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.

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device.

BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Comparison between manufacturing sites shows differential adhesion, activation, and GPIbα expression of cryopreserved platelets.

BACKGROUND: Transfusion of cryopreserved platelets (cryoplatelets) is not common but may replace standard liquid-preserved platelets (PLTs) in specific circumstances. To better understand cryoplatelet function, frozen concentrates from different manufacturing sites were compared. STUDY DESIGN AND METHODS: Cryoplatelets from Denver, Colorado (DEN); Sydney, Australia (SYD); and Ghent, Belgium (GHE) were compared (n = 6). A paired noncryopreserved control was included in Ghent. Microfluidic-flow chambers were used to study PLT adhesion and fibrin deposition in reconstituted blood. Receptor expression was measured by flow cytometry. Coagulation in static conditions was evaluated by rotational thromboelastometry (ROTEM). RESULTS: Regardless of the manufacturing site, adhesion of cryoplatelets under shear flow (1000/sec) was significantly (p < 0.05) reduced compared to control. Expression of GPIbα was decreased in a subpopulation of cryoplatelets comprising 45% ± 11% (DEN), 63% ± 9% (GHE), and 94% ± 6% (SYD). That subpopulation displayed increased annexin V binding and decreased integrin activation. PLT adhesion, agglutination, and aggregation were moreover decreased in proportion to that subpopulation. Fibrin deposition under shear flow was normal but initiated faster (546 ± 163 sec GHE) than control PLTs (631 ± 120 sec, p < 0.01), only in the absence of tissue factor. In static conditions, clotting time was faster, but clot firmness decreased compared to control. Coagulation was not different between manufacturing sites. CONCLUSION: Cryopreservation results in a subset of PLTs with enhanced GPIbα shedding, increased phosphatidylserine expression, reduced integrin response, and reduced adhesion to collagen in microfluidic models of hemostasis. The proportion of this phenotype is different between manufacturing sites. The clinical effects, if any, will need to be verified.

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing.

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 µm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 µg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 µm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

Allosteric activation of ADAMTS13 by von Willebrand factor.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

Ultraviolet C light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro.

BACKGROUND: Ultraviolet (UV) light illumination in the presence of exogenously added photosensitizers has been used to inactivate pathogens in platelet (PLT) concentrates for some time. The THERAFLEX UV-C system, however, illuminates PLT concentrates with UV-C light without additional photoactive compounds. In this study residual PLT function is measured in a comprehensive paired analysis of UV-C-treated, gamma-irradiated, and untreated control PLT concentrates. STUDY DESIGN AND METHODS: A pool-and-split design was used with buffy coat-derived PLT concentrates in 65% SSP+ additive solution. Thrombus formation kinetics in microfluidic flow chambers onto immobilized collagen was investigated with real-time video microscopy. PLT aggregation, membrane markers, and cellular metabolism were determined concurrently. RESULTS: Compared to gamma-treated and untreated controls, UV-C treatment significantly affected thrombus formation rates on Days 5 and 7, not Day 2. PLT degranulation (P-selectin) and PLT apoptosis (annexin V binding) was slightly but significantly increased from Day 2 on. UV-C treatment moreover induced integrin αIIb ß3 conformational changes reminiscent of activation. However, subsequent integrin activation by either PAR1-activating hexapeptide (PAR1AP) or convulxin was unaffected. This was confirmed by PLT aggregation studies induced with collagen, PAR1AP, and ristocetin at two different agonist concentrations. Finally, UV-C slightly increased lactic acid production rates, resulting in significantly decreased pH on Days 5 and 7, but never dropped below 7.2. CONCLUSION: UV-C pathogen inactivation treatment slightly but significantly increases PLT activation markers but does not profoundly influence activatability nor aggregation. The treatment does, however, attenuate thrombus formation kinetics in vitro in microfluidic flow chambers, especially after storage.

Observational study of corrected count increments after transfusion of platelets treated with riboflavin pathogen reduction technology in additive solutions.

BACKGROUND: Mirasol pathogen reduction technology (PRT) treatment inactivates bacteria, viruses, and parasites in plasma products and platelets (PLTs) suspended in plasma and PLT additive solutions (PAS). Few clinical studies exist documenting transfusions with PAS. This study objective was to evaluate the count increments of PRT-treated PAS-C and PAS-E buffy coat (BC) PLTs in routine use observational settings. STUDY DESIGN AND METHODS: PLT pools of five or six BCs were collected, processed, and suspended in PAS-C or PAS-E, respectively. Products were exposed to ultraviolet light in the presence of riboflavin and then transfused into 19 patients with hematologic diseases. Patients were monitored for PLT corrected count increment (CCI) at 1 and 24 hours and for any adverse events in the 72 hours after transfusion. Sterility monitoring was performed with a microbial detection system (BacT/ALERT, bioMérieux). RESULTS: The PAS-E products had significantly higher PLT concentrations and counts than the PAS-C products. The mean CCIs of per-protocol (PP) units at 1 and 24 hours were 11,900 (n=27) and 5500 (n=30), respectively. Seventy-eight percent of PP transfusions classify as successful with CCIs at 1 hour of higher than 7500, and 63% higher than 4500 at 24 hours. One patient was excluded from all analyses as she was refractory to Mirasol-treated PLT transfusions and follow-up untreated transfusion products. No adverse events were observed and no contaminated products were detected by BacT/ALERT. CONCLUSION: PRT-treated BC PLTs in PAS-C or PAS-E demonstrate PLT transfusion success rates in hematology patients with thrombocytopenia that are comparable to previous studies examining PLTs stored in plasma.

Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.