PF4 activates the c-Mpl-Jak2 pathway in platelets.

ABSTRACT: Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet α-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets. This leads to the activation of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5, leading to platelet aggregation. Inhibition of the c-Mpl-JAK2 pathway inhibits platelet aggregation to PF4, VITT sera, and the combination of PF4 and IgG isolated from VITT patient plasma. The results support a model in which PF4-based immune complexes activate platelets through binding of the Fc domain to FcγRIIA and PF4 to c-Mpl.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Dual pathology: paraneoplastic sarcoidosis emerging at the time of progression from smouldering to symptomatic myeloma.

Sarcoidosis is a systemic disease of unknown aetiology, which is diagnosed based on the presence of non-caseating granulomas on histology. The occurrence of sarcoidosis or a sarcoidosis-like reaction with malignancy has been recognised for several years. Although it has been established that there is an increased risk of lymphoproliferative disorder with sarcoidosis, the association between multiple myeloma and sarcoidosis has rarely been reported. Here, we report the case of woman in her mid-50s with an established diagnosis of smouldering myeloma, who presented with gradually worsening shortness of breath and fatigue after 15 months of active observation. A CT scan of her thorax showed mediastinal lymphadenopathy and the nodes were metabolically active on positron emission tomography CT scan. Endobronchial ultrasound with transbronchial needle aspiration confirmed the diagnosis of sarcoidosis. Further evaluation showed preserved lung function on spirometry. Blood analysis showed a simultaneous rise in the serum lambda-free light chain level from 377 mg/L at initial diagnosis up to 807 mg/L with the kappa/lambda ratio falling to 0.012. Repeat bone marrow aspirate and trephine biopsy showed a 15%-20% infiltrate of lambda light chain-restricted plasma cells with aberrant cyclin D1 expression and abundant sarcoid-like non-necrotising giant cell granulomata. Thus, a diagnosis of paraneoplastic sarcoidosis was established.

Blood Coagulation and Beyond: Position Paper from the Fourth Maastricht Consensus Conference on Thrombosis.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

UK media reporting of NICE recommendation of crizanlizumab for patients with sickle cell disease.

Crizanlizumab was recommended for use in patients with sickle cell disease in the UK in October 2021 and received widespread media coverage. Accuracy of reporting is paramount in building trust with this group of patients who are often wary of the medical profession. We carried out an analysis of internet-based news articles and applied a validated scoring system to assess quality. 21 articles from 19 media organisations were identified. 71% of articles stated unproven benefits of the drug and only 14% were of satisfactory quality. The former was largely due to quoting of two NHS England press releases. Overstating of drug efficacy may be detrimental to the need to address healthcare inequalities.

Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia: Who, When, and How?

Although the majority of patients with acute myeloid leukemia (AML) treated with intensive chemotherapy achieve a complete remission (CR), many are destined to relapse if treated with intensive chemotherapy alone. Allogeneic stem cell transplant (allo-SCT) represents a pivotally important treatment strategy in fit adults with AML because of its augmented anti-leukemic activity consequent upon dose intensification and the genesis of a potent graft-versus-leukemia effect. Increased donor availability coupled with the advent of reduced intensity conditioning (RIC) regimens has dramatically increased transplant access and consequently allo-SCT is now a key component of the treatment algorithm in both patients with AML in first CR (CR1) and advanced disease. Although transplant related mortality has fallen steadily over recent decades there has been no real progress in reducing the risk of disease relapse which remains the major cause of transplant failure and represents a major area of unmet need. A number of therapeutic approaches with the potential to reduce disease relapse, including advances in induction chemotherapy, the development of novel conditioning regimens and the emergence of the concept of post-transplant maintenance, are currently under development. Furthermore, the use of genetics and measurable residual disease technology in disease assessment has improved the identification of patients who are likely to benefit from an allo-SCT which now represents an increasingly personalized therapy. Future progress in optimizing transplant outcome will be dependent on the successful delivery by the international transplant community of randomized prospective clinical trials which permit examination of current and future transplant therapies with the same degree of rigor as is routinely adopted for non-transplant therapies.

Treatment of paediatric eczema with narrowband ultraviolet light B therapy.

BACKGROUND: Narrowband ultraviolet B phototherapy (nbUVB) is a well-established, well-tolerated and efficacious treatment for eczema. There is a distinct lack of literature surrounding the therapeutic use of nbUVB in eczema in children and especially in children with higher skin phototypes (III to VI). METHODS: We undertook a retrospective review of children aged 18 years and under with eczema who had undergone nbUVB in our department between 1 January 2011 and 31 December 2017. Abstracted data included sex, age, skin phototype, severity as graded by a paediatric dermatologist, cumulative dose, response to treatment and subsequent remission. RESULTS: In total, 60 children had nbUVB. Of those, 56 had more than 10 nbUVB exposures. Complete or near-complete clearance was achieved in 31 children (52%). Of those, 24 (77%) had a skin phototype of III or greater. Clinical remission rates of these patients were 100%, 87%, 57% and 52% at 0, 3, 6 and 12 months, respectively. Seventeen patients (28%) suffered side effects. Most commonly these were mild side effects such as erythema and xerosis. CONCLUSION: We have demonstrated that nbUVB is a safe, well-tolerated and efficacious form of treatment for children with atopic eczema. We have shown it to be effective in those with skin phototypes greater than III and shown that they are a group that may derive greater long-term efficacy. In clinical practice, preference for nbUVB as second-line treatment, over oral systemics, should always be considered.

Biomarkers in Human Anaphylaxis: A Critical Appraisal of Current Evidence and Perspectives.

Anaphylaxis is a type I hypersensitivity reaction that is potentially fatal if not promptly treated. It is a clinical diagnosis, although measurement of serial serum total mast cell tryptase (MCT) is gold standard and may help differentiate anaphylaxis from its mimics. The performance characteristics of MCT assays in anaphylaxis has been variable in previous studies, due to multiple factors including differences in the definition of anaphylaxis, methods of MCT interpretation, clinical setting of anaphylaxis, causative agents, and timing of blood sample. An international consensus equation for MCT to interpret mast cell activation has been proposed and recently validated in the context of peri-operative anaphylaxis during general anesthesia. There has been an interest in the detection of newer biomarkers in anaphylaxis including platelet activation factor (PAF), chymase, carboxypeptidase A3, dipeptidyl peptidase I (DPPI), basogranulin, and CCL-2. The key determinants of an ideal biomarker in anaphylaxis are half-life, sample handling and processing requirements, and cost. There may be a role for metabolomics and systems biology in the exploration of novel biomarkers in anaphylaxis. Future studies applying these approaches might provide greater insight into factors determining severity, clinical risk stratification, identification of mast cell disorders and improving our understanding of this relatively complex acute immunological condition. Post mortem MCT evaluation is used in Forensic Medicine during autopsy for cases involving sudden death or suspected anaphylaxis. Interpretation of post mortem MCT is challenging since there is limited published evidence and the test is confounded by multiple variables largely linked to putrefaction and site of sampling. Thus, there is no international consensus on a reference range. In this state of the art review, we will focus on the practical challenges in the laboratory diagnosis of anaphylaxis and critically appraise (a) performance characteristics of MCT in anaphylaxis in different clinical scenarios (b) the role for novel biomarkers and (c) post mortem MCT and its role in fatal anaphylaxis.

Clinical indications and scanning protocols for chest CT in children with cystic fibrosis: a survey of UK tertiary centres.

OBJECTIVES: Chest CT is increasingly used to monitor disease progression in children with cystic fibrosis (CF) but there is no national guideline regarding its use. Our objective was to assess the indications for undertaking chest CT and the protocols used to obtain scans. DESIGN SETTING AND PARTICIPANTS: An electronic questionnaire was developed to assess clinicians views on chest CT in children with CF. It included general questions on perceived benefits and specific questions about its role in five clinical scenarios. It was sent to the clinical lead in 27 UK paediatric CF centres. A separate questionnaire was developed to collect the technical details of chest CT in children with CF. It was sent to the superintendent radiographer at each of the 27 centres. RESULTS: Responses were obtained from 27 (100%) clinical leads and 22 (81%) superintendent radiographers. 93% clinicians reported chest CT useful in monitoring disease progression and 70% said it frequently altered management. Only 5 (19%) undertook routine scans. To aid diagnosis, 81% performed chest CT in non-tuberculous mycobacterial disease and 15% in allergic bronchopulmonary aspergillosis. There was wide variation in the perceived need for and/or timing of chest CT in children with reduced lung function with no benefit from intravenous antibiotics, new cystic changes on chest X-ray, and lobar collapse. The radiographers reported using a mixture of helical (volumetric) and axial scans depending on the clinical question, the age and the cooperation of the child. When indicated, 6 (27%) used sedation and 16 (73%) general anaesthetic. Only 1 (5%) used intravenous contrast routinely and 3 (14%) obtained expiratory images routinely. CONCLUSIONS: There is marked variation in the use of chest CT in children with CF and in the scan protocols. The lack of a national guideline is likely to be contributing to this lack of standardisation.

Anaphylaxis and Clinical Utility of Real-World Measurement of Acute Serum Tryptase in UK Emergency Departments.

BACKGROUND: British guidelines recommend that serial acute serum tryptase measurements be checked in all adults and a subset of children presenting with anaphylaxis. This is the first study reporting the clinical utility of acute serum tryptase in a "real-world" emergency department (ED) setting following the publication of the World Allergy Organization (WAO) criteria for anaphylaxis. OBJECTIVES: To (1) assess sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of acute serum tryptase in anaphylaxis; (b) determine factors associated with higher acute serum tryptase levels; and (c) audit compliance of acute serum tryptase measurement in the ED. METHODS: The methods used were retrospective electronic search for ED admissions to 3 acute care hospitals in Birmingham, UK, with anaphylaxis in 2012 using wide search terms followed by scrutiny of electronic clinical records and application of the WAO diagnostic criteria for anaphylaxis. Patients with an acute serum tryptase measurement were included in the analysis. RESULTS: Acute serum tryptase level was measured in 141 of 426 (33.1%) cases. Mean time from the onset of symptoms to the measurement of acute serum tryptase level was 4 hours 42 minutes (SD ± 05:03 hours) and no patients had serial measurements conforming to British guidelines. Acute serum tryptase level of more than 12.4 ng/mL (75th centile) was associated with a sensitivity, specificity, PPV, and NPV of 28%, 88%, 0.93, and 0.17, respectively. Multiple regression analysis showed that male sex (odds ratio, 2.66; P = .003) and hypotension (odds ratio, 7.08; P = .001) predicted higher acute serum tryptase level. CONCLUSIONS: An acute serum tryptase level of more than 12.4 ng/mL in an ED setting carries high PPV and specificity, but poor sensitivity and NPV.

Tolerability and Clinical Activity of Post-Transplantation Azacitidine in Patients Allografted for Acute Myeloid Leukemia Treated on the RICAZA Trial.

Disease relapse is the major causes of treatment failure after allogeneic stem cell transplantation (SCT) in patients with acute myeloid leukemia (AML). As well as demonstrating significant clinical activity in AML, azacitidine (AZA) upregulates putative tumor antigens, inducing a CD8(+) T cell response with the potential to augment a graft-versus-leukemia effect. We, therefore, studied the feasibility and clinical sequelae of the administration of AZA during the first year after transplantation in 51 patients with AML undergoing allogeneic SCT. Fourteen patients did not commence AZA either because of transplantation complications or withdrawal of consent. Thirty-seven patients commenced AZA at a median of 54 days (range, 40 to 194 days) after transplantation, which was well tolerated in the majority of patients. Thirty-one patients completed 3 or more cycles of AZA. Sixteen patients relapsed at a median time of 8 months after transplantation. No patient developed extensive chronic graft-versus-host disease. The induction of a post-transplantation CD8(+) T cell response to 1 or more tumor-specific peptides was studied in 28 patients. Induction of a CD8(+) T cell response was associated with a reduced risk of disease relapse (hazard ratio [HR], .30; 95% confidence interval [CI], .10 to .85; P = .02) and improved relapse-free survival (HR, .29; 95% CI, .10 to .83; P = .02) taking into account death as a competing risk. In conclusion, AZA is well tolerated after transplantation and appears to have the capacity to reduce the relapse risk in patients who demonstrate a CD8(+) T cell response to tumor antigens. These observations require confirmation in a prospective clinical trial.