Fresh Platelets Research Articles

Autophagy-enabled protein degradation: Key to platelet activation and ANGII production in patients with type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2DM) presents a thrombotic environment, contributing to diabetic macroangiopathy and microangiopathy. In this study, the regulation of microthrombosis in T2DM was assessed. MethodsPlatelets from T2DM patients and healthy controls were analyzed using 4D label-free proteomics and bioinformatics. The role of autophagy in T2DM platelet activation and conversion of platelet-derived angiotensinogen (AGT) was investigated. ResultsThe results showed that complement and coagulation cascades, platelet activation, metabolic pathways, endocytosis, autophagy, and other protein digestion-related pathways were enriched. The levels of the key protein AGT were increased in T2DM platelets. Chloroquine (CQ) inhibited ADP- or arachidonic acid (AA)-stimulated platelet aggregation and granule release in a dose-dependent manner, while the effects were less pronounced or even reversed for the proteasome inhibitor PYR-41 and the endocytosis inhibitor Pitstop 2. This indicated the dependence of platelet activation and the accompanying protein digestion on the autophagy-lysosome pathway. Mitophagy occurred in fresh T2DM platelets and ADP- or storage-stimulated platelets; mitophagy was inhibited by CQ. However, the mitophagy inhibitor Mdivi-1 failed to show effects similar to those of CQ. AGT, which could be transformed into ANGII in vitro by ADP-stimulated platelets, was upregulated in T2DM platelets and in MEG-01 cell-derived platelets cultured in a high-glucose medium. Finally, microthrombosis was alleviated as indicated by a reduction in the levels of red blood cells in the liver, spleen, heart, and kidney tissues of db/db mice treated with CQ or valsartan. ConclusionIn platelets, macroautophagy promotes protein digestion, subsequently facilitating platelet activation, ANGII-mediated vasoconstriction, and microthrombosis. Our results suggested that lysosome is a promising therapeutic target for antithrombotic treatment in T2DM.

In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Plain Language SummaryThe identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies.

Platelet transcription factors license the pro-inflammatory cytokine response of human monocytes

In humans, blood Classical CD14+ monocytes contribute to host defense by secreting large amounts of pro-inflammatory cytokines. Their aberrant activity causes hyper-inflammation and life-threatening cytokine storms, while dysfunctional monocytes are associated with ‘immunoparalysis’, a state of immune hypo responsiveness and reduced pro-inflammatory gene expression, predisposing individuals to opportunistic infections. Understanding how monocyte functions are regulated is critical to prevent these harmful outcomes. We reveal platelets’ vital role in the pro-inflammatory cytokine responses of human monocytes. Naturally low platelet counts in patients with immune thrombocytopenia or removal of platelets from healthy monocytes result in monocyte immunoparalysis, marked by impaired cytokine response to immune challenge and weakened host defense transcriptional programs. Remarkably, supplementing monocytes with fresh platelets reverses these conditions. We discovered that platelets serve as reservoirs of key cytokine transcription regulators, such as NF-κB and MAPK p38, and pinpointed the enrichment of platelet NF-κB2 in human monocytes by proteomics. Platelets proportionally restore impaired cytokine production in human monocytes lacking MAPK p38α, NF-κB p65, and NF-κB2. We uncovered a vesicle-mediated platelet-monocyte-propagation of inflammatory transcription regulators, positioning platelets as central checkpoints in monocyte inflammation.

Evaluation of activation characteristics of a canine platelet concentrate produced by a commercial double centrifugation system

IntroductionIn veterinary medicine there are few readily available products for platelet transfusion to patients with thrombocytopenia. Commercial tabletop platelet concentrating systems have recently become available to veterinarians, primarily directed towards uses associated with regenerative medicine. These systems could potentially be used to produce fresh concentrated platelets for use in transfusion medicine. This study evaluated the concentration, activation, and sterility of a double centrifugation platelet concentrate (PC) produced by a commercial benchtop system.MethodsTen healthy dogs were studied. Whole blood was collected and mixed with ACD-A in a 1:7.6 ratio of ACD-A to whole blood. 12 mL of this mixture was processed into PC via single centrifugation, while 60 mL of the anticoagulated whole blood was processed via a commercial double centrifugation system. Both types of PC were evaluated for platelet concentration, CD62P expression with and without thrombin stimulation, and for sterility.ResultsMean platelet count in the double centrifuged PC was 863 ± 352 × 103/μL, with very low white blood cell contamination (median of 0.47 × 103 leukocyte/μL (range 0.15–2.18 × 103/μL)). The double-centrifuged PC had similar baseline activation characteristics (as determined by P-selectin expression) as the single centrifuge PC (0.76% vs. 0.72% unstimulated, 30.5% vs. 34.9% stimulated, p = 0.432).DiscussionThe benchtop PC system studied here did not cause activation of platelets during production and produced a sterile product that can be further investigated as a source of fresh platelet concentrates for transfusion purposes.

Analysis And Reason For Discard Of Blood Products: A Retrospective Study In A Tertiary Care Hospital Blood Bank From Parul Sevashram Hospital, Vadodara.

Background: A life-saving procedure is a blood transfusion, but there is often a shortage in blood supply due to various factors. Despite the enormous advancements in medical research, there is still no substitute for blood, making blood transfusions a crucial component of contemporary healthcare. The study aimed to evaluate the discard reasons for blood components, particularly Fresh Frozen Plasma, Platelet Concentration, and Red Cell Concentration and to suggest possible strategies to reduce wastage. Methods: The Retrospective analysis of discarded blood components was carried out from July, 1st 2023 to December 31st 2023 in the blood bank of Parul Sevashram. Data was collected using a discard register and computer data of discard available in blood bank and was analysed in SPSS software version 26.0 and Microsoft Excel. The wastage rate was calculated with a percentage of total number of units wastage against the total number of collections. Results: A total of 4990 blood component were prepared out of which 706 blood component was discarded. The total discard ratio of the study was 14.14%. Total replacement donors were 83.91% and 16.31% were voluntary donations. The discard rate for PC, RCC, and FFP was 86.30%, 6.82%, and 2.01%. HbsAg was the most common reason for the discard due to seropositivity of TTIs. Conclusion: The Most common component discarded was platelet and the reason for discard was the date of expiry due to non-utilization. Seropositivity was the reason for discarding red cell concentration and leakage was the most common reason for fresh frozen plasma. The wastage can be reduced by implementing proper strategies such as FIFO policy, avoiding Mega camps, proper staff training and wastage reduced protocol.

Characterization of a novel mouse platelet transfusion model.

Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

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.

Coagulation in Patients with Immune Thrombocytopenia and Other Associated Autoimmune Disorders

Introduction Chronic immune thrombocytopenia (ITP) is characterized by perturbations of immune homeostasis with hyperactivated effector cells as well as defective regulation of the adaptive immune system. It has been reported that, with varying frequency, patients with ITP may also have associated other autoimmune disorders, with circulating autoantibodies such as lupus anticoagulant antibodies (LACs), anti-nuclear antibodies (ANA), anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-beta2GPI), anti-thyroid peroxidase (ATPA), and rheumatoid factor (RF). Objectives To evaluate coagulation in patients with ITP with autoantibodies associated to other autoimmune diseases. Methods This is a prospective study that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. Adult ITP patients (n=182) without and with associated autoantibodies such as LACs, ANA, aCL, anti-beta2GPI, ATPA, and RF and healthy controls (n=180) were recruited. To evaluate the kinetics of clot formation and fibrinolysis, ROTEM® was performed on fresh platelet rich plasma adjusted to 25x10 9 platelets/L with the subjects own platelet poor plasma (PPP). Recalcification (NATEM) was used to induce coagulation. Clotting time (CT = time from start of measurement until initiation of clotting, in seconds); alpha angle (tangent to the curve at 2 mm amplitude, in degrees, which reflects the rate of fibrin polymerization); maximum clot firmness (MCF = maximum clot firmness, in mm, which reflects the maximum tensile strength of the thrombus); and lysis at 60 min (LI60) were recorded. Thrombin generation was measured in citrated PPP by calibrated automated thrombogram (CAT). To evaluate procoagulant capacity of plasma, coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/L of recombinant human tissue factor (TF) and 4 µmol/L of phospholipid mixture (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). Procoagulant activity of microparticles (MPs) associated with their content of either TF or phosphatidylserine (PS) was determined, respectively, with MP reagent (4 µM phospholipids) or PRP reagent (1 pM of recombinant human TF) by CAT. The following parameters were determined: Lag time (LT), or time from the start of the assay until 10 nM thrombin is formed, in min; time-to-peak (ttPeak), or time required to reach the maximum thrombin concentration, in min; peak height (Peak), or maximum thrombin concentration reached, in nM; and endogenous thrombin potential (ETP), or total amount of thrombin generated over time, in nMxmin. Results Fifty of the 182 patients with ITP (27.5%) were positive for autoantibodies associated with other autoimmune diseases, distributed as shown in Table 1. ANA were the most frequent antibodies present in these patients, and 22% had more than one kind of associated antibodies. Patients with ITP without antibodies had a shorter clot formation time (CT) than healthy controls. Presence of associated autoimmune diseases prolongs CT and resulting in values similar to controls. Both groups of patients with ITP had a diminished fibrinolysis (Table 2). Thrombin generation triggered by TF plus phospholipids as well as that dependent on TF content of microparticles (MP-reagent) was impaired in patients with other associated autoimmune disorders as shown by the prolonged LT and the diminution in either ETP or peak of generated thrombin (Table 2). When thrombin generation relayed on phospholipids of MPs, all patients with ITP generated less thrombin. Conclusion Association of other autoimmune diseases with ITP seemed to produce a delay in clot formation and in thrombin generation whereas did not affect the diminished fibrinolysis observed in ITP patients. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

MANAGEMENT OF CRITICALLY ILL PATIENT FOLLOWING MULTIPLE ABDOMINAL TRAUMA WITH MASSIVE HEMORRHAGE : CASE REPORT

Background: Most patients with severe abdominal trauma with massive bleeding require intensive care. On most occasions patients will be admitted to Intensive Care after surgery and require close monitoring of vital signs. Care in the ICU will focus on monitoring and supportive measures. The management of bleeding in critically ill patients is a dynamic and complex process. In addition, transfusion strategies during bleeding may involve several concurrent strategies to monitor coagulopathy, transfusion of blood products, and administration of drugs to correct coagulation disorders. Case report : A 48-year-old male was admitted to the ICU after a traffic accident. The patient came to the emergency room with bleeding shock and fluid resuscitation was performed to restore MAP 60 mmHg. Damage control surgery and massive transfusion were performed to save the patient. After the first operation, the patient returned to shock, producing 1500 cc hepatal and subphrenic drain in 3 hours. Re-laparotomy abdominal re-breaking surgery was performed again with additional packed red blood cells (PRBCs), Fresh Frozen Plasma (FFP) and platelet transfusion during surgery. Post-second surgery, the patient's hemodynamics were stable and vasopressor drugs could be weaned. The patient was admitted to the ICU with invasive monitoring and mechanical ventilator. After the 4th day, the patient experienced volume overload and pulmonary edema. Furosemide was given to eliminate fluid accumulation. The patient was extubated on day 6 and moved to the ward on day 7. Discussion : In cases of intra-abdominal trauma, Focused Assessment With Sonography For Trauma (FAST) examination can be performed quickly so that the decision to perform surgical resuscitation can be made immediately. Damage control surgery is performed in conditions where bleeding cannot be controlled through definitive procedures. Abdominal packing can be performed to prevent further bleeding while waiting for hemodynamic stabilization and coagulopathy in the ICU. Giving massive transfusions such as PRBCs, FFP and platelets according to protocol prevents the occurrence of lethal triad in massive bleeding patients. On the other hand, massive transfusion also brings side effects and complications such as volume overload, electrolyte disturbances, acidosis, transfusion reactions and infection in patients. The administration of blood and fluid transfusion in post-traumatic patients in the ICU must be done carefully to prevent these complications. Conclusion : In cases of intra-abdominal trauma patients with massive bleeding, early diagnosis, damage control surgery and proper management of massive transfusion are the management that must be done to save the patient.

PB0910 Evaluation of the Clot Formation Tranfusing Fresh, Cold and Frozen Platelets. Advantages for the Military Environment

PB0910 Evaluation of the Clot Formation Tranfusing Fresh, Cold and Frozen Platelets. Advantages for the Military Environment

Comparative Study of Autogenous Fresh Tooth Graft and Platelet Rich Fibrin Mixture versus Autogenous Fresh Tooth Graft Only For Immediate Dental Implant Placement

Comparative Study of Autogenous Fresh Tooth Graft and Platelet Rich Fibrin Mixture versus Autogenous Fresh Tooth Graft Only For Immediate Dental Implant Placement

Role of antibody screening in high titre group O donors

Red Cells, Fresh Frozen Plasma (FFP) and Platelets (RDP/SDP) prepared from donors with group O may have high titers of anti-A, anti-B and/or anti-A,B antibodies. When such components given to non-group O patients, this high titre can result in haemolytic transfusion reactions (HTRs). Such group O donors are generally termed ‘high-titer group O donors. Significant amounts of ABO antigen being present on the platelet surface and anti-ABO iso-agglutinins being present in the donor’s plasma, Platelet transfusions need to take into account the issues. Although relatively rare, but acute intravascular haemolytic transfusion reaction has been caused by passive transfer of anti-A and anti-B antibodies, present in group O donors. This study aimed to identify the prevalence of high titre antibodies in group O donors. Thus, titre are performed in randomly selected group O donors from different gender and age group to identify best available source of products for Platelet and follow up transfusions.: Randomly selected 100 O blood group donors were included in the study. Samples from these donors were tested for ABO antibody (both IgM and IgG) titres using conventional tube technique at RT and 37°C at Indirect Antiglobulin phase. Statistical analysis was performed using two sample t-test and anova test.Donors included in the present study were mainly male (85%) donors. ABO antibody titres ranged from 0 to 2048. In the present study, Anti-B (IgG) titer is significantly higher than other antibodies in both genders.There are a large proportion of group O donors having high titres of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic transfusion reaction by issuing only low titre components for out of group transfusions.Each Blood Transfusion Service should establish a policy for testing and issue of group O platelets to non-O group recipient to avoid chances of development of adverse transfusion reaction.

High incidence of adult respiratory distress syndrome associated with amphetamine use in the burn population: a retrospective cohort study.

A total of 188 patients age 18 and older with total body surface area (TBSA) between 20 and 60% were sampled over 5 years. To capture the moderate to severe burn population, a lower limit of 20% was chosen while 60% was used as the upper limit to exclude patients likely to die from the burns alone. Patients eligible to be include in the study had to meet the TBSA criteria. Demographic data was ascertained. Patients were placed into two cohorts: the amphetamine positive group (AmPOS), the amphetamine negative group (AmNEG). Key endpoints included hospital mortality, length of ICU stay, development of ARDS, and cardiac output parameters. Nonparametric data was evaluated with the Mann-Whitney test and categorical variables were compared using χ 2. Data from 49 patients with ARDS were collected retrospectively out of the 188 patients in this TBSA range. The incidence of amphetamine abuse in these burn patients is 14.9%. The average age of AmPOS and AmNEG patients was 36 and 34 and the average TBSA of burns in the AmPOS and AmNEG group was 51.8 and 45.2%. The mean onset of ARDS was 2.2 days for the AmPOS versus 3.3 days for the AmNEG (P=0.19). At admission, patients with amphetamine use exhibited less inhalational injury and a lower Acute Physiology and Chronic Health Evaluation II (APACHE II) score. In AmPOS, 64% developed ARDS compared to 19% in AmNEG (P<0.001). Mortality, time on a ventilator, ICU days, packed Red Blood Cell, Fresh Frozen Plasm, platelets transfused, and initial cardiac parameters all were not statistically significant. On the initial date of ARDS diagnosis, there was no statistically significant difference with PaO2/FiO2 and compliance (P=0.67), but positive end expiratory pressure requirements was higher in AmPOS (P=0.018). Amphetamine Use was associated with increased risk of developing ARDS in the burn population. This is despite having a better APACHE II and lower incidence of inhalational injury in the AmPOS group, supporting amphetamine as an independent risk factor for ARDS.

Characterization of the human platelet N- and O-glycome upon storage using tandem mass spectrometry

AbstractChanges in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature–stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.

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.

Cryopreserved platelets and amotosalen-treated plasma in an experimental clot formation set-up.

Amotosalen treatment of plasma and cryopreservation of platelets affect the quality and potentially the interplay between platelets and coagulation factors. We set up an experimental clot formation study to test the hypothesis that amotosalen treatment of plasma affects the interaction with different platelet preparations. Pooled plasma units (n=16) were subjected to coagulation tests before and after pathogen inactivation with amotosalen treatment (PI) and aliquots were frozen at -80°C. Fresh and cryopreserved platelets were analyzed for phenotypic and activity markers. Finally, coagulation properties of different combinations of platelets and plasma, before and after PI, were analyzed by viscoelastography (ROTEM). PI of plasma reduced the concentration of several coagulation factors (p<0.01). Cryopreservation altered phenotypic expression and reduced the platelets' ability to respond to agonists (p<0.0001). The interplay between all plasma derivatives and cryopreserved platelets resulted in shortened coagulation time (p<0.0001) but prolonged clot formation time and reduced clot strength (p<0.0001) as compared to the interaction between fresh platelets with different plasma variants. PI of the plasma does not seem to have a major impact on coagulation time, clot formation time or clot strength. Our data show that the reduced concentration of coagulation factors after PI treatment of plasma are negligible measured by viscoelastography, with fresh and cryopreserved platelets in this experimental clot formation setup, and that platelets play a more pronounced role. Cryopreserved platelets are more activated and result in reduced clot stability.

Retrospective evaluation of fresh platelet concentrate administration in dogs: Patient characteristics, outcomes, and transfusion practices in 189 transfusion episodes (2008-2019).

To describe patient characteristics, underlying disease processes, clinical outcomes, transfusion dose and type (therapeutic or prophylactic), platelet count changes, and adverse events associated with platelet concentrate (PC) administration in dogs. Retrospective study. University teaching hospital. A total of 149 dogs, representing 189 PC transfusion episodes. None. In this population, 39 of 149 dogs (26.2%) were diagnosed with primary immune-mediated thrombocytopenia, 22 of 149 (14.8%) had decreased bone marrow production, 12 of 149 (8.0%) received PC during a massive transfusion, 3 of 149 (2.0%) had congenital thrombocytopathia, 59 of 149 (39.6%) had severe thrombocytopenia of other causes, and 14 of 149 (9.4%) underwent transfusion for miscellaneous causes without a documented severe thrombocytopenia. In 117 of 149 dogs (78.5%), >1 site of hemorrhage was noted. The most common sites of hemorrhage were the gastrointestinal (GI) tract in 89 of 149 (59.7%) and the skin in 78 of 149 (52.3%). Overall survival to discharge was 59.1% (88/149). The median PC dose was 0.8 units per 10kg of body weight per transfusion episode (range: 0.2-6.7). Of 189 episodes, 29 of 189 (15.7%) were prophylactic, and 158 of 189 (83.6%) were therapeutic. For 99 of 189 transfusion episodes, paired pre- and postplatelet counts were available within 24hours. The median platelet count change was 5.0×109 /L (5000/μL; range: -115×109 /L to 158×109 /L [-115,000 to 158,000/μL]); the posttransfusion platelet count was significantly higher than pretransfusion (P<0.0001). The increase in platelet count after transfusion was greater in the prophylactic group than the therapeutic group (P=0.0167). Transfusion reactions were suspected during 2 of 168 episodes (1.2%). Immune-mediated thrombocytopenia was the most common disease process that resulted in PC transfusion. PC was more frequently administered to animals with active hemorrhage rather than prophylactically, and most dogs had evidence of hemorrhage in multiple organ systems, particularly the GI tract and skin. PC transfusions typically appeared safe, and the median platelet count increased after transfusion.

The effects of several forms of platelet concentrates combined with biomaterial collapan on segmental tibial bone defect regeneration

Objective: the purpose of the study was to evaluate the effects of three forms of allogeneic platelet concentrates on segmental tubular bone defect osseous regeneration.Material and Methods. In 145 laboratory rats tibial shaft segment was resected and stable osteosynthesis of the bone ends was performed. In group 1 animals the bone defect was left graftless, in group 2 animals biomaterial Collapan and shattered orthotopic autologous bone mixture into the bone defect site was placed. In group 3 animals as part of Collapan-autobone mixture defect filling fresh allogeneic platelet rich plasma was added, in group 4 animals allogeneic cryodestructed platelet rich plasma to the Collapan-bone mixture was added and group 5 animals received Collapan-bone mixture with allogeneic platelet rich fibrin added.Results. According to gross dissection data in 23 of the 32 group 3 rats, in 25 of the 37 group 4 rats and in 19 of the 27 group 5 rats complete visual tibial shaft defect healing occurred. In group 2 on visual inspection of tibial bone in 5 of the 10 rats defect healing was noted and in group 1 rats the defect healed in 15 of the 39 animals only. Bone defect healing rates in groups 3, 4 and 5 differed from group 1 healing rate significantly with χ2 = 7,889, χ2 = 6,452 and χ2 = 6,504 respectively, group 2 bone defect healing rate did not differ from that of group 1 (χ2 = 0,439). Histological analysis of specimens from groups 3, 5 and especially from group 4, at 3 weeks after plasty demonstrated active intermediary callus formation at bonesaw-line and neoformed osteoid bone on Collapan-bone chips aggregations surfaces; no intermediary callus formation in specimens from group 2 was found. At 6 to 12 weeks after plasty the most mature osseous tissue in specimens from group 4 was observed, the least mature neoformed osseous tissue amount was common to specimens from group 2.Conclusion. All three allogeneic Collapan-immobilized platelet concentrates studied promoted bone formation and healing of considerable segmental tibial shaft bone defect in rat model. The most evident osteogenesis activating effect was histologically confirmed in cases where cryodestructed platelet rich plasma to Collapan-bone chips mixture was added.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry

Cryopreservation of platelets, at − 80 °C with 5–6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1−/CD42b+/CD62P+) and a novel population (AnnV+/PAC1−/CD42b+/CD62P−) that did not align with the phenotype of aggregatory (AnnV−/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1−/CD42b−/CD62P−) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents.

During long-term antiplatelet agents (APAs) administration, patients with thrombotic diseases take a fairly high risk of life-threatening bleeding, especially when in need of urgent surgery. Rapid functional reversal of APAs remains an issue yet to be efficiently resolved by far due to the lack of any specific reversal agent in the clinic, which greatly restricts the use of APAs. Although platelet transfusion has become a common practice to counteract the function of APAs, personalized dosage of platelet transfusion is difficult to be judged by assessing the trade-off between its effectiveness and additional thrombotic risk. Here we reported a platelet-mimicking perfluorocarbon-based nanosponge, which potently reversed the antiplatelet effect of APAs by competitively binding with APAs. Platelet-mimicking perfluorocarbon-based nanosponges showed high binding affinity comparable to fresh platelets in vitro with first-line APAs, ticagrelor and tirofiban, and efficiently reversed their function in both tail bleeding and postischemic-reperfusion models. Moreover, the deficiency of platelet intrinsic thrombotic activity diminished the risk of thrombogenesis. In summary, our results demonstrated the safety and effectiveness of the platelet-mimicking nanosponge in ameliorating the bleeding risk of different APAs, which offers a promising strategy for the management of bleeding complications induced by antiplatelet therapy.