platelets frozen with 6% dimethylsulphoxide (DMSO) can be stored at -80 degrees C for 2 years, and platelets frozen with 5% DMSO can be stored at -150 degrees C for 3 years. Studies are being conducted to determine the effects of lyophilization of platelets. In the present study, we assessed the survival of autologous lyophilized-reconstituted platelets in the baboon. We studied fresh baboon platelets and baboon platelets that had been treated with paraformaldehyde, frozen, lyophilized, thawed and reconstituted. Aliquots of these platelets were labelled with (111)In-oxine or biotin-X-N-hydroxysuccinimide (biotin-X-NHS) before autotransfusion, and measurements were made of the in vivo recovery and lifespan. We also evaluated the response of fresh and lyophilized platelets to in vitro agonists by measuring the level of platelet surface markers and heterotypic aggregates in the peripheral blood following the autotransfusions. The (111)In-oxine- or biotin-X-NHS-labelled lyophilized, reconstituted platelets exhibited survival times of less than 15 min. These platelets did not respond to stimulation with agonists to decrease platelet GPIb and increase platelet P-selectin and platelet GPIIb-IIIa levels 1 min post-transfusion and they accumulated more procoagulant factor V than did the fresh platelets. Lyophilized reconstituted baboon platelets labelled with (111)In-oxine or biotin-X-NHS before autotransfusion exhibited an in vivo circulation time of less than 15 min. Further study of the lyophilized, reconstituted platelets is required to evaluate their haemostatic function.

Comparison of the Procoagulant Activity of Freeze-Dried and Fresh Platelets with Phospholipid in Three Phospholipid Dependent Assays.

Background and Objective: Platelet activation and coagulation together determine the haemostatic activity of plasma. When activated, platelets expose phosphatidylserine (PS) at their outer surface, on which the tenase and prothrombinase complexes assemble, forming FXa and thrombin, respectively. In in-vitro tests phospholipid vesicles are normally used as a platelet substitute. However it is not clear if PL vesicles behave in the same way as platelets. The objective of the study was to compare and contrast the procoagulant activity (PCA) of lyophilized platelets, a bovine brain phospholipid standard (NIBSC 91/542) against fresh platelets using three phospholipid (PL) dependent assays. Inhibition of this PCA by Annexin A5 (AA5) and an anti-PS antibody (3G4) was also studied.Methods and Materials: Lyophilised fixed platelets were obtained from Bio/Data corporation. Fresh platelets were collected into ACD-A from healthy donors and then gel-filtered through Sepharose 2B. Fresh platelets were activated by A23187 (10μM) before use in the assay systems. Platelet counts over a range of 10–200 x 109/L were used in the assays. FXa and prothrombinase generation were measured in purified chromogenic systems, and calibrated against FXa and IIa standards. The time course of thrombin generated in plasma activated by FIXa was monitored, and peak thrombin levels and the total amount of free thrombin measured over time (ETP). AA5 and 3G4 at concentrations of 3.3–33 μM and 50–400 μg/mL respectively were used to inhibit the assembly and function of macromolecular coagulant enzyme complexes.Results: Fresh platelets were 2.3 and 2.8-fold more potent than lyophilized platelets and PL standard respectively, in supporting FXa generation. When prothrombinase activity was measured, fresh platelets were 3.4 and 38-fold more potent than the lyophilized platelets and PL standard, respectively. Lyophilised platelets were 11-fold more potent than the PL standard. When thrombin generation was investigated fresh platelets were 3.1 (peak thrombin) and 3.7-fold (ETP) more potent than lyophilized platelets and 2.2 (peak thrombin) and 7-fold (ETP) more active than the PL standard. Lyophilised platelets were 0.7 (peak thrombin) and 1.9-fold more active than the PL standard. The tenase and prothrombinase PCA supported by the PL standard and fresh platelets were more susceptible to AA5 (8.3μM) inhibition (>70%) than lyophilized platelets (45 – 59%) though this activity was totally inhibited at 33μM. In the same tests 3G4 (400μg/mL) completely inhibited the PL standard but was only able to inhibit fresh and lyophilized platelets by 40–80%. Thrombin generation supported by the PL standard was completely inhibited by AA5 (16.5μM). Lyophilised platelets were more susceptible to the action of AA5 than fresh platelets. However 3G4 (400μg/mL) was unable to completely inhibit thrombin generation activity supported by PL standard or platelet preparations. 3G4 for all preparations delayed the time taken for peak thrombin levels to be reached.Conclusions: Lyophilized platelets showed similar activity patterns to the PL standard in supporting tenase and thrombin formation/generation, though they were more active than the PL standard. Fresh-platelets were significantly better able to support these complexes, probably because of the presence of specific receptors for FVa and FXa and the maintenance of membrane fluidity.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

Intracellular function in rehydrated lyophilized platelets.

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.

Intracellular function in rehydrated lyophilized platelets

This study aimed to evaluate the effect of cross‐linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross‐linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein‐bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus‐response functions of fresh platelets and are capable of feedback amplification in coagulation.

Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion.

Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.

Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion.

Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion.

Determinants of von Willebrand Factor Activity Elicited by Ristocetin and Botrocetin: Studies on a Human von Willebrand Factor-Binding Antibody

The three main probes for functional vWF activity--ristocetin, botrocetin, and the PAggF test--and similarities and differences in their elicited vWF activities have been reviewed. Emphasis has been placed on the technologies dependent on these probes, with a brief description of a series of relatively simple and sensitive tests developed in this laboratory. These tests include the development of the PAF test for vWF in certain animal plasmas; the development and use of fixed lyophilized platelets that retain receptor activity for vWF; the purification of botrocetin (venom coagglutinin) freed of thrombinlike enzymes and its use in vWF assays; the development of macroscopic platelet aggregation tests for screening and assay of vWF; and the application of the macroscopic test for rapid screening and quantitation of human plasmas for acquired inhibitors of vWF utilizing each of the three probes. Historically, the similarities of the ristocetin and botrocetin probes were first observed. For normal human plasmas and for patients with classic vWD, both homozygous or heterozygous, similar values for vWF were obtained with these two probes. Similar platelet binding of vWF in the presence of the two probes was likewise noted. However, further studies of these two probes revealed striking differences. Especially important for study of animal plasmas generally as well as a canine model of vWD was the observation that the vWF in all animal plasmas tested with botrocetin was highly reactive, whereas with ristocetin nearly all plasmas were resistant. Similarly, all animal platelets tested for vWF-dependent aggregation with the two probes were highly reactive with botrocetin, but inactive with ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporary suppression of circulating antiplatelet alloantibodies by the massive infusion of fresh stored or lyophilized platelets

Temporary suppression of circulating antiplatelet alloantibodies by the massive infusion of fresh stored or lyophilized platelets