Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients.

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index - EI) as a function of pO2 . Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3-diphosphoglycerate showed a significant correlation (respectively R = -0.993 and R = 0.980) with Point of Sickling (PoS5% ), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.

Anti-ß2-glycoprotein I and anti-prothrombin antibodies cause lupus anticoagulant through different mechanisms of action.

BACKGROUND: The presence of lupus anticoagulant (LA) is an independent risk factor for thrombosis. This laboratory phenomenon is detected as a phospholipid-dependent prolongation of the clotting time and is caused by autoantibodies against beta2-glycoprotein I (ß2GPI) or prothrombin. How these autoantibodies cause LA is unclear. OBJECTIVE: To elucidate how anti-ß2GPI and anti-prothrombin antibodies cause the LA phenomenon. METHODS: The effects of monoclonal anti-ß2GPI and anti-prothrombin antibodies on coagulation were analyzed in plasma and with purified coagulation factors. RESULTS: Detection of LA caused by anti-ß2GPI or anti-prothrombin antibodies required the presence of the procofactor factor V (FV) in plasma. LA effect disappeared when FV was replaced by activated FV (FVa), both in a model system and in patient plasma, although differences between anti-ß2GPI and anti-prothrombin antibodies were observed. Further exploration of the effects of the antibodies on coagulation showed that the anti-ß2GPI antibody attenuated FV activation by activated faxtor X (FXa), whereas the anti-prothrombin antibody did not. Binding studies showed that ß2GPI--antibody complexes directly interacted with FV with high affinity. Anti-prothrombin complexes caused the LA phenomenon through competition for phospholipid binding sites with coagulation factors as reduced FXa binding to lipospheres was observed with flow cytometry in the presence of these antibodies. CONCLUSION: Anti-ß2GPI and anti-prothrombin antibodies cause LA through different mechanisms of action: While anti-ß2GPI antibodies interfere with FV activation by FXa through a direct interaction with FV, anti-prothrombin antibodies compete with FXa for phospholipid binding sites. These data provide leads for understanding the paradoxical association between thrombosis and a prolonged clotting time in the antiphospholipid syndrome.

Thrombotic Events in COVID-19 Are Associated With a Lower Use of Prophylactic Anticoagulation Before Hospitalization and Followed by Decreases in Platelet Reactivity.

Background: Coronavirus disease of 2019 (COVID-19) is associated with a prothrombotic state and a high incidence of thrombotic event(s) (TE). Objectives: To study platelet reactivity in hospitalized COVID-19 patients and determine a possible association with the clinical outcomes thrombosis and all-cause mortality. Methods: Seventy nine hospitalized COVID-19 patients were enrolled in this retrospective cohort study and provided blood samples in which platelet reactivity in response to stimulation with ADP and TRAP-6 was determined using flow cytometry. Clinical outcomes included thrombotic events, and all-cause mortality. Results: The incidence of TE in this study was 28% and all-cause mortality 16%. Patients that developed a TE were younger than patients that did not develop a TE [median age of 55 vs. 70 years; adjusted odds ratio (AOR) = 0.96 per 1 year of age, 95% confidence interval (CI) 0.92-1.00; p = 0.041]. Furthermore, patients using preexisting thromboprophylaxis were less likely to develop a thrombotic complication than patients that were not (18 vs. 54%; AOR = 0.19, 95% CI 0.04-0.84; p = 0.029). Conversely, having asthma strongly increased the risk on TE development (AOR = 6.2, 95% CI 1.15-33.7; p = 0.034). No significant differences in baseline P-selectin expression or platelet reactivity were observed between the COVID-19 positive patients (n = 79) and COVID-19 negative hospitalized control patients (n = 21), nor between COVID-19 positive survivors or non-survivors. However, patients showed decreased platelet reactivity in response to TRAP-6 following TE development. Conclusion: We observed an association between the use of preexisting thromboprophylaxis and a decreased risk of TE during COVID-19. This suggests that these therapies are beneficial for coping with COVID-19 associated hypercoagulability. This highlights the importance of patient therapy adherence. We observed lowered platelet reactivity after the development of TE, which might be attributed to platelet desensitization during thromboinflammation.

Erythrocytosis associated with a novel missense mutation in the HIF2A gene.

The ERYTHROPOIETIN (EPO) gene is regulated by the transcription factor Hypoxia Inducible Factor-alpha (HIF-alpha). In this pathway, Prolyl Hydroxylase Domain protein 2 (PHD2) hydroxylates two prolyl residues in HIF-alpha, which in turn promotes HIF-alpha degradation by the von Hippel Lindau (VHL) protein. Evidence that HIF-2alpha is the important isoform for EPO regulation in humans comes from the recent observation that mutations in the HIF2A gene are associated with cases of erythrocytosis. We report here a new erythrocytosis-associated mutation, p.Asp539Glu, in the HIF2A gene. Similar to all reported cases, the affected residue is in close vicinity and C-terminal to the primary hydroxylation site in HIF-2alpha, Pro531. This mutation, however, is notable in producing a rather subtle amino acid substitution. Nonetheless, we find that this mutation compromises binding of HIF-2alpha to both PHD2 and VHL, and we propose that this mutation is the cause of erythrocytosis in this individual.

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: structural implications of amino acid substitutions in PK.

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK-deficient patients from 35 unrelated families. Twenty-nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in-frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N-terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three-dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme-linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype-to-phenotype correlation in PK deficiency as well as the enzyme's structure and function.

Ex vivo analysis of aberrant splicing induced by two donor site mutations in PKLR of a patient with severe pyruvate kinase deficiency.

Two single-nucleotide substitutions in PKLR constituted the molecular basis underlying pyruvate kinase (PK) deficiency in a patient with severe haemolytic anaemia. One novel mutation, IVS5+1G>A, abolished the intron 5 donor splice site. The other mutation, c.1436G>A, altered the intron 10 donor splice site consensus sequence and, moreover, encoded an R479H substitution. We studied the effects on PKLR pre-mRNA processing, using ex vivo-produced nucleated erythroid cells from the patient. Abolition of the intron 5 splice site initiated two events in the majority of transcripts: skipping of exon 5 or, surprisingly, simultaneous skipping of exon 5 and 6 (Delta5,6). Subcellular localization of transcripts suggested that no functional protein was produced by the IVS5+1A allele. The unusual Delta5,6 transcript suggests that efficient inclusion of exon 6 in wild-type PKLR mRNA depends on the presence of splice-enhancing elements in exon 5. The c.1436G>A mutation caused skipping of exon 10 but was mainly associated with a severe reduction in transcripts although these were, in general, normally processed. Accordingly, low amounts of PK were detected in nucleated erythroid cells of the patient, thus correlating with the patient's PK-deficient phenotype. Finally, several low-abundant transcripts were detected that represent the first examples of "leaky-splicing" in PKLR.