Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.

Flow cytometry detection of neutrophil NETs markers is feasible in whole blood and correlates with serum markers of NETs. We propose the flow cytometry detection of MPO/H3cit positive neutrophils and serum dsDNA as simple methods to quantify cellular and extracellular NET markers in patients with thrombosis and sepsis.

Key Points• GPR84 simultaneously augments b-catenin signaling and an oncogenic transcription program essential for establishment of MLL.• Our study demonstrates a strong dependence of hematopoietic stem cell-derived MLL leukemic cells on GPR84 for disease maintenance in vivo.b-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of b-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel b-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active b-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and b-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant b-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/b-catenin signaling axis may represent a novel therapeutic strategy for AML. (Blood. 2014;124(22):3284-3294) IntroductionThe ability for self-renewal, migration/invasion, and drug resistance are fundamental properties of malignant stem cells that drive both disease progression and relapse. Identification of pathways and their molecular components essential for the regulation of abnormally acquired stem cell-like properties is a prerequisite for understanding the underlying mechanisms of oncogenesis and designing effective anticancer therapeutic strategies. We and others have previously shown that mixed-lineage leukemia (MLL) fusion proteins can aberrantly activate Wnt/b-catenin signaling in hematopoietic stem cells (HSCs) or more differentiated granulocyte-macrophage progenitors (GMPs) for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia subtype M5 (AML-M5) 1,2 ; however, the mechanisms involved remain obscure. It has also been noted that activation of b-catenin is observed in tumors without clear mutations in major components of this pathway or an increase in Wnt signaling.3 This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of b-catenin.G protein-coupled receptors (GPCRs), the largest family of cell-surface molecules with key roles in transmitting signals to downstream effectors, ...

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Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34 cells under suitable conditions. The protocol detailed here describes isolation of CD34 cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Heparin-induced thrombocytopenia (HIT) is associated with severe and potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and NADPH oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes induced ROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant NETs and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis and thrombosis using human blood. Inhibition of NOX2 with diphenyleneiodonium chloride or GSK2795039 abrogated HIT-induced thrombi in vivo using FcγRIIa+/hPF4+ transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils were also confirmed using fresh blood from patients with active HIT. Our findings show that ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT. This enhances our understanding of the processes driving thrombosis in HIT and identifies NOX2 as a potential new therapeutic target for antithrombotic treatment for HIT.

Background The notable discrepancy between platelet count and bleeding manifestations in immune thrombocytopenia (ITP) patients with acquired Glanzmann thrombasthenia (GT) has been described. Objectives We aimed to examine the mechanisms responsible for thrombocytopenia and the bleeding phenotype in a patient with acquired GT. Patient, methods, and results A patient with primary ITP underwent splenectomy due to steroid intolerance. Despite platelet count normalization, bleeding continued. Platelet aggregometry was abnormal with all agonists except for ristocetin. Flow cytometry demonstrated the presence of antiplatelet antibody, which caused dose‐dependent inhibition of fibrinogen and PAC‐1 binding, induction of neuraminidase‐1 expression as well as platelet desialylation in donor platelets. Indirect monoclonal antibody immobilization of platelet specific antigen assay (MAIPA) confirmed specificity to αIIbβ3 only, corroborated by binding on Chinese hamster ovary (CHO) cells expressing human glycoprotein αIIbβ3 but not GP Ib/IX. Both desialylation and neuraminidase expression were observed with plasma adsorbed on Ib/IX CHO cells and with the immunoglobulin G (IgG) fraction. Desialylation was inhibited in the presence of anti‐Fc‐gamma receptor IIa (FcγRIIa) antibody. A nonobese diabetic/severe combined immunodeficient ITP murine model was established, which showed rapid hepatic donor platelet clearance in the presence of patient IgG. Treatment of mice with the neuraminidase inhibitor oseltamivir significantly reduced antibody‐induced platelet destruction. Conclusions We report the first case of a patient with acquired GT due to ITP with FcγRIIa mediated platelet desialylation, independent of platelet activation. Treatment with neuraminidase inhibitor may prevent platelet clearance by anti‐αIIbβ3 antibodies.