Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia.

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor 4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n = 5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n = 10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptor IIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.

Adjunct Immune Globulin for Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The use of high-dose intravenous immune globulin (IVIG) plus anticoagulation is recommended for the treatment of vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare side effect of adenoviral vector vaccines against coronavirus disease 2019 (Covid-19). We describe the response to IVIG therapy in three of the first patients in whom VITT was identified in Canada after the receipt of the ChAdOx1 nCoV-19 vaccine. The patients were between the ages of 63 and 72 years; one was female. At the time of this report, Canada had restricted the use of the ChAdOx1 nCoV-19 vaccine to persons who were 55 years of age or older on the basis of reports that VITT had occurred primarily in younger persons. Two of the patients in our study presented with limb-artery thrombosis; the third had cerebral venous and arterial thrombosis. Variable patterns of serum-induced platelet activation were observed in response to heparin and platelet factor 4 (PF4), indicating the heterogeneity of the manifestations of VITT in serum. After the initiation of IVIG, reduced antibody-induced platelet activation in serum was seen in all three patients. (Funded by the Canadian Institutes of Health Research.).

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

Evaluating Diagnostic Algorithms for Heparin-Induced Thrombocytopenia using Two Combined Automated Rapid Immunoassays.

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder caused by antibodies against platelet factor 4 (PF4) and heparin complexes. Rapid immunoassays (IAs) for detection of these antibodies mark a milestone in HIT diagnosis, despite a higher false-positive rate compared with functional platelet-activation assays. However, combining different rapid IAs may help to improve their diagnostic specificity. Here, we compared the individual performance of the latex immunoturbidimetric assay (LIA; HemosIL HIT-Ab [PF4-H]; sensitivity 91.7%, specificity 68.4%) and chemiluminescence immunoassay (CLIA; HemosIL AcuStarHIT-Ab [PF4-H]; sensitivity 92.4%, specificity 85.8%) with their combined performance using two unique diagnostic algorithms in a single prospective cohort of suspected HIT patients. Using the simultaneous algorithm adapted from Warkentin et al, the combined LIA-CLIA had a sensitivity of 99.0% and specificity of 64.3%. The sequential algorithm adapted from Rittener-Ruff et al was applied in two theoretical scenarios to reflect real-world circumstances in diagnostic laboratories where access to clinical information is limited: (1) assuming all patients had an intermediate 4Ts score and (2) assuming all patients had a high 4Ts score. This algorithm correctly predicted HIT in 94.5% (high 4Ts) and 96.0% (intermediate 4Ts) and excluded HIT in 82.6% (high 4Ts) and 80.1% (intermediate 4Ts) of patients in either scenario, respectively. Although both combined algorithms improved diagnostic performance of individual IAs, the simultaneous algorithm showed fewer false predictions (7.9%) than the sequential algorithm (intermediate 4Ts: 37.6% and high 4Ts: 41.5%) and proved more practical as it does not rely on physician evaluations. Our findings highlight the importance of accounting for clinician and interlaboratory variability when evaluating diagnostic tests for HIT.

Bleeding self-assessments by patients with immune thrombocytopenia (ITP): An agreement study.

We designed anagreement study to compare the results of bleeding assessments done in tandem by ITP patients and trained research staff. We used a modified version of the ITP Bleeding Scale, which captured the patients' worst bleeding event at any of nine anatomical sites since the time of the last assessment. Interrater agreement was determined using the 2-way kappa for the assessment of severe vs. non-severe bleeds. We analyzed 108 consecutive patients with ITP from the McMaster ITP Registry who had duplicate bleeding assessments. Two-way agreement was excellent for gynecological (k = 0.86, 95% CI 0.71-1.02), gastrointestinal (k = 1), genitourinary (k = 1), pulmonary (k = 1) and intracranial (k = 1) bleeds; good for skin (k = 0.68, 95% CI, 0.54-0.82), oral (k = 0.76, 95% CI, 0.53-0.98) and ocular (k = 0.66, 95% CI, 0.04-1-28) bleeds; and moderate for epistaxis (k = 0.58, 95% CI, 0.21-0.95). Bleeding self-assessments by ITP patients were similar to trained research staff, but disagreements in severity grades were more frequent with skin bleeds, oral bleeds and epistaxis. Bleeding self-assessments could simplify bleeding assessments in clinical trials.

Cytomegalovirus Seropositivity in Older Adults Changes the T Cell Repertoire but Does Not Prevent Antibody or Cellular Responses to SARS-CoV-2 Vaccination.

Chronic infection with human CMV may contribute to poor vaccine efficacy in older adults. We assessed the effects of CMV serostatus on Ab quantity and quality, as well as cellular memory recall responses, after two and three SARS-CoV-2 mRNA vaccine doses, in older adults in assisted living facilities. CMV serostatus did not affect anti-Spike and anti-receptor-binding domain IgG Ab levels, nor neutralization capacity against wild-type or ß variants of SARS-CoV-2 several months after vaccination. CMV seropositivity altered T cell expression of senescence-associated markers and increased effector memory re-expressing CD45RA T cell numbers, as has been previously reported; however, this did not impact Spike-specific CD4+ T cell memory recall responses. CMV-seropositive individuals did not have a higher incidence of COVID-19, although prior infection influenced humoral immunity. Therefore, CMV seropositivity may alter T cell composition but does not impede the durability of humoral protection or cellular memory responses after SARS-CoV-2 mRNA vaccination in older adults.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Circulating anti-nuclear autoantibodies in COVID-19 survivors predict long COVID symptoms.

BACKGROUND: Autoimmunity has been reported in patients with severe coronavirus disease 2019 (COVID-19). We investigated whether anti-nuclear/extractable-nuclear antibodies (ANAs/ENAs) were present up to a year after infection, and if they were associated with the development of clinically relevant post-acute sequalae of COVID-19 (PASC) symptoms. METHODS: A rapid-assessment line immunoassay was used to measure circulating levels of ANAs/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6 and 12 months post-recovery. Patient-reported fatigue, cough and dyspnoea were recorded at each time point. Multivariable logistic regression model and receiver operating curves were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines. RESULTS: Compared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3 months post-recovery (p<0.001). The mean number of ANA autoreactivities per individual decreased between 3 and 12 months (from 3.99 to 1.55) with persistent positive titres associated with fatigue, dyspnoea and cough severity. Antibodies to U1-snRNP and anti-SS-B/La were both positively associated with persistent symptoms of fatigue (p<0.028, area under the curve (AUC) 0.86) and dyspnoea (p<0.003, AUC=0.81). Pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and C-reactive protein predicted the elevated ANAs at 12 months. TNF-α, D-dimer and interleukin-1ß had the strongest association with symptoms at 12 months. Regression analysis showed that TNF-α predicted fatigue (ß=4.65, p=0.004) and general symptomaticity (ß=2.40, p=0.03) at 12 months. INTERPRETATION: Persistently positive ANAs at 12 months post-COVID are associated with persisting symptoms and inflammation (TNF-α) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.

Early humoral and cellular responses after bivalent SARS-CoV-2 mRNA-1273.214 vaccination in long-term care and retirement home residents in Ontario, Canada: An observational cohort study.

Immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) bivalent mRNA-1273.214 vaccine (Original/Omicron B.1.1.529 [BA.1]) is underreported in vulnerable older adults in congregate care settings. In residents of 26 long-term care and retirement homes in Ontario, Canada, humoral (i.e., serum anti-spike and anti-receptor binding domain [anti-RBD]) IgG and IgA antibodies and live SARS-CoV-2 neutralization) and cellular (i.e., CD4+ and CD8+ activation-induced marker spike-specific T cell memory) responses were assessed 7-120 days postvaccination with four monovalent mRNA vaccines (n = 494) or subsequent bivalent mRNA-1273.214 vaccination (fifth vaccine) (n = 557). Within 4 months, anti-spike and anti-RBD antibody levels were similar after monovalent and bivalent vaccination in infection-naïve individuals. Hybrid immunity (i.e., vaccination and natural infection) generally increased humoral responses. After bivalent vaccination, compared to monovalent vaccination, residents with hybrid immunity had elevated anti-spike and anti-RBD IgG and IgA antibodies. Omicron BA.1 antibody-mediated neutralization, and CD8+ T cell memory responses to the Omicron BA.1 spike protein, were also higher after bivalent vaccination. Humoral and cellular responses were, therefore, noninferior within 4 months of bivalent mRNA-1273.214 vaccination compared to monovalent mRNA vaccination. Waning of humoral but not cellular immunity was particularly evident in individuals without hybrid immunity. Continued monitoring of vaccine-associated and hybrid immunity against emerging Omicron variants of concern is necessary to assess longevity of protection.

Clinical and laboratory predictors of fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the most common cause of intracranial hemorrhage (ICH) in thrombocytopenic term infants. We investigated clinical and laboratory predictors of severe FNAIT in a tertiary care referral center. STUDY DESIGN AND METHODS: Retrospective cohort study over a 30-year period. We defined FNAIT as recurrence of neonatal thrombocytopenia in a subsequent pregnancy; and severe outcomes as any of: (1) a birth platelet count below 20 × 109 /L; (2) ICH or (3) fetal death. We used a generalized estimating equations analysis and classification tree analysis to identify risk factors for severe FNAIT in a subsequent pregnancy. RESULTS: During index pregnancies (n = 135 in 131 mothers), 71 infants (52.6%) had severe outcomes including a platelet count <20 × 109 /L (n = 45), fetal or neonatal ICH (n = 32), or fetal death (n = 4). During subsequent pregnancies (n = 72), 15 infants (20.8%) had severe outcomes including birth platelets <20 × 109 /L (n = 10), ICH (n = 2), or death (n = 3). Forty-two women (58.3%) received antenatal intravenous immune globulin (IVIG) during subsequent pregnancies. Eight mothers (n = 9 infants) had severe FNAIT outcomes despite receiving antenatal IVIG. Maternal antibodies to human platelet antigens (HPA) was the only independent predictor of severe FNAIT in a subsequent pregnancy (OR = 25.3, p = .004). Nevertheless, one of 43 infants from antibody-negative mothers had a severe outcome. CONCLUSIONS: The presence of anti-HPA is highly indicative of the diagnosis of severe FNAIT; however, we observed one infant who had severe FNAIT recurrence, defined using strict clinical criteria, without a maternal antibody. Improved diagnostic and therapeutic strategies are needed to prevent severe FNAIT in high-risk mothers.

T cell-mediated autoimmunity in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a low platelet count and an increased risk of bleeding. In addition to anti-platelet autoantibodies, CD8+ T cells have been implicated as a mechanism of platelet destruction. The current evidence for the existence of platelet-specific CD8+ T cells in ITP is inconclusive. The purpose of this review is to summarize the studies that investigated CD8+ T cells in ITP and to review the methods that have been used to detect autoreactive CD8+ T cells in other autoimmune diseases.

Performance characteristics of platelet autoantibody testing for the diagnosis of immune thrombocytopenia using strict clinical criteria.

Misclassification of immune thrombocytopenia (ITP) is common, which might undermine the value of platelet autoantibody testing. We determined the sensitivity and specificity of platelet autoantibody testing using the direct antigen capture assay for anti-glycoprotein (GP) IIb/IIIa or anti-GPIbIX in patients with 'definite ITP', defined as those with a documented treatment response. Sensitivity of platelet autoantiboody testing increased from 48·3% [95% confidence interval (CI) 43·5-53·2] for all ITP patients to 64·7% (95% CI 54·6-73·9) for definite ITP patients. Specificity was unchanged [75·3% (95% CI 67·5-82·1)]. High optical density values (>0·8) improved the specificity of platelet autoantibody testing but lowered sensitivity. In patients with a high pretest probability, platelet autoantibodies can aid in the diagnosis of ITP and may be most prevalent in certain patient subsets.

Serotonin-release assay-positive but platelet factor 4-dependent enzyme-immunoassay negative: HIT or not HIT?

IgG-specific and polyspecific PF4-dependent enzyme-immunoassays (EIAs) have exceptionally high sensitivity (≥99%) for diagnosis of heparin-induced thrombocytopenia (HIT), a drug reaction caused by platelet-activating antibodies detectable by serotonin-release assay (SRA). The IgG-specific EIAs are recommended for screening, as their high sensitivity is accompanied by relatively high specificity vis-à-vis polyspecific EIAs. We investigated the frequency of SRA-positive/EIA-negative (SRA+/EIA-) HIT, prompted by referral to our reference HIT laboratory of serial blood samples from a patient ("index case") with false-negative IgG-specific EIAs. Despite initial clinical suspicion for HIT, repeat negative IgG-specific EIAs prompted heparin resumption, which triggered recurrent thrombocytopenia and near-fatal cardiac arrest, indicating likely post-heparin HIT-associated anaphylactoid reaction. Further investigations revealed a strong-positive SRA, whether performed with heparin alone, PF4 alone, or PF4/heparin, with inhibition by Fc receptor-blocking monoclonal antibody (indicating IgG-mediated platelet activation); however, five different IgG-specific immunoassays yielded primarily negative (or weak-positive) results. To investigate the frequency of SRA+/EIA- HIT, we reviewed the laboratory and clinical features of patients with this serological profile during a 6-year period in which our reference laboratory investigated for HIT using both SRA and IgG-specific EIA. Although ~0.2% of 8546 patients had an SRA+/EIA- profile, further review of 15 such cases indicated clerical/laboratory misclassification or false-positive SRA in all, with no SRA+/EIA- HIT case identified. We conclude that while SRA+/EIA- HIT is possible-as shown by our index case-this clinical picture is exceptionally uncommon. Moreover, the requirement for a positive EIA is a useful quality control maneuver that reduces risk of reporting a false-positive SRA result.

Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and Assembly.

Association of platelet factor 4 (PF4) with heparin is a first step in formation of aggregates implicated in the development of heparin-induced thrombocytopenia (HIT), a potentially fatal immune disorder affecting 1-5% of patients receiving heparin. Despite being a critically important element in HIT etiology, relatively little is known about the specific molecular mechanism of PF4-heparin interactions. This work uses native mass spectrometry to investigate PF4 interactions with relatively short heparin chains (up to decasaccharides). The protein is shown to be remarkably unstable at physiological ionic strength in the absence of polyanions; only monomeric species are observed, and the extent of multiple charging of corresponding ions indicates a partial loss of conformational integrity. The tetramer signal remains at or below the detection threshold in the mass spectra until the solution's ionic strength is elevated well above the physiological level, highlighting the destabilizing role played by electrostatic interactions vis-à-vis quaternary structure of this high-pI protein. The tetramer assembly is dramatically facilitated by relatively short polyanions (synthetic heparin-mimetic pentasaccharide), with the majority of the protein molecules existing in the tetrameric state even at physiological ionic strength. Each tetramer accommodates up to six pentasaccharides, with at least three such ligands required to guarantee the higher-order structure integrity. Similar results are obtained for PF4 association with longer and structurally heterogeneous heparin oligomers (decamers). These longer polyanions can also induce PF4 dimer assembly when bound to the protein in relatively low numbers, lending support to a model of PF4/heparin interaction in which the latter wraps around the protein, making contacts with multiple subunits. Taken together, these results provide a more nuanced picture of PF4-glycosaminoglycan interactions leading to complex formation. This work also advocates for a greater utilization of native mass spectrometry in elucidating molecular mechanisms underlying HIT, as well as other physiological processes driven by electrostatic interactions.

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor.

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.

Serotonin-release assay-negative heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Pathogenic HIT antibodies can be detected by the serotonin-release assay (SRA), a platelet activation test. We have regarded the SRA performed in our medical community ("McMaster" SRA) as having high sensitivity and specificity. Recently, the concept of "SRA-negative HIT" has been proposed for enzyme-immunoassay (EIA)-positive/SRA-negative patients with a HIT-compatible clinical picture, who test positive in a PF4-enhanced platelet activation assay. After identifying an index case of SRA-negative HIT, we estimated the frequency of this condition by performing the "PF4-SRA" (modified SRA using high concentrations of added PF4 rather than heparin) in EIA-positive patients from a cohort study evaluating clinical and laboratory diagnosis of HIT. We defined SRA-negative HIT as patients meeting three criteria: clinical picture compatible with HIT (4Ts ≥ 4 points); EIA-positive (≥1.00 units); and PF4-SRA-positive. Among 430 patients, 35 were EIA-positive/SRA-positive and 27 were EIA-positive/SRA-negative. Among these 27 SRA-negative patients, three were found to have subthreshold levels of platelet-activating antibodies by PF4-SRA, of whom one met clinical criteria for SRA-negative HIT. Thus, based on identifying one patient with SRA-negative HIT within a cohort study that found 35 SRA-positive HIT patients, we estimate the sensitivity of the McMaster SRA for diagnosis of HIT to be 35/36 (97.2%; 95% CI, 85.8-99.9%). Although the McMaster SRA is highly sensitive for HIT, occasional SRA-negative but EIA-positive patients strongly suspected of having HIT can have this diagnosis supported by a PF4-enhanced activation assay such as the PF4-SRA.

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia.

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = -0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by 14C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure.

BACKGROUND: Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis. METHODS: Using flow cytometry and/or fluorescence microscopy, we analyzed desialylation patterns and eryptotic alterations in erythrocytes exposed to Clostridium perfringens-derived neuraminidase. RESULTS: Exogenous bacterial neuraminidase significantly augmented membrane PS exposure and cytosolic Ca2+ levels in a dose- and time-dependent manner. Neuraminidase treatment significantly reduced fluorescence-tagged agglutinin binding, an effect temporally preceding the increase in PS externalization. Neuraminidase-induced PS exposure was significantly curtailed by pretreatment with the pan-sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. Neuraminidase treatment further induced hemolysis but did not significantly impact erythrocyte volume, ceramide abundance, or the generation of reactive oxygen species. CONCLUSION: Collectively, our data reveal that alteration of erythrocyte sialylation status by bacterial neuraminidase favors eryptotic cell death, an effect potentially contributing to reduced erythrocyte lifespan and anemia in sepsis.