Mortimer Poncz

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

FLI1 level during megakaryopoiesis affects thrombopoiesis and platelet biology

Friend leukemia virus integration 1 (FLI1), a critical transcription factor (TF) during megakaryocyte differentiation, is among genes hemizygously deleted in Jacobsen syndrome, resulting in a macrothrombocytopenia termed Paris-Trousseau syndrome (PTSx). Recently, heterozygote human FLI1 mutations have been ascribed to cause thrombocytopenia. We studied induced-pluripotent stem cell (iPSC)-derived megakaryocytes (iMegs) to better understand these clinical disorders, beginning with iPSCs generated from a patient with PTSx and iPSCs from a control line with a targeted heterozygous FLI1 knockout (FLI1+/-). PTSx and FLI1+/- iMegs replicate many of the described megakaryocyte/platelet features, including a decrease in iMeg yield and fewer platelets released per iMeg. Platelets released in vivo from infusion of these iMegs had poor half-lives and functionality. We noted that the closely linked E26 transformation-specific proto-oncogene 1 (ETS1) is overexpressed in these FLI1-deficient iMegs, suggesting FLI1 negatively regulates ETS1 in megakaryopoiesis. Finally, we examined whether FLI1 overexpression would affect megakaryopoiesis and thrombopoiesis. We found increased yield of noninjured, in vitro iMeg yield and increased in vivo yield, half-life, and functionality of released platelets. These studies confirm FLI1 heterozygosity results in pleiotropic defects similar to those noted with other critical megakaryocyte-specific TFs; however, unlike those TFs, FLI1 overexpression improved yield and functionality.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.

Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL‐VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody‐mediated animal model would be useful for their development. Previously, TTP patient anti‐ADAMTS13 single‐chain variable‐region fragments (scFvs) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFvs is explored in vivo by transfecting mice with inhibitory antibody cDNA.

Platelet-Specific Chemokines Contribute to the Pathogenesis of Acute Lung Injury

&NA; Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet‐derived chemokines in the pathogenesis of ALI using Cxcl7−/− and Cxcl4−/− knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N‐terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7−/− mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4−/− mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet‐derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

Endothelial antigen assembly leads to thrombotic complications in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (Fc&ggr;RIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

Role of monocytes and endothelial cells in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder characterised by thrombocytopenia and thrombosis. The mechanisms leading to platelet destruction are complex and the thrombotic complications of HIT appear to be due to multiple different intravascular targets. The dual binding of HIT antibodies to platelet surface PF4/GAG complexes and to FcγRIIA likely leads to both platelet clearance and to their direct activation. Monocytes and endothelial cells bind PF4 with higher avidity than platelets and are more resistant to competitive removal of surface-bound PF4 in the presence of heparin. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface on these cells leads to their activation and increased procoagulant activity. Binding of higher levels of PF4 released from activated platelets to the endothelium may lead to changes of the anticoagulant properties of the glycocalyx and target the endothelial cells for HIT antibodies. Pathogenic antibodies bound to endothelial cells further promote prothrombotic conditions by a mechanism that is independent of FcγR activation, yet not completely understood. A more detailed understanding of the role of monocytes and endothelium may identify new targets for intervention to mitigate the risk of thrombosis with less impact on systemic haemostasis than current approaches to treatment for this serious disorder.

Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis.

Abstract Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.

Serpin functions in host-pathogen interactions

Serpins are a broadly distributed superfamily of protease inhibitors that are present in all kingdoms of life. The acronym, serpin, is derived from their function as potent serine proteases inhibitors. Early studies of serpins focused on their functions in haemostasis since modulating serine proteases activities are essential for coagulation. Additional research has revealed that serpins function in infection and inflammation, by modulating serine and cysteine proteases activities. The aim of this review is to summarize the accumulating findings and current understanding of the functions of serpins in host-pathogen interactions, serving as host defense proteins as well as pathogenic factors. We also discuss the potential crosstalk between host and pathogen serpins. We anticipate that future research will elucidate the therapeutic value of this novel target.

2-O, 3-O desulfated heparin mitigates murine chemotherapy- and radiation-induced thrombocytopenia

Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4s effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.

Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

In vitro-grown megakaryocytes for generating platelets may have value in meeting the increasing demand for platelet transfusions. Remaining challenges have included the poor yield and quality of in vitro-generated platelets. We have shown that infusing megakaryocytes leads to intrapulmonary release of functional platelets. A Src kinase inhibitor (SU6656), a Rho-associated kinase inhibitor (Y27632), and an aurora B kinase inhibitor (AZD1152) have been shown to increase megakaryocyte ploidy and in vitro proplatelet release. We now tested whether megakaryocytes generated from CD34+ hematopoietic cells in the presence of these inhibitors could enhance functional platelet yield following megakaryocyte infusion. As expected, all inhibitors increased megakaryocyte ploidy, size, and granularity, but these inhibitors differed in whether they injured terminal megakaryocytes: SU6656 was protective, whereas Y27632 and AZD1152 increased injury. Upon infusion, inhibitor-treated megakaryocytes released threefold to ninefold more platelets per initial noninjured megakaryocyte relative to control, but only SU6656-treated megakaryocytes had a significant increase in platelet yield when calculated based on the number of initial CD34+ cells; this was fourfold over nontreated megakaryocytes. The released platelets from drug-treated, but healthy, megakaryocytes contained similar percentages of young, uninjured platelets that robustly responded to agonists and were well incorporated into a growing thrombus in vivo as controls. These studies suggest that drug screens that select megakaryocytes with enhanced ploidy, cell size, and granularity may include a subset of drugs that can enhance the yield and function of platelets, and may have clinical application for ex vivo-generated megakaryocytes and platelet transfusion.