Agitation-dependent biomechanical forces modulate GPVI receptor expression and platelet adhesion capacity during storage

Background Continuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition. Methods Platelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage. Results While MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability. Conclusion Given the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.

Role of 12-LOX in the Platelet Storage Lesion

Background: 12-lipoxygenase (12-LOX) is an enzyme abundant in platelets which can contribute to the platelet storage lesion by oxidizing polyunsaturated fatty acids (PUFAs) released from phospholipid membranes. We and others have shown that the PUFA arachidonic acid (AA) and its lipid oxidation products, such as 12-hydroxyeicosatetraenoic acid (12-HETE), accumulate during storage and have inhibitory effects on platelet recovery, survival, and function. However, several PUFAs are substrates for 12-LOX, and their resulting oxylipins may have different effects. We used targeted metabolomics to quantify PUFAs and oxylipins and platelet function assays to characterize function of fresh and stored wild-type (WT) and 12-LOX -/- platelets. Methods: Blood from WT and 12-LOX -/- mice was collected by retro-orbital bleeding. Platelet-rich plasma (PRP) was generated from whole blood. After fresh samples were aliquoted, the remaining PRP was separated in two groups. One group was stored at room temperature with agitation (RT) for 24 hours, and the other for 48 hours. Metabolites were extracted from samples and quantified by targeted metabolomics as described previously. We assessed platelet function by αIIbβ3 integrin activation by flow cytometry. In vivo recovery of function was measured by transfusing stored platelets into UBiC-GFP mice and stimulating platelets with agonists, followed by gating for transfused (GFP-negative) platelets by flow cytometry. For recovery and survival, we traced biotinylated fresh, 24h, or 48h-stored platelets after transfusion in vivo. Results: We quantified metabolites present in platelets by targeted metabolomics to monitor their changes in concentration over storage time. Among the 10 PUFAs and 28 related oxylipins we analyzed, 15 of 38 analytes showed a significant difference in PRP from WT and 12-LOX-/- mouse samples. The major metabolites of 12-LOX include 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA), from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). 12-HETE, 12-HEPE, and 14-HDHA were only detected at <8 nmol/L levels in fresh PRP from 12-LOX -/- mice compared to 668 ± 409nM, 149 ± 85nM, and 295 ± 154nM from WT mice, respectively. After 24 hours of storage at RT, 12-HETE, 12-HEPE, and 14-HDHA dramatically increased to 29.0±4.2µM, 3.7±1.1µM, and 6.3±0.8µM in PRP from WT mice, respectively. As expected, these same metabolites remained at low nmol/L levels in 12-LOX-/- samples during storage accompanied by a significant increase of their precursors AA, EPA, and DHA due to lack of 12-LOX activity. Interestingly, there was also a significant reduction in 15-HETE, 17-HDHA, and 13-hydroxyoctadecadienoic acid (13-HODE) in the 12-LOX -/- mice compared to the WT mice, which are primarily produced by the 15-LOX enzyme. Additionally, we observed a significant decrease of metabolites mediated via the cyclooxygenase (COX) pathway in PRP from 12-LOX-/- mice, including prostaglandin E2 (PGE2), PGD2, thromboxane B2, and 12-hydroxyheptadecatrienoic acid (12-HHTrE). Function-wise, fresh 12-LOX -/- platelets were less responsive to agonists compared to WT platelets. Surprisingly, after transfusion of fresh 12-LOX -/- platelets, we found comparable αIIbβ3-integrin activation results after 1, 4, and 24h of circulation time. In contrast, 24h and 48h of storage of 12-LOX -/- platelets led to significantly lower pre-activation at baseline and a significantly lower activation response than WT platelets after 1h and 4h of circulation time. No significant differences were observed after 24h of circulation time. We observed a clear trend for longer survival after 24 and 48h of storage. Conclusions: We found many metabolic changes between 12-LOX -/- and WT mice during storage. While the 12-LOX -/- mouse model highlights the primary metabolic differences that occur without 12-LOX activity, other changes, such as differences in COX or additional LOX isoform activity, may attenuate oxylipin production. Functionally, we observed less pre-activation and better survival in functional studies, but this may be due to a combined effect of each of these individual metabolites. Future studies will have to determine the roles of individual oxylipins. Disclosures Stolla: Cerus: Research Funding.

A Review Study of Dendrimer Nanoparticles Influences on Stored Platelet in Order to Treat Patients (2001-2020)

Background: Platelets are sensitive to chilling, therefore, the optimal storage temperature for maintaining normal function and structure in platelets is 22-24 °C up to 3-5 days. Introduction: Platelets are important blood cells involved in immunity, inflammation, and thrombosis. Today, platelet products are widely used to prevent bleeding in patients with thrombocytopenia and coagulopathy disorders. As a result, maintaining the quality of these products is very important. Method: In this review study, the reported influences of various dendrimers on platelets from 2001 to 2020 were investigated. Result: The results showed that positively charged dendrimers could cause platelet aggregation and activation during platelet storage time through their amine residues. In addition to surface charge, high generations, molecular weight and concentration are not recommended in the field of platelet storage and treatment. In contrast, negatively charged dendrimers, usually used at lower generations with proper molecular weight, lower size (less than 100 nm) and their carboxyl residues, cannot induce adverse effects on platelets during storage time. In addition, the results of this study revealed that PEGylation of dendrimers and platelets could improve platelet storage conditions. Conclusion: As anionic dendrimers can improve platelet storage time without inducing significant changes in morphology and function of platelets, they are recommended in the field of platelet storage and treatment.

Current Understanding of the Relationship between Blood Donor Variability and Blood Component Quality

While differences among donors has long challenged meeting quality standards for the production of blood components for transfusion, only recently has the molecular basis for many of these differences become understood. This review article will examine our current understanding of the molecular differences that impact the quality of red blood cells (RBC), platelets, and plasma components. Factors affecting RBC quality include cytoskeletal elements and membrane proteins associated with the oxidative response as well as known enzyme polymorphisms and hemoglobin variants. Donor age and health status may also be important. Platelet quality is impacted by variables that are less well understood, but that include platelet storage sensitive metabolic parameters, responsiveness to agonists accumulating in storage containers and factors affecting the maintenance of pH. An increased understanding of these variables can be used to improve the quality of blood components for transfusion by using donor management algorithms based on a donors individual molecular and genetic profile.

A zinc transporter, transmembrane protein 163 (TMEM163), is critical for the biogenesis of platelet dense granules

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as Ca2+, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet Zn2+ transporter TMEM163, which was significantly reduced in BLOC-1 (Dtnbp1 sdy and Pldn pa), BLOC-2 (Hps6 ru) or AP-3 (Ap3b1 pe) deficient mice and HPS patients (HPS2, 3, 5, 6, or 9). We observed similar platelet DG defects and abnormal intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome (LE) marker-positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis, and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.

Platelet storage pool disorder in pregnancy: Utilising thromboelastography to guide a risk-based delivery plan

We present a case of a 33-year-old woman in her third pregnancy diagnosed with platelet storage pool disorder who had previously suffered two postpartum major obstetric haemorrhages. Platelet storage pool disorder is a rare bleeding disorder where the platelet count is normal but platelet function is impaired due to deficiency of dense granules. A peripartum plan devised by an extensive multi-disciplinary team using principles for managing other bleeding and platelet function disorders helped minimise her risk of major haemorrhage. We also describe how point-of-care thromboelastography can help guide management and enable an individualised risk-benefit discussion with the woman about her anaesthetic choices.

An Investigation of ABO Blood Type and the Platelet Delta Granule Storage Pool

Individuals with bleeding tendencies are more likely to have blood type O than blood types A, B, or AB. Platelet storage pool deficiencies are a lesser-known group of bleeding disorders which often go undiagnosed and may account for a significant number of patients with unexplained bleeding defects. We hypothesized that patients with platelet δ-storage pool deficiency might also have a predominance of type O blood. A retrospective review of medical records of 2,020 patients with unexplained bleeding and evaluated for δ-storage pool deficiency was performed. Correlations between dense granule numbers, blood type, and von Willebrand factor were analyzed for statistical differences. 51.5% of blood samples were blood type O compared to an incidence of 44.0% in the U.S. population. There was a significant association of vWF and blood type O but not with the delta storage pool. There is a preponderance of blood type O in the study population compared to the U.S. population. There is no statistically significant link between blood type O and lower dense granule numbers in this study.