Extension of the circulatory half-life of recombinant ecallantide via albumin fusion without loss of anti-kallikrein activity.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP.

ABSTRACT: Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody's Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum-sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

Minimal impact of anticoagulant on in vitro whole blood quality throughout a 35-day cold-storage regardless of leukoreduction timing.

BACKGROUND: There is increasing interest in leukoreduced whole blood (WB) as a transfusion product for trauma patients. In some jurisdictions, few leukoreduced filters are approved or appropriate for WB leukoreduction and quality information is therefore limited. This study assessed the impact of filtration timing of WB collected in CPDA-1 versus CPD on in vitro quality. STUDY DESIGN AND METHODS: WB was collected in CPDA-1 or CPD and leukoreduction filtered either after 3-8 h (early) or 18-24 h (late) from stop bleed time. In vitro quality was assessed after filtration and throughout 5 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation. RESULTS: Although there were some data points which showed statistically significant differences associated with anticoagulant choices or the filtration timing, no general trend in inferiority/performance could be discerned. After 35 days' storage, only clotting time, alpha angle and factor II in the early filtration arm comparing anticoagulants and prothrombin time and factor II in the CPDA-1 study arm comparing filtration timing showed a significant difference. CONCLUSION: In vitro WB quality seems to be independent on the choice of anticoagulant and filtration timing supporting WB hold-times to up to 24 h, increasing operational flexibility for transfusion services.

Spotlight on animal models of acute traumatic coagulopathy: an update.

Critically injured persons suffer trauma, hemorrhage, and high mortality. A subset of such patients develops early coagulation dysfunction characterized as acute traumatic coagulopathy (ATC), with a poor prognosis. The mechanisms contributing to ATC remain incompletely understood. Notwithstanding some successes in conducting clinical trials in early traumatic coagulopathy, conducting clinical research in ATC is ethically and logistically challenging. In vitro studies cannot capture the complex pathophysiological interplay between blood, vasculature, and organ systems relevant to ATC. Animal models are therefore vital for understanding ATC and to test interventions. Previous systematic reviews of animal models of ATC covered progress up to 2014. The current review aimed to extend that coverage to the end of 2021. A structured systematic search of MEDLINE/PubMed was carried out and identified 56 relevant publications. Unlike in previous reviews, where pig models predominated, rat and pig models contributed equally (19 studies each), and non-human primate models entered the field. Most studies now featured defined trauma (39 of 56), and hemorrhage controlled by pressure or volume (42 studies), with some documenting that both were necessary to induce ATC. Most studies documented coagulopathy using clotting or viscoelastometric assays and created an endogenous coagulopathy not dependent on iatrogenic dilution. As before, the diversity of species and experimental protocols may limit the translatability of the identified studies. Thus, while animal research has become more aligned to clinical realities since 2014, further efforts are required to unravel ATC mechanisms and enable the prediction and evaluation of optimal clinical interventions.

Challenging the 30-min rule for thawed plasma.

BACKGROUND AND OBJECTIVES: Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS: Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS: Testing results of all quality parameters were comparable between T30 and T60 units (p < 0.05). Serratia liquefaciens proliferated in cold-stored plasma, while P. putida showed variable viability. Serratia epidermidis and P. aeruginosa survived but did not grow in cold-stored plasma. Positive and negative controls showed expected results. Overall, no statistical differences in bacterial concentration between T30 and T60 units were observed (p < 0.05). CONCLUSION: Multiple RT exposures for 30 or 60 min do not affect the stability of coagulation factors or promote bacterial growth in thawed plasma stored for 5 days. It is therefore safe to expose thawed plasma to uncontrolled temperatures for limited periods of 60 min.

Retention of hemostatic and immunological properties of frozen plasma and COVID-19 convalescent apheresis fresh-frozen plasma produced and freeze-dried in Canada.

BACKGROUND: Randomized clinical trial data show that early plasma transfusion may save lives among trauma patients. Supplying plasma in remote environments is logistically challenging. Freeze-dried plasma (FDP) offers a possible solution. STUDY DESIGN AND METHODS: A Terumo BCT plasma freeze-drying system was evaluated. We compared pooled frozen plasma (FP) units with derived Terumo BCT FDP (TFDP) units and pooled COVID-19 convalescent apheresis fresh-frozen plasma (CC-AFFP) with derived CC-TFDP units. Parameters measured were: coagulation factors (F) II; V; VII; VIII; IX; XI; XIII; fibrinogen; Proteins C (PC) and S (PS); antithrombin (AT); α2 -antiplasmin (α2 AP); ADAMTS13; von Willebrand Factor (vWF); thrombin-antithrombin (TAT); D-dimer; activated complement factors 3 (C3a) and 5 (C5a); pH; osmolality; prothrombin time (PT); and activated partial thromboplastin time (aPTT). Antibodies to SARS-CoV-2 in CC-AFFP and CC-TFDP units were compared by plaque reduction assays and viral protein immunoassays. RESULTS: Most parameters were unchanged in TFDP versus FP or differed ≤15%. Mean aPTT, PT, C3a, and pH were elevated 5.9%, 6.9%, 64%, and 0.28 units, respectively, versus FP. CC-TFDP showed no loss of SARS-CoV-2 neutralization titer versus CC-AFFP and no mean signal loss in most pools by viral protein immunoassays. CONCLUSION: Changes in protein activities or clotting times arising from freeze-drying were <15%. Although C3a levels in TFDP were elevated, they were less than literature values for transfusable plasma. SARS-CoV-2-neutralizing antibody titers and viral protein binding levels were largely unaffected by freeze-drying. In vitro characteristics of TFDP or CC-TFDP were comparable to their originating plasma, making future clinical studies appropriate.

Cold-stored leukoreduced whole blood: Extending the time between donation and filtration has minimal impact on in vitro quality.

BACKGROUND: Leukoreduced whole blood (LR-WB) has received renewed attention as alternative to component-based transfusion in trauma. According to the manufacturer's instructions, leukoreduction should be carried out within 8 h after collection. This study assessed impact of (1) WB collection bag, (2) LR filtration, and (3) timing of filtration on in vitro quality. STUDY DESIGN AND METHODS: WB collected into different vendor bags was held at room temperature for <8 h or >16 h but <24 h prior to LR. In vitro quality was assessed before and after filtration, and throughout 3 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation, and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation analyzer or quantitative immunoblotting. RESULTS: Bag type had no impact on WB in vitro quality. LR by filtration had some impact, but is aligned with data in the literature. The time between donation and filtration resulted in some statistically significant differences in metabolic activity, platelet yield, platelet activation, and factor protein activity initially; however, these differences in in vitro quality attributes decreased throughout 21-day cold storage. CONCLUSION: WB hold time showed only a minor impact on WB in vitro quality, so it may be possible for blood processing facilities to explore extended hold times prior to filtration in order to provide greater operational flexibility.

Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant.

Alpha-1 antitrypsin (AAT, also known as alpha-1 proteinase inhibitor or SERPINA1) is the most abundant member of the serpin superfamily found in human plasma. The naturally occurring variant AAT M358R, altered at the P1 position of the critical reactive center loop (RCL), is re-directed away from inhibition of AAT's chief natural target, neutrophil elastase, and toward accelerated inhibition of thrombin (FIIa), kallikrein (Kal), and other proteases such as factor XIa (FXIa). FXIa is an emerging target for the development of antithrombotic agents, since patients with FXI deficiency are protected from thromboembolic disease and do not exhibit a strong bleeding tendency. Previously, we used phage display, bacterial lysate screening, and combinatorial mutagenesis to identify AAT-RC, an engineered AAT M358R with additional changes between RCL positions P7-P3', CLEVEPR-STE [with changes bolded and the P1-P1' (R358-S359) reactive center shown as R-S]. AAT-RC was 279- and 16-fold more selective for FXIa/IIa or FXIa/Kal than AAT M358R; the increased selectivity came at a cost of a 2.3-fold decrease in the rate of FXIa inhibition and a 3.3-fold increase in the stoichiometry of inhibition (SI). Here, we asked which alterations in AAT-RC were most important for the observed increases in selectivity for FXIa inhibition. We back-mutated AAT-RC to AAT-RC-1 (P7-P3' FLEVEPRSTE), AAT-RC-2 (P7-P3' FLEAEPRSTE), and AAT RC-3 (P7-P3' FLEAIPR-STE). Proteins were expressed as cleavable, hexahistidine-tagged glutathione sulfotransferase fusion proteins in E. coli and purified by proteolytic elution from glutathione agarose, with polishing on nickel chelate agarose. Selectivity for FXIa over Kal of AAT-RC-1, -2, and -3 was 14, 21, and 2.3, respectively. AAT-RC-2 inhibited FXIa 31% more rapidly than AAT M358R, with the same SI, and enhanced selectivity for FXIa over Kal, FXa, FXIIa, activated protein C, and FIIa of 25-, 130-, 420-, 440-, and 470-fold, respectively. Structural modeling of the AAT-RC-2/FXIa encounter complex suggested that both E (Glu) substitutions at P3 and P3' may promote FXIa binding via hydrogen bonding to K192 in FXIa. AAT-RC-2 is the most selective and active AAT variant reported to date for FXIa inhibition and will be tested in animal models of thrombosis and bleeding.

Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis.

Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis.

Selection and in vitro and in vivo characterization of a Kunitz protease inhibitor domain of protease nexin 2 variant that inhibits factor XIa without inhibiting plasmin.

The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.

Correction: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin.

[This corrects the article DOI: 10.1371/journal.pone.0084491.].

Prothrombin, alone or in complex concentrates or plasma, reduces bleeding in a mouse model of blood exchange-induced coagulopathy.

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

Extending the pre-processing holding time of whole blood beyond 48 h reduces coagulation FVIII activity and immunoglobulin G content of recovered plasma.

BACKGROUND: Plasma obtained via whole blood (WB) donation may be used either for transfusion or as recovered plasma (RP) for pooling and fractionation. In Canada, transfusable plasma must be processed within 24 h of phlebotomy, while the limit for RP processing is 72 h. We assessed the quality of RP produced by two WB processing methods and as a function of processing time. STUDY DESIGN AND METHODS: RP units produced via the buffy coat method (BCM, n = 26) or whole blood filtration (WBF, n = 52) were tested for: the activities of prothrombin, fibrinogen, von Willebrand Factor (VWF), FV, FVII, and FVIII; the prothrombin time (PT); and total protein and IgG concentration. WBF RP units were evenly divided between those processed <48 h of phlebotomy (shorter-processed) or 48-72 h after phlebotomy (longer-processed). RESULTS: WBF-RP did not differ significantly from BCM-RP in any tested parameter except for FV and FVIII, which exhibited mean reductions of 10.2% and 20%, respectively. Longer-processed WBF-RP did not differ significantly from shorter-processed WBF-RP in any tested parameter except for FVIII activity and IgG concentration, which exhibited mean reductions of 30.1% and 14.3%, respectively. CONCLUSIONS: Canadian RP is currently fractionated into IgG, albumin, fibrinogen, and FVII/VWF concentrates irrespective of its method or time of processing. Our results supported the current approach of fractionating both BCM- and WBF-derived RP, but suggest that greater yields of immunoglobulin and FVIII/VWF products could be obtained if the maximum processing time was reduced from 72 h to 48 h.

Serpin Phage Display: The Use of a T7 System to Probe Reactive Center Loop Libraries with Different Serine Proteinases.

Phage display is a protein engineering approach that involves construction of libraries of variant proteins displayed on the surface of bacteriophage as capsid fusion proteins and their screening for binding and inhibitory function through the use of bait proteins. Recently, we adapted a commercially available T7 phage display system to create phage-displayed serpin libraries hypervariable in up to five positions in their reactive center loop (RCL). The RCL is a key determinant in serpin specificity, the relationship between the structure of a given serpin and which target proteinase(s) it inhibits. In this chapter, we describe protocols to assess the feasibility of this method for different serpin/proteinase combinations and share experience with this technology gathered in the course of studying two serpins and multiple proteinases with this powerful iterative screening approach.

A factor XIa-activatable hirudin-albumin fusion protein reduces thrombosis in mice without promoting blood loss.

BACKGROUND: Hirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects. Because hirudin's N-terminus must engage thrombin's active site for effective inhibition, fusing a cleavable peptide at this site may improve hirudin's risk/benefit ratio as a therapeutic agent. Previously we engineered a plasmin cleavage site (C) between human serum albumin (HSA) and hirudin variant 3 (HV3) in fusion protein HSACHV3. Because coagulation factor XI (FXI) is more involved in thrombosis than hemostasis, we hypothesized that making HV3 activity FXIa-dependent would also improve HV3's potential therapeutic profile. We combined albumin fusion for half-life extension of hirudin with positioning of an FXIa cleavage site N-terminal to HV3, and assessed in vitro and in vivo properties of this novel protein. RESULTS: FXIa cleavage site EPR was employed. Fusion protein EPR-HV3HSA but not HSAEPR-HV3 was activated by FXIa in vitro. FVIIa, FXa, FXIIa, or plasmin failed to activate EPR-HV3HSA. FXIa-cleavable EPR-HV3HSA reduced the time to occlusion of ferric chloride-treated murine arteries and reduced fibrin deposition in murine endotoxemia; noncleavable mycHV3HSA was without effect. EPR-HV3HSA elicited less blood loss than constitutively active HV3HSA in murine liver laceration or tail transection but extended bleeding time to the same extent. EPR-HV3HSA was partially activated in citrated human or murine plasma to a greater extent than HSACHV3. CONCLUSIONS: Releasing the N-terminal block to HV3 activity using FXIa was an effective way to limit hirudin's bleeding side-effects, but plasma instability of the exposed EPR blocking peptide rendered it less useful than previously described plasmin-activatable HSACHV3.

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2.

BACKGROUND/AIMS: The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. METHODS: Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography. Clearance profiles in mice were determined, as well as the effects of KPI or KPIHSA administration on FeCl3-induced vena cava thrombus size or carotid artery time to occlusion, respectively. RESULTS: Fusion to HSA increased the mean terminal half-life of KPI by 8-fold and eliminated its interaction with the low density lipoprotein receptor-related protein. KPI and KPIHSA similarly reduced thrombus size and occlusion in both venous and arterial thrombosis models when administered at the time of injury, but only KPI was effective when administered one hour before injury. CONCLUSIONS: Albumin fusion deflects KPI from rapid in vivo clearance without impairing its antithrombotic properties and widens its potential therapeutic window.

Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa.

Factor XIa (FXIa) is a serine protease that catalyzes the activation of Factor IX (FIX) in the blood coagulation cascade. FXIa and its precursor FXI are emergent therapeutic targets for the development of safer anticoagulant agents. Here, we sought a novel DNA-based agent to inhibit FXIa. Towards this goal, an 80 base, single-stranded DNA aptamer library (containing a 40 base randomized core) was screened for FXIa-binding candidates, using ten rounds of positive and negative selection. After selection, 6 of 89 different sequences inhibited FXIa-mediated chromogenic substrate S2366 cleavage. The most active anti-FXIa aptamer had a hypervariable central sequence 5'-AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT-3' and was designated Factor ELeven Inhibitory APtamer (FELIAP). FELIAP, but not a scrambled aptamer control (SCRAPT), competitively inhibited FXIa-catalyzed S2366 cleavage, FIX activation, and complex formation with antithrombin. No effect of FELIAP on FXI activation was observed. FELIAP inhibited plasma clotting and thrombin generation assays to a significantly greater extent than SCRAPT. Immobilized FELIAP bound FXIa with strong affinity and an equilibrium binding constant (KD) in the low nanomolar range determined using surface plasmon resonance. FELIAP is the first FXIa-inhibitory aptamer to be described and constitutes a lead compound to develop related aptamers for in vivo use.

Pathogen inactivation by riboflavin and ultraviolet light illumination accelerates the red blood cell storage lesion and promotes eryptosis.

BACKGROUND: Pathogen reduction treatment using riboflavin and ultraviolet light illumination (Mirasol) effectively reduces the risk of transfusion-transmitted infections. This treatment is currently licensed for only platelets and plasma products, while its application to whole blood (WB) to generate pathogen-inactivated red blood cells (RBCs) is under development. RBC storage lesion, constituting numerous morphologic and biochemical changes, influences RBC quality and limits shelf life. Stored RBCs further show enhanced susceptibility to RBC programmed cell death (eryptosis) characterized by increased cytosolic Ca2+ -provoked membrane phosphatidylserine (PS) externalization. STUDY DESIGN AND METHODS: Using a "pool-and-split" approach, we examined multiple variables of RBC storage lesion and eryptosis in RBC units, derived from Mirasol-treated or untreated WB, after 4 to 42 days of storage, under blood bank conditions. RESULTS: In comparison to untreated RBC units, Mirasol treatment significantly altered membrane microvesiculation, supernatant hemoglobin, osmotic fragility, and intracellular adenosine triphosphate levels but did not influence membrane CD47 expression and 2,3-diphosphoglycerate levels. Mirasol-treated RBCs showed significantly higher PS exposure after 42, but not after not more than 21, days of storage, which was accompanied by enhanced cytosolic Ca2+ activity, ceramide abundance, and oxidative stress, but not p38 kinase activation. Mirasol treatment significantly augmented PS exposure, Ca2+ entry, and protein kinase C activation after energy depletion, a pathophysiologic cell stressor. Mirasol-treated RBCs were, however, more resistant to cell shrinkage. CONCLUSIONS: Prolonged storage of Mirasol-treated RBCs significantly increases the proportion of eryptotic RBCs, while even short-term storage enhances the susceptibility of RBCs to stress-induced eryptosis, which could reduce posttransfusion RBC recovery in patients.