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.

Small ubiquitin-related modifier (SUMO) is a protein moiety that is

Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Here we describe two additional Ikaros family proteins, Eos and Pegasus. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. Our results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues.

hThe CACCC-box binding protein erythroid Krüppel-like factor (EKLF/KLF1) is a master regulator that directs the expression of many important erythroid genes. We have previously shown that EKLF drives transcription of the gene for a second KLF, basic Krüppel-like factor, or KLF3. We have now tested the in vivo role of KLF3 in erythroid cells by examining Klf3 knockout mice. KLF3-deficient adults exhibit a mild compensated anemia, including enlarged spleens, increased red pulp, and a higher percentage of erythroid progenitors, together with elevated reticulocytes and abnormal erythrocytes in the peripheral blood. Impaired erythroid maturation is also observed in the fetal liver. We have found that KLF3 levels rise as erythroid cells mature to become TER119 ؉ . Consistent with this, microarray analysis of both TER119 ؊ and TER119 ؉ erythroid populations revealed that KLF3 is most critical at the later stages of erythroid maturation and is indeed primarily a transcriptional repressor. Notably, many of the genes repressed by KLF3 are also known to be activated by EKLF. However, the majority of these are not currently recognized as erythroid-cell-specific genes. These results reveal the molecular and physiological function of KLF3, defining it as a feedback repressor that counters the activity of EKLF at selected target genes to achieve normal erythropoiesis.