Shedding light on GPIbα shedding.

PURPOSE OF REVIEW: Ectodomain shedding has been investigated since the late 1980s. The abundant and platelet specific GPIbα receptor is cleaved by ADAM17 resulting in the release of its ectodomain called glycocalicin. This review will address the role of glycocalicin as an end-stage marker of platelet turnover and storage lesion and will consider a potential function as effector in processes beyond hemostasis. RECENT FINDINGS: Glycocalicin has been described as a marker for platelet senescence, turnover and storage lesion but is not routinely used in a clinical setting because its diagnostic value is nondiscriminatory. Inhibition of glycocalicin shedding improves posttransfusion recovery but little is known (yet) about potential hemostatic improvements. In physiological settings, GPIbα shedding is restricted to the intracellular GPIbα receptor subpopulation suggesting a role for shedding or glycocalicin beyond hemostasis. SUMMARY: So far, all evidence represents glycocalicin as an end-stage biomarker of platelet senescence and a potential trigger for platelet clearance. The extensive list of interaction partners of GPIbα in fields beyond hemostasis opens new possibilities to investigate specific effector functions of glycocalicin.

Towards standardized human platelet lysate production in Europe: An initiative of the European Blood Alliance.

Human platelet lysate (hPL) is a supplement for cell culture media that can be derived from platelet concentrates. As not-for-profit blood establishments, we endorse the evolution of maximally exploiting the potential of donated blood and its derived components, including platelets. The decision to use platelet concentrates to supply hPL as a cell culture supplement should align with the principles and values that blood establishments hold towards the use of donated blood components in transfusion. As a consequence, questions on ethics, practical standardization of hPL production and logistics as well as on assuring hPL quality and safety need careful consideration. We therefore propose an opinion on some of these matters based on available literature and on discussions within the proceedings of the Working Group on Innovation and New Products of the European Blood Alliance. In addition, we propose collaboration among European blood establishments to streamline efforts of hPL supply to maximize the potential of hPL and its application in the wider field of medicine.

Single step method for high yield human platelet lysate production.

BACKGROUND: We aimed to develop a single step method for the production of human platelet lysate (hPL). The method must result in high hPL yields, be closed system and avoid heparin use. STUDY DESIGN AND METHODS: The method aimed at using glass beads and calcium. An optimal concentration of calcium and glass beads was determined by serial dilution. This was translated to a novel method and compared to known methods: freeze-thawing and high calcium. Quality outcome measures were transmittance, fibrinogen and growth factor content, and cell doubling time. RESULTS: An optimal concentration of 5 mM Ca2+ and 0.2 g/ml glass beads resulted in hPL with yields of 92% ± 1% (n = 50) independent of source material (apheresis or buffy coat-derived). The transmittance was highest (56% ± 9%) compared to known methods (<39%). The fibrinogen concentration (7.0 ± 1.1 µg/ml) was well below the threshold, avoiding the need for heparin. Growth factor content was similar across hPL production methods. The cell doubling time of adipose derived stem cells was 25 ± 1 h and not different across methods. Batch consistency was determined across six batches of hPL (each n = 25 constituting concentrates) and was <11% for all parameters including cell doubling time. Calcium precipitation formed after 4 days of culturing stem cells in media with hPL prepared by the high (15 mM) Ca2+ method, but not with hPL prepared by glass bead method. DISCUSSION: The novel method transforms platelet concentrates to hPL with little hands-on time. The method results in high yield, is closed system, without heparin and non-inferior to published methods.

Quality assessment and harmonization of laboratories across Europe for multiple SARS-CoV-2 serology assays.

BACKGROUND AND OBJECTIVES: There is a need for conversion of SARS-CoV-2 serology data from different laboratories to a harmonized international unit. We aimed to compare the performance of multiple SARS-CoV-2 antibody serology assays among 25 laboratories across 12 European countries. MATERIALS AND METHODS: To investigate this we have distributed to all participating laboratories a panel of 15 SARS-CoV-2 plasma samples and a single batch of pooled plasma calibrated to the WHO IS 20/136 standard. RESULTS: All assays showed excellent discrimination between SARS-CoV-2 seronegative plasma samples and pre-vaccinated seropositive plasma samples but differed substantially in raw antibody titres. Titres could be harmonized to binding antibody units per millilitre by calibration in relation to a reference reagent. CONCLUSION: The standardization of antibody quantification is of paramount importance to allow interpretation and comparison of serology data reported in clinical trials in order to identify donor cohorts from whom the most effective convalescent plasma can be collected.

Open ADAMTS13, induced by antibodies, is a biomarker for subclinical immune-mediated thrombotic thrombocytopenic purpura.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.

A novel competition ELISA for the rapid quantification of SARS-CoV-2 neutralizing antibodies in convalescent plasma.

BACKGROUND: COVID-19 convalescent plasma (CCP) ideally contains high titers of (neutralizing) anti-SARS-CoV-2 antibodies. Several scalable immunoassays for CCP selection have been developed. We designed an enzyme-linked immunosorbent assay (ELISA) that measures neutralizing antibodies (of all isotypes) in plasma by determining the level of competition between CCP and a mouse neutralizing antibody for binding to the receptor binding domain (RBD) of SARS-CoV-2. METHODS: Plasma was collected from 72 convalescent individuals and inhibition of viral infection was determined by plaque reduction neutralization (PRNT50). The level of neutralizing antibodies was measured in the novel competition ELISA and in a commercially available ELISA that measures inhibition of recombinant ACE2 binding to immobilized RBD. These results were compared with a high throughput chemiluminescent microparticle immunoassay (CMIA). RESULTS: The results from both ELISAs were correlating, in particular for high titer CCP (PRNT50 ≥ 1:160) (Spearman r = .73, p < .001). Moderate correlation was found between the competition ELISA and CMIA (r = .57 for high titer and r = .62 for low titer CCP, p < .001). Receiver operator characteristic analysis showed that the competition ELISA selected CCP with a sensitivity and specificity of 61% and 100%, respectively. However, discrimination between low and high titer CCP had a lower resolution (sensitivity: 34% and specificity: 89%). CONCLUSION: The competition ELISA screens for neutralizing antibodies in CCP by competition for just a single epitope. It exerts a sensitivity of 61% with no false identifications. These ELISA designs can be used for epitope mapping or for selection of CCP.

SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.

The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion.

BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase life-span of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased two-fold from 4.8 ± 0.5 fmol (mean ± SD, n = 6) to 2.1 ± 1.8 fmol per 103 control cells, but increased almost 10-fold to 41.5 ± 4.1 fmol per 103 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin αIIb ß3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet life-span cannot be prolonged using this strategy.

Platelet Biochemistry and Morphology after Cryopreservation.

Platelet cryopreservation has been investigated for several decades as an alternative to room temperature storage of platelet concentrates. The use of dimethylsulfoxide as a cryoprotectant has improved platelet storage and cryopreserved concentrates can be kept at -80 °C for two years. Cryopreserved platelets can serve as emergency backup to support stock crises or to disburden difficult logistic areas like rural or military regions. Cryopreservation significantly influences platelet morphology, decreases platelet activation and severely abrogates platelet aggregation. Recent data indicate that cryopreserved platelets have a procoagulant phenotype because thrombin and fibrin formation kicks in earlier compared to room temperature stored platelets. This happens both in static and hydrodynamic conditions. In a clinical setting, low 1-h post transfusion recoveries of cryopreserved platelets represent fast clearance from circulation which may be explained by changes to the platelet GPIbα receptor. Cryopreservation splits the concentrate in two platelet subpopulations depending on GPIbα expression levels. Further research is needed to unravel its physiological importance. Proving clinical efficacy of cryopreserved platelets is difficult because of the heterogeneity of indications and the ambiguity of outcome measures. The procoagulant character of cryopreserved platelets has increased interest for use in trauma stressing the need for double-blinded randomized clinical trials in actively bleeding patients.

Impact of cold storage on platelets treated with Intercept pathogen inactivation.

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22°C), cold (4°C, n = 6), or cryopreservation (-80°C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS: Platelet aggregation of cold-stored AS-PCT PLTs was 44% ± 11% compared to 57% ± 14% for cold-stored standard PLTs and 58% ± 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% ± 9% compared to 77% ± 6% for cold-stored standard PLTs and 69% ± 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 ± 140 sec) compared to cold-stored standard PLTs (960 ± 192 sec) and RT-stored AS-PCT PLTs (1134 ± 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 ± 47 nmol/L thrombin compared to 159 ± 33 nmol/L for cold-stored standard PLTs and 83 ± 25 nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% ± 10%) was not significantly different from cryopreserved standard PLTs (25% ± 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation.